{"doi":"10.1101/2025.04.21.649828","title":"Whole-Proteome Screening and Multi-Modal Profiling of Antigen-Specific CD4+ T Cells at Single-Cell Resolution","abstract":"Abstract Systematic whole-proteome screening and comprehensive profiling of antigen-specific CD4+ T cells are crucial for advancing our understanding of CD4+ T cell immunity, yet such efforts remain technically challenging. Here, we present a high-throughput platform that employs large-scale class II single-chain trimer libraries to detect antigen-specific CD4+ T cells, while simultaneously profiling their antigen specificity, TCRα/β sequences, MHC restriction, whole transcriptomes, and patient/timepoint origins at single-cell resolution. We benchmarked SCTs against conventional pMHCs and validated the SCT library-based approach in direct ex vivo identification of antigen-specific CD4+ T cells in healthy donors. We then applied the platform to screen the entire SARS-CoV-2 receptor-binding domain in a longitudinal patient cohort, identifying 2,188 antigen-specific CD4+ T cells and revealing key features that define antigen immunogenicity. Extending to cancer, we performed whole-proteome screening of HPV-16 E6/E7 for TCR repertoire profiling in a precancerous cohort, uncovering functional heterogeneity of HPV-specific TCRs. By integrating high-throughput antigen screening with high-dimensional, multi-modal cellular characterization, our approach offers an unprecedented window into CD4+ T cell immunity across diverse disease contexts and empowers the development of new therapies.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":564708,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9488,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":838837,"name":"Qi Jin","orcid":"0000-0002-6536-621X","position":1,"is_corresponding":false},{"id":1409554,"name":"Michaela McKasson","orcid":null,"position":2,"is_corresponding":false},{"id":109700,"name":"Jongchan Choi","orcid":"0000-0003-0394-9978","position":3,"is_corresponding":false},{"id":1467969,"name":"Vanessa Gutiérrez","orcid":"0009-0008-4881-6195","position":4,"is_corresponding":false},{"id":1468371,"name":"Conor Brennan","orcid":null,"position":5,"is_corresponding":false},{"id":109717,"name":"Sunga Hong","orcid":"0000-0003-4412-2811","position":6,"is_corresponding":false},{"id":226349,"name":"William Chour","orcid":"0000-0003-1817-0123","position":7,"is_corresponding":false},{"id":632850,"name":"Raymond T. Ng","orcid":"0000-0003-3692-8524","position":8,"is_corresponding":false},{"id":109721,"name":"Jingyi Xie","orcid":"0000-0002-8959-4268","position":9,"is_corresponding":false},{"id":109698,"name":"Dan Yuan","orcid":"0000-0001-9651-8020","position":10,"is_corresponding":false},{"id":1409082,"name":"Andrew Webster","orcid":"0000-0003-3420-8689","position":11,"is_corresponding":false},{"id":1468372,"name":"Simranjeet K. Sidhu","orcid":null,"position":12,"is_corresponding":false},{"id":1148057,"name":"Abby Anderson","orcid":null,"position":13,"is_corresponding":false},{"id":109697,"name":"Daniel Chen","orcid":"0000-0001-6660-6257","position":14,"is_corresponding":false},{"id":109716,"name":"Rick Edmark","orcid":null,"position":15,"is_corresponding":false},{"id":109715,"name":"Kim M. 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Brown","orcid":"0000-0003-4915-8207","position":31,"is_corresponding":false},{"id":58599,"name":"Antoni Ribas","orcid":"0000-0003-3669-8458","position":32,"is_corresponding":false},{"id":1468375,"name":"Deborah Wong","orcid":null,"position":33,"is_corresponding":false},{"id":434374,"name":"Kelly G. Paulson","orcid":"0000-0002-0033-3708","position":34,"is_corresponding":false},{"id":490053,"name":"Charles W. Drescher","orcid":"0000-0002-6579-0416","position":35,"is_corresponding":false},{"id":226367,"name":"Cristina Puig-Saus","orcid":"0000-0002-3014-3649","position":36,"is_corresponding":false},{"id":109738,"name":"Jason D. Goldman","orcid":"0000-0002-3825-6832","position":37,"is_corresponding":false},{"id":457985,"name":"Cornelia L. Trimble","orcid":null,"position":38,"is_corresponding":false},{"id":6311,"name":"James R. 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