Kohlgruber et al. developed TCR-MAP – a system in which Jurkat cells transduced with TCRs of unknown specificity are screened against barcoded peptide libraries that are expressed, processed, and presented in live APCs on patient- or mouse-specific MHC-I and MHC-II alleles. Upon TCR/cognate antigen recognition, SrtA-CD40L gets expressed on the surface of Jurkat cells and covalently biotinylates CD40-G5 on the cognate APC. TCR-MAP accurately captured viral, cancer, and self antigens with high throughput and sensitivity, identifying both high- and low-affinity TCR antigens and cross-reactivities, without relying on freshly collected cells or killing of target cells.
Contributed by Lauren Hitchings
ABSTRACT: Antigen discovery technologies have largely focused on major histocompatibility complex (MHC) class I-restricted human T cell receptors (TCRs), leaving methods for MHC class II-restricted and mouse TCR reactivities relatively undeveloped. Here we present TCR mapping of antigenic peptides (TCR-MAP), an antigen discovery method that uses a synthetic TCR-stimulated circuit in immortalized T cells to activate sortase-mediated tagging of engineered antigen-presenting cells (APCs) expressing processed peptides on MHCs. Live, tagged APCs can be directly purified for deconvolution by sequencing, enabling TCRs with unknown specificity to be queried against barcoded peptide libraries in a pooled screening context. TCR-MAP accurately captures self-reactivities or viral reactivities with high throughput and sensitivity for both MHC class I-restricted and class II-restricted TCRs. We elucidate problematic cross-reactivities of clinical TCRs targeting the cancer/testis melanoma-associated antigen A3 and discover targets of myocarditis-inciting autoreactive T cells in mice. TCR-MAP has the potential to accelerate T cell antigen discovery efforts in the context of cancer, infectious disease and autoimmunity.
Author Info: (1) Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. Department of Genetics, Harvard University Medical School, Boston, MA, USA. Divisio
Author Info: (1) Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. Department of Genetics, Harvard University Medical School, Boston, MA, USA. Division of Immunology, Boston Children's Hospital, Boston, MA, USA. (2) Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. Department of Genetics, Harvard University Medical School, Boston, MA, USA. (3) Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. Department of Genetics, Harvard University Medical School, Boston, MA, USA. (4) Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. Department of Genetics, Harvard University Medical School, Boston, MA, USA. Department of Pathology, Massachusetts General Hospital, Boston, MA, USA. (5) Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. Department of Genetics, Harvard University Medical School, Boston, MA, USA. Society of Fellows, Harvard University, Cambridge, MA, USA. (6) Department of Genetics and Genomic Sciences and Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. (7) Institute for Cell Engineering, Division of Immunology, Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD, USA. (8) Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. selledge@genetics.med.harvard.edu. Department of Genetics, Harvard University Medical School, Boston, MA, USA. selledge@genetics.med.harvard.edu. Howard Hughes Medical Institute, Chevy Chase, MD, USA. selledge@genetics.med.harvard.edu.
