Rongyu Zhang, Jingqi Qi, Michaela McKasson, Jongchan Choi, Vanessa Gutierrez, Conor Brennan, Sunga Hong, William Chour, Rachel H. Ng, Jingyi Xie, Dan Yuan, Andrew Webster, Simranjeet Sidhu, Abby Anderson, Daniel Chen, Rick Edmark, Kim M. Murray, Sarah Li, Connor McDonald, Lee Rowen, Shuo Wang, Yusuf Rasheed, Yapeng Su, Jamie R. Wagner, Jia Ming Chen, Karla Nawaly, Jie Fu, Alexandria Duven, Stephen J. Forman, Mihae Song, Saul J. Priceman, Christine E. Brown, Antoni Ribas, Deborah J. Wong, Kelly G. Paulson, Charles W. Drescher, Cristina Puig-Saus, Jason D. Goldman, Cornelia L. Trimble, James R. Heath
Systematic whole-protein screening and comprehensive profiling of antigen-specific CD4+ T cells are crucial for advancing vaccine design and cancer immunotherapies, yet remain technically challenging. Here, we present a high-throughput platform that utilizes 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. Upon rigorous platform validation, we screened the full SARS-CoV-2 spike receptor binding domain in a longitudinal cohort of 22 participants, identifying 2,188 antigen-specific CD4+ T cells and showing key metrics defining the immunogenicity of class II-restricted viral antigens. We further extended the platform to whole-protein screening of HPV-16 E6/E7 in a cohort of precancerous patients, indicating HPV-specific CD4 TCRs that, upon extensive characterization, demonstrate strong therapeutic potential. By integrating high-throughput antigen screening with high-dimensional, multi-modal cellular characterization, our approach provides detailed insight into CD4+ T cell immunity, potentially guiding vaccine design and next-generation TCR-based cancer immunotherapies. Rapid methods to identify antigen-specific T cells are essential for developing targeted immunotherapies. Here the authors present a high-throughput MHC class II single-chain trimer platform for the comprehensive profiling of CD4+ T cells, enabling the rapid identification and characterization of virus- and tumour-specific T cell receptors (TCR) at single-cell resolution.