{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T22:38:28Z","timestamp":1786747108783,"version":"build-2736575974"},"reference-count":35,"publisher":"Oxford University Press (OUP)","issue":"8","license":[{"start":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T00:00:00Z","timestamp":1786665600000},"content-version":"vor","delay-in-days":13,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Institutes of Health\/National Institute of Allergy and Infectious Diseases","award":["R01AI158830"],"award-info":[{"award-number":["R01AI158830"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,8,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The gastrointestinal pathogen Clostridioides difficile is a major burden for health systems due to high rates of recurrence. C. difficile pathogenesis is mediated by two virulence factors, toxin A (TcdA) and toxin B (TcdB). Antibodies specific for TcdA and TcdB are correlated with protection from symptomatic recurrence; however, the role for CD4+ T cells is poorly understood in part due to the lack of tools to study the toxin-specific CD4+ T cell response. Our group recently demonstrated the antibody and CD4+ T cell response to C. difficile toxins is impaired via the glucosyltransferase activity of the toxins; however, tools do not exist to study the protective capacity and the phenotype of toxin-specific CD4+ T cells. Therefore, we developed a major histocompatibility complex class II (MHC-II) tetramer to identify TcdB-specific CD4+ T cells via flow cytometry. Herein, we identified an immunodominant epitope (TcdB1961\u20131975) in the CROPs region of TcdB and optimized an MHC-II tetramer for use in tracking and phenotyping TcdB-specific CD4+ T cell responses following multiple different immunization strategies in mice. Utilizing the tetramer, TcdB-specific T follicular helper cells were detected following TcdB-CROPs messenger RNA lipid nanoparticle vaccination validating the advantage of the tetramer. Furthermore, using a modular messenger RNA vector expressing the TcdB1961 peptide covalently bound to the beta chain of MHC-II, we were able to generate a robust population of TcdB-specific CD4+ T cells. These data outline the generation of new tools for the C. difficile field and lay the groundwork for future studies of toxin-specific CD4+ T cell responses.<\/jats:p>","DOI":"10.1093\/jimmun\/vkag152","type":"journal-article","created":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T22:24:11Z","timestamp":1786746251000},"source":"Crossref","is-referenced-by-count":0,"title":["Generation and characterization of a novel MHC-II tetramer for tracking and characterization of toxin B\u2013specific CD4+ T cell responses"],"prefix":"10.1093","volume":"215","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6982-5946","authenticated-orcid":false,"given":"Jeffrey R","family":"Maslanka","sequence":"first","affiliation":[{"name":"Department of Microbiology, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA,","place":["United States"]},{"name":"Immunology Graduate Group, University of Pennsylvania , Philadelphia, PA,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qianxuan","family":"She","sequence":"additional","affiliation":[{"name":"Department of Pathology and Laboratory Medicine, Children\u2019s Hospital of Philadelphia , Philadelphia, PA,","place":["United States"]},{"name":"Center for Microbial Medicine, Children\u2019s Hospital of Philadelphia , Philadelphia, PA,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kathleen S","family":"Krauss","sequence":"additional","affiliation":[{"name":"Department of Pathology and Laboratory Medicine, Children\u2019s Hospital of Philadelphia , Philadelphia, PA,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emily N","family":"Bitsko","sequence":"additional","affiliation":[{"name":"Department of Medicine, Drexel University College of Medicine , Philadelphia, PA,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nile U","family":"Bayard","sequence":"additional","affiliation":[{"name":"Department of Pathology and Laboratory Medicine, Children\u2019s Hospital of Philadelphia , Philadelphia, PA,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer A","family":"Londregan","sequence":"additional","affiliation":[{"name":"Immunology Graduate Group, University of Pennsylvania , Philadelphia, PA,","place":["United States"]},{"name":"Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohamad-Gabriel","family":"Alameh","sequence":"additional","affiliation":[{"name":"Department of Pathology and Laboratory Medicine, Children\u2019s Hospital of Philadelphia , Philadelphia, PA,","place":["United States"]},{"name":"Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA,","place":["United States"]},{"name":"Penn Institute for RNA Innovation, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laurence C","family":"Eisenlohr","sequence":"additional","affiliation":[{"name":"Department of Pathology and Laboratory Medicine, Children\u2019s Hospital of Philadelphia , Philadelphia, PA,","place":["United States"]},{"name":"Institute for Immunology and Immune Health, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michele A","family":"Kutzler","sequence":"additional","affiliation":[{"name":"Department of Medicine, Drexel University College of Medicine , Philadelphia, PA,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3228-3055","authenticated-orcid":false,"given":"Joseph P","family":"Zackular","sequence":"additional","affiliation":[{"name":"Department of Pathology and Laboratory Medicine, Children\u2019s Hospital of Philadelphia , Philadelphia, PA,","place":["United States"]},{"name":"Center for Microbial Medicine, Children\u2019s Hospital of Philadelphia , Philadelphia, PA,","place":["United States"]},{"name":"Institute for Immunology and Immune Health, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA,","place":["United States"]},{"name":"Division of Protective Immunity, Children\u2019s Hospital of Philadelphia , Philadelphia, PA,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7351-2061","authenticated-orcid":false,"given":"Michael C","family":"Abt","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA,","place":["United States"]},{"name":"Institute for Immunology and Immune Health, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,8,14]]},"reference":[{"key":"2026081418240737500_vkag152-B1","doi-asserted-by":"publisher","first-page":"3241","DOI":"10.3390\/jcm9103241","article-title":"Enhanced humoral immune responses against toxin A and B of clostridium difficile is associated with a milder disease manifestation","volume":"9","author":"Na\u2019amnih","year":"2020","journal-title":"J Clin Med"},{"key":"2026081418240737500_vkag152-B2","doi-asserted-by":"publisher","first-page":"1323","DOI":"10.1111\/1469-0691.12769","article-title":"Humoral immune response as predictor of recurrence in Clostridium difficile infection","volume":"20","author":"Bauer","year":"2014","journal-title":"Clin Microbiol Infection"},{"key":"2026081418240737500_vkag152-B3","doi-asserted-by":"publisher","first-page":"398","DOI":"10.1002\/eji.201747295","article-title":"Adaptive immune response to Clostridium difficile infection: a perspective for prevention and therapy","volume":"48","author":"Rees","year":"2018","journal-title":"Eur J Immunol"},{"key":"2026081418240737500_vkag152-B4","doi-asserted-by":"publisher","first-page":"390","DOI":"10.1056\/NEJM200002103420604","article-title":"Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A","volume":"342","author":"Kyne","year":"2000","journal-title":"N Engl J Med"},{"key":"2026081418240737500_vkag152-B5","doi-asserted-by":"publisher","first-page":"e74452","DOI":"10.1371\/journal.pone.0074452","article-title":"Circulating antibody and memory B-cell responses to C. difficile toxins A and B in patients with C. difficile-associated diarrhoea, inflammatory bowel disease and cystic fibrosis","volume":"8","author":"Monaghan","year":"2013","journal-title":"PLoS One"},{"key":"2026081418240737500_vkag152-B6","doi-asserted-by":"publisher","first-page":"97","DOI":"10.1007\/BF01642866","article-title":"Serum antibody response to Clostridium difficile toxins in patients with Clostridium difficile diarrhoea","volume":"13","author":"Aronsson","year":"1985","journal-title":"Infection"},{"key":"2026081418240737500_vkag152-B7","doi-asserted-by":"publisher","first-page":"730","DOI":"10.1093\/cid\/ciw364","article-title":"Antibodies to toxin B are protective against clostridium difficile infection recurrence","volume":"63","author":"Gupta","year":"2016","journal-title":"Clin Infect Dis"},{"key":"2026081418240737500_vkag152-B8","doi-asserted-by":"publisher","first-page":"189","DOI":"10.1016\/S0140-6736(00)03592-3","article-title":"Association between antibody response to toxin A and protection against recurrent Clostridium difficile diarrhoea","volume":"357","author":"Kyne","year":"2001","journal-title":"Lancet"},{"key":"2026081418240737500_vkag152-B9","doi-asserted-by":"publisher","first-page":"e00829-19","DOI":"10.1128\/IAI.00829-19","article-title":"Clostridioides difficile infection induces an inferior IgG response to that induced by immunization and is associated with a lack of T follicular helper cell and memory B cell expansion","volume":"88","author":"Amadou Amani","year":"2020","journal-title":"Infect Immun"},{"key":"2026081418240737500_vkag152-B10","doi-asserted-by":"publisher","first-page":"1410","DOI":"10.1053\/j.gastro.2020.11.043","article-title":"Recurrent clostridioides difficile infection is associated with impaired T helper type 17 immunity to C difficile toxin B","volume":"160","author":"Cook","year":"2021","journal-title":"Gastroenterology"},{"key":"2026081418240737500_vkag152-B11","doi-asserted-by":"publisher","first-page":"1271","DOI":"10.1016\/j.mucimm.2025.08.004","article-title":"Clostridioides difficile toxin A and toxin B inhibit toxin-specific adaptive immune responses through glucosyltransferase-dependent activity","volume":"18","author":"Maslanka","year":"2025","journal-title":"Mucosal Immunol"},{"key":"2026081418240737500_vkag152-B12","doi-asserted-by":"publisher","first-page":"755","DOI":"10.1038\/s41467-020-20793-x","article-title":"Host immunity modulates the efficacy of microbiota transplantation for treatment of Clostridioides difficile infection","volume":"12","author":"Littmann","year":"2021","journal-title":"Nat Commun"},{"key":"2026081418240737500_vkag152-B13","doi-asserted-by":"publisher","first-page":"27","DOI":"10.1016\/j.chom.2015.06.011","article-title":"Innate immune defenses mediated by two ILC subsets are critical for protection against acute clostridium difficile infection","volume":"18","author":"Abt","year":"2015","journal-title":"Cell Host Microbe"},{"key":"2026081418240737500_vkag152-B14","unstructured":"Theiller E, Moustafa A. \u00a0C.difficile - WhatsGNU ortholog database. 10.5281\/zenodo.13387715"},{"key":"2026081418240737500_vkag152-B15","doi-asserted-by":"publisher","first-page":"1043","DOI":"10.1101\/gr.186072.114","article-title":"CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes","volume":"25","author":"Parks","year":"2015","journal-title":"Genome Res"},{"key":"2026081418240737500_vkag152-B16","doi-asserted-by":"publisher","first-page":"124","DOI":"10.12688\/wellcomeopenres.14826.1","article-title":"Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications","volume":"3","author":"Jolley","year":"2018","journal-title":"Wellcome Open Res"},{"key":"2026081418240737500_vkag152-B17","doi-asserted-by":"publisher","first-page":"2864","DOI":"10.1038\/ismej.2017.126","article-title":"dRep: a tool for fast and accurate genomic comparisons that enables improved genome recovery from metagenomes through de-replication","volume":"11","author":"Olm","year":"2017","journal-title":"Isme J"},{"key":"2026081418240737500_vkag152-B18","author":"Sebaihia","year":"2005"},{"key":"2026081418240737500_vkag152-B19","doi-asserted-by":"publisher","first-page":"D10","DOI":"10.1093\/nar\/gkaa892","article-title":"Database resources of the National Center for Biotechnology Information","volume":"49","author":"Sayers","year":"2021","journal-title":"Nucleic Acids Res"},{"key":"2026081418240737500_vkag152-B20","doi-asserted-by":"publisher","first-page":"000685","DOI":"10.1099\/mgen.0.000685","article-title":"Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification","volume":"7","author":"Schwengers","year":"2021","journal-title":"Microb Genom"},{"key":"2026081418240737500_vkag152-B21","doi-asserted-by":"publisher","first-page":"180","DOI":"10.1186\/s13059-020-02090-4","article-title":"Producing polished prokaryotic pangenomes with the Panaroo pipeline","volume":"21","author":"Tonkin-Hill","year":"2020","journal-title":"Genome Biol"},{"key":"2026081418240737500_vkag152-B22","doi-asserted-by":"publisher","first-page":"1530","DOI":"10.1093\/molbev\/msaa015","article-title":"IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era","volume":"37","author":"Minh","year":"2020","journal-title":"Mol Biol Evol"},{"key":"2026081418240737500_vkag152-B23","doi-asserted-by":"publisher","first-page":"86","DOI":"10.1007\/BF02101990","article-title":"A new method for calculating evolutionary substitution rates","volume":"20","author":"Lanave","year":"1984","journal-title":"J Mol Evol"},{"key":"2026081418240737500_vkag152-B24","doi-asserted-by":"publisher","first-page":"306","DOI":"10.1007\/BF00160154","article-title":"Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: approximate methods","volume":"39","author":"Yang","year":"1994","journal-title":"J Mol Evol"},{"key":"2026081418240737500_vkag152-B25","doi-asserted-by":"publisher","first-page":"W78","DOI":"10.1093\/nar\/gkae268","article-title":"Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool","volume":"52","author":"Letunic","year":"2024","journal-title":"Nucleic Acids Res"},{"key":"2026081418240737500_vkag152-B26","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1126\/science.adn4955","article-title":"A multivalent mRNA-LNP vaccine protects against Clostridioides difficile infection","volume":"386","author":"Alameh","year":"2024","journal-title":"Science"},{"key":"2026081418240737500_vkag152-B27","doi-asserted-by":"publisher","first-page":"4080","DOI":"10.1128\/IAI.01950-14","article-title":"An optimized, synthetic DNA vaccine encoding the toxin A and toxin B receptor binding domains of Clostridium difficile induces protective antibody responses in vivo","volume":"82","author":"Baliban","year":"2014","journal-title":"Infect Immun"},{"key":"2026081418240737500_vkag152-B28","doi-asserted-by":"publisher","first-page":"vkaf318","DOI":"10.1093\/jimmun\/vkaf318","article-title":"An mRNA platform to create isolated, monospecific Th1 responses","volume":"215","author":"Krauss","year":"2026","journal-title":"J Immunol"},{"key":"2026081418240737500_vkag152-B29","doi-asserted-by":"publisher","first-page":"26","DOI":"10.1016\/j.omtn.2019.02.018","article-title":"A facile method for the removal of dsRNA contaminant from in vitro-transcribed mRNA","volume":"15","author":"Baiersd\u00f6rfer","year":"2019","journal-title":"Mol Ther Nucleic Acids"},{"key":"2026081418240737500_vkag152-B30","doi-asserted-by":"publisher","first-page":"1933","DOI":"10.1038\/s41590-023-01644-5","article-title":"Cryptic MHC-E epitope from influenza elicits a potent cytolytic T cell response","volume":"24","author":"Hogan","year":"2023","journal-title":"Nat Immunol"},{"key":"2026081418240737500_vkag152-B31","doi-asserted-by":"publisher","first-page":"e1000048","DOI":"10.1371\/journal.pcbi.1000048","article-title":"A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach","volume":"4","author":"Wang","year":"2008","journal-title":"PLoS Comput Biol"},{"key":"2026081418240737500_vkag152-B32","doi-asserted-by":"publisher","first-page":"568","DOI":"10.1186\/1471-2105-11-568","article-title":"Peptide binding predictions for HLA DR, DP and DQ molecules","volume":"11","author":"Wang","year":"2010","journal-title":"BMC Bioinformatics"},{"key":"2026081418240737500_vkag152-B33","doi-asserted-by":"publisher","first-page":"347","DOI":"10.1038\/s42003-020-1078-y","article-title":"Subtyping analysis reveals new variants and accelerated evolution of Clostridioides difficile toxin B","volume":"3","author":"Shen","year":"2020","journal-title":"Commun Biol"},{"key":"2026081418240737500_vkag152-B34","doi-asserted-by":"publisher","first-page":"4706","DOI":"10.4049\/jimmunol.164.9.4706","article-title":"DO11.10 and OT-II T cells recognize a C-terminal ovalbumin 323\u2013339 epitope1","volume":"164","author":"Robertson","year":"2000","journal-title":"J Immunol"},{"key":"2026081418240737500_vkag152-B35","doi-asserted-by":"publisher","first-page":"114245","DOI":"10.1016\/j.celrep.2024.114245","article-title":"Clostridioides difficile toxin B subverts germinal center and antibody recall responses by stimulating a drug-treatable CXCR4-dependent mechanism","volume":"43","author":"Norman","year":"2024","journal-title":"Cell Rep"}],"container-title":["The Journal of Immunology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/jimmunol\/article-pdf\/215\/8\/vkag152\/70653623\/vkag152.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/jimmunol\/article-pdf\/215\/8\/vkag152\/70653623\/vkag152.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T22:24:12Z","timestamp":1786746252000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/jimmunol\/article\/doi\/10.1093\/jimmun\/vkag152\/8761892"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8]]},"references-count":35,"journal-issue":{"issue":"8","published-print":{"date-parts":[[2026,8,4]]}},"URL":"https:\/\/doi.org\/10.1093\/jimmun\/vkag152","relation":{},"ISSN":["0022-1767","1550-6606"],"issn-type":[{"value":"0022-1767","type":"print"},{"value":"1550-6606","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2026,8]]},"published":{"date-parts":[[2026,8]]},"article-number":"vkag152"}}