We demonstrate TScan-II's potential for basic and translational research by identifying a non-canonical antigen for a cancer-reactive CD4 + T cell clone. Leveraging genome-scale human, virome, and epitope mutagenesis libraries, TScan-II facilitates de novo antigen discovery and deep exploration of TCR specificity. This platform seamlessly integrates the endogenous HLA-II antigen-processing machinery in synthetic antigen-presenting cells and TCR signaling in T cells, enabling the simultaneous screening of multiple HLAs and TCRs. Here, we introduce TScan-II, a highly sensitive genome-scale CD4 + antigen discovery platform. However, our inability to associate peptide human leukocyte antigen class-II (HLA-II) complexes with their cognate T cell receptors (TCRs) in an unbiased manner has hampered our understanding of CD4 + T cell function and role in pathologies. Electronic address: onĬD4 + T cells play fundamental roles in orchestrating immune responses and tissue homeostasis. 12 Division of Genetics, Department of Medicine, Howard Hughes Medical Institute, Brigham and Women's Hospital, Boston, MA, USA Department of Genetics, Harvard Medical School, Boston, MA, USA.11 Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.10 Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Departmentment of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.9 Genes and Human Disease Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.8 Genes and Human Disease Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.7 Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Departmentment of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.6 Salivary Disorders Unit, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA.5 Genes and Human Disease Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.4 Department of Medicine, Harvard Medical School, Boston, MA, USA.3 Department of Genetics, Harvard Medical School, Boston, MA, USA Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, USA Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA.2 Adeno-Associated Virus Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA.1 Division of Genetics, Department of Medicine, Howard Hughes Medical Institute, Brigham and Women's Hospital, Boston, MA, USA Department of Genetics, Harvard Medical School, Boston, MA, USA.

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