dance research paper sample

The  is a peer-reviewed premiere publication for dance scholarship of international reach and includes articles, book reviews, and lists books received.

 is published three times per year by Cambridge University Press.  Published articles address dance history, theory, politics, ethnography, and intersections with cultural, gender, critical race, and diasporic studies among others.  is committed to cross-disciplinary research with a dance perspective.  Contributions for publication consideration are open to both members and nonmembers of DSA, and will be accepted any time. 

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(excerpt from Editors' Note) 

This issue converges around the theme of the trace and its role in the scholarly research process. Our interdisciplinary offerings here situate dance as the center of inquiry and discovery. Each of the five revelatory articles illuminates the novel application of a basic research method in humanistic studies, which is discovering a pattern and identifying a trace—the means of tracking that pattern over sets of data and/or the materials under investigation. In short, the articles we have assembled offer striking examples of how identifying something to trace can connect scholarly practice with evidentiary praxis. All the articles in this issue tangle with precedents, conventions, and prevailing notions—in short, cultural work and workings that hold structural and cognitive (intellectual) systems in place. Our authors organize their research around identifying and tracking such stabilizing factors as a way of unveiling contradictions and entanglements. (Rebekah J. Kowal)

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Dance Research

Dance Research  is the Society of Dance Research’s peer-reviewed journal, which is published twice a year by  Edinburgh University Press  and contains articles and book reviews reflecting the range of our members’ interests. It is edited and published in Britain with the assistance of a distinguished group of editorial consultants based in Europe and the USA.

Dance Research  is essential reading for those involved in the study and practice of dance. The journal provides an international forum for the presentation and discussion of contemporary dance research and contains a section of comprehensive book and journal reviews.

Dance Research  is addressed to scholars and practitioners working within the many disciplines which constitute Dance Studies, such as but not limited to:

–         Studies of Dancers –         Ballet –         Choreography –         Dance and Movement –         Dance and Music –         Dance History and Social History –         Folk Dance –         Dance Science –         Performance Technique –         Sociology of Dance –         Anthropology of Dance –         Philosophy and Dance

Members of the SDR receive a subscription to the journal as part of their membership. For more information please visit the  Join Us  page and the Edinburgh University Press  website . Sign up for Table of Contents alerts here .

Dance 260: Introduction to Dance: Dance Topics

• About Dance 260
• Research Help
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Other Guides that May Help Your Research

What to write about?

You may be interested in discussing a particular form or genre of dance in your research paper. You can combine the discussion of a form or genre with a specific time, place or person.

Examples of forms and genres:

• Click here to see a longer list

Sample research questions:What are the differences and similarities between disco style in the 1970s and disco revival in the 2000s? How was the career of Sammy Davis Jr. important to the evolution of tap dance style in America?

You may wish to develop your topic with a specific style in mind, such as ballroom or ballet, or specific techniques such as en pointe or gancho. You can combine the discussion of a style or technique with a specific time, place or person.

Examples of styles and techniques:

• aplomb (ballet)
• enpointe (ballet)
• bourree (ballet)
• Guapacha timing (ballroom)
• Promenade position (ballroom)
• Headspin (hip-hop)
• The worm (hip-hop)
• jazz piroutte (turn)
• grand jete (leap)
• Graham (Contemporary dance technique)

Sample research questions: What are differences and similarities in the swing dance styles on the east and west coasts? What are the differences and similarities of the release technique as taught by (person) and (person)?

If your interest is dance history, styles and periods are central concepts to consider. You may wish to use historical time periods such as Baroque, Medieval, and Renaissance as organizing concepts. Sometimes specific groups of dancers are associated with movements or genres within a specific time period.

Examples of periods and styles:

• Renaissance
• English Regency
• Polka craze (mid 19th century)
• Roaring twenties
• Swing (30's and 40's)
• Contemporary

Sample research questions: Although they derive from quite different cultures, how are the dances of Japan and Afghanistan similar?  Explore the relationship between superstitions and fears and the dances of a particular culture.

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Dance Analysis: Social and Cultural Context Research Paper

• To find inspiration for your paper and overcome writer’s block
• As a source of information (ensure proper referencing)
• As a template for you assignment

Dance is a form of art that is represented in form of performance in which a person moves rhythmically to some musical tone in order to communicate some latent meaning to the audience. It is a way of exploring the world, understanding its ideas and emotions that are elicited by unique body movement patterns. It comprises of elements, such as time, space, force, and shape that can be viewed in both theory and practice. However, the analysis of dance mostly depends on the social and cultural context of performance.

A study of dance involves connection of ideas between the past and the present with an aim of improving the quality of performance in regards to other social practices that arise from cultural beliefs. Besides, dance is important in the modern society because one gets inspirations from the legendary past that helps people improve their compositions. The latter creates awareness, and also offers a sense of belonging to the whole humanity.

Dance analysis is a very important aspect of studying because it enables one to observe the movements that he/she makes in the current dancing styles, and also provides several solutions to challenges arising. For instance, it assists one in appreciating the fact that dance can be used to improve people’s unique sense of identity by offering enough physical exercises. People also benefit from the aesthetic appeal through the use of different props.

In the African continent, dance is ceremonial, and, thus, plays a major role in society since it helps in boosting productivity alongside molding expertise skills and competences among different social groups. This kind of benefit was also realized during the Renaissance period.

On the other hand, historians view dance as a form of art through which rituals are passed during rites of passages. For instance, in the times of ancient civilizations (such as the age of Absolutionism, for example,), most people considered dancing as an important element in ritual ceremonies.

This was also evident in Egypt whereby women would dance at funerals to express their grief. In addition, their paintings created an outward appeal of dancing as a form of entertainment. Moreover, there are some dances, which existed in the Middle Ages that are evident through different works of literature.

However, it is pertinent to observe that quite a number of dances that originated from Spain replicate the influence of age. Later on, a lot of dances emerged in different eras even as their significance continued to change. In the mid 19 th century, dance became popular among different societies.

Popularity continued to grow to the late 19 th century, although it was practiced by a small number of people because there were some changes that created some difficulty in practicing new styles of dancing. Hence, only a few people were interested in dancing.

However, the art of dance gained momentum towards the close of 1920s as it was highly appreciated by the youth. They embraced it with great enthusiasm. Dancing at that time was equal to real life experiences in the society. Myriads of dancing styles were shared across the board. These dances were also aimed at presenting the inner self instead of borrowing movements from other cultures that had established themselves.

Bibliography

Adshead-Lansdale, Janet. “Dance Analysis in Performance”. The Journal of the Society for Dance Research 12, no. 2(1994): 15-19.

Ambrosio, Nora. Learning about dance: dance as an art form and entertainment . Dubuque, Iowa: Kendall/Hunt, 2008.

Dils, Ann. Moving history, dancing cultures: a dance history reader . Middletown, Conn: Wesleyan University Press, 2001.

William, McNeill. Dance and Drill in Human History . New York: Harvard University Press, 1997.

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IvyPanda. (2018, November 30). Dance Analysis: Social and Cultural Context. https://ivypanda.com/essays/analysis-of-dance/

"Dance Analysis: Social and Cultural Context." IvyPanda , 30 Nov. 2018, ivypanda.com/essays/analysis-of-dance/.

IvyPanda . (2018) 'Dance Analysis: Social and Cultural Context'. 30 November.

IvyPanda . 2018. "Dance Analysis: Social and Cultural Context." November 30, 2018. https://ivypanda.com/essays/analysis-of-dance/.

1. IvyPanda . "Dance Analysis: Social and Cultural Context." November 30, 2018. https://ivypanda.com/essays/analysis-of-dance/.

IvyPanda . "Dance Analysis: Social and Cultural Context." November 30, 2018. https://ivypanda.com/essays/analysis-of-dance/.

Choreographic Analysis as Dance Studies Methodology

• In book: Dance Research Methodologies (pp.113-133)
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How to do Research on Theater & Dance

In addition to combing through general humanities and arts sources, the focus of your research paper can include books, periodicals, and videos, art history and studio art materials, music, sound recordings, and musical scores, and special collections of theatrical manuscripts and rare print materials, all specific to your topic or subject of interest. Described in this article are selected sources and references for research papers on theater and dance.

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Get 10% off with 24start discount code, selected subject headings.

Listed below is a sample of a few broad Library of Congress subject headings—made up of one word or more representing concepts under which all library holdings are divided and subdivided by subject—which you can search under and use as subject terms as well when searching online library catalogs for preliminary and/or additional research, such as books, audio and video recordings, and other references, related to your research paper topic. When researching materials on your topic, subject heading searching may be more productive than searching using simple keywords. However, keyword searching when using the right search method (Boolean, etc.) and combination of words can be equally effective in finding materials more closely relevant to the topic of your research paper.

Suggested Research Topics for Theater:

• Children’s Theater
• Drama Technique
• Improvisation Acting
• Method Acting
• Movement Acting
• One-Act Plays
• Playwriting
• Psychodrama
• Puppet Plays
• Radio Plays
• Stage Fighting
• Stage Fright
• Stage Lighting
• Stage Management
• Theater and Society
• Theater—Audiences
• Theater—History
• Theater—Production and direction
• Theatrical Makeup
• Women—Drama

Suggested Research Topics for  Dance:

• Choreographers
• Choreography
• Dance for Children
• Dance—History
• Dance—Music
• Dance—Notation
• Dance—Periodicals
• Dance—Philosophy
• Dance—Physiological aspects
• Dance—Social aspects
• Dancers Biography
• Dance Therapy
• Dancing Injuries
• Folk Dancing
• Improvisation in Dance
• Modern Dance
• Movement—Aesthetics of Rhythm

Selected Keyword Search Strategies and Guides

If your research paper topic is “the mental and physical benefits of dance therapy,” for example, enter “benefits” and “dance therapy” with “and” on the same line to locate sources directly compatible with the primary focus of your paper. To find research on more specific aspects of your topic, alternate with one new keyword at a time with “and” in between (for example, “disabilities [or disorders] and dance therapy,” “history and dance therapy,” “principles and dance therapy,” “qualifications and dance therapy,” “treatments and dance therapy,” etc.).

For additional help with keyword searching, navigation or user guides for online indexes and databases by many leading providers—including Cambridge Scientific Abstracts, EBSCO, H.W. Wilson, OCLC, Ovid Technologies, ProQuest, and Thomson Gale—are posted with direct links on library Web sites to guides providing specific instruction to using whichever database you want to search. They provide additional guidance on how to customize and maximize your searching, including advanced searching techniques and grouping of words and phrases using the Boolean search method—of your topic, of bibliographic records, and of full-text articles, and other documents related to the subject of your research paper. Many libraries, under the “Help” sections of their Web sites, post their own tutorials on subject and keyword searching, which you can also consult.

Selected Source and Subject Guides

As part of your preliminary research to find appropriate resources for your paper, information source and research guides are available at most public and academic libraries and are keyword searchable through your library’s online catalog (to search and locate guides, enter your “subject” followed by these keywords one search at a time: “information sources,” “reference sources,” and “research guide”). Printed guides available for this subject area include

American Theater and Drama Research: An Annotated Guide to Information Sources, 1945–199 0, by Irene Shaland (Jefferson, N.C.: McFarland & Co, 1991)

The American Stage to World War I: A Guide to Information Sources , by Don B. Wilmeth, 269 pages (Detroit, Mich.: Gale Research Co., 1978)

Performing Arts Research: A Guide to Information Sources , by Marion K. Whalon, 280 pages (Detroit, Mich.: Gale Research Co., 1976)

Research in Dance: A Guide to Resources , by Mary S. Bopp, 296 pages (New York: G. K. Hall; Toronto: Maxwell Macmillan Canada; and New York: Maxwell Macmillan International, 1994)

Theatre and Cinema Architecture: A Guide to Information Sources , by Richard Stoddard, 368 pages (Detroit, Mich.: Gale Research Co., 1978)

In addition to these sources of research, most college and university libraries offer online subject guides arranged by subject on the library’s Web page; others also list searchable course-related “LibGuides” by subject. Each guide lists more recommended published and Web sources—including books and references, journal, newspaper and magazines indexes, full-text article databases, Web sites, and even research tutorials—you can access to expand your research on more specific issues and relevant to the subject of your research paper.

Selected Books and References

General references.

American Musical Theatre—A Chronicle , 3rd ed., by Gerald Bordman, 936 pages (New York: Oxford University Press, 2000)

Written by Gerald Bordman, author of Oxford University Press’s acclaimed American Theatre series, this exhaustively researched guide offers detailed summaries of musical theater productions, including musical comedies, operettas, reviews, and one-man and one-woman shows, from 1866 to 1960. A detailed show, song, and people index is included.

American Theatre—A Chronicle of Comedy and Drama 1869–1914 , by Gerald Bordman, Vol. 1, 808 pages (New York: Oxford University Press, 1994)

The first of four volumes, this well-written and researched book examines American theater history, from post–Civil War era to the start of World War I. Every Broadway show is fully chronicled by season, including plot summaries, details of the production and its stars, and other characteristics.

American Theatre: A Chronicle of Comedy and Drama 1914–1930 , by Gerald Bordman, Vol. 2, 464 pages (New York: Oxford University Press, 1995)

This second volume in Oxford’s American Theatre series chronicles every American theater production in chronological order from 1914 to 1930, what many historians believe to be the richest period in American theater. Covering the works of such noted playwrights as George Kaufman, Eugene O’Neill, and Elmer Rice and the era’s biggest stars, such as John and Ethel Barrymore and Alfred Lunt, entries include plot summaries, production details, cast and character names, and critical reviews.

American Theatre: A Chronicle of Comedy and Drama 1930–1969 , by Gerald Bordman, Vol. 3, 480 pages (New York: Oxford University Press, 1996)

In the third volume of Oxford’s American Theatre series, Gerald Bordman once again surveys American theater, this time focusing on nonmusical theater—comedy and drama—between 1930 and 1969. Following the same format as previous editions, Bordman vividly details Broadway productions offering plotlines, historical context, cast and credits, critical response, and more. This premier history also chronicles the best work of playwrights from this era, including Arthur Miller (Death of a Salesman), Eugene O’Neill (Long Day’s Journey into Night), Tennessee Williams (A Streetcar Named Desire), and many others.

American Theatre: A Chronicle of Comedy and Drama 1969–2000 , by Thomas S. Hischak, Vol. 4, 520 pages (New York: Oxford University Press, 2001)

This last edition and final volume of Oxford’s American Theatre series, written by State University of New York professor of theater Thomas S. Hischak, coauthor of The Oxford Companion to American Theatre, offers a fascinating look at Broadway productions through the end of the 20th century.

The Cambridge Guide to American Theatre , 2nd ed., edited by Don B. Wilmeth, 786 pages (New York: Cambridge University Press, 2009)

This alphabetically arranged, second-edition chronicle offers extensive coverage of American theater history, including major plays and all forms of theater, including burlesque, circuses, and vaudeville, from their beginnings through 2008. Featuring the contributions of more than 80 experts, this book covers more than 2,700 subjects, including biographical sketches of theater personalities, entries for individual plays, essays on production companies and theaters, historical sketches of theater, and related subjects, from overviews of Asian-American theater to Shakespearean stage productions. This volume includes a list of 1,000 additional sources for further reading, and a biographical index of more than 3,200 names.

A Chronology of American Musical Theater , by Richard C. Norton, 3 vols., 3,078 pages (New York: Oxford University Press, 2002)

Named an Outstanding Reference Source for 2003 by the American Library Association, this three-volume reference set surveys more than 3,000 musicals by year and by season, from the 1860s through 2001. Each entry provides considerable detail about the productions, including information about the cast and crew, composers, lyricists, set designers, and songs, and every kind of production—Broadway and off-Broadway musicals, operettas, revues, and other stage works. Contents are extensively indexed.

Contemporary Dramatists , 6th ed., edited by Thomas Riggs, 897 pages (Detroit: St. James Press, 1998)

This fully revised and updated sixth edition offers 450 entries examining the lives and works of the most famous living playwrights in the English language. Entries include biographies, bibliographies, and critical essays on the most studied dramatists in the world of theater.

Contemporary Theatre, Film, and Television: A Biographical Guide Featuring Performers, Directors, Writers, Producers, Designers, Managers, Choreographers, Technicians, Composers, Executives, Dancers, and Critics in the United States and Great Britain , 97 vols. (Detroit, Mich.: Gale Group, 2009)

This first-rate biographical reference, also available in e-book format through the online library database Gale Virtual Reference Library, details the lives and careers of some 20,000 entertainment industry professionals, such as choreographers, critics, designers, directors, executives, producers, technicians, and writers from the United States and Great Britain. Entries provide personal and career vitals, including birth dates, education and professional training, and political and religious affiliations. This series was published as a supplement to  Who’s Who in Theatre , which ceased publication in 1981.

International Encyclopedia of Dance , 2nd ed., edited by Selma Jeanne Cohen, 6 vols., 4,000 pages (New York: Oxford University Press, 2004)

This heavily illustrated six-volume encyclopedia features more than 2,000 topical essays exploring all forms of dance throughout the world and its cultural and social significance.

The New Penguin Dictionary of the Theatre , edited by Jonathan Law, David Pickering, and Richard Helfer, 668 pages (New York: Penguin USA, 2001)

An invaluable reference guide for students and aficionados of drama, containing more than 5,000 articles exploring all aspects, styles, and developments in theater.

The Oxford Companion to American Theatre , 3rd ed., by Gerald Bordman and Thomas S. Hischak, 696 pages (New York: Oxford University Press, 2004)

Fully updated and expanded, this encyclopedia is an authoritative source of information about all aspects of American theater, from its beginnings to the late 20th century. More than 3,000 entries highlight great American playwrights, producers, and directors, Broadway stage productions, composers and lyricists, theater companies and organizations, performers, and some foreign plays. Entries include such celebrated plays as Cat on a Hot Tin Roof, Cats, The Iceman Cometh, Arsenic and Old Lace, My Fair Lady, and Who’s Afraid of Virginia Woolf?, and the greatest names in theater, such as Clifford Odets, Lillian Hellman, George Gershwin, Neil Simon, Florenz Ziegfeld, Mae West, Lee Strasberg, and Jessica Tandy.

Theatre Backstage From A to Z , 4th ed., by Warren C. Lounsbury and Norman C. Boulanger, 231 pages (Seattle: University of Washington Press, 1999)

An ideal source for amateurs and professionals alike, this copiously illustrated revised and expanded manual covers the technical aspects of theater production, including construction, design, lighting, painting, stage managing, and more.

Theatre World , by John Willis, et al., 65 vols. (New York: Daniel C. Blum, 1945–50; Crown Publishers, 1966–91; Applause Theatre and Cinema Books, 1992– )

Annual survey of the American theater offering brief summaries and cast lists for Broadway, off-Broadway, and regional theater productions. Information covered includes awards, biographies, and obituaries for the previous year. Between 1944 and 1965, the series was published by various publishers, including its first, Daniel C. Blum, which issued the first six volumes in the series through 1950. Eighteen additional annual volumes followed. Publication continued under the Crown Publishers imprint in 1966, when the seventh volume in the series was released. Crown became the principal publisher of this series, continuing publication through the 1989–90 season (Volume 46). In 1973, the publication was renamed after its author John Willis’ Theatre World before returning to its former title in 1982. In 1992, Applause Theatre and Cinema Books acquired the series, publishing its first edition, Volume 47 (1990–91), that same year.

Variety Obituaries , 15 vols. (New York: Garland Publishing Co., 1905–94)

This 15-volume set reprinted show business obituaries of well-known celebrities in the performing arts from 1905 through 1993–94.

Who’s Who in Theatre: A Biographical Record of the Contemporary Stage , 17th ed., 4 vols. (London: Pitman; Detroit, Mich.: Gale Research, 1981– )

No longer in print but still available at some school and public libraries, this important four-volume set was the first reference of its kind featuring biographies of people from all aspects of theater from 1912 to 1981. Largely emphasizing London theater in earlier volumes, the entire set offers biographical entries on leading actors, composers, critics, dramatists, designers, and historians from both the London and New York stage. In addition, playbills from various productions are included.

Who Was Who in the Theatre, 1912–1976: A Biographical Dictionary of Actors, Actresses, Directors, Playwrights, and Producers of the English-Speaking Theatre , 15 vols., 2,664 pages (Detroit, Mich.: Gale Research, 1978)

Now out of print but still useful, this 15-volume reference is a “who was who” of noted theater actors, actresses, directors, producers, and playwrights. Biographical sketches offer personal and career information on each subject.

The World Encyclopedia of Contemporary Theatre , by Don Rubin, 6 vols., 3,470 pages (London and New York: Routledge, 1994–2000)

This six-volume set, called by its publisher “the largest international cooperative publication in the history of world theater,” covers the theater productions and performances of 30 countries around the world. Coverage includes dramatists, plays, and theatrical companies and the cultural, political, and religious impact of their work and performances.

Reviews/Criticism

Critical Survey of Drama , 2nd ed., edited by Frank N. Magill, revised edition edited by Carl Rollyson, 8 vols. (Pasadena, Calif.: Salem Press, 2003)

Eight-volume set of alphabetically arranged articles about major playwrights and their plays comprises the original seven volumes edited by Frank N. Magill, formerly called Critical Survey of Drama: English Language Series (1985) and Critical Survey of Drama: Foreign Language Series (1986). This acclaimed reference series features biographical and critical essays on important English-language dramatists from ancient times to the present and such areas as Africa, Australia, Britain, Canada, West Indies, and the United States. Each entry discusses the subject’s achievements and principal dramas combining critical analysis and bibliographies for further reading. Additional essays also focus on other aspects of the development and presentation of drama, such as acting, costumes, lighting, and more. Cumulated author and title indexes accompany the set.

Critics’ Theatre Reviews , 3 vols. (New York: Critics Theatre Reviews, 1940–42)

This three-volume set offers full text of critical reviews for stage productions from 1940 to 1942. The series continued publication under two different names: New York Theatre Critics’ Reviews and National Theatre Critics’ Reviews, each containing full-text reviews from 1943 through 1996.

Dramatic Criticism Index: A Bibliography of Commentaries on Playwrights from Ibsen to the Avant-Garde , compiled and edited by Paul F. Breed and Florence M. Sniderman, 1,022 pages (Detroit, Mich.: Gale Research Co., 1972)

A good source of criticism and lists of plays by modern playwrights from Ibsen to the early 1970s, this comprehensive index includes approximately 12,000 critical articles, essays, and books on individual plays, arranged alphabetically by playwright. Indexed are both American and foreign 20th-century playwrights. Title and critic’s name indexes are provided for cross-referencing of subjects.

A Guide to Critical Reviews , 3rd Ed., by James M. Salem, 2 vols. (Metuchen, N.J.: Scarecrow Press, 1984–91)

This two-volume, three-part series indexes in alphabetical order critical reviews published in magazines, newspapers, and theater journals by subject. Following the name of each entry, the title of the production, debut date, number of performances, and reviews are listed by publication and date published. Part I of the series indexes reviews of American drama from 1909 to 1982; Part II, musicals from 1909 to 1989, and Part III, foreign dramas from 1909 to 1977.

International Bibliography of Theatre , by Benito Ortolani (Brooklyn: Theatre Research Data Center, Brooklyn College, City University of New York, 1982–1999)

Updated annually from 1982 to 1999, its last edition, this popular reference is considered by librarians as one of the best indexes in theatre. This annual lists international publications in theater and performing arts and more than 5,000 entries in all, covering all time periods and geographic areas of theater arts. Coverage includes books, articles in scholarly journals, articles in literary and theater magazines, criticism and interviews, and reviews published around the world and in several languages. This directory is divided into two parts: a list of resources and a subject index. Unlike the MLA International Bibliography and other arts and humanities indexes, this publication does not list reviews in newspapers and popular media.

Modern Drama Scholarship and Criticism 1966–1980: An International Bibliography , by Charles A. Carpenter, 587 pages (Toronto, Canada, and Buffalo, N.Y.: University of Toronto Press, 1986)

Suitable for researching international criticism on contemporary and modern playwrights, this first of two volumes covers nearly 25,000 international publications arranged by topic. Books and articles are arranged by geographic area, and playwrights’ names are conveniently indexed in the front of the book. Content includes criticism and interviews through 1980.

Modern Drama Scholarship and Criticism, 1981–1990: An International Bibliography , by Charles A. Carpenter, 632 pages (Toronto, Canada, and Buffalo, N.Y.: University of Toronto Press in association with Modern Drama, 1997)

This follow-up volume to Modern Drama Scholarship and Criticism, 1966–1980 adheres to the same format as its predecessor, chronicling 25,200 additional periodicals since 1981, including books, articles, criticisms, and interviews on contemporary and modern playwrights.

National Theatre Critics’ Reviews , 2 vols. (Woodside, N.Y.: Critics’ Theatre Reviews, 1995–96)

Formerly known as Critics’ Theatre Reviews, 1940–1942 and New York Theatre Critics’ Reviews, 1943–1994, this two-volume set reproduces full-text reviews from 1995 to 1996.

New York Theatre Critics’ Reviews , 52 vols. (New York: Critics’ Theatre Reviews, 1943–95)

The best source for Broadway and off-Broadway show reviews, this 52-volume index features full-text New York theater critic reviews from 1943 to 1995. The last volume indexes actors, choreographers, directors, and other personnel from the shows reviewed.

The New York Times Index , 95 vols. (New York: The New York Times Co., 1851–2007)

Indexes citations from 1857 to the present (under the subject heading “Theater”) of articles about theater and reviews of individual plays listed alphabetically by title corresponding with The New York Times microfilm collection.

The New York Times Theater Reviews  (New York: The New York Times, 1870–2001)

Ceasing publication with the 1999–2000 edition, this set of indexes contains the full texts of theater reviews published in The New York Times since 1870.

Selected Full-Text Article Databases

Academic Search Elite  (Ipswich, Mass.: EBSCO Publishing, EBSCOHost, indexing: 1980– , full text: 1990– )

Covering a variety of disciplines, Academic Search Elite is a good source for reviews and general articles on theater and stage personalities culled from popular magazines and some scholarly journals.

Academic Search Premier  (Ipswich, Mass.: EBSCO Publishing, EBSCOHost, indexing: 1972– , full text: 1972– )

Another good source of entertainment reviews, this major database contains full-text articles from many academic journals, such as Cineaste and Journal of Performance and Art, and popular magazines, including People, Time, and Rolling Stone. Coverage varies by title.

Art Full Text  (Bronx, N.Y.: H.W. Wilson Co., Wilson Disc, WilsonWeb/Ovid Technologies, Inc., indexing: September 1984– , abstracting, Spring 1984/– , full text: 1997– )

Abstracts and indexes, with full text of 98 journals, a wide array of peer-reviewed journals that are international in scope, plus links to Web sites of many articles, from 1997 to the present. As with the print version, this online edition indexes only a handful of journals, mostly geared towards the technical side of theater arts. Also offered on CD-ROM and online as Wilson Art Full Text.

Expanded Academic ASAP  (Farmington Hills, Mich.: Thomson Gale InfoTrac, 1980– )

One of the best sources for theater reviews and theater criticism, Expanded Academic ASAP contains citations and full-text articles from 1,900 popular magazines, scholarly journals, and major newspapers. Articles cover a wide range of theater topics, as well as related subjects in the arts, humanities, and sciences. Subjects are keyword searchable; to search, combine the name of the production with the phrase “theater reviews.” This database includes full-text access to the following magazines and journals: American Theatre, Back Stage, Dance Magazine, Down Beat, Early Music, Opera News, Performing Arts Journal, TDR, Theatre Journal, Theatre Research International, among others, from 1994 to the present.

JSTOR  (Ann Arbor, Mich.: Journal Storage Project, 1996– )

This electronic archive contains the complete back files of 119 scholarly journals in the arts, humanities, and the social sciences, with many titles extending back to the 1800s. Options allow users to browse journals online or retrieve full text using title or subject search. The journals in this collection have been digitized back to the first issue published, and more than 4.5 million pages are available.

LexisNexis Academic Universe  (Dayton, Ohio: LexisNexis, 1977– )

LexisNexis Academic Universe includes full-text articles from virtually thousands of newspapers, magazines, trade journals, industry publications, and more published in the United States and abroad. This easy-to-use database is a good source for finding current information on actors, performers, and reviews of performances. Reviews are searchable under the “General News Topics” category, and under “News/Arts and Sports/Book, Movie, Music and Play Reviews.”

Periodical Abstracts Research II  (Ann Arbor, Mich.: UMI ProQuest, 1986)

Another solid source is this full-text edition of Periodical Abstracts Research featuring abstracts and some full-text articles to reviews and general articles about stage and theater. Out of some 1,600 general reference publications represented, PAR indexes articles from approximately 396 humanities periodicals in the field.

ProjectMUSE  (Baltimore, Md.: Johns Hopkins University Press, 1990– )

One of the best collections on the Web today, with access to almost 250 electronic journals published by major universities, including film, television, mass media, and theater arts studies. Coverage dates back 10 years but varies by journal. Users can browse or search journals by title or subjects. Among the full-text journals featured are Asian Theatre Journal (1999– ), Discourse (2000– ), Performing Arts Journal (1996– ), TDR: The Drama Review (1999– ), Theater Journal (1996– ), and Theatre Topics (1996– ).

ProQuest Direct  (Ann Arbor, Mich.: ProQuest/UMI, indexing: 1971– , full text: 1986– )

Indexes more than 1,100 Web-searchable scholarly periodicals, as well as newspapers and general-interest magazines, from 1986 to the present. In most cases, full-text articles are available from such journals as American Theater and Theatre Journal.

Selected Periodicals

American Theatre  (New York: Theatre Communications Group, 1984– , monthly)

This highly regarded monthly, published by the nonprofit Theatre Communications Groups of New York, primarily focuses on professional theater. Published in each issue are two or three major features, including actor profiles, articles on legal and professional issues affecting the theater arts community, and articles covering trends and events in theater. Online access is provided to certain articles from back issues since January 2000. To access, visit  https://www.americantheatre.org/ .

Back Stage  (New York: Back Stage Publications, 1960– , weekly)

Providing an inside look into the performing arts since 1960, this weekly trade paper covers East Coast theater. Coverage includes the latest industry news, feature stories, reviews, casting notices, and advice articles for industry professionals and students. A collection of feature stories is accessible online at  https://www.backstage.com/ .

Drama: The Journal of National Drama  (Shaftesbury, U.K.: National Drama, 1993– , biannual)

Designed for educators and practitioners in theater arts, Drama is the official publication of National Drama in Great Britain. This scholarly journal, published biannually, offers a forum for educators and practitioners worldwide to discuss theories and practices, opinions and criticisms, debate key issues, and share new research in the field.

Early Theatre  (Hamilton, Canada: McMaster University, 1998– , biannual)

A journal associated with the records of early English drama, Early Theatre is a peer-reviewed print journal, now published biannually by the Department of English at McMaster University in Hamilton, Ontario, devoted to drama and theater history of England, Scotland, Ireland, and Wales. This scholarly journal publishes research studies, articles, and notes on the performance history, as well as literary and analytical articles about individual performances.

Performing Arts Journal  (New York: Performing Arts Journal Publications, 1976–97; Baltimore, Md.: Johns Hopkins University Press for PAJ Publications, 1998– , three times a year)

Originally titled the Performing Arts Journal from 1976 to 1997 (Volumes 1–19), this scholarly journal, renamed PAJ: A Journal of Performance and Art in January 1998, explores new directions and new work in dance, film, music, performance, theater, and the visual arts. Published three times yearly, PAJ includes essays and critical commentaries, interviews and book reviews, artists’ writings and festival reports, and performance texts and plays in the performing arts. Contents of current and past issues are abstracted and indexed in such popular online indexes as Arts and Humanities Citation Index, ISI Current Contents, Film Literature Index, Humanities Index, and International Index to the Performing Arts.

Playbill  (New York: American Theatre Press, 1982– , monthly)

This monthly print magazine covers the professional theater, especially New York theater. Each issue contains feature articles and columns by or about theater personalities, engaging editorials, and travel, fashion, and dining news aimed at active theatergoers. In addition, Playbill offers free access to information published on its Web site ( http://www.playbill.com/ ), such as news, features, theater listings, and other resources, including box-office grosses and a theater awards database.

Shakespeare Quarterly  (Washington, D.C.: Folger Shakespeare Library, 1950– , quarterly)

First published in 1950 by the Shakespeare Association of America, this quarterly journal is the foremost publication covering all aspects of Shakespeare studies, including play criticisms and theater histories. Issues include essays and research studies, reviews of books, films, and stage productions, and criticism and scholarship of Shakespeare-related works.

Stage Directions  (West Sacramento, Calif.: SMW Communications; New York: Lifestyle Media, 1988– , monthly)

With members of community, regional, and academic theater including producers, lightning technicians, and set designers among its readers, Stage Directions offers practical help to people involved in theater production, featuring articles about new strategies, ideas, and solutions to common problems, as well as book, CD, and play reviews.

TDR: The Drama Review  (New York: New York University, School of Arts, 1968–87; Cambridge, Mass.: MIT Press, 1988– , quarterly)

This widely read, highly acclaimed scholarly journal is devoted to serious study and debate on various kinds of performances, including dance, theater, performance art, popular entertainment, and sports. Published by the New York University School of Arts from 1967 to 1997 and by MIT Press since 1988, each issue features articles, commentaries, interviews, texts of performances, and translations of important works on contemporary performing arts and performing theory. Bibliographic citations with abstracts and full-text articles, in some cases, are indexed in such well-known library databases as Arts and Humanities Citation Index, Expanded Academic ASAP, Humanities Index, International Index to the Performing Arts, and MLA International Bibliography.

Theatre Journal  (Baltimore, Md.: Johns Hopkins University Press in cooperation with Association for Theatre in Higher Education, 1941– , quarterly)

The scholarly Theatre Journal, published quarterly by Johns Hopkins University Press in association with the Association of Theater in Higher Education, offers a global view of all aspects of theater arts. Issues feature many social and historical studies and critical reviews of productions, written by noted scholars and practitioners. Issues and contents are indexed and abstracted in ProjectMUSE.

Theatre Topics  (Baltimore, Md.: Johns Hopkins University Press in cooperation with Association for Theatre in Higher Education, 1991– , semi-annual)

Hailed as “an excellent addition to literature of drama,” this scholarly, peer-reviewed electronic journal, accessible through ProjectMUSE (1996– ), features timely articles on a vast number of practical, performance-oriented subjects of interest to theater educators and practitioners and scholars and students of theater. First published in March 1991, it offers articles that reflect the theory and practice of acting, community-based theater, design, directing, dramaturgy, performance studies, and theater pedagogy. Theatre Topics is indexed and abstracted in several article databases including American Humanities Index, Annual Bibliography of English Language and Literature, Education Index, and MLA International Bibliography.

Variety  (New York: Variety Pub. Co., 1905– , weekly)

Published weekly, Variety, the longest-running industry trade paper, features news and reviews covering all areas of show business, including Broadway and off-Broadway players and shows abroad, as well as regional and New York theater productions. It is a good source for theater industry news casting and box-office gross information each week.

Selected Web Sites

American Variety Stage  ( http://memory.loc.gov/ammem/vshtml/vshome.html )

This multimedia online anthology, presented by the Library of Congress, displays various stage and theater holdings from its collection of popular entertainment material, including vaudeville, from 1870 to 1920. Items include English- and Yiddish-language play scripts, theater playbills and programs, motion pictures and sound recordings, and photographs and other memorabilia.

A Brief Guide to Internet Resources in Theatre and Performance Studies  ( https://www2.stetson.edu/creative-arts/resources/theatre-arts/mccoy-theatre-guide.html )

Celebrating its 16th anniversary in 2009, this Web site is actually one of the most comprehensive listings of content and links on theater and performance studies.

Federal Theatre Project  ( http://memory.loc.gov/ammem/fedtp/fthome.html )

This Web presentation of the Library of Congress’ Federal Theatre Project Collection features more than 13,000 images of stage and costume designs, still photographs, posters, and scripts (for Orson Welles’s productions of Macbeth and The Tragical History Dr. Faustus) from 1935 to 1939. The FTPC was originally established by President Franklin D. Roosevelt during the New Deal era.

Internet Broadway Database (IBDB)  ( http://www.ibdb.com/ )

This official database for Broadway archives features searchable records of theater information for stage productions from its early New York beginnings to the present.

Performing Arts in America 1875–1923  ( http://digital.nypl.org/lpa/nypl/lpa_home4.html )

Partially funded by the National Endowment of the Arts and part of the New York Public Library for Performing Arts, Performing Arts in America 1875–1923 is a searchable online database, funded in part by the National Endowment for the Arts, of 16,000 items representative of this period of performing arts, including newspaper clippings; photographs; music sheet samples (popular music, show tunes, jazz, and dance music); photographs of artists and theater, dance, and popular performances; and movie posters and lobby cards.

Theatre Reviews Limited  ( http://www.theatrereviews.com/ )

Online source providing free access to reviews of recent Broadway, Off-Broadway, and Off-Off Broadway shows in New York City.

The WWW Virtual Library Theatre and Drama  ( http://www.vl-theatre.com/ )

This multicultural, multilingual Web site features resources from more than 50 countries on all aspects of theater, designed for amateurs, professionals, and students of all ages.

Careers Related to Theater and Dance

Dance Career Field ( http://career.iresearchnet.com/career-fields/dance-career-field/ )

Dance is one of the oldest of the arts. Anthropologists believe the first formal dances were probably symbolic dances performed by early tribal societies as part of ritual ceremonies held to ask spirits or gods for success in hunting or in battle. Some anthropologists think that dancing and music originally came from the same mating-display impulses that occur in other species. The Egyptians used dance to honor their leaders and during parades, funerals, and religious ceremonies. Israelites performed circle dances, processional chain dances, and energetic stamping- and-jumping dances at religious festivals. Greek children learned to dance as part of their education; adults performed dances in festivals honoring the god Dionysus and as part of Greek comedy and tragedy. Many of these dances were adopted and developed by the Romans as well.

Theater Career Field ( http://career.iresearchnet.com/career-fields/theater-career-field/ )

Theatrical performance is among the oldest of human art forms. It probably began with storytelling to recount recent and historical events in small communities. Ancient peoples often performed elaborate rituals to ask spirits or gods for success in hunting or in battle. Sometimes community leaders or religious officials wore masks and colorful costumes. Ceremonies also were held to pray for the health of individuals, mourn the dead, ward off evil spirits, or promote the welfare of the society. Later societies enacted the myths and stories involving their gods and heroes. For example, Egyptian dramas often centered around the god Osiris.

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319 Dance Essay Topics & Research Questions on Hip Hop, Ballet, & More

Dancing is a universal form of expression and movement. It has been an integral part of human culture for centuries. From traditional cultural dances to contemporary urban dance styles, this art form transcends language barriers and brings people together.

But dancing is not just about entertainment. It is significant in various aspects of society, from its role in expressing emotions to its impact on mental health.

In this article, our expert team delves into the diverse world of dance. Below, you’ll find interesting dance topics to write about and explore the cultural, social, emotional, and physical dimensions of dance. Read till the end to find a writing guide with examples.

🔝 Top 10 Dance Essay Topics

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• Cultural significance of dance.
• Mental health benefits of dancing.
• Gender roles in dance.
• Dance as nonverbal communication.
• Technological innovations in dance.
• Social impact of dance activism.
• Dance education in schools.
• Emotional expression in dance.
• Dance and cultural identity.
• Politics of dance movements.

Why I Love Dance: Essay Prompt

Are you passionate about dance and want to write about it? Consider including the following points in your essay:

• Explain your deep-rooted passion for dance. Reflect on how dance has influenced your life, shaped your identity, and impacted your personal growth.
• Describe the specific styles or dance genres you are passionate about and explain why they hold a special place in your heart. Share experiences that have strengthened your love for dance, such as memorable performances, challenges , or inspirational moments.
• Discuss how dance has enriched your life and contributed to your overall well-being. Convey your dedication to dance and the importance of this art form in your life.

What Does Dance Mean to You: Essay Prompt

This essay is your chance to reflect on the place of dance in your life. Here are some ideas to include in your paper:

• Express your understanding and connection to dance. Describe what dance means to you on a deeply personal and emotional level. Share your unique perspective on the effects of dance on your life choices and identity.
• Discuss how dance has impacted your physical, mental, and emotional well-being. Write about its contribution to your personal growth and development. Describe the emotions and sensations you experience while dancing and how they enrich your life. Discuss any memorable dance experiences, performances, or interactions with fellow dancers that impressed you.
• Depict your passion and appreciation for dance. Articulate the role of dancing in your life. Consider how dance has allowed you to express yourself , connect with others, and navigate life.

Hip Hop Essay Prompt

Are you a fan of hip-hop dance? Then you might enjoy writing an essay about it! Consider the points below to cover in your paper:

• Explore the art and culture of hip-hop. Discuss the origins and evolution of hip-hop dance, including its roots in African, Caribbean, and African-American dance traditions. Study the key elements of hip-hop dance, including its unique style, movement vocabulary, and musicality.
• Discuss the impact of hip-hop dance on popular culture , including its influence on music videos, films, fashion, and the entertainment industry. Analyze the social and cultural significance of hip-hop dance, including its role as a form of self-expression, community-building, and activism . Discuss the diversity and inclusion within hip-hop dance, including its ability to bring people of different backgrounds together.
• Describe any personal experiences or connections with hip-hop dance , and discuss how it has influenced your understanding of dance and culture. Convey your appreciation for hip-hop dance’s artistry, creativity, and cultural relevance. Provide insights into this dynamic and influential dance form.

Why Dance Is a Sport: Essay Prompt

There’s been a long-lasting debate about whether dance should be considered an art or a sport. You can present your opinion on this issue in your essay. Here is a prompt to guide you:

• Study the arguments on both sides of the debate and articulate your stance. Discuss the physical demands of dance, including athleticism, strength, and endurance. Compare them to the criteria used to define sports.
• Explore the competitive nature of dance , including competitions, rankings, and performance expectations . Discuss the training, dedication, and discipline required for dance and compare them to other recognized sports.
• Examine the artistic and creative elements of dance and how they may differentiate dance from traditional sports. Discuss any challenges or controversies surrounding the classification of dance as a sport. It might be the lack of standardized rules, funding, and recognition.
• Consider the cultural, historical, and societal factors influencing perceptions of dance as a sport. Present your argument supported by evidence and examples. Provide a nuanced perspective on this complex and debated topic.

Below, you’ll find plenty of ideas for a college essay about dance. Read on to find a suitable topic for your paper!

Modern Dance Essay: Topics

Modern dance significantly differs from traditional dance forms like ballet. Nowadays, dancers prioritize emotional expression instead of following rigid rules. Here are some interesting dance titles & ideas for your essay on modern styles:

• The diversity of contemporary dance styles.
• Psychological determinants of choosing a particular dance style.
• Creative Movement and Dance in My School.
• Sociopolitical concerns through the prism of dance.
• Modern dance as a form of protest against classical dance.
• Modern Black dance: race in motion.
• Significant people who contributed to the modernization of dance.
• Features of post-modern dance.
• Art Therapy: Creativity as a Critical Part of Art Therapy.
• The new American dance forms.
• Modern dance in the US vs. Europe: differences and similarities.
• Injuries in modern dance: career options for dancers.
• The concepts of modernity, race, and nation in the early modern dance forms.
• Oxygen uptake during a modern dance class.
• Physical Therapy Services for Sports Injuries.
• Socio-demographic correlates of modern dance genre preferences.
• Indian modern dance and feminism.
• Self-expression in modern dance.
• National and transnational modern dance.
• Principles of modern dance therapy.
• Regular engagement in modern dance and depression treatment.
• Modern dance pioneers who revolutionized this art: a case of Lester Horton.
• Incorporating Physical Activity into Lesson Plans.
• Mood changes of people attending dance classes.
• Modern dance classes: can one learn to dance online?
• Evolving gay stereotypes in modern dance.
• Dance as a reflection of culture.
• Depression and Physical Exercise.
• German modern dance during the Nazi rule.
• Body image and modern dance engagement.
• Eating disorders in professional modern dancers.
• Significant elements of modern dance.
• Dance as a hobby and professional activity.
• Art Education, Its Role and Benefits.
• Examples of popular modern dance styles.
• Controversial dance practices.
• Is folk dance considered modern dance?
• Does modern dance come with serious choreography similar to classical dance?
• Class and ethnicity reflected in dance.
• Physical Activity and Sports Team Participation.
• Modern dance in France.
• The transformative power of flamenco dancing.

Dance Critique Essay: Topics

A dance critique essay requires you to evaluate carefully a particular dance performance or a specific aspect of this art form. Check out some dance titles for an essay to inspire your critical response :

• Degradation of dance quality in the 21st century.
• Passion versus physical characteristics in dance performance.
• Racism and gender discrimination at the dance competitions.
• Gender inequality in professional dance.
• Subway dance – a subculture or simply a weird place for practice?
• Dancing and Its Effects on Self-Esteem .
• How did industrialization contribute to the departure from classical dance?
• The rise of burlesque as a rebellion against ballet.
• The Role of Music and Movement in Education.
• The ideology and importance of radical dance.
• The Wigman technique of dance.
• José Limón’s contribution to contemporary dance.
• The place and influence of the American Dance Festival in modern American dance traditions.
• Art, Music, and Dance in Therapeutic Treatment .
• Combination of choreography and multimedia in Alwin Nikolais’s works.
• The flaws and loopholes of early modern dance.
• Popularization of African American dance across the world.
• Ballet elements in contemporary dance.
• The Importance of Arts in the School Curriculum .
• The contribution of Grete Wiesenthal to modern dance development.
• What place does concert dance occupy in the dance typology?
• Place of women in dance – now and then.
• Differences between modern and contemporary dance styles.
• Pole dance’s potential for inclusion in the Olympic Games list.

Dance Reflection Essay: Topic Ideas

Are you looking for thought-provoking dance topics to talk about in a reflection paper ? Check out the list below:

• What features and character traits should a good dance teacher possess?
• What does it mean to be a dance revolutionary?
• What did modern dance pioneers do to create new dance types?
• Should professional dance be taught in college?
• Sports Psychologist: Working With Athletes.
• The national dance of my country.
• My first experience in a dance class.
• My music preferences for dancing.
• Dances with Wolves Essay – Movie Analysis .
• Dancing as a personal form of relaxation and meditation.
• Why do some people prefer dancing alone at home and others need an audience?
• My favorite movie about dancing.
• Ballet as my passion and my greatest disappointment.
• My opinion of dancing as a profession.
• Music in the Service of Social Movements.
• Importance of dancing in my culture.
• What I think makes a good and bad dancer.
• My experience of participating in a dance contest.
• The role of dance in my family.
• My fascination with Indian dance.
• My dream is to become a hip-hop dancer.
• Benefits I see in regular dancing.
• The pros and cons of professional engagement in dance.

There are some controversial issues related to dance you can explore in your paper. Below, you’ll discover a list of dance topics to express your reasoned opinion about.

• Dance can be a tool for social change .
• How can culture be reflected through dance?
• Should College Athletes Be Paid?
• The impact of the environment on dance perception.
• The importance of dance styles and types for specific cultures.
• Dance as an intangible cultural heritage.
• Is dance a kind of sport?
• Sports-Related Problems and Conflicts .
• Why do people start to dance?
• Why is the audience attracted to dance performances?
• What is the motivation behind starting to dance at an early age?
• Why is contemporary dance more popular than classical dance today?
• The Cognitive Performance and Physical Activity Link.
• Are modern dance styles, like hip hop, easier than ballet dancing?
• Dance training is highly traumatic and shouldn’t be practiced at an early age.
• The risk of trauma and permanent injury among professional ballet dancers.
• Street dance is not serious dance.
• Exercise – The Mind and Body Connection.
• The impact of motivation and self-confidence on dance performance.
• Dancing positively affects the physical and cognitive health of aging adults.
• Dance is a tool against aging .
• Different dance styles and genres require different physical endurance and energy expenditure.
• Music Incorporation into Classes.
• Importance of choosing your dance style according to your BMI and bodily peculiarities.
• Partner dancing is healthier for motor control and coordination than solo dancing.
• Benefits of ballroom dancing.
• The economic effect of dance development.
• What is the hardest dance style?
• Cultural Appropriation in Music .
• Ghost dance as a religious movement.
• Electronic dance as a new subculture.
• Techniques of dancing to jazz music.
• Flamenco dance as a reflection of Spanish passion.
• Students’ Motivation and Satisfaction of Music Festival .
• Cultural significance of feminist dance films.
• The impact of proper nutrition on dance performance.
• The art of staging a dance performance.
• The social significance of street dance.
• Creative Art Therapy for Mental Illness.
• What does dance have to do with math?
• Belly dance as a recreational activity.
• Interpretative dance as a strong therapeutic tool.
• The use of dance therapy for individuals with Down syndrome.

Hip-hop dance is a popular street dance style that emerged in the second half of the 20th century. It encourages freedom of expression and is characterized by freestyle movements, particularly drops to the ground and sharp turns. If you seek to write a paper on hip-hop, here are catchy dance titles to inspire you:

• The role of dance in hip-hop culture.
• Chinese Hip Hop and Identity .
• Hip-hop dance and body power.
• Hip-hop as an African diaspora dance style.
• Meanings and messages in hip-hop dance.
• Genders, Sexuality, and Hip-Hop .
• Origins of hip-hop.
• Injury incidence in hip-hop training and performance.
• Commercialization of hip-hop dance.
• Core elements of hip-hop dancing.
• The Uprising of Hip-Hop: Music History .
• Hip-hop dance in modern ballet choreography.
• Low back pain in hip-hop dancers.
• B-boying in hip-hop.
• Motivations of hip-hop dancers for professional engagement with this dance style.
• The “breaks” concept in break dancing.
• The Evolution of Hip-Hop Culture .
• The roots of hip-hop choreography.
• Battling among hip-hop crews.
• Hip-hop scandals – sex and violence in hip-hop content.
• Theorizing hip-hop dance.
• Hip-Hop’s Response to Crime Analysis .
• Hip-hop dance consumption: who is the target audience?
• Globalization and hip-hop dancing.
• Principles of hip-hop dance codification.
• Hip-hop as a black social dance.
• Metaphors in hip-hop dance moves.
• Black feminism in hip-hop dance.
• Concepts of Elite Culture and Popular Culture .
• US Ebonics as the language of American hip-hop choreography.
• Extreme kinematics in hip-hop performances.
• What injuries are the most common among hip-hop dancers?
• Can hip-hop be a professional career?
• Basic rhythmic movements of hip-hop dance.
• Hip-hop practice’s impact on the dancer’s mood.
• Body poetics in hip-hop performances.
• Standards for judging hip-hop contests.
• Cultural Movement: Hip-Hop Related Films.
• Le hip hop – a French hip-hop subculture.
• The hip-hop culture in New York.
• Perceptions of race in the hip-hop community: white vs. black dancers.
• Hip-hop theater in London: a new level of dance style’s legitimization.

Do you want to learn more about dance? Then why not write a research paper on that? Below, you’ll discover a list of engaging dance topics to research.

• Novel approaches to dance: a blend with architecture at the Barnes Foundation’s exhibition.
• Dance from an anthropological perspective .
• Politics and poetics in dance.
• The biology of dance movements’ language.
• Teaching dance: should it be art or sport?
• Dance ethnology.
• Women in dance since antiquity : the images of sylphs and sirens.
• Philosophy of the dance: universal or nationally bound?
• What is a dance movement?
• Dancing and the brain.
• Psychology of dance.
• Dance as a mighty psychotherapeutic tool.
• Should national dance be included in the school curriculum?
• Living through psychological trauma in dance.
• The role of dance improvisations.
• Reform and revival of old dance styles.
• The role of space and subjectivity in dance.
• Shakespeare and the dance tradition of England.
• The social anthropology of dance performances.
• Theories and methodologies in dance research.
• A Eurocentric approach to the study of dance.
• Dance and the body.
• Dance as a method of self-study.
• Computer technology and dance.
• The evolution of Irish dance.
• The contribution of Merce Cunningham to modern dance.
• How does dance stimulate social fantasy?
• Partner dancing as a way to strengthen relationships in a couple.
• Verbal language of dancers and choreographers.
• The political side of national dancing.
• Dance and physics.
• The mind-body concept in dance.
• What can be regarded as contemporary dance?
• Dance imagery in various dance styles.
• What makes people dance? The philosophy of bodily movement.
• Dancing out one’s emotions: the dance of anger or happiness.
• The neural basis of human dance movements.
• Isadora Duncan’s revolutionary dance theory.
• The challenges of the interactive dance genre.
• Dance from the position of existential phenomenology.
• Dualism and body-soul separation: a dance perspective.
• Dance and embodiment.
• Dance and self: a philosophical perspective.
• Body as object vs. subject in dance.
• Freedom and intention in body movements during dance.
• Staged dance: the concept of created body.
• A tension between the personal and the universal in dance.
• Health benefits of recreational and professional dance.
• Japanese dance as an embodiment of culture.
• Turning body and identity into dance choreography.

The history of dance dates back millennia. So, there is so much to explore! Check out possible dance research project ideas for your history paper:

• Approaches to rethinking dance history.
• The elements of early European modern dance.
• Feminist perspectives on dance history.
• Dance at the dawn of history.
• History of somatic education and its relationship to dance.
• History of classic theatrical dancing.
• New media use in dance history reconstructions.
• The evolution of dance and sexuality relationship.
• Tools for capturing dance from the past.
• Dances in prehistory.
• Early records of dance performances in historical manuscripts.
• Ancient Greek dance traditions and forms.
• Dancing in Ancient Rome.
• Dance at court: the 16th-17th centuries in Europe.
• Evolution of dance from royal court to theater in the 18th century.
• The early dancing traditions in the 17th-century US.
• Dancing genres in Medieval Europe.
• The history of waltz: origin, evolution of dance elements, and traditions.
• How did the tango dance emerge?
• The emergence of belly dance.
• Flamenco dance history: a style born in Andalusia.
• Historical perspectives on dance research.
• The Ojibwa dance drum: history of the practice.
• Yoeme performs as a narrative of Yaqui history.
• The evolution of African American dance.
• The African roots of Latin American popular dance.
• Ethnographic elements of modern dance performances.
• The dance halls of Britain, 1918-1960.
• A historical overview of social dance.
• Global history of dance development.
• Dance in the French baroque opera.
• The history and meaning of tango dancing in Argentina.
• What materials to use when studying dance history?
• A cultural history of dance in the troubled areas: a case study of Palestine.
• The hidden history of capoeira: intersections of battle craft and dance.

Ballet is a highly formalized dance form with rigid rules and predefined positions. If you’re interested in this classical type of choreography, check out the dance research topics below:

• Is free dance a subtype of ballet?
• The evolution of ballet traditions by Isadora Duncan.
• Russo-American ballet at the start of the 20th century.
• Emerging American ballet: the 1930s onwards.
• Romantic and classical ballet traditions in the 19th century.
• Classical ballet in Russia.
• Classical ballet vs. modern dance: key similarities.
• Injuries in professional ballet.
• Static and functional balance in ballet dance.
• Ballet as a form of ethnic dance.
• The impact of ballet dance attire on female dancers’ self-perception.
• Endurance of pain among ballet dancers.
• The concept of the ideal ballet body.
• Methods of preventing hip and knee injuries in professional ballet.
• Physiological eligibility characteristics for classical ballet.
• Evolution of ballet dancer identity in the process of training.
• Incidence of sprained ankles in ballet dancers.
• Physiological responses to active ballet exercise among dancers of different ages.
• Importance of artistic performance ability in ballet dancers.
• Disordered eating patterns among ballet dancers.
• Management of stress fractures in ballet dancers.
• Ballet dancer career: an international perspective.
• Incidence of scoliosis in young ballet dancers.
• Travesty dancing in the 19th-century ballet tradition.
• Gender issues in ballet.
• The cultural power of ballet.
• Degenerative joint disease risks among female ballet trainees.
• Hip arthrosis as a long-term consequence of ballet training.
• History of the “Apollo’s Angels” ballet.
• Sleep quality in professional ballet dancers.
• Heteromasculinity images among male ballet dancers.
• Postural stability before and after a ballet injury.
• Dance classicism as an ideology.
• Royal Ballet’s dancers and body perception.
• How do professional ballet dancers perceive injury and aging?

Are you looking for research topics about dance in the form of questions? We’ve got you covered! Look through the list below to find good dance topics to research:

• How does dance education foster creativity in children?
• How do feminists view belly dance?
• What is the impact of technology on dance performances?
• How to preserve indigenous peoples’ traditional dance forms?
• How have traditional gender roles been challenged through dance?
• What creative strategies do choreographers employ?
• What is the impact of dance on community building?
• What factors contribute to the commercialization of dance?
• How does dance function as a means of storytelling?
• What is the link between dance and spirituality in different religions?
• What is the role of costumes and stage design in dance performances?
• What are the cognitive benefits of dance for older adults?
• How has ballet adapted to modern artistic movements?
• How has globalization affected the cross-cultural exchange of dance styles?
• What dance styles have been influenced by flamenco?

Do you need help writing an essay on dance? Below, we’ve prepared a short guide with examples. Read on to learn how to write each section of your dance essay.

Dance Essay Introduction

The introduction is the opening paragraph of an essay that should engage the readers. Use a hook to grab the readers’ interest and introduce your topic. It can be a catchy quote, interesting question, or controversial statement.

Hook example : Imagine a world where bodies move in perfect harmony, telling stories without words. That’s the magic of dance, an art form that transcends language and culture, captivating audiences for centuries.

After that, you can provide some background information about the subject. End your introduction with a thesis statement .

Thesis Statement about Dance

The thesis statement outlines the central argument or purpose of the essay. It is the last sentence of your introduction, summarizing the whole paper. Your thesis should include all the main points mentioned in your writing in the same sequence.

Dance thesis example : Dance is not only a form of artistic expression but also a means of communication, a tool for personal growth, and a reflection of cultural identity.

Essay about Dance: Body Paragraphs

The essay’s body contains paragraphs that provide evidence and support for the thesis statement. Each section should begin with a topic sentence that presents a point related to your central argument.

Topic sentence example : One significant aspect of dance is its ability to convey emotions and stories through movement.

Evidence, such as examples, facts, or research, should support the topic sentence. The evidence should be analyzed and explained to show how it supports the thesis statement.

Evidence example : In classical ballet, dancers’ graceful movements and facial expressions can portray a wide range of emotions, from love and joy to sorrow and anger.

Dancing Essay: Conclusion

The conclusion is the final part that summarizes the main points made in the essay’s body. It should also restate the thesis statement in a paraphrased form without introducing new information. The conclusion should leave a lasting impression and a sense of closure.

Conclusion example : In conclusion, dance is a powerful form of expression beyond mere movement. It is a universal language conveying emotions, facilitating personal growth, and reflecting cultural identity.

How to Describe Dancing: Words & Tips

When describing dancing in an essay, use vivid and sensory language to convey the experience. Words such as “graceful,” “rhythmic,” “expressive,” and “dynamic” can capture the movement and style of dancing.

Emotive words like “joy,” “passion,” “energy,” and “freedom” can convey the emotional impact of dancing. Describing the physical sensations of dancing can make the description more immersive. You can write about the feeling of one’s body in motion, the sound of feet tapping, the swish of skirts, or the rush of adrenaline.

Metaphors or similes, such as “floating like a butterfly,” can add depth and creativity. Overall, using vivid language that engages the senses and emotions will benefit your paper.

We hope you found a perfect essay topic in this article. Use our free online title generator to get even more creative topics about dance and writing inspiration.

• Dance 260: Introduction to Dance: Dance Topics | BYU Library
• Dance: Topics in Dance and Dance History | Utah Tech University Library
• Research Areas | UCI Claire Trevor School of the Arts
• Dance | PBS Learning Media
• Dancing Styles | Boston University
• Dance 260: Introduction to Dance: Research Help | BYU Library
• Writing a Dance Critique | Utah Tech University Library
• Glossary for Dance | Connecticut’s Official State Website
• Guidelines For Writing a Dance Review | Human Kinetics

414 Proposal Essay Topics for Projects, Research, & Proposal Arguments

725 research proposal topics & title ideas in education, psychology, business, & more.

How to Write a Research Proposal: (with Examples & Templates)

Table of Contents

Before conducting a study, a research proposal should be created that outlines researchers’ plans and methodology and is submitted to the concerned evaluating organization or person. Creating a research proposal is an important step to ensure that researchers are on track and are moving forward as intended. A research proposal can be defined as a detailed plan or blueprint for the proposed research that you intend to undertake. It provides readers with a snapshot of your project by describing what you will investigate, why it is needed, and how you will conduct the research.  

Your research proposal should aim to explain to the readers why your research is relevant and original, that you understand the context and current scenario in the field, have the appropriate resources to conduct the research, and that the research is feasible given the usual constraints.  

This article will describe in detail the purpose and typical structure of a research proposal , along with examples and templates to help you ace this step in your research journey.  

What is a Research Proposal ?  

A research proposal¹ ,²  can be defined as a formal report that describes your proposed research, its objectives, methodology, implications, and other important details. Research proposals are the framework of your research and are used to obtain approvals or grants to conduct the study from various committees or organizations. Consequently, research proposals should convince readers of your study’s credibility, accuracy, achievability, practicality, and reproducibility.   

With research proposals , researchers usually aim to persuade the readers, funding agencies, educational institutions, and supervisors to approve the proposal. To achieve this, the report should be well structured with the objectives written in clear, understandable language devoid of jargon. A well-organized research proposal conveys to the readers or evaluators that the writer has thought out the research plan meticulously and has the resources to ensure timely completion.  

Purpose of Research Proposals  

A research proposal is a sales pitch and therefore should be detailed enough to convince your readers, who could be supervisors, ethics committees, universities, etc., that what you’re proposing has merit and is feasible . Research proposals can help students discuss their dissertation with their faculty or fulfill course requirements and also help researchers obtain funding. A well-structured proposal instills confidence among readers about your ability to conduct and complete the study as proposed.  

Research proposals can be written for several reasons:³  

• To describe the importance of research in the specific topic  
• Address any potential challenges you may encounter  
• Showcase knowledge in the field and your ability to conduct a study  
• Apply for a role at a research institute  
• Convince a research supervisor or university that your research can satisfy the requirements of a degree program  
• Highlight the importance of your research to organizations that may sponsor your project  
• Identify implications of your project and how it can benefit the audience  

What Goes in a Research Proposal?    

Research proposals should aim to answer the three basic questions—what, why, and how.  

The What question should be answered by describing the specific subject being researched. It should typically include the objectives, the cohort details, and the location or setting.  

The Why question should be answered by describing the existing scenario of the subject, listing unanswered questions, identifying gaps in the existing research, and describing how your study can address these gaps, along with the implications and significance.  

The How question should be answered by describing the proposed research methodology, data analysis tools expected to be used, and other details to describe your proposed methodology.   

Research Proposal Example  

Here is a research proposal sample template (with examples) from the University of Rochester Medical Center. 4 The sections in all research proposals are essentially the same although different terminology and other specific sections may be used depending on the subject.  

Structure of a Research Proposal  

If you want to know how to make a research proposal impactful, include the following components:¹  

1. Introduction  

This section provides a background of the study, including the research topic, what is already known about it and the gaps, and the significance of the proposed research.  

2. Literature review  

This section contains descriptions of all the previous relevant studies pertaining to the research topic. Every study cited should be described in a few sentences, starting with the general studies to the more specific ones. This section builds on the understanding gained by readers in the Introduction section and supports it by citing relevant prior literature, indicating to readers that you have thoroughly researched your subject.  

3. Objectives  

Once the background and gaps in the research topic have been established, authors must now state the aims of the research clearly. Hypotheses should be mentioned here. This section further helps readers understand what your study’s specific goals are.  

4. Research design and methodology  

Here, authors should clearly describe the methods they intend to use to achieve their proposed objectives. Important components of this section include the population and sample size, data collection and analysis methods and duration, statistical analysis software, measures to avoid bias (randomization, blinding), etc.  

5. Ethical considerations  

This refers to the protection of participants’ rights, such as the right to privacy, right to confidentiality, etc. Researchers need to obtain informed consent and institutional review approval by the required authorities and mention this clearly for transparency.  

6. Budget/funding  

Researchers should prepare their budget and include all expected expenditures. An additional allowance for contingencies such as delays should also be factored in.  

7. Appendices  

This section typically includes information that supports the research proposal and may include informed consent forms, questionnaires, participant information, measurement tools, etc.  

8. Citations  

Important Tips for Writing a Research Proposal  

Writing a research proposal begins much before the actual task of writing. Planning the research proposal structure and content is an important stage, which if done efficiently, can help you seamlessly transition into the writing stage. 3,5  

The Planning Stage  

• Manage your time efficiently. Plan to have the draft version ready at least two weeks before your deadline and the final version at least two to three days before the deadline.
• What is the primary objective of your research?  
• Will your research address any existing gap?  
• What is the impact of your proposed research?  
• Do people outside your field find your research applicable in other areas?  
• If your research is unsuccessful, would there still be other useful research outcomes?  

  The Writing Stage  

• Create an outline with main section headings that are typically used.  
• Focus only on writing and getting your points across without worrying about the format of the research proposal , grammar, punctuation, etc. These can be fixed during the subsequent passes. Add details to each section heading you created in the beginning.   
• Ensure your sentences are concise and use plain language. A research proposal usually contains about 2,000 to 4,000 words or four to seven pages.  
• Don’t use too many technical terms and abbreviations assuming that the readers would know them. Define the abbreviations and technical terms.  
• Ensure that the entire content is readable. Avoid using long paragraphs because they affect the continuity in reading. Break them into shorter paragraphs and introduce some white space for readability.  
• Focus on only the major research issues and cite sources accordingly. Don’t include generic information or their sources in the literature review.  
• Proofread your final document to ensure there are no grammatical errors so readers can enjoy a seamless, uninterrupted read.  
• Use academic, scholarly language because it brings formality into a document.  
• Ensure that your title is created using the keywords in the document and is neither too long and specific nor too short and general.  
• Cite all sources appropriately to avoid plagiarism.  
• Make sure that you follow guidelines, if provided. This includes rules as simple as using a specific font or a hyphen or en dash between numerical ranges.  
• Ensure that you’ve answered all questions requested by the evaluating authority.  

Key Takeaways   

Here’s a summary of the main points about research proposals discussed in the previous sections:  

• A research proposal is a document that outlines the details of a proposed study and is created by researchers to submit to evaluators who could be research institutions, universities, faculty, etc.  
• Research proposals are usually about 2,000-4,000 words long, but this depends on the evaluating authority’s guidelines.  
• A good research proposal ensures that you’ve done your background research and assessed the feasibility of the research.  
• Research proposals have the following main sections—introduction, literature review, objectives, methodology, ethical considerations, and budget.  

Frequently Asked Questions  

Q1. How is a research proposal evaluated?  

A1. In general, most evaluators, including universities, broadly use the following criteria to evaluate research proposals . 6  

• Significance —Does the research address any important subject or issue, which may or may not be specific to the evaluator or university?  
• Content and design —Is the proposed methodology appropriate to answer the research question? Are the objectives clear and well aligned with the proposed methodology?  
• Sample size and selection —Is the target population or cohort size clearly mentioned? Is the sampling process used to select participants randomized, appropriate, and free of bias?  
• Timing —Are the proposed data collection dates mentioned clearly? Is the project feasible given the specified resources and timeline?  
• Data management and dissemination —Who will have access to the data? What is the plan for data analysis?  

Q2. What is the difference between the Introduction and Literature Review sections in a research proposal ?  

A2. The Introduction or Background section in a research proposal sets the context of the study by describing the current scenario of the subject and identifying the gaps and need for the research. A Literature Review, on the other hand, provides references to all prior relevant literature to help corroborate the gaps identified and the research need.  

Q3. How long should a research proposal be?  

A3. Research proposal lengths vary with the evaluating authority like universities or committees and also the subject. Here’s a table that lists the typical research proposal lengths for a few universities.  

Q4. What are the common mistakes to avoid in a research proposal ?  

A4. Here are a few common mistakes that you must avoid while writing a research proposal . 7  

• No clear objectives: Objectives should be clear, specific, and measurable for the easy understanding among readers.  
• Incomplete or unconvincing background research: Background research usually includes a review of the current scenario of the particular industry and also a review of the previous literature on the subject. This helps readers understand your reasons for undertaking this research because you identified gaps in the existing research.  
• Overlooking project feasibility: The project scope and estimates should be realistic considering the resources and time available.   
• Neglecting the impact and significance of the study: In a research proposal , readers and evaluators look for the implications or significance of your research and how it contributes to the existing research. This information should always be included.  
• Unstructured format of a research proposal : A well-structured document gives confidence to evaluators that you have read the guidelines carefully and are well organized in your approach, consequently affirming that you will be able to undertake the research as mentioned in your proposal.  
• Ineffective writing style: The language used should be formal and grammatically correct. If required, editors could be consulted, including AI-based tools such as Paperpal , to refine the research proposal structure and language.  

Thus, a research proposal is an essential document that can help you promote your research and secure funds and grants for conducting your research. Consequently, it should be well written in clear language and include all essential details to convince the evaluators of your ability to conduct the research as proposed.  

This article has described all the important components of a research proposal and has also provided tips to improve your writing style. We hope all these tips will help you write a well-structured research proposal to ensure receipt of grants or any other purpose.  

References  

• Sudheesh K, Duggappa DR, Nethra SS. How to write a research proposal? Indian J Anaesth. 2016;60(9):631-634. Accessed July 15, 2024. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5037942/  
• Writing research proposals. Harvard College Office of Undergraduate Research and Fellowships. Harvard University. Accessed July 14, 2024. https://uraf.harvard.edu/apply-opportunities/app-components/essays/research-proposals  
• What is a research proposal? Plus how to write one. Indeed website. Accessed July 17, 2024. https://www.indeed.com/career-advice/career-development/research-proposal  
• Research proposal template. University of Rochester Medical Center. Accessed July 16, 2024. https://www.urmc.rochester.edu/MediaLibraries/URMCMedia/pediatrics/research/documents/Research-proposal-Template.pdf  
• Tips for successful proposal writing. Johns Hopkins University. Accessed July 17, 2024. https://research.jhu.edu/wp-content/uploads/2018/09/Tips-for-Successful-Proposal-Writing.pdf  
• Formal review of research proposals. Cornell University. Accessed July 18, 2024. https://irp.dpb.cornell.edu/surveys/survey-assessment-review-group/research-proposals  
• 7 Mistakes you must avoid in your research proposal. Aveksana (via LinkedIn). Accessed July 17, 2024. https://www.linkedin.com/pulse/7-mistakes-you-must-avoid-your-research-proposal-aveksana-cmtwf/  

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Get accurate academic translations, rewriting support, grammar checks, vocabulary suggestions, and generative AI assistance that delivers human precision at machine speed. Try for free or upgrade to Paperpal Prime starting at US$19 a month to access premium features, including consistency, plagiarism, and 30+ submission readiness checks to help you succeed.  

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Peer-reviewed

Research Article

Archaeology in space: The Sampling Quadrangle Assemblages Research Experiment (SQuARE) on the International Space Station. Report 1: Squares 03 and 05

Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review & editing

* E-mail: [email protected]

Affiliations Department of Art, Chapman University, Orange, CA, United States of America, Space Engineering Research Center, University of Southern California, Marina del Rey, CA, United States of America

Roles Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Affiliation Department of History, Carleton University, Ottawa, ON, United States of America

Roles Conceptualization, Data curation, Methodology, Project administration, Supervision, Writing – review & editing

Affiliation College of Humanities, Arts and Social Sciences, Flinders University, Adelaide, Australia

Roles Software, Writing – original draft

Roles Investigation, Writing – original draft

Affiliation Archaeology Research Center, University of Southern California, Los Angeles, CA, United States of America

• Justin St. P. Walsh, 
• Shawn Graham, 
• Alice C. Gorman, 
• Chantal Brousseau, 
• Salma Abdullah

• Published: August 7, 2024
• https://doi.org/10.1371/journal.pone.0304229
• Reader Comments

Between January and March 2022, crew aboard the International Space Station (ISS) performed the first archaeological fieldwork in space, the Sampling Quadrangle Assemblages Research Experiment (SQuARE). The experiment aimed to: (1) develop a new understanding of how humans adapt to life in an environmental context for which we are not evolutionarily adapted, using evidence from the observation of material culture; (2) identify disjunctions between planned and actual usage of facilities on a space station; (3) develop and test techniques that enable archaeological research at a distance; and (4) demonstrate the relevance of social science methods and perspectives for improving life in space. In this article, we describe our methodology, which involves a creative re-imagining of a long-standing sampling practice for the characterization of a site, the shovel test pit. The ISS crew marked out six sample locations (“squares”) around the ISS and documented them through daily photography over a 60-day period. Here we present the results from two of the six squares: an equipment maintenance area, and an area near exercise equipment and the latrine. Using the photographs and an innovative webtool, we identified 5,438 instances of items, labeling them by type and function. We then performed chronological analyses to determine how the documented areas were actually used. Our results show differences between intended and actual use, with storage the most common function of the maintenance area, and personal hygiene activities most common in an undesignated area near locations for exercise and waste.

Citation: Walsh JSP, Graham S, Gorman AC, Brousseau C, Abdullah S (2024) Archaeology in space: The Sampling Quadrangle Assemblages Research Experiment (SQuARE) on the International Space Station. Report 1: Squares 03 and 05. PLoS ONE 19(8): e0304229. https://doi.org/10.1371/journal.pone.0304229

Editor: Peter F. Biehl, University of California Santa Cruz, UNITED STATES OF AMERICA

Received: March 9, 2024; Accepted: May 7, 2024; Published: August 7, 2024

Copyright: © 2024 Walsh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: JW was the recipient of funding from Chapman University’s Office of Research and Sponsored Programs to support the activities of Axiom Space as implementation partner for the research presented in this article. There are no associated grant numbers for this financial support. Axiom Space served in the role of a contractor hired by Chapman University for the purpose of overseeing logistics relating to our research. In-kind support in the form of ISS crew time and access to the space station’s facilities, also awarded to JW from the ISS National Laboratory, resulted from an unsolicited proposal, and therefore there is no opportunity title or number associated with our work. No salary was received by any of the investigators as a result of the grant support. No additional external funding was received for this study.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The International Space Station Archaeological Project (ISSAP) aims to fill a gap in social science investigation into the human experience of long-duration spaceflight [ 1 – 3 ]. As the largest, most intensively inhabited space station to date, with over 270 visitors from 23 countries during more than 23 years of continuous habitation, the International Space Station (ISS) is the ideal example of a new kind of spacefaring community—“a microsociety in a miniworld” [ 4 ]. While it is possible to interview crew members about their experiences, the value of an approach focused on material culture is that it allows identification of longer-term patterns of behaviors and associations that interlocutors are unable or even unwilling to articulate. In this respect, we are inspired by previous examples of contemporary archaeology such as the Tucson Garbage Project and the Undocumented Migration Project [ 5 – 7 ]. We also follow previous discussions of material culture in space contexts that highlight the social and cultural features of space technology [ 8 , 9 ].

Our primary goal is to identify how humans adapt to life in a new environment for which our species has not evolved, one characterized by isolation, confinement, and especially microgravity. Microgravity introduces opportunities, such as the ability to move and work in 360 degrees, and to carry out experiments impossible in full Earth gravity, but also limitations, as unrestrained objects float away. The most routine activities carried out on Earth become the focus of intense planning and technological intervention in microgravity. By extension, our project also seeks to develop archaeological techniques that permit the study of other habitats in remote, extreme, or dangerous environments [ 10 , 11 ]. Since it is too costly and difficult to visit our archaeological site in person, we have to creatively re-imagine traditional archaeological methods to answer key questions. To date, our team has studied crew-created visual displays [ 12 , 13 ], meanings and processes associated with items returned to Earth [ 14 ], distribution of different population groups around the various modules [ 15 ], and the development of machine learning (ML) computational techniques to extract data about people and places, all from historic photographs of life on the ISS [ 16 ].

From January to March 2022, we developed a new dataset through the first archaeological work conducted off-Earth. We documented material culture in six locations around the ISS habitat, using daily photography taken by the crew which we then annotated and studied as evidence for changes in archaeological assemblages of material culture over time. This was the first time such data had been captured in a way that allowed statistical analysis. Here, we present the data and results from Squares 03 and 05, the first two sample locations to be completed.

Materials and methods

Square concept and planning.

Gorman proposed the concept behind the investigation, deriving it from one of the most traditional terrestrial archaeological techniques, the shovel test pit. This method is used to understand the overall characteristics of a site quickly through sampling. A site is mapped with a grid of one-meter squares. Some of the squares are selected for initial excavation to understand the likely spatial and chronological distribution of features across the entire site. In effect, the technique is a way to sample a known percentage of the entire site systematically. In the ISS application of this method, we documented a notional stratigraphy through daily photography, rather than excavation.

Historic photography is a key dataset for the International Space Station Archaeological Project. Tens of thousands of images have been made available to us, either through publication [ 17 ], or through an arrangement with the ISS Research Integration Office, which supplied previously unpublished images from the first eight years of the station’s habitation. These photographs are informative about the relationships between people, places, and objects over time in the ISS. However, they were taken randomly (from an archaeological perspective) and released only according to NASA’s priorities and rules. Most significantly, they were not made with the purpose of answering archaeological questions. By contrast, the photographs taken during the present investigation were systematic, representative of a defined proportion of the habitat’s area, and targeted towards capturing archaeology’s primary evidence: material culture. We were interested in how objects move around individual spaces and the station, what these movements revealed about crew adherence to terrestrial planning, and the creative use of material culture to make the laboratory-like interior of the ISS more habitable.

Access to the field site was gained through approval of a proposal submitted to the Center for the Advancement of Science in Space (also known as the ISS National Laboratory [ISS NL]). Upon acceptance, Axiom Space was assigned as the Implementation Partner for carriage of the experiment according to standard procedure. No other permits were required for this work.

Experiment design

Since our work envisioned one-meter sample squares, and recognizing the use of acronyms as a persistent element of spacefaring culture, we named our payload the Sampling Quadrangle Assemblages Research Experiment (SQuARE). Permission from the ISS NL to conduct SQuARE was contingent on using equipment that was already on board the space station. SQuARE required only five items: a camera, a wide-angle lens, adhesive tape (for marking the boundaries of the sample locations), a ruler (for scale), and a color calibration card (for post-processing of the images). All of these were already present on the ISS.

Walsh performed tests on the walls of a terrestrial art gallery to assess the feasibility of creating perfect one-meter squares in microgravity. He worked on a vertical surface, using the Pythagorean theorem to determine where the corners should be located. The only additional items used for these tests were two metric measuring tapes and a pencil for marking the wall (these were also already on the ISS). While it was possible to make a square this way, it also became clear that at least two people were needed to manage holding the tape measures in position while marking the points for the corners. This was not possible in the ISS context.

Walsh and Gorman identified seven locations for the placement of squares. Five of these were in the US Orbital Segment (USOS, consisting of American, European, and Japanese modules) and two in the Russian Orbital Segment. Unfortunately, tense relations between the US and Russian governments meant we could only document areas in the USOS. The five locations were (with their SQuARE designations):

• 01—an experimental rack on the forward wall, starboard end, of the Japanese Experiment Module
• 02—an experimental rack on the forward wall, port end, of the European laboratory module Columbus
• 03—the starboard Maintenance Work Area (workstation) in the US Node 2 module
• 04—the wall area “above” (according to typical crew body orientation) the galley table in the US Node 1 module
• 05—the aft wall, center location, of the US Node 3 module

Our square selection encompassed different modules and activities, including work and leisure. We also asked the crew to select a sixth sample location based on their understanding of the experiment and what they thought would be interesting to document. They chose a workstation on the port wall of the US laboratory module, at the aft end, which they described in a debriefing following their return to Earth in June 2022 as “our central command post, like our shared office situation in the lab.” Results from the four squares not included here will appear in future publications.

Walsh worked with NASA staff to determine payload procedures, including precise locations for the placement of the tape that would mark the square boundaries. The squares could not obstruct other facilities or experiments, so (unlike in terrestrial excavations, where string is typically used to demarcate trench boundaries) only the corners of each square were marked, not the entire perimeter. We used Kapton tape due to its bright yellow-orange color, which aided visibility for the crew taking photographs and for us when cropping the images. In practice, due to space constraints, the procedures that could actually be performed by crew in the ISS context, and the need to avoid interfering with other ongoing experiments, none of the locations actually measured one square meter or had precise 90° corners like a trench on Earth.

On January 14, 2022, NASA astronaut Kayla Barron set up the sample locations, marking the beginning of archaeological work in space ( S1 Movie ). For 30 days, starting on January 21, a crew member took photos of the sample locations at approximately the same time each day; the process was repeated at a random time each day for a second 30-day period to eliminate biases. Photography ended on March 21, 2022. The crew were instructed not to move any items prior to taking the photographs. Walsh led image management, including color and barrel distortion correction, fixing the alignment of each image, and cropping them to the boundaries of the taped corners.

Data processing—Item tagging, statistics, visualizations

We refer to each day’s photo as a “context” by analogy with chronologically-linked assemblages of artifacts and installations at terrestrial archaeological sites ( S1 and S2 Datasets). As previously noted, each context represented a moment roughly 24 hours distant from the previous one, showing evidence of changes in that time. ISS mission planners attempted to schedule the activity at the same time in the first month, but there were inevitable changes due to contingencies. Remarkably, the average time between contexts in Phase 1 was an almost-perfect 24h 0m 13s. Most of the Phase 1 photos were taken between 1200 and 1300 GMT (the time zone in which life on the ISS is organized). In Phase 2, the times were much more variable, but the average time between contexts during this period was still 23h 31m 45s. The earliest Phase 2 photo was taken at 0815 GMT, and the latest at 2101. We did not identify any meaningful differences between results from the two phases.

Since the “test pits” were formed of images rather than soil matrices, we needed a tool to capture information about the identity, nature, and location of every object. An open-source image annotator platform [ 18 ] mostly suited our needs. Brousseau rebuilt the platform to work within the constraints of our access to the imagery (turning it into a desktop tool with secure access to our private server), to permit a greater range of metadata to be added to each item or be imported, to autosave, and to export the resulting annotations. The tool also had to respect privacy and security limitations required by NASA.

The platform Brousseau developed and iterated was rechristened “Rocket-Anno” ( S1 File ). For each context photograph, the user draws an outline around every object, creating a polygon; each polygon is assigned a unique ID and the user provides the relevant descriptive information, using a controlled vocabulary developed for ISS material culture by Walsh and Gorman. Walsh and Abdullah used Rocket-Anno to tag the items in each context for Squares 03 and 05. Once all the objects were outlined for every context’s photograph, the tool exported a JSON file with all of the metadata for both the images themselves and all of the annotations, including the coordinate points for every polygon ( S3 Dataset ). We then developed Python code using Jupyter “notebooks” (an interactive development environment) that ingests the JSON file and generates dataframes for various facets of the data. Graham created a “core” notebook that exports summary statistics, calculates Brainerd-Robinson coefficients of similarity, and visualizes the changing use of the square over time by indicating use-areas based on artifact types and subtypes ( S2 File ). Walsh and Abdullah also wrote detailed square notes with context-by-context discussions and interpretations of features and patterns.

We asked NASA for access to the ISS Crew Planner, a computer system that shows each astronaut’s tasks in five-minute increments, to aid with our interpretation of contexts, but were denied. As a proxy, we use another, less detailed source: the ISS Daily Summary Reports (DSRs), published on a semi-regular basis by NASA on its website [ 19 ]. Any activities mentioned in the DSRs often must be connected with a context by inference. Therefore, our conclusions are likely less precise than if we had seen the Crew Planner, but they also more clearly represent the result of simply observing and interpreting the material culture record.

The crew during our sample period formed ISS Expedition 66 (October 2021-March 2022). They were responsible for the movement of objects in the sample squares as they carried out their daily tasks. The group consisted of two Russians affiliated with Roscosmos (the Russian space agency, 26%), one German belonging to the European Space Agency (ESA, 14%), and four Americans employed by NASA (57%). There were six men (86%) and one woman (14%), approximately equivalent to the historic proportions in the ISS population (84% and 16%, respectively). The Russian crew had their sleeping quarters at the aft end of the station, in the Zvezda module. The ESA astronaut slept in the European Columbus laboratory module. The four NASA crew slept in the US Node 2 module (see below). These arrangements emphasize the national character of discrete spaces around the ISS, also evident in our previous study of population distributions [ 15 ]. Both of the sample areas in this study were located in US modules.

Square 03 was placed in the starboard Maintenance Work Area (MWA, Fig 1 ), one of a pair of workstations located opposite one another in the center of the Node 2 module, with four crew berths towards the aft and a series of five ports for the docking of visiting crew/cargo vehicles and two modules on the forward end ( Fig 2 ). Node 2 (sometimes called “Harmony”) is a connector that links the US, Japanese, and European lab modules. According to prevailing design standards when the workstation was developed, an MWA “shall serve as the primary location for servicing and repair of maximum sized replacement unit/system components” [ 20 ]. Historic images published by NASA showing its use suggested that its primary function was maintenance of equipment and also scientific work that did not require a specific facility such as a centrifuge or furnace.

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An open crew berth is visible at right. The yellow dotted line indicates the boundaries of the sample area. Credit: NASA/ISSAP.

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Credit: Tor Finseth, by permission, modified by Justin Walsh.

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Square 03 measured 90.3 cm (top) x 87.8 (left) x 89.4 (bottom) x 87.6 (right), for an area of approximately 0.79 m 2 . Its primary feature was a blue metal panel with 40 square loop-type Velcro patches arranged in four rows of ten. During daily photography, many items were attached to the Velcro patches (or held by a clip or in a resealable bag which had its own hook-type Velcro). Above and below the blue panel were additional Velcro patches placed directly on the white plastic wall surface. These patches were white, in different sizes and shapes and irregularly arranged, indicating that they had been placed on the wall in response to different needs. Some were dirty, indicating long use. The patches below the blue panel were rarely used during the sample period, but the patches above were used frequently to hold packages of wet wipes, as well as resealable bags with electrostatic dispersion kits and other items. Outside the sample area, the primary features were a crew berth to the right, and a blue metal table attached to the wall below. This table, the primary component of the MWA, “provides a rigid surface on which to perform maintenance tasks,” according to NASA [ 21 ]. It is modular and can be oriented in several configurations, from flat against the wall to horizontal ( i . e ., perpendicular to the wall). A laptop to the left of the square occasionally showed information about work happening in the area.

In the 60 context photos of Square 03, we recorded 3,608 instances of items, an average of 60.1 (median = 60.5) per context. The lowest count was 24 in context 2 (where most of the wall was hidden from view behind an opaque storage bag), and the highest was 75 in both contexts 20 and 21. For comparison between squares, we can also calculate the item densities per m 2 . The average count was 76.1/m 2 (minimum = 30, maximum = 95). The count per context ( Fig 3(A)) began much lower than average in the first three contexts because of a portable glovebag and a stowage bag that obscured much of the sample square. It rose to an above-average level which was sustained (with the exception of contexts 11 and 12, which involved the appearance of another portable glovebag) until about context 43, when the count dipped again and the area seemed to show less use. Contexts 42–59 showed below-average numbers, as much as 20% lower than previously.

(a) Count of artifacts in Square 03 over time. (b) Proportions of artifacts by function in Square 03. Credit: Rao Hamza Ali.

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74 types of items appeared at least once here, belonging to six categories: equipment (41%), office supplies (31%), electronic (17%), stowage (9%), media (1%), and food (<1%). To better understand the significance of various items in the archaeological record, we assigned them to functional categories ( Table 1 , Fig 3(B)) . 35% of artifacts were restraints, or items used for holding other things in place; 12% for tools; 9% for containers; 9% for writing items; 6% for audiovisual items; 6% for experimental items; 4% for lights; 4% for safety items; 4% for body maintenance; 4% for power items; 3% for computing items; 1% for labels; and less than 1% drinks. We could not identify a function for two percent of the items.

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One of the project goals is understanding cultural adaptations to the microgravity environment. We placed special attention on “gravity surrogates,” pieces of (often simple) technology that are used in space to replicate the terrestrial experience of things staying where they are placed. Gravity surrogates include restraints and containers. It is quite noticeable that gravity surrogates comprise close to half of all items (44%) in Square 03, while the tools category, which might have been expected to be most prominent in an area designated for maintenance, is less than one-third as large (12%). Adding other groups associated with work, such as “experiment” and “light,” only brings the total to 22%.

Square 05 (Figs 2 and 4 ) was placed in a central location on the aft wall of the multipurpose Node 3 (“Tranquility”) module. This module does not include any specific science facilities. Instead, there are two large pieces of exercise equipment, the TVIS (Treadmill with Vibration Isolation Stabilization System, on the forward wall at the starboard end), and the ARED (Advanced Resistive Exercise Device, on the overhead wall at the port end). Use of the machines forms a significant part of crew activities, as they are required to exercise for two hours each day to counteract loss of muscle mass and bone density, and enable readjustment to terrestrial gravity on their return. The Waste and Hygiene Compartment (WHC), which includes the USOS latrine, is also here, on the forward wall in the center of the module, opposite Square 05. Finally, three modules are docked at Node 3’s port end. Most notable is the Cupola, a kind of miniature module on the nadir side with a panoramic window looking at Earth. This is the most popular leisure space for the crew, who often describe the hours they spend there. The Permanent Multipurpose Module (PMM) is docked on the forward side, storing equipment, food, and trash. In previous expeditions, some crew described installing a curtain in the PMM to create a private space for changing clothes and performing body maintenance activities such as cleaning oneself [ 22 , 23 ], but it was unclear whether that continued to be its function during the expedition we observed. One crew member during our sample period posted a video on Instagram showing the PMM interior and their efforts to re-stow equipment in a bag [ 24 ]. The last space attached to Node 3 is an experimental inflatable module docked on the aft side, called the Bigelow Expandable Activity Module (BEAM), which is used for storage of equipment.

The yellow dotted line indicates the boundaries of the sample area. The ARED machine is at the far upper right, on the overhead wall. The TVIS treadmill is outside this image to the left, on the forward wall. The WHC is directly behind the photographer. Credit: NASA/ISSAP.

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Square 05 was on a mostly featureless wall, with a vertical handrail in the middle. Handrails are metal bars located throughout the ISS that are used by the crew to hold themselves in place or provide a point from which to propel oneself to another location. NASA’s most recent design standards acknowledge that “[t]hey also serve as convenient locations for temporary mounting, affixing, or restraint of loose equipment and as attachment points for equipment” [ 25 ]. The handrail in Square 05 was used as an impromptu object restraint when a resealable bag filled with other bags was squeezed between the handrail and the wall.

The Brine Processing Assembly (BPA), a white plastic box which separates water from other components of urine for treatment and re-introduction to the station’s drinkable water supply [ 26 ], was fixed to the wall outside the square boundaries at lower left. A bungee cord was attached to both sides of the box; the one on the right was connected at its other end to the handrail attachment bracket. Numerous items were attached to or wedged into this bungee cord during the survey, bringing “gravity” into being. A red plastic duct ran through the square from top center into the BPA. This duct led from the latrine via the overhead wall. About halfway through the survey period, in context 32, the duct was wrapped in Kapton tape. According to the DSR for that day, “the crew used duct tape [ sic ] to make a seal around the BPA exhaust to prevent odor permeation in the cabin” [ 27 ], revealing an aspect of the crew’s experience of this area that is captured only indirectly in the context photograph. Permanently attached to the wall were approximately 20 loop-type Velcro patches in many shapes and sizes, placed in a seemingly random pattern that likely indicates that they were put there at different times and for different reasons.

Other common items in Square 05 were a mirror, a laptop computer, and an experimental item belonging to the German space agency DLR called the Touch Array Assembly [ 28 ]. The laptop moved just three times, and only by a few centimeters each time, during the sample period. The Touch Array was a black frame enclosing three metal surfaces which were being tested for their bacterial resistance; members of the crew touched the surfaces at various moments during the sample period. Finally, and most prominent due to its size, frequency of appearance, and use (judged by its movement between context photos) was an unidentified crew member’s toiletry kit.

By contrast with Square 03, 05 was the most irregular sample location, roughly twice as wide as it was tall. Its dimensions were 111 cm (top) x 61.9 (left) x 111.4 (bottom) x 64.6 (right), for an area of approximately 0.7 m 2 , about 89% of Square 03. We identified 1,830 instances of items in the 60 contexts, an average of 30.5 (median = 32) per context. The minimum was 18 items in context 5, and the maximum was 39 in contexts 24, 51, and 52. The average item density was 43.6/m 2 (minimum = 26, maximum = 56), 57% of Square 03.

The number of items trended upward throughout the sample period ( Fig 5(A)) . The largest spike occurred in context 6 with the appearance of the toiletry kit, which stored (and revealed) a number of related items. The kit can also be linked to one of the largest dips in item count, seen from contexts 52 to 53, when it was closed (but remained in the square). Other major changes can often be attributed to the addition and removal of bungee cords, which had other items such as carabiners and brackets attached. For example, the dip seen in context 25 correlates with the removal of a bungee cord with four carabiners.

(a) Count of artifacts and average count in Square 05 over time. (b) Proportions of artifacts by function in Square 05. Credit: Rao Hamza Ali.

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41 different item types were found in Square 05, about 55% as many as in Square 03. These belonged to five different categories: equipment (63%), electronic (17%), stowage (10%), office supplies (5%), and food (2%). The distribution of function proportions was quite different in this sample location ( Table 2 and Fig 5(B)) . Even though restraints were still most prominent, making up 32% of all items, body maintenance was almost as high (30%), indicating how strongly this area was associated with the activity of cleaning and caring for oneself. Computing (8%, represented by the laptop, which seems not to have been used), power (8%, from various cables), container (7%, resealable bags and Cargo Transfer Bags), and hygiene (6%, primarily the BPA duct) were the next most common items. Experiment was the function of 4% of the items, mostly the Touch Array, which appeared in every context, followed by drink (2%) and life support (1%). Safety, audiovisual, food, and light each made up less than 1% of the functional categories.

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Tracking changes over time is critical to understanding the activity happening in each area. We now explore how the assemblages change by calculating the Brainerd-Robinson Coefficient of Similarity [ 29 , 30 ] as operationalized by Peeples [ 31 , 32 ]. This metric is used in archaeology for comparing all pairs of the contexts by the proportions of categorical artifact data, here functional type. Applying the coefficient to the SQuARE contexts enables identification of time periods for distinct activities using artifact function and frequency alone, independent of documentary or oral evidence.

Multiple phases of activities took place in the square. Moments of connected activity are visible as red clusters in contexts 0–2, 11–12, 28–32, and 41 ( Fig 6(A)) . Combining this visualization with close observation of the photos themselves, we argue that there are actually eight distinct chronological periods.

• Contexts 0–2: Period 1 (S1 Fig in S3 File ) is a three-day period of work involving a portable glovebag (contexts 0–1) and a large blue stowage bag (context 2). It is difficult to describe trends in functional types because the glovebag and stowage bag obstruct the view of many objects. Items which appear at the top of the sample area, such as audiovisual and body maintenance items, are overemphasized in the data as a result. It appears that some kind of science is happening here, perhaps medical sample collection due to the presence of several small resealable bags visible in the glovebag. The work appears particularly intense in context 1, with the positioning of the video camera and light to point into the glovebag. These items indicate observation and oversight of crew activities by ground control. A white cargo transfer bag for storage and the stowage bag for holding packing materials in the context 2 photo likely relate to the packing of a Cargo Dragon vehicle that was docked to Node 2. The Dragon departed from the ISS for Earth, full of scientific samples, equipment, and crew personal items, a little more than three hours after the context 2 photo was taken [ 33 ].
• Contexts 3–10: Period 2 (S2 Fig in S3 File ) was a “stable” eight-day period in the sample, when little activity is apparent, few objects were moved or transferred in or out the square, and the primary function of the area seems to be storage rather than work. In context 6, a large Post-It notepad appeared in the center of the metal panel with a phone number written on it. This number belonged to another astronaut, presumably indicating that someone on the ISS had been told to call that colleague on the ground (for reasons of privacy, and in accordance with NASA rules for disseminating imagery, we have blurred the number in the relevant images). In context 8, the same notepad sheet had new writing appear on it, this time reading “COL A1 L1,” the location of an experimental rack in the European lab module.
• Contexts 11–12: Period 3 (S3 Fig in S3 File ) involves a second appearance of a portable glovebag (a different one from that used in contexts 0–1, according to its serial number), this time for a known activity, a concrete hardening experiment belonging to the European Space Agency [ 34 , 35 ]. This two-day phase indicates how the MWA space can be shared with non-US agencies when required. It also demonstrates the utility of this flexible area for work beyond biology/medicine, such as material science. Oversight of the crew’s activities by ground staff is evident from the positioning of the video camera and LED light pointing into the glovebag.
• Contexts 13–27: Period 4 (S4 Fig in S3 File ) is another stable fifteen-day period, similar to Period 2. Many items continued to be stored on the aluminum panel. The LED light’s presence is a trace of the activity in Period 3 that persists throughout this phase. Only in context 25 can a movement of the lamp potentially be connected to an activity relating to one of the stored items on the wall: at least one nitrile glove was removed from a resealable bag behind the lamp. In general, the primary identifiable activity during Period 4 is storage.
• Contexts 28–32: Period 5 (S5 Fig in S3 File ), by contrast, represents a short period of five days of relatively high and diverse activity. In context 28, a Microsoft Hololens augmented reality headset appeared. According to the DSR for the previous day, a training activity called Sidekick was carried out using the headset [ 36 ]. The following day, a Saturday, showed no change in the quantity or type of objects, but many were moved around and grouped by function—adhesive tape rolls were placed together, tools were moved from Velcro patches into pouches or straightened, and writing implements were placed in a vertical orientation when previously they were tilted. Context 29 represents a cleaning and re-organization of the sample area, which is a common activity for the crew on Saturdays [ 37 ]. Finally, in context 32, an optical coherence tomography scanner—a large piece of equipment for medical research involving crew members’ eyes—appeared [ 38 ]. This device was used previously during the sample period, but on the same day as the ESA concrete experiment, so that earlier work seems to have happened elsewhere [ 39 ].
• Contexts 33–40: Period 6 (S6 Fig in S3 File ) is the third stable period, in which almost no changes are visible over eight days. The only sign of activity is a digital timer which was started six hours before the context 39 image was made and continued to run at least through context 42.
• Context 41: Period 7 (S7 Fig in S3 File ) is a single context in which medical sample collection may have occurred. Resealable bags (some holding others) appeared in the center of the image and at lower right. One of the bags at lower right had a printed label reading “Reservoir Containers.” We were not able to discern which type of reservoir containers the label refers to, although the DSR for the day mentions “[Human Research Facility] Generic Saliva Collection,” without stating the location for this work [ 40 ]. Evidence from photos of other squares shows that labeled bags could be re-used for other purposes, so our interpretation of medical activity for this context is not conclusive.
• Contexts 42–60: Period 8 (S8 Fig in S3 File ) is the last and longest period of stability and low activity—eighteen days in which no specific activity other than the storage of items can be detected. The most notable change is the appearance for the first time of a foil water pouch in the central part of the blue panel.

Visualization of Brainerd-Robinson similarity, compared context-by-context by item function, for (a) Square 03 and (b) Square 05. The more alike a pair of contexts is, the higher the coefficient value, with a context compared against itself where a value of 200 equals perfect similarity. The resulting matrix of coefficients is visualized on a scale from blue to red where blue is lowest and red is highest similarity. The dark red diagonal line indicates complete similarity, where each context is compared to itself. Dark blue represents a complete difference. Credit: Shawn Graham.

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In the standards used at the time of installation, “stowage space” was the sixth design requirement listed for the MWA after accessibility; equipment size capability; scratch-resistant surfaces; capabilities for electrical, mechanical, vacuum, and fluid support during maintenance; and the accommodation of diagnostic equipment [ 20 ]. Only capabilities for fabrication were listed lower than stowage. Yet 50 of the 60 contexts (83%) fell within stable periods where little or no activity is identifiable in Square 03. According to the sample results, therefore, this area seems to exist not for “maintenance,” but primarily for the storage and arrangement of items. The most recent update of the design standards does not mention the MWA, but states, “Stowage location of tool kits should be optimized for accessibility to workstations and/or maintenance workbenches” [ 25 ]. Our observation confirms the importance of this suggestion.

The MWA was also a flexible location for certain science work, like the concrete study or crew health monitoring. Actual maintenance of equipment was hardly in evidence in the sample (possibly contexts 25, 39, and 44), and may not even have happened at all in this location. Some training did happen here, such as review of procedures for the Electromagnetic Levitator camera (instructions for changing settings on a high-speed camera appeared on the laptop screen; the day’s DSR shows that this camera is part of the Electromagnetic Levitator facility, located in the Columbus module [ 41 ]. The training required the use of the Hololens system (context 28 DSR, cited above).

Although many item types were represented in Square 03, it became clear during data capture how many things were basically static, unmoving and therefore unused, especially certain tools, writing implements, and body maintenance items. The MWA was seen as an appropriate place to store these items. It may be the case that their presence here also indicates that their function was seen as an appropriate one for this space, but the function(s) may not be carried out—or perhaps not in this location. Actualization of object function was only visible to us when the state of the item changed—it appeared, it moved, it changed orientation, it disappeared, or, in the case of artifacts that were grouped in collections rather than found as singletons, its shape changed or it became visibly smaller/lesser. We therefore have the opportunity to explore not only actuality of object use, but also potentiality of use or function, and the meaning of that quality for archaeological interpretation [ 42 , 43 ]. This possibility is particularly intriguing in light of the archaeological turn towards recognizing the agency of objects to impact human activity [ 44 , 45 ]. We will explore these implications in a future publication.

We performed the same chronological analysis for Square 05. Fig 6(B) represents the analysis for both item types and for item functions. We identified three major phases of activity, corresponding to contexts 0–5, 6–52, and 53–59 (S9-S11 Figs in S3 File ). The primary characteristics of these phases relate to an early period of unclear associations (0–5) marked by the presence of rolls of adhesive tape and a few body maintenance items (toothpaste and toothbrush, wet wipes); the appearance of a toiletry kit on the right side of the sample area, fully open with clear views of many of the items contained within (6–52); and finally, the closure of the toiletry kit so that its contents can no longer be seen (53–59). We interpret the phases as follows:

• Contexts 0–5: In Period 1 (six days, S9 Fig in S3 File ), while items such as a mirror, dental floss picks, wet wipes, and a toothbrush held in the end of a toothpaste tube were visible, the presence of various other kinds of items confounds easy interpretation. Two rolls of duct tape were stored on the handrail in the center of the sample area, and the Touch Array and laptop appeared in the center. Little movement can be identified, apart from a blue nitrile glove that appeared in context 1 and moved left across the area until it was wedged into the bungee cord for contexts 3 and 4. The tape rolls were removed prior to context 5. A collection of resealable bags was wedged behind the handrail in context 3, remaining there until context 9. Overall, this appears to be a period characterized by eclectic associations, showing an area without a clear designated function.
• Contexts 6–52: Period 2 (S10 Fig in S3 File ) is clearly the most significant one for this location due to its duration (47 days). It was dominated by the number of body maintenance items located in and around the toiletry kit, especially a white hand towel (on which a brown stain was visible from context 11, allowing us to confirm that the same towel was present until context 46). A second towel appeared alongside the toiletry kit in context 47, and the first one was fixed at the same time to the handrail, where it remained through the end of the sample period. A third towel appeared in context 52, attached to the handrail together with the first one by a bungee cord, continuing to the end of the sample period. Individual body maintenance items moved frequently from one context to the next, showing the importance of this type of activity for this part of Node 3. For reasons that are unclear, the mirror shifted orientation from vertical to diagonal in context 22, and then was put back in a vertical orientation in context 31 (a Monday, a day which is not traditionally associated with cleaning and organization). Collections of resealable bags appeared at various times, including a large one labeled “KYNAR BAG OF ZIPLOCKS” in green marker at the upper left part of the sample area beginning of context 12 (Kynar is a non-flammable plastic material that NASA prefers for resealable bags to the generic commercial off-the-shelf variety because it is non-flammable; however, its resistance to heat makes it less desirable for creating custom sizes, so bags made from traditional but flammable low-density polyethylene still dominate on the ISS [ 14 ]). The Kynar bag contained varying numbers of bags within it over time; occasionally, it appeared to be empty. The Touch Array changed orientation on seven of 47 days in period 2, or 15% of the time (12% of all days in the survey), showing activity associated with scientific research in this area. In context 49, a life-support item, the Airborne Particulate Monitor (APM) was installed [ 46 ]. This device, which measures “real-time particulate data” to assess hazards to crew health [ 47 ], persisted through the end of the sample period.
• Contexts 53–59: Period 3 (S11 Fig in S3 File ) appears as a seven-day phase marked by low activity. Visually, the most notable feature is the closure of the toiletry kit, which led to much lower counts of body maintenance items. Hardly any of the items on the wall moved at all during this period.

While body maintenance in the form of cleaning and caring for oneself could be an expected function for an area with exercise and excretion facilities, it is worth noting that the ISS provides, at most, minimal accommodation for this activity. A description of the WHC stated, “To provide privacy…an enclosure was added to the front of the rack. This enclosure, referred to as the Cabin, is approximately the size of a typical bathroom stall and provides room for system consumables and hygiene item stowage. Space is available to also support limited hygiene functions such as hand and body washing” [ 48 ]. A diagram of the WHC in the same publication shows the Cabin without a scale but suggests that it measures roughly 2 m (h) x .75 (w) x .75 (d), a volume of approximately 1.125 m 3 . NASA’s current design standards state that the body volume of a 95th percentile male astronaut is 0.99 m 3 [ 20 ], meaning that a person of that size would take up 88% of the space of the Cabin, leaving little room for performing cleaning functions—especially if the Cabin is used as apparently intended, to also hold “system consumables and hygiene item[s]” that would further diminish the usable volume. This situation explains why crews try to adapt other spaces, such as storage areas like the PMM, for these activities instead. According to the crew debriefing statement, only one of them used the WHC for body maintenance purposes; it is not clear whether the toiletry kit belonged to that individual. But the appearance of the toiletry kit in Square 05—outside of the WHC, in a public space where others frequently pass by—may have been a response to the limitations of the WHC Cabin. It suggests a need for designers to re-evaluate affordances for body maintenance practices and storage for related items.

Although Square 03 and 05 were different sizes and shapes, comparing the density of items by function shows evidence of their usage ( Table 3 ). The typical context in Square 03 had twice as many restraints and containers, but less than one-quarter as many body maintenance items as Square 05. 03 also had many tools, lights, audiovisual equipment, and writing implements, while there were none of any of these types in 05. 05 had life support and hygiene items which were missing from 03. It appears that flexibility and multifunctionality were key elements for 03, while in 05 there was emphasis on one primary function (albeit an improvised one, designated by the crew rather than architects or ground control), cleaning and caring for one’s body, with a secondary function of housing static equipment for crew hygiene and life support.

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As this is the first time such an analysis has been performed, it is not yet possible to say how typical or unusual these squares are regarding the types of activities taking place; but they provide a baseline for eventual comparison with the other four squares and future work on ISS or other space habitats.

Some general characteristics are revealed by archaeological analysis of a space station’s material culture. First, even in a small, enclosed site, occupied by only a few people over a relatively short sample period, we can observe divergent patterns for different locations and activity phases. Second, while distinct functions are apparent for these two squares, they are not the functions that we expected prior to this research. As a result, our work fulfills the promise of the archaeological approach to understanding life in a space station by revealing new, previously unrecognized phenomena relating to life and work on the ISS. There is now systematically recorded archaeological data for a space habitat.

Squares 03 and 05 served quite different purposes. The reasons for this fact are their respective affordances and their locations relative to activity areas designated for science and exercise. Their national associations, especially the manifestation of the control wielded by NASA over its modules, also played a role in the use of certain materials, the placement of facilities, and the organization of work. How each area was used was also the result of an interplay between the original plans developed by mission planners and habitat designers (or the lack of such plans), the utility of the equipment and architecture in each location, and the contingent needs of the crew as they lived in the station. This interplay became visible in the station’s material culture, as certain areas were associated with particular behaviors, over time and through tradition—over the long duration across many crews (Node 2, location of Square 03, docked with the ISS in 2007, and Node 3, location of Square 05, docked in 2010), and during the specific period of this survey, from January to March 2022. During the crew debriefing, one astronaut said, “We were a pretty organized crew who was also pretty much on the same page about how to do things…. As time went on…we organized the lab and kind of got on the same page about where we put things and how we’re going to do things.” This statement shows how functional associations can become linked to different areas of the ISS through usage and mutual agreement. At the same time, the station is not frozen in time. Different people have divergent ideas about how and where to do things. It seems from the appearance of just one Russian item—a packet of generic wipes ( salfetky sukhiye ) stored in the toiletry kit throughout the sample period—that the people who used these spaces and carried out their functions did not typically include the ISS’s Russian crew. Enabling greater flexibility to define how spaces can be used could have a significant impact on improving crew autonomy over their lives, such as how and where to work. It could also lead to opening of all spaces within a habitat to the entire crew, which seems likely to improve general well-being.

An apparent disjunction between planned and actual usage appeared in Square 03. It is intended for maintenance as well as other kinds of work. But much of the time, there was nobody working here—a fact that is not captured by historic photos of the area, precisely because nothing is happening. The space has instead become the equivalent of a pegboard mounted on a wall in a home garage or shed, convenient for storage for all kinds of items—not necessarily items being used there—because it has an enormous number of attachment points. Storage has become its primary function. Designers of future workstations in space should consider that they might need to optimize for functions other than work, because most of the time, there might not be any work happening there. They could optimize for quick storage, considering whether to impose a system of organization, or allow users to organize as they want.

We expected from previous (though unsystematic) observation of historic photos and other research, that resealable plastic bags (combined with Velcro patches on the bags and walls) would be the primary means for creating gravity surrogates to control items in microgravity. They only comprise 7% of all items in Square 03 (256 instances). There are more than twice as many clips (572—more than 9 per context) in the sample. There were 193 instances of adhesive tape rolls, and more than 100 cable ties, but these were latent (not holding anything), representing potentiality of restraint rather than actualization. The squares showed different approaches to managing “gravity.” While Square 03 had a pre-existing structured array of Velcro patches, Square 05 showed a more expedient strategy with Velcro added in response to particular activities. Different needs require different affordances; creating “gravity” is a more nuanced endeavor than it initially appears. More work remains to be done to optimize gravity surrogates for future space habitats, because this is evidently one of the most critical adaptations that crews have to make in microgravity (44% of all items in Square 03, 39% in 05).

Square 05 is an empty space, seemingly just one side of a passageway for people going to use the lifting machine or the latrine, to look out of the Cupola, or get something out of deep storage in one of the ISS’s closets. In our survey, this square was a storage place for toiletries, resealable bags, and a computer that never (or almost never) gets used. It was associated with computing and hygiene simply by virtue of its location, rather than due to any particular facilities it possessed. It has no affordances for storage. There are no cabinets or drawers, as would be appropriate for organizing and holding crew personal items. A crew member decided that this was an appropriate place to leave their toiletry kit for almost two months. Whether this choice was appreciated or resented by fellow crew members cannot be discerned based on our evidence, but it seems to have been tolerated, given its long duration. The location of the other four USOS crew members’ toiletry kits during the sample period is unknown. A question raised by our observations is: how might a function be more clearly defined by designers for this area, perhaps by providing lockers for individual crew members to store their toiletries and towels? This would have a benefit not only for reducing clutter, but also for reducing exposure of toiletry kits and the items stored in them to flying sweat from the exercise equipment or other waste particles from the latrine. A larger compartment providing privacy for body maintenance and a greater range of motion would also be desirable.

As the first systematic collection of archaeological data from a space site outside Earth, this analysis of two areas on the ISS as part of the SQuARE payload has shown that novel insights into material culture use can be obtained, such as the use of wall areas as storage or staging posts between activities, the accretion of objects associated with different functions, and the complexity of using material replacements for gravity. These results enable better space station design and raise new questions that will be addressed through analysis of the remaining four squares.

Supporting information

S1 movie. nasa astronaut kayla barron installs the first square for the sampling quadrangle assemblages research experiment in the japanese experiment module (also known as kibo) on the international space station, january 14, 2022..

She places Kapton tape to mark the square’s upper right corner. Credit: NASA.

https://doi.org/10.1371/journal.pone.0304229.s001

S1 Dataset.

https://doi.org/10.1371/journal.pone.0304229.s002

S2 Dataset.

https://doi.org/10.1371/journal.pone.0304229.s003

S3 Dataset. The image annotations are represented according to sample square in json formatted text files.

The data is available in the ‘SQuARE-notebooks’ repository on Github.com in the ‘data’ subfolder at https://github.com/issarchaeologicalproject/SQuARE-notebooks/tree/main ; archived version of the repository is at Zenodo, DOI: 10.5281/zenodo.10654812 .

https://doi.org/10.1371/journal.pone.0304229.s004

S1 File. The ‘Rocket-Anno’ image annotation software is available on Github at https://github.com/issarchaeologicalproject/MRE-RocketAnno .

The archived version of the repository is at Zenodo, DOI: 10.5281/zenodo.10648399 .

https://doi.org/10.1371/journal.pone.0304229.s005

S2 File. The computational notebooks that process the data json files to reshape the data suitable for basic statistics as well as the computation of the Brainerd-Robinson coefficients of similarity are in the.ipynb notebook format.

The code is available in the ‘SQuARE-notebooks’ repository on Github.com in the ‘notebooks’ subfolder at https://github.com/issarchaeologicalproject/SQuARE-notebooks/tree/main ; archived version of the repository is at Zenodo, DOI: 10.5281/zenodo.10654812 . The software can be run online in the Google Colab environment ( https://colab.research.google.com ) or any system running Jupyter Notebooks ( https://jupyter.org/ ).

https://doi.org/10.1371/journal.pone.0304229.s006

https://doi.org/10.1371/journal.pone.0304229.s007

Acknowledgments

We thank Chapman University’s Office of Research and Sponsored Programs, and especially Dr. Thomas Piechota and Dr. Janeen Hill, for funding the Implementation Partner costs associated with the SQuARE payload. Chapman’s Leatherby Libraries’ Supporting Open Access Research and Scholarship (SOARS) program funded the article processing fee for this publication. Ken Savin and Ken Shields at the ISS National Laboratory gave major support by agreeing to sponsor SQuARE and providing access to ISS NL’s allocation of crew time. David Zuniga and Kryn Ambs at Axiom Space were key collaborators in managing payload logistics. NASA staff and contractors were critical to the experiment’s success, especially Kristen Fortson, Jay Weber, Crissy Canerday, Sierra Wolbert, and Jade Conway. We also gratefully acknowledge the help and resources provided by Dr. Erik Linstead, director of the Machine Learning and Affiliated Technology Lab at Chapman University. Aidan St. P. Walsh corrected the color and lens barrel distortion in all of the SQuARE imagery. Rao Hamza Ali produced charts using accessible color combinations for Figs 3 and 5 . And finally, of course, we are extremely appreciative of the efforts of the five USOS members of the Expedition 66 crew on the ISS—Kayla Barron, Raja Chari, Thomas Marshburn, Matthias Maurer, and Mark Vande Hei—who were the first archaeologists in space.

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• Published: 07 August 2024

Molecular mimicry in multisystem inflammatory syndrome in children

• Aaron Bodansky   ORCID: orcid.org/0000-0001-8943-8233 1   na1 ,
• Robert C. Mettelman   ORCID: orcid.org/0000-0002-3527-5616 2   na1 ,
• Joseph J. Sabatino Jr 3 , 4 ,
• Sara E. Vazquez 5 ,
• Janet Chou 6 , 7 ,
• Tanya Novak   ORCID: orcid.org/0000-0002-7115-7545 8 , 9 ,
• Kristin L. Moffitt 7 , 10 ,
• Haleigh S. Miller 5 , 11 ,
• Andrew F. Kung 5 , 11 ,
• Elze Rackaityte   ORCID: orcid.org/0000-0003-3889-8082 5 ,
• Colin R. Zamecnik   ORCID: orcid.org/0000-0002-9477-1388 3 , 4 ,
• Jayant V. Rajan 5 ,
• Hannah Kortbawi 5 , 12 ,
• Caleigh Mandel-Brehm 5 ,
• Anthea Mitchell 13 ,
• Chung-Yu Wang 13 ,
• Aditi Saxena 13 ,
• Kelsey Zorn   ORCID: orcid.org/0000-0003-1227-2137 5 ,
• David J. L. Yu 14 ,
• Mikhail V. Pogorelyy 2 ,
• Walid Awad 2 ,
• Allison M. Kirk   ORCID: orcid.org/0000-0002-4286-3678 2 ,
• James Asaki 15 ,
• John V. Pluvinage   ORCID: orcid.org/0000-0002-9607-2783 4 ,
• Michael R. Wilson   ORCID: orcid.org/0000-0002-8705-5084 3 , 4 ,
• Laura D. Zambrano 16 ,
• Angela P. Campbell   ORCID: orcid.org/0000-0002-2576-482X 16 ,
• Overcoming COVID-19 Network Investigators ,
• Paul G. Thomas   ORCID: orcid.org/0000-0001-7955-0256 2   na2 ,
• Adrienne G. Randolph 7 , 8 , 9   na2 ,
• Mark S. Anderson   ORCID: orcid.org/0000-0002-3093-4758 14 , 17   na2 &
• Joseph L. DeRisi   ORCID: orcid.org/0000-0002-4611-9205 5 , 13   na2  

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• Autoimmunity
• Autoinflammatory syndrome
• Immune tolerance
• Inflammation
• Viral infection

Multisystem inflammatory syndrome in children (MIS-C) is a severe, post-infectious sequela of SARS-CoV-2 infection 1 , 2 , yet the pathophysiological mechanism connecting the infection to the broad inflammatory syndrome remains unknown. Here we leveraged a large set of samples from patients with MIS-C to identify a distinct set of host proteins targeted by patient autoantibodies including a particular autoreactive epitope within SNX8, a protein involved in regulating an antiviral pathway associated with MIS-C pathogenesis. In parallel, we also probed antibody responses from patients with MIS-C to the complete SARS-CoV-2 proteome and found enriched reactivity against a distinct domain of the SARS-CoV-2 nucleocapsid protein. The immunogenic regions of the viral nucleocapsid and host SNX8 proteins bear remarkable sequence similarity. Consequently, we found that many children with anti-SNX8 autoantibodies also have cross-reactive T cells engaging both the SNX8 and the SARS-CoV-2 nucleocapsid protein epitopes. Together, these findings suggest that patients with MIS-C develop a characteristic immune response to the SARS-CoV-2 nucleocapsid protein that is associated with cross-reactivity to the self-protein SNX8, demonstrating a mechanistic link between the infection and the inflammatory syndrome, with implications for better understanding a range of post-infectious autoinflammatory diseases.

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Diverse functional autoantibodies in patients with COVID-19

SARS-CoV-2 immune repertoire in MIS-C and pediatric COVID-19

Immunology of SARS-CoV-2 infection in children

Children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections typically have mild disease 3 , 4 , but can develop a rare life-threatening post-infectious complication known as MIS-C 1 , 2 . MIS-C presents with a distinctive inflammatory signature indicative of altered innate immune responses 5 , 6 , including dysregulation of the mitochondrial antiviral signalling (MAVS) protein pathway 7 . Aberrant adaptive immunity is also involved, with multiple MIS-C-associated autoantibodies reported 8 , 9 , 10 , 11 , 12 . Furthermore, T cell signatures have also been associated with development of MIS-C 13 , 14 , 15 , 16 , which are accompanied by autoimmune-associated B cell expansions 8 . Some autoimmune diseases have been shown to involve tandem cross-reactive B cell and T cell responses. In multiple sclerosis, for example, cross-reactive B cells and T cells have been shown to respond to Epstein–Barr virus protein (EBNA1) and antigens in the human nervous system 17 , 18 , 19 . Decades of research into paraneoplastic autoimmune encephalitis has also demonstrated that autoreactive B cells and T cells can cause disease through coordinated targeting of a shared intracellular antigen and, in certain cases, a shared epitope 20 , 21 , 22 , 23 , 24 , 25 , 26 . Despite intense interest, a pathophysiological link between SARS-CoV-2 and MIS-C remains enigmatic, and identification of disease-specific autoantigens remains incompletely explored. Here children previously infected with SARS-CoV-2 with ( n  = 199) and without ( n  = 45) MIS-C were enrolled and comprehensively evaluated for differential autoreactivity to the entire human and SARS-CoV-2 proteome. Patients with MIS-C were found to have both cross-reactive antibodies and T cells targeting an epitope motif shared by the viral nucleocapsid protein and human SNX8, a protein involved in MAVS antiviral function 27 . These findings suggest that many cases of MIS-C may be triggered by molecular mimicry and could provide a framework for identifying potential cross-reactive epitopes in other autoimmune and inflammatory diseases with predicted viral triggers such as Kawasaki disease 28 , type 1 diabetes mellitus (T1DM) 29 and multiple sclerosis.

Patients with MIS-C have a distinct set of autoreactivities

To explore the hypothesis that MIS-C is driven by an autoreactive process, we evaluated the proteome-wide autoantibody profiles of children with MIS-C ( n  = 199) and children convalescing following asymptomatic or mild SARS-CoV-2 infection without MIS-C ( n  = 45, hereafter referred to as ‘at-risk controls’) using our custom phage immunoprecipitation and sequencing (PhIP-seq) 30 library, which has previously been used to define novel autoimmune syndromes and markers of disease for various conditions 12 , 24 , 25 , 31 , 32 , 33 . Given the inherently heterogeneous nature of antibody repertoires among individuals 34 , the identification of disease-associated autoreactive antigens requires the use of large numbers of cases and controls 12 . To minimize spurious hits, this study includes substantially more patients with MIS-C and controls than similar, previously published studies 8 , 9 , 10 , 12 (Fig. 1a ). Clinical characteristics of this cohort are described in Extended Data Table 1 .

a , Design of the PhIP-seq experiment comparing patients with MIS-C ( n  = 199) and at-risk controls ( n  = 45; children with SARS-CoV-2 infection at least 5 weeks before sample collection without symptoms of MIS-C). Schematics in panel a were created using BioRender ( https://www.biorender.com ). b , Venn diagram highlighting the number of autoantigens identified with statistically significant PhIP-seq enrichment (‘enrichment set’: grey circle; P  < 0.01 on one-sided Kolmogorov–Smirnov test with false discovery rate correction) and autoantigens identified, which contribute to a logistic regression classifier of MIS-C relative to at-risk controls (‘classifier set’: purple circle). There are 35 autoantigens present in both the classifier set and the enrichment set (pink; union of the Venn diagram) of which 30 are exclusive to MIS-C and referred to as the ‘MIS-C set’ (no two controls have low reactivity as defined by the fold-change (FC) signal over the mean of protein A/G beads only (FC > mock-IP) of 3 or greater, and no single control has high reactivity defined as FC > mock-IP greater than 10). LR, logistic regression. c , Receiver operating characteristic curve for the logistic regression classifier showing upper and lower bounds of performance through 1,000 iterations. d , Bar plots with error bars showing logistic regression coefficients for the top 10 autoantigens across 1,000 iterations. The whiskers extend to 1.5 times the interquartile range (IQR) from the quartiles. The boxes represent the IQR, and the centre lines represent the median. e , Hierarchically clustered (Pearson) heatmap showing the PhIP-seq enrichment (FC > mock-IP) for the 30 autoantigens in the MIS-C set in each patient with MIS-C and each at-risk plasma control.

Source Data

For a given set of samples, PhIP-seq can yield dozens to thousands of differential enrichments of phage-displayed peptides. Here logistic regression machine learning was used as an initial unbiased measure of how accurately a set of differentially enriched peptides could classify people with MIS-C and controls—an approach that has been used to classify people with autoimmune polyglandular syndrome type 1 using PhIP-seq data 12 . In all, 107 proteins had logistic regression coefficients greater than zero (‘classifier set’; Fig. 1b ). As this is an unbalanced dataset with a random accuracy less than 50%, we also generated a receiver operating characteristic (ROC) curve. ROC analysis iterated 1,000 times and yielded an average area under the curve (AUC) of 0.94 (Fig. 1c ). Examination of the logistic regression coefficients associated with MIS-C revealed the largest contributions from peptides derived from the ETS repressor factor-like (ERFL), sorting nexin 8 (SNX8) and KDEL endoplasmic reticulum protein retention receptor 1 (KDELR1) coding sequences (Fig. 1d ).

In parallel, a Kolmogorov–Smirnov test was used to define a set of 661 autoreactivities statistically enriched after false discovery rate adjustment for multiple comparisons ( q  < 0.01; ‘enrichment set’). To avoid false positives, the intersection of the classifier set and enrichment set were considered further. Of these 35 hits, peptides derived from 30 different proteins satisfied an additional set of conservative criteria, requiring that none was enriched (fold change over mock-immunoprecipitation (IP) of more than 3) in more than a single control, or was enriched more than 10-fold in any control (‘MIS-C set’; Fig. 1e ).

Previously reported MIS-C autoantibodies

To date, at least 34 autoantigen candidates have been reported to associate with MIS-C 8 , 9 , 10 , 12 . However, we found that only UBE3A (a ubiquitously expressed ubiquitin protein ligase) was differentially enriched in our MIS-C dataset, whereas the remaining 33 were present in a similar proportion of cases with MIS-C and at-risk controls (Extended Data Fig. 1a ). Autoreactivity to UBE3A was independently identified in this study as part of both the classifier and the enrichment sets, but was not included in the final MIS-C set due to the low positive signal present in two controls.

In addition, autoantibodies to the receptor antagonist IL-1RA have been previously reported in 13 of 21 (62%) patients with MIS-C 11 . In this cohort, anti-IL-1RA antibodies were detected by PhIP-seq ( z score > 6 over at-risk control) in six patient samples. To further examine immune reactivity to full-length IL-1RA, sera from 196 of the 199 patients in this study were used to immunoprecipitate [35S]-methionine-radiolabelled IL-1RA (radioligand-binding assay (RLBA)). Positive immunoprecipitation of IL-1RA (defined as more than 3 s.d. above mean of controls) was found in 39 of 196 (19.9%) patients with MIS-C. However, many patients with MIS-C were treated with intravenous immunoglobulin (IVIG), a blood product shown to contain autoantibodies 35 . After removing samples from patients treated with IVIG (61 remaining), the difference between samples from patients with MIS-C (5 of 61, 8.2%) and at-risk controls (1 of 45, 2.2%) was not significant ( P  = 0.299; Extended Data Fig. 1b ).

MIS-C autoantigens lack tissue-specific associations with clinical phenotypes

Consistent with previous MIS-C reports 1 , 5 , this cohort was clinically heterogeneous (Extended Data Table 2 ). To determine whether specific phenotypes, including myocarditis and the requirement of vasopressors, might be associated with specific autoantigens present in the MIS-C set, tissue expression levels were assigned to each autoantigen 36 (Human Protein Atlas; https://proteinatlas.org ), including the amount of expression in cardiomyocytes and the cardiac endothelium. The PhIP-seq signal for patients with MIS-C with a particular phenotype was compared with those patients with MIS-C without the phenotype. Autoantigens with tissue specificity were not enriched in those patients with MIS-C with phenotypes involving said tissue. Similarly, autoantigens associated with myocarditis or vasopressor requirements did not correlate with increased cardiac expression (Extended Data Fig. 1c ).

Orthogonal validation of PhIP-seq autoantigens

Peptides derived from ERFL, SNX8 and KDELR1 carried the largest logistic regression coefficients in the MIS-C classifier. The PhIP-seq results were orthogonally confirmed by RLBAs using full-length ERFL, SNX8 and KDELR1 proteins. Relative to at-risk controls, samples from patients with MIS-C significantly enriched each of the three target proteins ( P  < 1 × 10 −10 for ERFL, SNX8 and KDELR1), consistent with the PhIP-seq assay (Extended Data Fig. 2a ). Using only the RLBA data for these three proteins, MIS-C could be confidently classified (ROC with fivefold cross-validation; 1,000 iterations) from at-risk control sera with an AUC of 0.93, suggesting the potential for molecular diagnostic purposes (Extended Data Fig. 2b ).

As noted, IVIG was administered to 138 of the 199 patients with MIS-C before sample collection and was absent from all 45 at-risk controls. The autoreactivity to the ERFL, SNX8 and KDELR1 proteins from the 61 patients with MIS-C who had not been treated with IVIG before sample collection were compared with the at-risk controls. In contrast to IL-1RA, the differential enrichment of these three proteins remained significant ( P  = 6.69 × 10 −10 , P  = 6.26 × 10 −5 and P  = 0.0001, respectively), suggesting that autoreactivity to ERFL, SNX8 and KDELR1 proteins was not confounded by IVIG treatment (Extended Data Fig. 2c ).

Independent MIS-C cohort validation

To further test the validity of these findings, an independent validation cohort consisting of samples from 24 different patients with MIS-C and 29 children with severe acute COVID-19 was evaluated (acquired via ongoing enrolment of the Overcoming COVID-19 study; Extended Data Table 3 ). Using RLBAs with full-length ERFL, SNX8 and KDELR1 proteins, we found that all three target proteins were significantly enriched compared with both the at-risk controls ( P  = 0.00022, P  = 3.68 × 10 −5 and P  = 2.36 × 10 −5 , respectively) and the patients with severe acute COVID-19 ( P  = 0.0066, P  = 0.00735 and P  = 0.00114, respectively; Extended Data Fig. 2d ). A logistic regression model, trained on the original cohort, classified MIS-C from at-risk controls with an AUC of 0.84, and from severe acute paediatric COVID-19 with an AUC of 0.78 (Extended Data Fig. 2e ). This suggests that autoreactivity to ERFL, SNX8 and KDELR1 is a significant feature of MIS-C that is separable from SARS-CoV-2 exposure and severe acute paediatric COVID-19.

MIS-C autoantibodies target a single epitope within the SNX8 protein

SNX8 is a protein that is 456 amino acids and belongs to a family of sorting nexins involved in endocytosis, endosomal sorting and signalling 37 . Publicly available expression data 36 (Human Protein Atlas) show that SNX8 is widely expressed across various tissues including the brain, heart, gastrointestinal tract, kidneys and skin, with the highest expression in undifferentiated cells and immune cells. Previous work has associated SNX8 with host defence against RNA viruses 27 . ERFL is a poorly characterized 354-amino acid protein. A survey of single-cell RNA sequencing (scRNA-seq) data 36 (Human Protein Atlas) suggests enrichment in plasma cells, B cells and T cells in some tissues. Using a Spearman correlation in principal component analysis (PCA) space based on tissue RNA-seq data 36 (Human Protein Atlas), SNX8 has the second closest expression pattern to ERFL compared with all other coding genes, with a correlation coefficient of 0.81. KDELR1 is a 212-amino acid endoplasmic reticulum–Golgi transport protein essential to lymphocyte development with low tissue expression specificity. All three proteins are predicted to be intracellular, suggesting that putative autoantibodies targeting these proteins are unlikely to be sufficient for disease pathology on their own. However, autoantibodies targeting intracellular antigens are often accompanied by autoreactive T cells specific for the protein from which that antigen was derived, and which targets cell types expressing the protein 22 , 25 , 26 , 38 . We selected SNX8 for further investigation, given its enrichment in immune cells and its putative role in regulating the MAVS pathway in response to RNA virus infection, a pathway implicated in MIS-C pathology 7 .

Full-length SNX8 is represented in this PhIP-seq library by 19 overlapping 49-mer peptides. For all but one patient sample, the peptide fragment spanning amino acid positions 25–73 was the most enriched in the PhIP-seq assay (Fig. 2a ), suggesting a common autoreactive site. A sequential alanine scan was performed to determine the minimal immunoreactive peptide sequence (Fig. 2b ; Methods ). Using samples from six individuals with MIS-C, we determined that the critical region for immunoreactivity was a nonamer spanning positions 51–59 (PSRMQMPQG). Using the wild-type 49-amino acid peptide and the version with the critical region mutated to alanine, 182 of the 199 patients with MIS-C (insufficient sample for the remaining 17) and all 45 controls were assessed for immunoreactivity using a split-luciferase-binding assay (SLBA). We found that samples from 31 of 182 (17.0%) patients with MIS-C immunoprecipitated the wild-type fragment. Of these, 29 (93.5%) failed to immunoprecipitate the mutated peptide, suggesting a common shared autoreactive epitope among nearly all of the patients with MIS-C with anti-SNX8 antibodies (Extended Data Fig. 2f ).

a , PhIP-seq signal (reads per 100,000) for each patient with MIS-C ( n  = 199) and each at-risk control ( n  = 45) across each of the 19 bacteriophage-encoded peptide fragments, which together tile the full-length SNX8 protein. b , SLBA enrichments (normalized antibody indices) for each sequential alanine mutagenesis construct. Constructs were designed with 10 amino acid alanine windows (highlighted in purple) shifted by 5 amino acids until the entire immunodominant SNX8 region (SNX8 fragment 2) was scanned. Values are averages of six separate patients with MIS-C. The identified autoantibody epitope is bounded by vertical grey dotted lines.

Patients with MIS-C have an altered antibody response to the SARS-CoV-2 nucleocapsid protein

To evaluate whether differences exist in the humoral immune response to SARS-CoV-2 infection in patients with MIS-C relative to at-risk controls, we repeated PhIP-seq with 181 of the original 199 patients with MIS-C and all 45 of the at-risk controls using a previously validated library specific for SARS-CoV-2 (ref. 39 ). To discover whether certain fragments were differentially enriched in either patients with MIS-C or at-risk controls, the enrichment of each phage encoded SARS-CoV-2 peptide (38 amino acids each) across all patients with MIS-C and at-risk controls was normalized to 48 healthy controls pre-COVID-19. Three nearly adjacent peptides derived from the SARS-CoV-2 nucleocapsid protein (fragments 5, 8 and 9) were significantly enriched (Kolmogorov–Smirnov test P  < 0.0001 for each). The first peptide (fragment 5), spanning amino acids 77–114, was significantly enriched in the at-risk controls (representing the typical serological response in children), whereas the next two fragments (fragments 8 and 9), spanning amino acids 134–190, were significantly enriched in patients with MIS-C (Fig. 3a,b ). The most differentially reactive region of the SARS-CoV-2 nucleocapsid protein in patients with MIS-C (fragment 8) was termed the MIS-C-associated domain of SARS-CoV-2 (MADS). The PhIP-seq results were orthogonally confirmed using an SLBA measuring the amount of MADS peptide immunoprecipitated with samples from 16 individuals, including 11 patients with MIS-C and 5 at-risk controls (Fig. 3c ). To precisely map the minimal immunoreactive region of MADS in MIS-C samples, peptides featuring a sliding window of ten alanine residues were used as the immunoprecipitation substrate for SLBAs, run in parallel with the SNX8 alanine scanning peptides using sera from three patients with MIS-C (Fig. 3d ). The critical regions identified here in both SNX8 and MADS were highly similar, represented by the (ML)Q(ML)PQG motif (Fig. 3e ).

a , Relative PhIP-seq signal (FC over the mean) of 48 controls who are pre-COVID-19 (FC > pre-COVID-19) in patients with MIS-C ( n  = 181) and at-risk controls ( n  = 45) using a custom phage display library expressing the entire SARS-CoV-2 proteome to different regions of SARS-CoV-2. Only regions with a mean antibody signal of more than 1.5-fold above pre-COVID-19 controls are shown. Antigenicity (sum of the mean FC > pre-COVID-19 in MIS-C and at-risk controls) are represented by darker shades. The length of the bars represents the statistical difference in signal between MIS-C and at-risk controls to a particular region (−log 10 of two-sided Kolmogorov–Smirnov test P values), with upward deflections representing enrichment in MIS-C versus at-risk controls, and downward deflections representing less signal in MIS-C. The asterisk indicates the differentially reactive region of the nucleocapsid (N) protein. b , Bar plots showing the PhIP-seq signal (FC > pre-COVID-19) across the specific region of the SARS-CoV-2 nucleocapsid protein (fragments 4–9) with the most divergent response in MIS-C samples ( n  = 181) relative to at-risk controls ( n  = 45), compared using a two-sided Kolmogorov–Smirnov test (exact P values are shown in the figure). The amino acid sequence of the region with the highest relative enrichment in MIS-C is highlighted in green and referred to as MADS. c , Strip plots and box plots showing MADS SLBA enrichments (normalized antibody indices) in patients with MIS-C ( n  = 11) relative to at-risk controls ( n  = 5). d , SLBA signal (normalized antibody indices) for full sequential alanine mutagenesis scans within the same three individuals for SNX8 (left) and MADS (right). Each identified epitope is bounded by black vertical dotted lines. e , Multiple sequence alignment of SNX8 and MADS epitopes with the amino acid sequence for the similarity region shown (for the text in colour, biochemically similar is in orange, and identical is in red). For the box plots ( b , c ), the whiskers extend to 1.5 times the IQR from the quartiles. The boxes represent the IQR, and the centre lines represent the median.

Patients with MIS-C have significantly increased SNX8 autoreactive T cells

In other autoimmune diseases, autoantibodies often arise to intracellular targets, yet the final effectors of cellular destruction are autoreactive T cells 22 , 26 , 40 . Given evidence that certain subsets of MIS-C are associated with HLA 16 , and that SNX8 is an intracellular protein, we hypothesized that patients with MIS-C with anti-SNX8 antibodies may, in addition to possessing SNX8 autoreactive B cells, also possess autoreactive T cells targeting SNX8-expressing cells. To test this hypothesis, T cells from nine patients with MIS-C (eight from SNX8 autoantibody-positive patients and one who was SNX8 autoantibody negative) and ten at-risk controls (chosen randomly) were exposed to a pool of 15-mer peptides with 11-amino acid overlaps tiling the full-length human SNX8 protein. T cell activation was measured by an activation-induced marker assay, which quantifies upregulation of three cell activation markers: OX40, CD69 and CD137 (ref. 41 ). The percent of T cells activated in response to SNX8 protein was significantly higher in patients with MIS-C than in controls ( P  = 0.00126). Using a positive cut-off of 3 s.d. above the mean of the controls, 7 of the 9 (78%) patients with MIS-C were positive for SNX8-expressing autoreactive T cells, whereas 0 of 10 (0%) controls met these criteria (Fig. 4a ). With respect to CD4 + and CD8 + subgroups, there was an increased signal in patients with MIS-C compared with controls, which did not meet significance ( P  = 0.0711 and P  = 0.0581, respectively; Extended Data Fig. 3a ). The patient with MIS-C who was seronegative for the SNX8 autoantibody was also negative for SNX8 autoreactive T cells.

a , Strip plots and box plots showing the distribution of T cells activated in response to either vehicle (culture media + 0.2% DMSO) or the SNX8 peptide pool (SNX8 peptide + culture media + 0.2% DMSO) in patients with MIS-C ( n  = 9) and controls ( n  = 10). The relative signal was compared using a two-sided Mann–Whitney U -test (exact P values are shown in the figure). The box plot whiskers extend to 1.5 times the IQR from the quartiles, the boxes represent the IQR, and the centre lines represent the median. The dashed line is 3 s.d. above the mean of the controls in the SNX8 pool condition. b , TCRdist similarity network of 48 unique, paired TCRαβ sequences ( n  = 259 sequences) obtained from four patients with MIS-C. CD8 + T cells were sorted from PBMCs directly ex vivo or after 10 days of peptide expansion and staining with A*02:01 or A*02:06 HLA class I tetramers loaded with MADS (LQLPQGITL) and SNX8 (MQMPQGNPL) peptides. Each node represents a unique TCR clonotype. Edges connect nodes with a TCRdist score of less than 150. The dashed lines surround TCR similarity clusters. The node size corresponds to the T cell clone size. Nodes are coloured based on the HLA experiment type (left) or patient (right). TCRs selected for further testing are numbered TCR 1–8. The convergent node is circled in green. c , Specificity of putative cross-reactive TCRs expressed in Jurkat-76 cells by HLA-A*02:01 or HLA-A*02:06 tetramers loaded with MADS (LQLPQGITL) and SNX8 (MQMPQGNPL) peptides. Jurkat-76 (TCR-null) cells were used as tetramer background staining controls. The gate values indicate the frequency of MADS–APC + and/or SNX8–BV421 + cells as the percentage of the total PE + cells (combination staining with MADS–PE and SNX8–PE tetramers). TCRs with confirmed cross-reactivity are indicated in red. Outliers are shown. Flow plots are representative of two independent evaluations. d , Summary of TCR sequencing results of the eight TCRs tested.

HLA type A*02 is more likely to present the shared epitope

MIS-C has been associated with HLA alleles A*02, B*35 and C*04 (ref. 16 ). The Immune Epitope Database and Analysis Resource ( https://IEDB.org ) 42 was used to rank the HLA class I (HLA-I) peptide presentation likelihoods for both SNX8 and SARS-CoV-2 nucleocapsid protein with respect to the MIS-C-associated HLA alleles. The distribution of predicted HLA-I-binding scores for nucleocapsid protein and SNX8 fragments matching the (ML)Q(ML)PQG SNX8/MADS motif relative to fragments lacking a match was compared. For HLA-A*02, predicted HLA-I binding was significantly higher ( P  = 8.78 × 10 −10 for nucleocapsid protein; P  = 0.0112 for SNX8) for fragments containing the putative autoreactive motif. There was no statistical difference for HLA-B*35 and HLA-C*04 predictions (Extended Data Fig. 3b,c ). Of note, of the seven patients with MIS-C with SNX8 autoreactive T cells, at least five were positive for HLA-A*02 (Extended Data Fig. 3a ). To experimentally validate HLA-I-binding predictions to SNX8 and MADS peptides, we measured peptide–HLA (pHLA) monomer stability using a β2 microglobulin (β2m) fold test, which is a proxy for pHLA-binding affinity in which anti-β2m staining reports on the strength of the pHLA complex 43 . SNX8 (MQMPQGNPL) and MADS (LQLPQGITL) peptides were loaded onto unfolded HLA-A*02:01, HLA-A*02:06 or HLA-B*35:01 monomers and stained with an anti-β2m fluorescent antibody. Consistent with the IEDB rankings, both HLA-A*02 alleles bound SNX8 and MADS peptides, with HLA-A*02:06 exhibiting the highest pHLA complex stability (Extended Data Fig. 3d ).

T cells from patients with MIS-C are cross-reactive to the SNX8 and nucleocapsid protein similarity regions

Given the prediction that HLA types associated with MIS-C preferentially display peptides containing the similarity regions for both SNX8 and the SARS-CoV-2 nucleocapsid protein, we sought to determine whether cross-reactive T cells were present and whether they were associated with MIS-C. We stimulated peripheral blood mononuclear cells (PBMCs) from three patients with MIS-C and three at-risk controls with peptides from either the SNX8 similarity region (MQMPQGNPL) or the MADS similarity region (LQLPQGITL) for 7 days to enrich for CD8 + T cells reactive to these epitopes. We then built differently labelled HLA-I tetramers loaded with either the SNX8 or MADS peptides and measured binding to T cells (Extended Data Fig. 4a ). We detected cross-reactive CD8 + T cells, which bound both peptide epitopes, in all three patients with MIS-C, whereas no cross-reactive CD8 + T cells were observed in at-risk controls (Extended Data Fig. 4b ).

As SNX8-responsive T cells were observed in patients with MIS-C, we next asked whether the region of SNX8 similar to the SARS-CoV-2 MADS region was sufficient to activate patient T cells. A pool of 20 10-mer peptides with 9-amino acid overlaps centred on the target motif from SNX8 (collectively spanning amino acids 44–72) was used to stimulate PBMCs from two patients with MIS-C and four at-risk controls. Both patients with MIS-C had activation of T cells, whereas none of the four controls had T cell activation (Extended Data Fig. 4c ).

Identification of ex vivo cross-reactive T cell receptors

Having determined that patients with MIS-C, but not controls, contained putative SNX8/MADS cross-reactive CD8 + T cells, we next sought to identify T cell receptor (TCR) sequences with specificity for both the SARS-CoV-2 MADS and the host SNX8 epitopes. To do this, PBMCs were obtained during the first 72 h of hospital admission from four study participants with HLA-A*02 and confirmed MIS-C (one individual previously identified as having putative cross-reactive T cells, and three new patients). Given that MIS-C PBMCs represent a scarce resource, we chose to expand one aliquot of PBMCs from each of the four participants (distinct from our previous peptide expansion protocol; see  Methods ) to maximize the chances of isolating putative cross-reactive TCRs. Although the frequency of ex vivo autoantigen-specific CD8 + T cells are extraordinarily low in peripheral blood, even for bona fide T cell-mediated autoimmune diseases such as T1DM 38 and multiple sclerosis 44 , 45 , we nevertheless utilized the remaining PBMCs from each participant for direct ex vivo analysis without previous expansion. To isolate the antigen-specific TCRs, participant cells (both ex vivo and following peptide expansion) were stained using the same tetramer-labelling strategy, which previously identified the putative cross-reactive TCRs (Extended Data Fig. 4a ); any cell exhibiting binding to at least two peptide-loaded tetramers was individually sorted and full-length paired TCRα and TCRβ sequences were determined. This resulted in 259 complete TCR sequences, comprising 30 and 18 unique T cell clones from the ex vivo and peptide expansion experiments, respectively. A complete list of TCR sequences is provided (Fig. 4 source data).

Next, we sought to validate the specificity of putative SNX8/MADS cross-reactive TCRs identified from the tetramer sorting, and further analyse features of the recovered TCRs. Because clusters of similar TCRs tend to recognize similar peptide antigens, a TCR similarity network was constructed from all 259 full-length TCR sequences using a previously established TCR distance metric (TCRdist) 46 , 47 (Fig. 4b and Extended Data Fig. 4d ). In two of the four patients, we identified unique populations of clonally expanded T cells expressing putative cross-reactive TCRs directly ex vivo, whereas each of the four patients had at least one ex vivo putative cross-reactive TCR (Fig. 4b ). To confirm the specificity of the TCRs identified in our tetramer sorting, we selected eight TCR sequences for additional validation and generated individual cell lines that stably expressed one TCR of interest (Extended Data Fig. 5a ). These Jurkat-TCR + cell lines were tetramer stained, and cross-reactivity was confirmed in three of the Jurkat-TCR + cell lines (TCR 1, 7 and 8; Fig. 4c ). Of these validated cross-reactive TCRs, two were obtained from ex vivo PBMCs from patients with MIS-C including TCR 7, which was clonally expanded. The minimum ex vivo frequency of TCR 7 alone was more than 1 in 25,000 (6 of 140,035) circulating CD8 + T cells. The two cross-reactive TCRs obtained from the ex vivo isolation were derived from the same participant, utilize the same TRAV gene ( TRAV1-2 ) with identical CDR3α sequences and clustered with three additional sequences in the TCRdist space, one of which was also clonally expanded, suggesting that this patient had an active expansion of a large cluster of SNX8/MADS cross-reactive CD8 + T cells (Fig. 4d ). Furthermore, we note a cluster of two similar TCRs obtained from ex vivo sampling of different participants (patients 2 and 4) with different HLA types (‘convergent node’; circled in green in Fig. 4b ). Although these putative cross-reactive TCRs were not evaluated further, the cluster suggests that TCR specificities to these epitopes may converge across individuals.

The remaining five Jurkat-TCR + cell lines (TCR 2–6) exhibited single specificity to the MADS tetramer with four of five coming from the peptide expansion. To evaluate possible interference between tetramers, which can arise when pHLA–TCR-binding affinities differ, Jurkat-TCR + cell lines were stained with individual tetramers. The results confirm that four of these TCRs are indeed reactive only to MADS (Extended Data Fig. 5b ). However, TCR 2, although showing strong binding preference to MADS, also bound the individual SNX8 tetramer, suggesting that the higher affinity for MADS may outcompete binding to the SNX8 tetramer in some cases. This observation is in line with the notion that autoreactive cross-reactive TCRs with lower relative affinities to autoantigens may escape thymic negative selection. Finally, because the original tetramer experiments were based on an early 2020 SARS-CoV-2 minor variant sequence (LQLPQGITL), all eight Jurkat-TCR + cell lines were also stained with HLA tetramers loaded with the SARS-CoV-2 Wuhan MADS sequence (LQLPQGTTL). In all cases, the Jurkat-TCR + cells bound the Wuhan MADS tetramer, consistent with the notion that T cells encoding these and other similar TCRs may be capable of responding to multiple SARS-CoV-2 strains (Extended Data Fig. 5c ).

RNA expression profile of SNX8  during SARS-CoV-2 infection

As previously discussed, SNX8 is expressed across multiple tissues, but is highest in immune cells, consistent with its role in defending against RNA viruses via recruitment of MAVS 27 . To further investigate the potential impact of combined B cell and T cell autoimmunity to SNX8 following SARS-CoV-2 infection, we used scRNA-seq to analyse SNX8 expression in PBMCs from patients with severe, mild or asymptomatic SARS-CoV-2 infection or influenza infection and uninfected healthy controls 48 . Following SARS-CoV-2 infection, SNX8 had the highest mean expression in classical and non-classical monocytes and B cells (Extended Data Fig. 6a,b ) and was elevated in individuals infected with SARS-CoV-2 compared with those who were uninfected (Extended Data Fig. 6c ). Within myeloid lineage cells, SNX8 expression correlated with MAVS expression and OAS1 and OAS2 (which encode two known regulators of the MAVS pathway implicated in MIS-C pathogenesis 7 ) expression (Extended Data Fig. 6d ). Conversely, SNX8 expression is inversely correlated to SARS-CoV-2 infection severity. This follows a similar pattern to OAS1 and OAS2 . However, unlike OAS1 , OAS2 and MAVS , SNX8 is preferentially expressed during SARS-CoV-2 infection compared with influenza virus infection (Extended Data Fig. 6e ).

The SARS-CoV-2 pandemic largely spared children from severe disease. One rare but notable exception is MIS-C, an enigmatic and life-threatening syndrome. Previous studies have surfaced numerous associations, but have failed to identify a direct mechanistic link between SARS-CoV-2 and MIS-C. In this study, 199 samples from patients with MIS-C and 45 paediatric at-risk controls were analysed using customized human and SARS-CoV-2 proteome PhIP-seq libraries. Targeted follow-up experiments from these assays ultimately revealed that patients with MIS-C preferentially had antibodies targeting the epitope motif (ML)Q(ML)PQG shared by both the SARS-CoV-2 nucleocapsid protein and the human protein SNX8. Cross-reactive CD8 + T cells targeting both regions were detected in patients with MIS-C, but not in controls, suggesting that these CD8 + T cells may contribute to immune dysregulation through the inappropriate targeting of immune cells expressing SNX8. We found evidence that the (ML)Q(ML)PQG epitope motif elicits both B cell and T cell reactivity; further study of this epitope convergence is warranted.

These findings help to connect several important known aspects of MIS-C pathophysiology and draw parallels to other diseases in which exposure to a new antigen leads to autoimmunity, such as paraneoplastic autoimmune disease or cross-reactive epitopes between Epstein–Barr virus and host proteins in multiple sclerosis 17 , 18 , 19 , 22 , 26 . An expansion of T cells expressing TCRβ variable gene 11-2 ( TRBV11-2 ) has been shown in MIS-C 8 , 15 , 16 ; however, the underlying driver remains unknown. Although we did not observe an overrepresentation of TRBV11-2 in our putative cross-reactive TCR dataset, we did identify two expanded TRBV11-2 + clones ( n  = 6 and n  = 2) sequenced directly from ex vivo samples. Although SNX8 is a relatively understudied protein, it has been linked to the function and activity of MAVS 27 . Dysregulation of the MAVS antiviral pathway, by inborn errors of immunity, has been shown to underlie certain cases of MIS-C 7 . The most straightforward connection linking MIS-C to SNX8 may be through an inappropriate autoimmune response against tissues with elevated MAVS pathway expression. These results are the first to directly link the initial SARS-CoV-2 infection and the subsequent development of MIS-C. We propose that MIS-C may be the result of multiple uncommon events converging. The initial insult is probably the formation of a combined B cell and T cell response that preferentially targets a particular motif within the MADS region of the SARS-CoV-2 nucleocapsid protein. In a subset of individuals, these B cell and T cell responses cross-react to the self-protein SNX8. This cross-reactive motif has strong binding characteristics for the MIS-C-associated HLA-A*02 (ref. 16 ), further indicating that this may be an important risk factor in the development of MIS-C.

Using conservative criteria (3 s.d. greater than controls by targeted immunoprecipitation of the epitope-containing peptide), at least 17% of sera from patients with MIS-C are autoreactive for SNX8; however, approximately 37% of sera from patients with MIS-C yielded detectable enrichment compared with controls in the entire dataset. Because we only tested for a single epitope target, we are unable to determine the upper limits of the in vivo frequency of cross-reactive CD8 + T cells in patients with MIS-C. Our results suggest that the frequency of these cross-reactive CD8 + T cells is within the range of 1 in 10,000–100,000 CD8 + T cells. This substantially exceeds the frequency of antigen-specific autoreactive CD8 + T cells found in peripheral circulation in bona fide T cell-mediated autoimmune diseases such as T1DM 38 and multiple sclerosis 44 , 45 . Similar to T1DM, the autoreactive and cross-reactive CD8 + T cells in patients with MIS-C may be found at far greater abundance within peripheral tissues known to be affected by the disease 38 . Even accounting for these limitations, our results describe a subset of MIS-C, indicating that other mechanisms probably exist. Antibodies to ERFL are present in many children with MIS-C who do not have autoreactivity to SNX8, and ERFL has a highly similar tissue RNA expression profile as SNX8 (second-most similar among all known proteins; Human Protein Atlas) 36 . If autoreactive T cells to ERFL indeed exist, they would be predicted to engage a nearly identical set of cells and tissues. It is important to also consider that MIS-C prevalence has rapidly decreased as an increasing number of children have developed immunity through vaccination and natural SARS-CoV-2 infection. We speculate that perhaps this could be related to the strong deviation of the anti-SARS-CoV-2 immune response away from the critical MADS region of the nucleocapsid protein that we have identified, to other major epitopes such as those in the spike protein through vaccination and past infection 49 . Supporting this notion is recent CDC surveillance, which noted that more than 80% (92 of 112) of individuals with MIS-C in 2023 were in unvaccinated children (but vaccine eligible), and that the majority of children who developed MIS-C despite previous vaccination probably had waned immunity 50 .

MIS-C is complex, and more work will be required to fully understand this syndrome. The results of this study, and specifically the development of combined cross-reactive B cells and T cells, build on other notable examples of molecular mimicry; however, the mechanisms by which the presence of a cross-reactive epitope forces a break in tolerance remain unclear. Our results shed light on how one post-infectious disease (MIS-C) develops, yielding insights that may help better explain, diagnose and ultimately treat a range of additional conditions associated with infections.

Patients were recruited through the prospectively enrolling multicentre Overcoming COVID-19 and Taking on COVID-19 Together study in the USA. All patients meeting clinical criteria were included in the study, and therefore no statistical methods were used to predetermine sample size and no blinding or randomization of subjects occurred. The study was approved by the central Boston Children’s Hospital Institutional Review Board (IRB) and reviewed by IRBs of participating sites with CDC IRB reliance. A total of 292 patients consented and were enrolled into one of the following independent cohorts between 1 June 2020 and 9 September 2021: 223 patients hospitalized with MIS-C (199 in the primary discovery cohort and 24 in a separate subsequent validation cohort), 29 patients hospitalized for COVID-19 in either an intensive care or step-down unit (referred to as ‘severe acute COVID-19’ in this study) and 45 outpatients (referred to as ‘at-risk controls’ in this study) post-SARS-CoV-2 infections associated with mild or no symptoms. The demographic and clinical data are summarized in Extended Data Tables 1 – 3 . The 2020 US CDC case definition was used to define MIS-C 51 . All patients with MIS-C had positive SARS-CoV-2 serology results and/or positive SARS-CoV-2 test results by reverse transcriptase quantitative PCR. All patients with severe COVID-19 or outpatient SARS-CoV-2 infections had a positive antigen test or nucleic acid amplification test for SARS-CoV-2. For outpatients, samples were collected from 36 to 190 days after the positive test (median of 70 days after a positive test; interquartile range of 56–81 days). For use as controls in the SARS-CoV-2-specific PhIP-seq, plasma from 48 healthy, pre-COVID-19 controls were obtained as deidentified samples from the New York Blood Center. These samples were part of retention tubes collected at the time of blood donations from volunteer donors who provided informed consent for their samples to be used for research.

DNA oligomers for SLBAs

DNA coding for the desired peptides for use in SLBAs were inserted into split luciferase constructs containing a terminal HiBiT tag and synthesized (Twist Biosciences) as DNA oligomers and verified by Twist Biosciences before shipment. Constructs were amplified by PCR using the 5′- AAGCAGAGCTCGTTTAGTGAACCGTCAGA-3′ and 5′-GGCCGGCCGTTTAAACGCTGATCTT-3′ primer pair.

For SNX8, the oligomers coded for the following sequences:

EADPPASDLPTPQAIEPQAIVQQVPAPSRMQMPQGNPLLLSHTLQELLA

AAAAAAAAAATPQAIEPQAIVQQVPAPSRMQMPQGNPLLLSHTLQELLA

EADPPAAAAAAAAAAEPQAIVQQVPAPSRMQMPQGNPLLLSHTLQELLA

EADPPASDLPAAAAAAAAAAVQQVPAPSRMQMPQGNPLLLSHTLQELLA

EADPPASDLPTPQAIAAAAAAAAAAAPSRMQMPQGNPLLLSHTLQELLA

EADPPASDLPTPQAIEPQAIAAAAAAAAAAQMPQGNPLLLSHTLQELLA

EADPPASDLPTPQAIEPQAIVQQVPAAAAAAAAAANPLLLSHTLQELLA

EADPPASDLPTPQAIEPQAIVQQVPAPSRMAAAAAAAAAASHTLQELLA

EADPPASDLPTPQAIEPQAIVQQVPAPSRMQMPQGAAAAAAAAAAELLA

EADPPASDLPTPQAIEPQAIVQQVPAPSRMQMPQGNPLLLAAAAAAAAA

For SARS-CoV-2 nucleocapsid protein, the oligomers coded for the following sequences:

ATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQA

AAAAAAAAAADHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQA

ATEGAAAAAAAAAAARNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQA

ATEGALNTPKAAAAAAAAAANAAIVLQLPQGTTLPKGFYAEGSRGGSQA

ATEGALNTPKDHIGTAAAAAAAAAALQLPQGTTLPKGFYAEGSRGGSQA

ATEGALNTPKDHIGTRNPANAAAAAAAAAAGTTLPKGFYAEGSRGGSQA

ATEGALNTPKDHIGTRNPANNAAIVAAAAAAAAAAKGFYAEGSRGGSQA

ATEGALNTPKDHIGTRNPANNAAIVLQLPQAAAAAAAAAAEGSRGGSQA

ATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPAAAAAAAAAAGSQA

ATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAAAAAAAAAA

DNA plasmids for RLBAs

For RLBAs, DNA expression plasmids under control of a T7 promoter and with a terminal Myc–DDK tag for the desired protein were utilized. For ERFL, a custom plasmid was ordered from Twist Bioscience in which a Myc–DDK-tagged full-length ERFL sequence under a T7 promoter was inserted into the pTwist Kan High Copy Vector (Twist Bioscience). Twist Bioscience verified a sequence-perfect clone by next-generation sequencing before shipment. Upon receipt, the plasmid was sequence verified by Primordium Labs. For SNX8, a plasmid containing the Myc–DDK-tagged full-length human SNX8 under a T7 promoter was ordered from Origene (RC205847) and was sequence verified by Primordium Labs upon receipt. For KDELR1, a plasmid containing the Myc–DDK-tagged full-length human KDELR1 under a T7 promoter was ordered from Origene (RC205880) and was sequence verified by Primordium Labs upon receipt. For IL1RN, a plasmid containing the Myc–DDK-tagged full-length human IL1RN under a T7 promoter was ordered from Origene (RC218518) and was sequence verified by Primordium Labs upon receipt.

Polypeptide pools for activation-induced marker assays

To obtain polypeptides tiling the full-length SNX8 protein, 15-mer polypeptide fragments with 11-amino acid overlaps were ordered from JPT Peptide Technologies and synthesized. Together, a pool of 130 of these polypeptides (referred to as the ‘SNX8 pool’) spanned all known translated SNX8 (the full-length 465-amino acid SNX8 protein, as well as a unique region of SNX8 isoform 3). A separate pool was designed to cover primarily the region of SNX8 with similarity to the SARS-CoV-2 nucleocapsid protein in high resolution (referred to as the ‘high-resolution epitope pool’). This pool contained 20 10-mers with 9-amino acid overlaps tiling amino acids 44–72 (IVQQVPAPSRMQMPQGNPLLLSHTLQELL) of the full-length SNX8 protein. The sequence of each of these 150 polypeptides was verified by mass spectrometry and purity was calculated by high-performance liquid chromatography (HPLC).

Peptides for tetramer assays

For use in loading tetramers, three peptides were ordered from Genemed Synthesis as 9-mers. LQLPQGTTL and LQLPQGITL correspond to the region of the SARS-CoV-2 nucleocapsid protein with similarity to human SNX8 in the ancestral sequence and a minor variant, respectively. This sequence was verified by mass spectrometry and purity was calculated as 96.61% by HPLC. The other sequence, MQMPQGNPL, corresponds to the region of human SNX8 protein with similarity to the SARS-CoV-2 nucleocapsid protein. This sequence was verified by mass spectrometry and purity was calculated as 95.83% by HPLC.

Human proteome PhIP-seq

Human proteome PhIP-seq was performed following our previously published vacuum-based PhIP-seq protocol 12 ( https://www.protocols.io/view/scaled-high-throughput-vacuum-phip-protocol-ewov1459kvr2/v1 ).

Our human peptidome library consists of a custom-designed phage library of 731,724 unique T7 bacteriophage each presenting a different 49-amino acid peptide on its surface. Collectively, these peptides tile the entire human proteome including all known isoforms (as of 2016) with 25-amino acid overlaps. Of the phage library, 1 ml was incubated with 1 μl of human serum overnight at 4 °C and immunoprecipitated with 25 μl of 1:1 mixed protein A and protein G magnetic beads (10008D and 10009D, Thermo Fisher). These beads were than washed, and the remaining phage–antibody complexes were eluted in 1 ml of Escherichia coli (BLT5403, EMD Millipore) at 0.5–0.7 OD and amplified by growing in a 37 °C incubator. This new phage library was then re-incubated with the serum from the same individual and the previously described protocol was repeated. DNA was then extracted from the final phage library, barcoded, PCR amplified and Illumina adaptors were added. Next-generation sequencing was performed using an Illumina sequencer (Illumina) to a read depth of approximately 1 million per sample.

Human proteome PhIP-seq analysis

All human peptidome analysis (except when specifically stated otherwise) was performed at the gene level, in which all reads for all peptides mapping to the same gene were summed, and 0.5 reads were added to each gene to allow inclusion of genes with zero reads in mathematical analyses. Within each individual sample, reads were normalized by converting to the percentage of total reads. To normalize each sample against background nonspecific binding, a fold change over mock-IP was calculated by dividing the sample read percentage for each gene by the mean read percentage of the same gene for the AG bead-only controls. This fold-change signal was then used for side-by-side comparison between samples and cohorts. Fold-change values were also used to calculate z scores for each patient with MIS-C compared with controls and for each control sample by using all remaining controls. These z scores were used for the logistic-regression feature weighting. In instances of peptide-level analysis, raw reads were normalized by calculating the number of reads per 100,000 reads.

SARS-CoV-2 proteome PhIP-seq

SARS-CoV-2 proteome PhIP-seq was performed as previously described 39 . In brief, 38 amino acid fragments tiling all open reading frames from SARS-CoV-2, SARS-CoV-1 and 7 other CoVs were expressed on T7 bacteriophage with 19-amino acid overlaps. Of the phage library, 1 ml was incubated with 1 μl of human serum overnight at 4 °C and immunoprecipitated with 25 μl of 1:1 mixed protein A and protein G magnetic beads (10008D and 10009D, Thermo Fisher). Beads were washed five times on a magnetic plate using a P1000 multichannel pipette. The remaining phage–antibody complexes were eluted in 1 ml of E. coli (BLT5403, EMD Millipore) at 0.5–0.7 OD and amplified by growing in 37 °C incubator. This new phage library was then re-incubated with the serum of the same individual and the previously described protocol was repeated for a total of three rounds of immunoprecipitations. DNA was then extracted from the final phage library, barcoded, PCR amplified and Illumina adaptors were added. Next-generation sequencing was then performed using an Illumina sequencer (Illumina) to a read depth of approximately 1 million per sample.

Coronavirus proteome PhIP-seq analysis

To account for differing read depths between samples, the total number of reads for each peptide fragment was converted to the number of reads per 100,000 (RPK). To calculate normalized enrichment relative to pre-COVID-19 controls (FC > pre-COVID-19), the RPK for each peptide fragment within each sample was divided by the mean RPK of each peptide fragment among all pre-COVID-19 controls. These FC > pre-COVID-19 values were used for all subsequent analyses as described in the text and figures.

RLBAs were performed as previously described 12 , 32 . In brief, DNA plasmids containing full-length cDNA under the control of a T7 promoter for each of the validated antigens (see ‘DNA plasmids for RLBAs’ above) were verified by Primordium Labs sequencing. The respective DNA templates were used in the T7 TNT in vitro transcription/translation kit (L1170, Promega) using [35S]-methionine (NEG709A, PerkinElmer). Respective protein was column purified on Nap-5 columns (17-0853-01, GE Healthcare), and equal amounts of protein (approximately 35,000 counts per minute) were incubated overnight at 4 °C with 2.5 μl of serum or 1 μl of anti-Myc-positive control antibody (1:10 dilution; 2272S, Cell Signaling Technology). Immunoprecipitation was then performed on 25 μl of Sephadex protein A/G beads (4:1 ratio; GE17-5280-02 and GE17-0618-05, Sigma-Aldrich) in 96-well polyvinylidene difluoride filtration plates (EK-680860, Corning). After thoroughly washing, the counts per minute of immunoprecipitated protein was quantified using a 96-well Microbeta Trilux liquid scintillation plate reader (Perkin Elmer).

SLBA was performed as previously described 52 . A detailed SLBA protocol is available on protocols.io ( https://doi.org/10.17504/protocols.io.4r3l27b9pg1y/v1 ).

In brief, the DNA oligomers listed above (see ‘DNA oligomers for SLBAs’) were amplified by PCR using the primer pairs listed above (see ‘DNA oligomers for SLBAs’). Unpurified PCR product was used as input in the T7 TNT in vitro transcription/translation kit (L1170, Promega) and the Nano-Glo HiBit Lytic Detection System (N3040, Promega) was used to measure relative luciferase units of translated peptides in a luminometre. Equal amounts of protein (in the range of 2 × 10 6 –2 × 10 7 relative luciferase units) were incubated overnight with 2.5 μl patient sera or 1 μl anti-HiBit-positive control antibody (1:10 dilution; CS2006A01, Promega) at 4 °C. Immunoprecipitation was then performed on 25 µl of Sephadex protein A/G beads (1:1 ratio; GE17-5280-02 and GE17-0618-05, Sigma-Aldrich) in 96-well polyvinylidene difluoride filtration plates (EK-680860, Corning). After thoroughly washing, luminescence was measured using the Nano-Glo HiBit Lytic Detection System (N3040, Promega) in a luminometre.

Activation-induced marker assay

PBMCs were obtained from ten patients with MIS-C and ten controls for use in the activation-induced marker assay. PBMCs were thawed, washed, resuspended in serum-free RPMI medium and plated at a concentration of 1 × 10 6 cells per well in a 96-well round-bottom plate. For each individual, PBMCs were stimulated for 24 h with either the SNX8 pool (see above) at a final concentration of 1 mg ml −1 per peptide in 0.2% DMSO or a vehicle control containing 0.2% DMSO only. For four of the controls and two of the patients with MIS-C, there were sufficient PBMCs for an additional stimulation condition using the SNX8 high-resolution epitope pool (see above) also at a concentration of 1 mg ml −1 per peptide in 0.2% DMSO for 24 h. Following the stimulation, cells were washed with FACS buffer (Dulbecco’s PBS without calcium or magnesium, 0.1% sodium azide, 2 mM EDTA and 1% FBS) and stained with the following antibody panel each at 1:100 dilution for 20 min at 4 °C, and then flow cytometry analysis was immediately performed.

For the antibody panel: anti-CD3 Alexa 647 (clone OKT3, 317312, BioLegend), anti-CD4 Alexa 488 (clone OKT4, 317420, BioLegend), anti-CD8 Alexa 700 (clone SK1, 344724, BioLegend), anti-OX-40 (also known as CD134) PE-Dazzle 594 (clone ACT35, 350020, BioLegend), anti-CD69 PE (clone FN-50, 310906, BioLegend), anti-CD137 (also known as 4-1BB) BV421 (clone 4B4-1, 309820, BioLegend), anti-CD14 PerCP-Cy5 (clone HCD14, 325622, BioLegend), anti-CD16 PerCP-Cy5 (clone B73.1, 360712, BioLegend), anti-CD19 PerCP-Cy5 (clone HIB19, 302230, BioLegend) and Live/Dead Dye eFluor 506 (65-0866-14, Invitrogen).

The activation-induced marker analysis was performed using FlowJo software using the gating strategy shown in Extended Data Fig. 7a . All gates were fixed within each condition of each sample. Activated CD4 T cells were defined as those that were co-positive for OX40 and CD137. Activated CD8 T cells were defined as those that were co-positive for CD69 and CD137. Gating thresholds for activation were defined by the outer limits of signal in the vehicle controls allowing for up to two outlier cells. Frequencies were calculated as a percentage of total CD3 + cells (T cells). Two MIS-C samples had insufficient total events captured by flow cytometry (total of 5,099 and 4,919 events, respectively) and were therefore removed from analysis.

Initial tetramer assay

For the initial tetramer assay, see Extended Data Fig. 4a . PBMCs from two patients with MIS-C with HLA-A*02:01 (HLA typed from PAXgene RNAseq, one confirmed by serotyping), one patient with MIS-C with HLA-B*35:01 (HLA typed from PAXgene RNAseq) and three at-risk controls with HLA-A*02.01 (all three identified by serotyping, two of three confirmed by PAXgene RNAseq HLA typing; the other sample did not have genomic DNA available for genotyping) were thawed, washed and put into culture with media containing recombinant human IL-2 at 10 ng ml −1 in 96-well plates. The peptide fragments (details above) LQLPQGITL and MQMPQGNPL were then added to PBMCs to a final concentration of 10 mg ml −1 per peptide and incubated (37 °C at 5% CO 2 ) for 7 days.

Following the 7 days of incubation, a total of eight pHLA class I tetramers were generated from UV-photolabile biotinylated monomers, four each from HLA-A*02:01 and HLA-B*35:01 (NIH Tetramer Core). Peptides were loaded via UV peptide exchange. Tetramerization was carried out using streptavidin conjugated to fluorophores PE and APC or BV421 followed by quenching with 500 µM d -biotin, similar to our previously published methods 44 , 53 . Tetramers were then pooled together as shown below:

For the HLA-A*02:01 pool, the MADS (LQLPQGITL)-loaded PE tetramer, MADS (LQLPQGITL)-loaded APC tetramer, SNX8 (MQMPQGNPL)-loaded PE tetramer and SNX8 (MQMPQGNPL)-loaded BV421 tetramer were used, all with HLA-A*02:01 restriction.

For the HLA-B*35:01 pool, the MADS (LQLPQGITL)-loaded PE tetramer, MADS (LQLPQGITL)-loaded APC tetramer, SNX8 (MQMPQGNPL)-loaded PE tetramer and SNX8 (MQMPQGNPL)-loaded BV421 tetramer were used, all with HLA-B*35:01 restriction.

All PBMCs were then treated with 100 nM dasatinib (StemCell) for 30 min at 37 °C followed by staining (no wash step) with the respective tetramer pool corresponding to their HLA restriction (final concentration of 2–3 µg ml −1 ) for 30 min at 25 °C. Cells were then stained with the following cell-surface markers each at 1:100 dilution for 20 min, followed by immediate analysis on a flow cytometer.

For the surface markers: anti-CD8 Alexa 700 (clone SK1, 357404, BioLegend), anti-CD4 PerCP-Cy5 (clone SK1, 300530, BioLegend), anti-CD14 PerCP-Cy5 (clone HCD14, 325622, BioLegend), anti-CD16 PerCP-Cy5 (clone B73.1, 360712, BioLegend), anti-CD19 PerCP-Cy5 (clone HIB19, 302230, BioLegend) and Live/Dead Dye eFluor 506 (65-0866-14, Invitrogen). Streptavidin was conjugated to PE (S866, Invitrogen), APC (S868, Invitrogen) and BV421 (405225, BioLegend).

The gating strategy is outlined in Extended Data Fig. 7b . A stringent tetramer gating strategy was used to identify cross-reactive T cells, in which CD8 + T cells were required to be triple positive for PE, APC and BV421 labels (that is, a single CD8 T cell bound to PE-conjugated LQLPQGITL and/or PE-conjugated MQMPQGNPL in addition to APC-conjugated LQLPQGITL and BV421-conjugated MQMPQGNPL).

Serotyping was performed using an anti-HLA-A2 antibody (1:100 dilution; FITC anti-human HLA-A2 antibody, clone BB7.2, 343303, BioLegend), and pertinent results are shown in Extended Data Fig. 7c .

Assembly of easYmer monomers and fold testing

For the assembly of HLA class I pHLA easYmer monomers and fold testing, see Fig. 4 . Unfolded, biotinylated easYmer monomers (Immudex) were obtained for HLA-A*02:01 and HLA-A*02:06. SARS-CoV-2 MADS (LQLPQGITL), SARS-CoV-2 Wuhan (LQLPQGTTL) and human SNX8 (MQMPQGNPL) peptides were commercially synthesized (Genscript), diluted to 1 mM in ddH 2 O or DMSO, and loaded onto each easYmer allele according to the manufacturer’s instructions at 18 °C for 48 h. Proper pHLA monomer formation and MADS and SNX8 peptide-binding strength were evaluated for each HLA using a ‘β2m fold test’ relative to negative (no peptide; unloaded monomer) and positive (strong binding peptide; CMV pp65 495–503 (NLVPMVATV)) controls as per the manufacturer’s protocol. In brief, peptide-loaded monomers with a concentration of 500 nM were serially diluted to 9 nM, 3 nM and 1 nM in dilution buffer (1× PBS with 5% glycerol; G5516, Sigma-Aldrich) and incubated with streptavidin beads (6–8 μm; SVP-60-5, Spherotech) at 37 °C for 1 h to allow binding of stable complexes to beads, then washed three times with FACS buffer (1× PBS, 0.5% BSA (A7030, Sigma-Aldrich) and 2 mM EDTA (15575-038, Thermo Fisher Scientific)). Samples were then stained with PE-conjugated anti-human β2m antibody (clone BBM.1, sc-13565, Santa Cruz Biotech) at 1:200 for 30 min at 4 °C, washed three times with FACS buffer and analysed on a 5 Laser 16UV-16V-14B-10YG-8R AURORA spectral cytometer (Cytek). pHLA-binding strength positively correlated with stability and concentration of the pHLA–β2m complex. Therefore, the geometric mean fluorescence intensity of anti-β2m staining in this assay reports on the strength of the pHLA binding compared with the positive and negative controls. We classified binding strength for each HLA and peptide combination based on the fold change in anti-β2m geometric mean fluorescence intensity over the no-peptide negative control at 9 nM. Strong binders were defined at more than 10-fold higher, moderate binders at more than 3-fold, weak binders at more than 1.5-fold and non-binders at less than 1.5-fold change over the negative control. Flow cytometry data were analysed using FlowJo version 10.7.2 software (BD Biosciences).

pHLA tetramer assembly

For the pHLA tetramer assembly, see Fig. 4 . pHLA tetramers were assembled from HLA-A*02:01 and HLA-A*02:06 easYmer monomers (Immudex) with confirmed peptide binding to SARS-CoV-2 MADS (LQLPQGITL), Wuhan (LQLPQGTTL) and SNX8 (MQMPQGNPL) peptides according to the manufacturer’s instructions. In brief, fluorochrome-conjugated streptavidin (0.2 mg ml −1 , PE, 405203, BioLegend; 0.2 mg ml −1 , APC, 405207, BioLegend; and BV421, 405226, BioLegend) was added to loaded monomers at 8 ng per 1 μl pHLA complex (500 nM) in three volumes. After each 1/3 volume addition, samples were mixed and incubated for 15 min at 4 °C in the dark. Assembled tetramers were stored at 4 °C in the dark until use.

Enhanced peptide-specific T cell expansion

For enhanced peptide-specific T cell expansion, see Fig. 4 . PBMCs from MIS-C confirmed participants with HLA-A*02:01 or HLA-A*02:06 were obtained for peptide-specific expansion according to published methods 54 before single-cell sorting of tetramer-positive T cells. On expansion day 0, PBMCs were thawed, counted and seeded onto 96-well round-bottom plates at 100,000 cells per well in 200 μl antigen-presenting cell differentiation media (X-VIVO 15 serum-free haematopoietic cell medium (04-418Q, Lonza) supplemented with human GM-CSF (1,000 IU ml −1 ; 130-095-372, Miltenyi Biotec), human IL-4 (500 IU ml −1 ; 204-IL-010, R&D Systems) and human Flt3-L (50 ng ml −1 ; 308-FKN-025, R&D Systems) final concentrations) and incubated for 24 h at 37 °C and 5% CO 2 . On day 1, 100 μl cell supernatant was replaced with 100 μl Adjuvant Solution (X-VIVO 15 supplemented with R848 (10 μM; tlrl-r848-5, InvivoGen), lipopolysaccharide ( Salmonella minnesota ; 100 ng ml −1 ; tlrl-smlps, InvivoGen) and human IL-1β (10 ng ml −1 ; 201-LB-010, R&D Systems) final concentrations) and pooled MADS (LQLPQGITL) and SNX8 (MQMPQGNPL) peptides at a final concentration of 10 μM each. No-peptide control wells were set up for each sample by adding a 1:2 dilution of DMSO in H 2 O to match the peptide volume and diluent. Cells were incubated for 24 h at 37 °C and 5% CO 2 . On days 2, 4, 7 and 9, 100 μl supernatant was replaced with 100 μl T cell expansion solution: RP-10 (RPMI 1640 (22400-089, Gibco), 10% heat-inactivated human serum AB (100-512, Gemini Bio-Products), 10 mM HEPES, 0.1 mg ml −1 gentamicin (15750-060, Thermo Fisher Scientific) and 1× GlutaMAX (35050-061, Gibco)) supplemented with human IL-2 (10 IU ml −1 ; 202-IL-050, R&D Systems), human IL-7 (10 ng ml −1 ; 207-IL-025, R&D Systems) and human IL-15 (10 ng ml −1 ; 200-15, PeproTech) final concentrations. On day 10, peptide-expanded cells from an individual participant were pooled; cells from no-peptide controls were collected separately.

Single-cell index sorting

Unexpanded PBMCs (direct ex vivo) or peptide-expanded T cells were obtained, washed in 1× PBS and treated with 100 nM dasatinib (CDS023389, Sigma-Aldrich) in 1× PBS for 30 min at 37 °C and 5% CO 2 (ref. 55 ). Cells were then pelleted and resuspended in 50 μl FACS buffer (1× PBS and 0.04% BSA) supplemented with human TruStain FcX blocking buffer (1:10 dilution; 422302, BioLegend), 500 μM d -biotin (B20656, Thermo Fisher Scientific) and a unique tetramer cocktail containing MADS–tetramer–PE (1:10 dilution), MADS–tetramer–APC (1:10 dilution), SNX8–tetramer–PE (1:10 dilution) and SNX8–tetramer–BV421 (1:10 dilution) based on participant HLA type (A*02:01 and A*02:06). Cells were incubated in the dark at 25 °C for 1 h followed by direct addition of 50 μl (100 μl total volume) of FACS supplemented with 500 μM d -biotin and an antibody cocktail containing FITC-conjugated anti-human CD3 (1:20 dilution; clone OKT3, lot B390808, 317306, BioLegend), BV605-conjugated anti-human CD8 (1:20 dilution; clone SK1, lot B371925, 344742, BioLegend), BV510-conjugated anti-human CD4 (1:20 dilution; clone OKT4, lot B375526, 317444, BioLegend), BV510-conjugated anti-human CD14 (1:20 dilution; clone 63D3, lot B390770, 367124, BioLegend), BV510-conjugated anti-human CD16 (1:20 dilution; clone 3G8, lot B372132, 302048, BioLegend), BV510-conjugated anti-human CD19 (1:20 dilution; clone HIB19, lot B390665, 302242, BioLegend) and Ghost Dye Violet 510 Viability Dye (1:400 dilution; lot D0870061322133, 13-0870-T500, Tonbo Biosciences) for 30 min in the dark at 4 °C. Cells were then pelleted, washed twice with 4 ml FACS buffer (containing 500 μM d -biotin), suspended in 500 μl FACS (containing 500 μM d -biotin) and passed through a 45-μM filter before proceeding to single-cell sorting on a Sony SY3200 cell sorter. Individual, live, BV510 dump gate (CD4, CD14, CD16 and CD19)-negative, CD3 + CD8 + T lymphocytes were gated to distinguish tetramer triple-positive cells (PE + APC + BV421 + ) as described in Extended Data Fig. 7d and sorted into individual wells of a 384-well plate loaded with Superscript VILO master mix (11754250, Thermo Fisher Scientific). After sorting, plates were centrifuged at 500 g and stored at −80 °C until processing.

Paired TCRαβ amplification and sequencing

Single-cell paired TCRα and TCRβ chain library preparation and sequencing was performed on T cells sorted into 384-well index plates as previously described 56 . In brief, after reverse transcription of cells sorted in Superscript VILO master mix, cDNA underwent two rounds of nested multiplex PCR amplification using a mix of human V-segment-specific forward primers and human TRAC and TRBC segment-specific reverse primers (see Supplementary Table 1 for primer details). Resulting TCRα and TCRβ amplicons were sequenced on an Illumina MiSeq at 2 × 150-bp read length.

All cultured cell lines were maintained at 37 °C and 5% CO 2 in a humidified incubator. HEK 293T cells (CRL-3216, American Type Culture Collection) were purchased from the American Type Culture Collection and verified commercially. HEK 293T cells were cultured in DMEM (11965-092, Gibco) supplemented with 10% FBS (16140-071, Gibco), 2 mM l -glutamine (25030-081, Gibco) and 100 U ml −1 penicillin–streptomycin (15140-122, Gibco). 2D3 Jurkat J76.7 cells 57 , 58 (TCR-null, CD8 + ) expressing an NFAT–eGFP reporter were kindly provided by F. Fujiki and were cultured in RPMI 1640 (22400-089, Gibco) supplemented with 10% FBS, 2 mM l -glutamine and 100 U ml −1 penicillin–streptomycin. All cell lines were confirmed to be mycoplasma negative during the course of experiments.

TCR repertoire analysis

TCR similarity networks were constructed as previously described 49 , 59 . In brief, to measure the distance between TCRαβ clonotypes, we used the TCRdist algorithm implementation from the CoNGA v0.1.2 Python package 47 . Further analysis was performed using the R language for statistical computing, with merging and subsetting of data performed using the dplyr v1.1.4 package. TCR similarity networks were built using stringdist v0.9.12 and igraph v2.0.3 (ref. 60 ) R packages, and visualized using gephi v0.9.7 (ref. 61 ) software.

TCR reconstruction and cloning

Full-length TCRαβ sequences were reconstructed from V/J gene usage and CDR3 sequences using Stitchr v1.0.0 (ref. 62 ) for each index-sorted T cell. TCRα and TCRβ chain sequences were modified to use murine constant regions and joined by a 2A element from thosea asigna virus (T2A). A sequence encoding mCherry was additionally appended by a 2A element from porcine teschovirus (P2A) as a fluorescent marker of transduction. The full-length gene fragment encoding TCRβ–T2A–TCRα–P2A–mCherry was synthesized and cloned commercially (Genscript) into the lentiviral vector pLVX-EF1α-IRES-Puro (631253, Takara).

Generation of TCR-expressing Jurkat cells

To generate transducing particles packaging individual TCRs of interest (Fig. 4c ), HEK 293T cells were transduced with a pLVX lentiviral vector encoding a unique TCRαβ–mCherry insert, psPAX2 packaging plasmid (plasmid #12260, Addgene) and an pMD2.G envelope plasmid (plasmid #12259, Addgene) at a ratio of 4:3:1. At 24 h and 48 h post-transfection, viral supernatants were harvested, passed through a 0.45-µm SFCA filter (723-9945, Thermo Fisher Scientific), concentrated using Lenti-X Concentrator (631232, Takara) and stored at −80 °C as single-use aliquots. To generate TCR-expressing Jurkat cell lines (Jurkat-TCR + ), 2D3 Jurkat J76.7 cells (TCR-null, CD8 + , NFAT–eGFP reporter) were seeded in a 12-well tissue-culture-treated plate at 1 × 10 6 cells per well in complete RPMI (RPMI 1640, 10% FBS, 2 mM l -glutamine, 100 U ml −1 penicillin–streptomycin) and transduced by adding concentrated lentivirus dropwise to each well. At 48–72 h post-tranduction, puromycin was added at 1 μg ml −1 and cultured for 1 week to select for transduced cells. Jurkat-TCR + cell lines were validated for the presence of correctly folded TCR on the cell surface by flow cytometry using a monoclonal antibody targeting the mouse TCRβ constant region (APC/Fire750-conjugated; clone H57-597, 109246, BioLegend; Extended Data Fig. 5a ). Flow cytometry data were collected on a custom-configured BD Fortessa using FACSDiva software (v8.0.1; Becton Dickinson) and analysed using FlowJo version 10.7.2 software (BD Biosciences).

Specificity validation of putative cross-reactive TCR sequences

The specificity of TCR-expressing Jurkat T cell lines was validated by tetramer staining using the same reagents used for single-cell sorting PBMCs (above). In brief, 1 × 10 6 Jurkat-TCR + cell lines or untransduced Jurkat J76.7 (TCR-null; background control) were washed in 1× PBS and resuspended in 50 μl FACS buffer (1× PBS and 0.04% BSA) and a unique tetramer cocktail containing MADS–tetramer–PE (1:10 dilution), MADS–tetramer–APC (1:10 dilution), SNX8–tetramer–PE (1:10 dilution) and SNX8–tetramer–BV421 (1:10 dilution) based on the restricting HLA type (A*02:01 and A*02:06). Tetramers conjugated to the Wuhan peptide sequence (LQLPQGTTL), including Wuhan–tetramer–PE (1:10 dilution) and Wuhan–tetramer–APC (1:10 dilution), were also tested. A second set of wells were set up in which each individual tetramer was used to stain cells. Cells were incubated in the dark at 25 °C for 30 min after which 50 µl of FACS buffer containing Ghost Dye Violet 510 Viability Dye (1:400 dilution; lot D0870061322133, 13-0870-T500, Tonbo Biosciences) was added for an additional 30-min incubation in the dark at 25 °C. Cells were then washed twice with 1 ml FACS buffer, suspended in 300 μl FACS and analysed by flow cytometry on a custom-configured BD Fortessa using FACSDiva software (v8.0.1; Becton Dickinson). Cell population gating and fluorescence analysis was performed using FlowJo version 10.7.2 software (BD Biosciences) as described in Extended Data Fig. 7e .

scRNA-seq analysis

To assess the cell-type specificity in a relevant disease context, we analysed SNX8 expression from a single-cell sequencing of PBMC samples from patients with severe, mild or asymptomatic COVID-19 infection, influenza virus infection and healthy controls 48 . Gene expression data from 59,572 pre-filtered cells were downloaded from the Gene Expression Omnibus database under accession GSE149689 for analysis and downstream processing with scanpy v1.10.0 (ref. 63 ). Cells with (1) less than 1,000 total counts, (2) less than 800 expressed genes, and (3) more than 3,000 expressed genes were filtered out as further quality control, leaving 42,904 cells for downstream analysis. Gene expression data were normalized to have 10,000 counts per cell and were log1p transformed. Highly variable genes were calculated using the scanpy function highly_variable_genes using Seurat flavor with the default parameters (min_mean = 0.0125, max_mean = 3, and min_disp = 0.5) 64 . Only highly variable genes were used for further analysis. The total number of counts per cell was regressed out, and the gene expression matrix was scaled using the scanpy function scale with max_value = 10. Dimensionality reduction was performed using principal components analysis with 50 principal components. Batch balanced k -nearest neighbours, implemented with scanpy’s function bbknn, was used to compute the top neighbours and normalize batch effects 65 . The batch-corrected cells were clustered using the Leiden algorithm and projected into two dimensions with uniform manifold approximation and projection for visualization. Initial cluster identity was determined by finding marker genes with differential expression analysis performed using a Student’s t -test on log1p-transformed raw counts with the scanpy function rank_genes_groups 66 , 67 .

Statistical methods

All statistical analysis was performed in Python using the Scipy Stats package unless otherwise indicated. For comparisons of distributions of PhIP-seq enrichment between two groups, a non-parametric Kolmogorov–Smirnov test was utilized. For logistic-regression feature weighting, the Scikit-learn package 68 was used, and logistic-regression classifiers were applied to z -scored PhIP-seq values from individuals with MIS-C versus at-risk controls. A liblinear solver was used with L1 regularization, and the model was evaluated using a five-fold cross-validation (four of the five for training, and one of the five for testing). For the RLBAs and SLBAs, first an antibody index was calculated as follows: (sample value – mean blank value)/(positive control antibody values – mean blank values). For the alanine mutagenesis scans, blank values of each construct were combined, and a single mean was calculated. A normalization function was then applied to the experimental samples only (excluding antibody-only controls) to create a normalized antibody index ranging from 0 to 1. Comparisons between two groups of samples were performed using a Mann–Whitney U -test. An antibody was considered to be ‘positive’ when the normalized antibody index in a sample was greater than 3 s.d. above the mean of controls. When comparing two groups of normally distributed data, a Student’s  t -test was performed.

Reporting summary

Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Data availability

The published article includes all datasets generated or analysed as a part of this study. Individual source data are provided with associated figures (where appropriate) per the data-sharing agreement stipulated under the Ruth L. Kirschstein National Research Service Award Individual Postdoctoral Fellowship (award no. F32AI157296 to R.C.M.). Raw flow cytometry source files can be made available on reasonable request. All PhIP-seq data are publicly available via a Dryad digital repository ( https://doi.org/10.7272/Q6SJ1HVH ). Raw TCR reads are available through the NCBI Sequence Read Archive (SRA) BioProject PRJNA1110271 , with associated BioSample accession numbers SAMN41334731 , SAMN41334732 , SAMN41334730 and SAMN41334729 .  Source data are provided with this paper.

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Acknowledgements

The following members of the Overcoming COVID-19 Network Investigators study group were all closely involved with the design, implementation and oversight of the Overcoming COVID-19 study, as well as collecting patient samples and data: M. Kong, H. Kelley, M. Murdock, C. Colston. K. V. Typpo, K. Irby, R. C. Sanders Jr, M. Yates, C. Smith, N. Z. Cvijanovich, M. S. Zinter, A. B. Maddux, E. Port, R. Mansour, S. Shankman, N. Baig, F. Zorensky, P. S. Espinal, B. Chatani, G. McLaughlin, K. M. Tarquinio, K. Jones, B. M. Coates, C. M. Rowan, A. G. Randolph, M. M. Newhams, S. Kucukak, T. Novak, E. R. McNamara, H. Kyung Moon, T. Kobayashi, J. Melo, S. R. Jackson, M. K. Echon Rosales, C. Young, S. R. Chen, J. Chou, R. Da Costa Aguiar, M. Gutierrez-Arcelus and M. Elkins. The Taking On COVID-19 Together team include: D. Williams, L. Williams, L. Cheng, Y. Zhang, D. Crethers, D. Morley, S. Steltz, K. Zakar, M. A. Armant, F. Ciuculescu, H. R. Flori, M. K. Dahmer, E. R. Levy, S. Behl, N. M. Drapeau, C. V. Hobbs, J. E. Schuster, A. Kietzman, S. Hill, M. L. Cullimore, R. J. McCulloh, S. J. Gertz, S. P. Schwartz, T. C. Walker, R. A. Nofziger, M. A. Staat, C. C. Rohlfs, J. C. Fitzgerald, R. Burnett, J. Bush, E. H. Mack, N. Reed, N. B. Halasa, L. L. Loftis, H. Crandall and K. K. Ampofo. Members of the US Centers for Disease Control and Prevention COVID-19 Response Team on the Overcoming COVID-19 study were L. D. Zambrano, M. M. Patel and A. P. Campbell. The authors acknowledge the New York Blood Center for contributing pre-COVID-19 healthy donor blood samples, which were used as controls for the SARS-CoV-2 library PhIP-seq. The authors acknowledge the contributions of W. Browne and S. Pleasure for their work investigating potential central nervous system-specific autoimmunity in MIS-C; T. Kharel for help designing the Python code used in the analysis; D. Blauvelt for ideas regarding the application of advanced statistics to PhIP-seq data analysis; and S. A. Schattgen for thoughtful discussion of TCR sequencing and TCR similarity network analysis and help with deposition of the TCR sequencing into the Sequence Read Archive. BioRender ( https://biorender.com ) was used to build graphics for Fig. 1a and Extended Data Fig. 4a . This work was supported by the Pediatric Scientist Development Program and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (K12-HD000850 to A.B.), and the Chan Zuckerberg Biohub SF (to J.L.D. and M.S.A.). Overcoming COVID-19 Study Network enrolment, patient data and specimen collections were supported by the CDC contracts 75D30120C07725, 75D30121C10297 and 75D30122C13330 from the Centers for Disease Control and Prevention to Boston Children’s Hospital to A.G.R. and the National Institute of Allergy and Infectious Diseases (R01AI154470) to A.G.R. Patient clinical data and specimens also collected at Boston Children’s Hospital for the Taking On COVID-19 Together (TOCT) study were supported in part by the Boston Children’s Hospital Emerging Pathogens and Epidemic Response Cluster of Clinical Research Excellence and the Institutional Centers for Clinical and Translational Research to A.G.R. and K.L.M. P.G.T. is supported by the American Lebanese Syrian Associated Charities at St. Jude Children’s Research Hospital (SJCRH) and funding from the National Institute of Allergy and Infectious Diseases (5R01AI154470-03, 2R01AI136514-06, 3P01AI165077-01S1, 75N93021C00016 and U01 AI144616). R.C.M. is supported by a Ruth L. Kirschstein National Research Service Award Individual Postdoctoral Fellowship award (F32AI157296).

Author information

These authors contributed equally: Aaron Bodansky, Robert C. Mettelman

These authors jointly supervised this work: Paul G. Thomas, Adrienne G. Randolph, Mark S. Anderson, Joseph L. DeRisi

Authors and Affiliations

Department of Pediatrics, Division of Critical Care, University of California San Francisco, San Francisco, CA, USA

Aaron Bodansky & Matt S. Zinter

Department of Host–Microbe Interactions, St. Jude Children’s Research Hospital, Memphis, TN, USA

Robert C. Mettelman, Mikhail V. Pogorelyy, Walid Awad, Allison M. Kirk & Paul G. Thomas

Weill Institute for Neurosciences, University of California San Francisco, San Francisco, CA, USA

Joseph J. Sabatino Jr, Colin R. Zamecnik & Michael R. Wilson

Department of Neurology, University of California San Francisco, San Francisco, CA, USA

Joseph J. Sabatino Jr, Colin R. Zamecnik, John V. Pluvinage & Michael R. Wilson

Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA

Sara E. Vazquez, Haleigh S. Miller, Andrew F. Kung, Elze Rackaityte, Jayant V. Rajan, Hannah Kortbawi, Caleigh Mandel-Brehm, Kelsey Zorn & Joseph L. DeRisi

Division of Immunology, Department of Pediatrics, Boston, MA, USA

Department of Pediatrics, Harvard Medical School, Boston, MA, USA

Janet Chou, Kristin L. Moffitt & Adrienne G. Randolph

Department of Anesthesiology, Critical Care and Pain Medicine, Boston Children’s Hospital, Boston, MA, USA

Tanya Novak & Adrienne G. Randolph

Department of Anesthesia, Harvard Medical School, Boston, MA, USA

Department of Pediatric, Division of Infectious Diseases, Boston Children’s Hospital, Boston, MA, USA

Kristin L. Moffitt

Biological and Medical Informatics Program, University of California San Francisco, San Francisco, CA, USA

Haleigh S. Miller & Andrew F. Kung

Medical Scientist Training Program, University of California San Francisco, San Francisco, CA, USA

Hannah Kortbawi

Chan Zuckerberg Biohub SF, San Francisco, CA, USA

Anthea Mitchell, Chung-Yu Wang, Aditi Saxena & Joseph L. DeRisi

Diabetes Center, School of Medicine, University of California San Francisco, San Francisco, CA, USA

David J. L. Yu & Mark S. Anderson

Biomedical Sciences Program, University of California San Francisco, San Francisco, CA, USA

James Asaki

COVID-19 Response Team and Coronavirus and Other Respiratory Viruses Division, Centers for Disease Control and Prevention, Atlanta, GA, USA

Laura D. Zambrano & Angela P. Campbell

Department of Medicine, Division of Endocrinology and Metabolism, University of California San Francisco, San Francisco, CA, USA

Mark S. Anderson

Department of Pediatrics, Division of Critical Care Medicine, Baylor College of Medicine, Houston, TX, USA

• Laura L. Loftis

Department of Pediatrics, Division of Infectious Diseases, University of Mississippi Medical Center, Jackson, MS, USA

Charlotte V. Hobbs

Department of Pediatrics, Division of Critical Care Medicine, Emory University School of Medicine, Children’s Healthcare of Atlanta, Atlanta, GA, USA

Keiko M. Tarquinio

Department of Pediatrics, Division of Pediatric Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA

Michele Kong

Department of Anesthesiology and Critical Care, Children’s Hospital of Philadelphia, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, USA

Julie C. Fitzgerald

Personalized Medicine and Health Outcomes Research, Nicklaus Children’s Hospital, Miami, FL, USA

Paula S. Espinal

Department of Pediatrics, University of North Carolina at Chapel Hill Children’s Hospital, Chapel Hill, NC, USA

Tracie C. Walker & Stephanie P. Schwartz

Department of Pediatrics, Division of Pediatric Critical Care, University of Utah, Primary Children’s Hospital, Salt Lake City, UT, USA

Hillary Crandall

Section of Pediatric Critical Care, Department of Pediatrics, Arkansas Children’s Hospital, Little Rock, AR, USA

Katherine Irby

Department of Pediatrics, Division of Infectious Diseases, University of Cincinnati and Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

Mary Allen Staat

Department of Pediatrics, Division of Pediatric Critical Care Medicine, Indiana University School of Medicine and Riley Hospital for Children, Indianapolis, IN, USA

Courtney M. Rowan

Department of Pediatrics, Division of Pediatric Infectious Diseases, Children’s Mercy Kansas City, Kansas City, MO, USA

Jennifer E. Schuster

Department of Pediatrics, Division of Pediatric Infectious Diseases, Vanderbilt University Medical Center, Nashville, TN, USA

Natasha B. Halasa

Department of Pediatrics, Division of Pediatric Critical Care, Cooperman Barnabas Medical Center, Livingston, NJ, USA

Shira J. Gertz

Division of Pediatric Critical Care Medicine, Medical University of South Carolina, Charleston, SC, USA

Elizabeth H. Mack

Department of Pediatrics, Section of Critical Care Medicine, University of Colorado School of Medicine and Children’s Hospital Colorado, Aurora, CO, USA

Aline B. Maddux

Division of Critical Care Medicine, UCSF Benioff Children’s Hospital Oakland, Oakland, CA, USA

Natalie Z. Cvijanovich

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Overcoming COVID-19 Network Investigators

• , Charlotte V. Hobbs
• , Keiko M. Tarquinio
• , Michele Kong
• , Julie C. Fitzgerald
• , Paula S. Espinal
• , Tracie C. Walker
• , Stephanie P. Schwartz
• , Hillary Crandall
• , Katherine Irby
• , Mary Allen Staat
• , Courtney M. Rowan
• , Jennifer E. Schuster
• , Natasha B. Halasa
• , Shira J. Gertz
• , Elizabeth H. Mack
• , Aline B. Maddux
• , Natalie Z. Cvijanovich
•  & Matt S. Zinter

Contributions

A.B., R.C.M., J.J.S.Jr, S.E.V., J.C., P.G.T., A.G.R., M.S.A. and J.L.D. conceptualized the study. A.B., R.C.M., J.J.S.Jr, S.E.V., E.R., C.R.Z., A.F.K., J.V.R., J.C., P.G.T., A.G.R., M.S.A. and J.L.D. curated the methodology. A.B., R.C.M., J.J.S.Jr, S.E.V., A.M., C.-Y.W., A.S., J.V.P., D.J.L.Y., H.K., W.A., A.M.K. and C.M.-B. performed or contributed to experiments. A.B., R.C.M., J.J.S.Jr, H.S.M., A.F.K., J.A. and M.V.P. performed the formal analysis. K.Z., T.N., L.D.Z., A.P.C., A.G.R., K.L.M. and the Overcoming COVID-19 Network Investigators acquired the patient sample and clinical data. T.N., A.G.R. and the Overcoming COVID-19 Network Investigators curated the clinical data. A.B., H.S.M. and J.L.D. wrote the original draft of the manuscript. A.B., R.C.M., J.C., T.N., H.S.M., L.D.Z., A.P.C., P.G.T., A.G.R., M.R.W., M.S.A. and J.L.D. reviewed and edited the manuscript. J.C., P.G.T., A.G.R., M.S.A. and J.L.D. supervised the study.

Corresponding authors

Correspondence to Mark S. Anderson or Joseph L. DeRisi .

Ethics declarations

Competing interests.

J.L.D. reports being a founder and paid consultant for Delve Bio, Inc., and a paid consultant for the Public Health Company and Allen & Co. M.A.S. receives unrelated research funding from the National Institutes of Health, the Centers for Disease Control and Prevention, Cepheid and Merck and unrelated honoria from UpToDate, Inc. M.R.W. receives unrelated research grant funding from Roche/Genentech and Novartis, and received speaking honoraria from Genentech, Takeda, WebMD and Novartis. J.C. reports consulting fees from GLG group, payments from Elsevier for work as an Associate Editor, a patent pending for methods and compositions for treating and preventing T cell-driven diseases, payments related to participation on a Data Safety Monitoring Board or Advisory Board for Enzyvant, and is a member of the Diagnostic Laboratory Immunology Committee of the Clinical Immunology Society. M.S.Z. receives unrelated funding from the National Heart, Lung, and Blood Institute and consults for Sobi. N.B.H. reports unrelated previous grant support from Sanofi and Quidel, and current grant support from Merck. C.V.H. reports being a speaker for Biofire and a reviewer for UpToDate, Inc. and Dynamed.com. A.G.R. receives royalties as a section editor for Pediatric Critical Care Medicine UpToDate, Inc., and received honoraria for MIS-C-related Grand Round Presentations. A.G.R. is also on the medical advisor board of Families Fighting Flu and is Chair of the International Sepsis Forum, which is supported by industry and has received reagents from Illumina, Inc. P.G.T. is on the Scientific Advisory Board of Immunoscape and Shennon Bio, has received research support and personal fees from Elevate Bio, and consulted for 10X Genomics, Illumina, Pfizer, Cytoagents, Merck and JNJ. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention nor the National Institute of Allergy and Infectious Diseases. All other authors declare no competing interests.

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Peer review information.

Nature thanks Shiv Pillai and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended data fig. 1 previously reported autoantigens and phenotypic associations of novel autoantigens..

a , Heatmap showing distribution of PhIP-Seq enrichments (FC > Mock-IP) of previously reported MIS-C autoantibodies in MIS-C patients ( n  = 199) and at-risk controls (n = 45). (b) Stripplots and boxplots showing distribution of signal (normalized antibody index) for antibodies targeting IL-1 receptor antagonist (IL-1Ra) measured by RLBA in at-risk controls (blue; n  = 45), MIS-C patient samples containing IVIG (red; n  = 135), and MIS-C patient samples without IVIG (green; n  = 61). Dotted line at 3 standard deviations above the mean of controls. Two-sided Mann-Whitney U testing was performed (exact P  values shown in figure). c , Heatmap of P  values (two-sided Kolmogorov-Smirnov testing) for differences in autoantibody enrichment for MIS-C patients ( n  = 199) with versus without each clinical phenotype (numbers vary for each phenotype and are shown in Extended Data Table 2 ). Significant P  values in the negative direction (in which there is increased signal in individuals without the phenotype) are masked (colored as P > 0.05). For each autoantigen, tissue RNA-sequencing data from Human Protein Atlas (Proteinatlas.org) is shown. Amount of expression in cardiac tissue in top row (Very high = nTPM >1000, High=nTPM 100-1000, Moderate=nTPM 10-100, Low=nTPM <10), and predominant tissue type in second-from-top row. Explanations of criteria for MIS-C phenotypes, and distribution of each phenotype within the cohort, can be found in Extended Data Table 2 .

Extended Data Fig. 2 Orthogonally validated autoantibodies classify MIS-C and can be epitope specific.

a , Stripplots and boxplots showing radioligand binding assay (RLBA) values (normalized antibody indices) for each of the top 3 autoantibodies identified by PhIP-Seq logistic regression in individuals with MIS-C ( n  = 197 for ERFL, n  = 196 for SNX8, n = 196 for KDELR1) and each at-risk control ( n  = 45 for ERFL, SNX8, and KDELR1). Two-sided Mann-Whitney U testing performed (exact P  values shown in figure). b , Logistic regression receiver operating characteristic (ROC) curve using RLBA values as input to distinguish MIS-C patients ( n  = 196) from at-risk controls ( n  = 45) iterated 1,000 times. c , Stripplots and boxplots showing RLBA enrichments (normalized antibody indices) only in those MIS-C samples without IVIG ( n  = 61 for ERFL, n  = 60 for SNX8, n  = 60 for KDELR1) relative to at-risk controls ( n  = 45 for ERFL, SNX8, and KDELR1). Two-sided Mann-Whitney U testing performed (exact P  values shown in figure). d , Stripplots abd boxplots showing RLBA enrichments (normalized antibody indices) for ERFL, SNX8, and KDELR1 in an independent cohort of children with MIS-C (red; n  = 24 for each RLBA) compared to children severely ill with acute COVID-19 (yellow; n  = 29 for each RLBA) and at-risk controls (blue; n  = 45 for each RLBA). Two-sided Mann-Whitney U testing performed (exact P  values shown in figure). e , Logistic regression ROC curves for classification of the independent MIS-C cohort ( n  = 24) versus at-risk controls ( n  = 45) (left) and the independent MIS-C ( n  = 24) cohort versus children severely ill with acute COVID-19 ( n  = 29) (right). f , Paired stripplots and boxplots showing SLBA enrichments (normalized antibody indices) in MIS-C patients ( n  = 182) and at-risk controls ( n  = 45) for the full 49 amino acid SNX8 wild-type (WT) polypeptide fragment (lavender) relative to the same SNX8 fragment with alanine mutagenesis of the [PSRMQMPQG] epitope (white). SNX8 WT fragment SLBA values are the means of technical replicates, SNX8 epitope mutagenesis values are from a single experiment. Two-sided Mann-Whitney U testing performed (exact P  values shown in figure). For all boxplots in the figure, the whiskers extend to 1.5 times the interquartile range (IQR) from the quartiles, the boxes represent the IQR, and centre lines represent the median.

Extended Data Fig. 3 HLA associations of SNX8 activated T cells and HLA binding characteristics of peptides containing SNX8/MADS shared epitope motif.

a , Stripplots and boxplots showing distribution of CD4 + , CD8 + , and total T cells which activate in response to either vehicle (culture media + 0.2% DMSO) or SNX8 peptide pool (SNX8 peptide + culture media + 0.2% DMSO) using AIM assay in MIS-C patients ( n  = 9) and controls ( n  = 10). Patient HLA type indicated by color of dot. HLA unpredicted means patient contained none of the MIS-C associated HLA types. Dotted line at 3 standard deviations above the mean of the SNX8 stimulated controls. Two-sided Mann-Whitney U testing was performed (exact P  values shown in figure). b , Computationally predicted HLA class I presentation scores (Immune Epitope Database; IEDB.org) for each possible peptide fragment of full-length SARS-CoV-2 N protein for each of the three MIS-C associated HLA types (A*02, B*35 and C*04) relative to a reference set of HLA-types encompassing over 99% of humans. Those fragments containing the MADS similarity region “LQLPQG” in orange. Data normally distributed; two-sided t-tests were performed (exact  P  values shown in figure). Percent of fragments within each specific HLA type with a score greater than 0.1 (likely to be presented) shown on right. c , Identical analysis but using full length SNX8 protein rather than SARS-CoV-2 NP, and the SNX8 similarity region “MGMPQG” rather than the MADS region “LQLPQG”. Data normally distributed; two-sided t-tests were performed (exact P  values shown in figure). d , HLA binding results from β2m folding assay for SARS-CoV-2 N and SNX8 peptides representative of two independent evaluations. Each peptide tested for binding in HLA-A*02:01, A*02:06, and B*35:01 class I monomers. Data presented as geometric mean fluorescence intensity (gMFI) of PE-conjugated anti-human β2m antibody staining of peptide-HLA monomers relative to negative (no peptide; unloaded HLA monomer) and positive (strong binding peptide; CMV pp65 495-503 [NLVPMVATV]) controls. For all boxplots in the figure, the whiskers extend to 1.5 times the interquartile range (IQR) from the quartiles, the boxes represent the IQR, and centre lines represent the median.

Extended Data Fig. 4 Identification, activation, and HLA restriction, of cross-reactive CD8+ T cells.

a , Gating strategy used to identify CD8 + T cells which bound to SNX8 epitope and/or MADS N protein epitope (CD8 + T cells positive for PE). Representative MIS-C patient and control showing each CD8 + T cell which bound to any tetramer (PE + ) and the relative binding of that T cell to both the SNX8 epitope (BV421 + ) and the MADS N protein epitope (APC + ) identifying cross-reactive T cells (PE + APC + BV421 + ). Schematics in panel a were created using BioRender ( https://www.biorender.com ). b , Stripplots and boxplots showing percentage of CD8 + T cells which are cross-reactive to both SNX8 and MADS in MIS-C patients ( n  = 3) and controls ( n  = 3). Insufficient numbers to perform robust statistical testing. c , Stripplots and boxplots showing percentage of total T cells which activate in response to either vehicle (culture media + 0.2% DMSO) or the SNX8 Epitope (SNX8 Epitope (Materials) + culture media + 0.2% DMSO) in MIS-C patients ( n  = 2) and at-risk controls ( n  = 4) measured by AIM assay. Insufficient numbers to perform robust statistical testing. Dotted line at 3 standard deviations above mean of SNX8 Epitope stimulated controls. d , TCRdist Similarity Network of 48 unique, paired TCRαβ sequences ( n  = 259 sequences) obtained from four patients with MIS-C. CD8 + T cells were sorted from PBMCs directly ex vivo or after 10-days of peptide expansion and staining with A*02:01 or A*02:06 HLA class I tetramers loaded with MADS [LQLPQGITL] and SNX8 [MQMPQGNPL] peptides. Each node represents a unique TCR clonotype. Edges connect nodes with a TCRdist score < 150. Dashed lines surround TCR similarity clusters. Node size corresponds to T cell clone size. Nodes are colored based on HLA restriction. TCRs selected for further testing are numbered TCR #1-8. Convergent node circled green. For all boxplots in the figure, the whiskers extend to 1.5 times the interquartile range (IQR) from the quartiles, the boxes represent the IQR, and centre lines represent the median.

Extended Data Fig. 5 Evaluation of Jurkat-TCR lines.

a , Jurkat-76 cells stably expressing putative cross-reactive TCRs (#1-8) stained with anti-murine TCRβ constant region (mTCRβc). Plots depict frequency of transduced (mCherry + ) Jurkat cells with presence of surface TCR (APC/Fire 750 + ) as a percentage of total live cells. b , Jurkat-TCR + cell lines expressing putative cross-reactive TCRs #1-8 stained with individual or combination of HLA-A*02:01 or A*02:06 tetramers loaded with MADS [LQLPQGITL] and SNX8 [MQMPQGNPL] peptides. Blue contour plots indicate staining with MADS-Tetramer (PE) and MADS-Tetramer (APC); purple contour plots indicate staining with SNX8-Tetramer (PE) and SNX8-Tetramer (BV421); red indicates combined staining with a pool of MADS/SNX8-Tetramer (PE), MADS-Tetramer (APC), and SNX8-Tetramer (BV421). Plots shown are gated from total PE + cells. Plots with confirmed cross-reactive TCRs outlined in red. c , Jurkat-TCR+ cell lines expressing putative cross-reactive TCRs #1-8 stained with individual HLA-A*02:01 or A*02:06 tetramers loaded with MADS Wuhan [LQLPQGTTL] peptide. Gate values indicate frequency of MADS-APC + cells as percentage of total MADS-PE + cells. Outliers shown in contour plots. Flow plots representative of two independent evaluations.

Extended Data Fig. 6 SNX8 expression during viral infection.

a , UMAPs showing SNX8 expression in various peripheral blood cell types during SARS-CoV-2 infection. b , Mean expression and percent of cells expressing SNX8 in peripheral blood subsets during SARS-CoV-2 infection. c , Mean expression and percent of cells expressing SNX8 averaged across all peripheral blood mononuclear cells from SARS-CoV-2 infected individuals without symptoms, with mild symptoms, or with severe disease compared to uninfected controls. d , Mean expression and percent of cells expressing SNX8 , OAS1 , OAS2 , and MAVS in peripheral blood subsets during SARS-CoV-2 infection. e , Relative expression of SNX8 , OAS1 , OAS2 , and MAVS during influenza virus infection compared to different severities of SARS-CoV-2 infection.

Extended Data Fig. 7 Representative flow cytometry gating.

a , Flow cytometry gating strategy for identifying CD4 positive and CD8 positive T cells for the AIM analysis with representative activation induced marker (AIM) assay flow cytometry gating strategy measuring percent of CD4 + T cells which activate (CD137 + OX40 + ) and percent of CD8 + T cells which activate (CD137 + CD69 + ) in response to SNX8 protein. b , Flow cytometry gating strategy for the initial SNX8/MADS tetramer cross-reactivity assay (Extended Data Fig. 4a,b ) showing isolation of PE-tetramer positive CD8 positive T cells. c , Flow cytometry plots showing results of serotyping for the PBMCs used in the initial SNX8/MADS tetramer cross-reactivity assay (Extended Data Fig. 4a,b ) which did not have sufficient cells for genotyping. Shown is the 1 MIS-C patient (far left) and 3 controls (middle 3) which are positive for HLA-A*02 and were used and one control negative for HLA-A*02 (far right) which was not used. d , Index sorting strategy for patient PBMCs from ex vivo and peptide expansion experiments for TCR sequencing. Single cells were sorted from live/lineage (CD4, CD14, CD16, CD19)-negative, CD3 + CD8 + T lymphocytes positive for MADS/SNX8-Tetramer (PE) and MADS-Tetramer (APC) and/or SNX8-Tetramer (BV421). e , Flow cytometry gating strategy to evaluate putative cross-reactive Jurkat-TCRs. Gates include single, live, transduced Jurkat lymphocytes triple positive for MADS/SNX8-(PE), MADS-(APC), and SNX8-(BV421) tetramers shown in Fig. 4 .

Supplementary information

Supplementary table 1.

The complete set of primers used for the single-cell T cell receptor (TCR) sequencing by nested multiplex polymerase chain reaction (PCR).

Reporting Summary

Peer review file, source data, source data fig. 1, source data fig. 2, source data fig. 3, source data fig. 4, source data extended data fig. 1, source data extended data fig. 2, source data extended data fig. 3, source data extended data fig. 4, source data extended data fig. 5, rights and permissions.

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Bodansky, A., Mettelman, R.C., Sabatino, J.J. et al. Molecular mimicry in multisystem inflammatory syndrome in children. Nature (2024). https://doi.org/10.1038/s41586-024-07722-4

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Received : 20 June 2023

Accepted : 14 June 2024

Published : 07 August 2024

DOI : https://doi.org/10.1038/s41586-024-07722-4

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