ABSTRACT
Although critical to T cell function, antigen specificity is often omitted in high-throughput multiomics-based T cell profiling due to technical challenges. We describe a high-dimensional, tetramer-associated T cell antigen receptor (TCR) sequencing (TetTCR-SeqHD) method to simultaneously profile cognate antigen specificities, TCR sequences, targeted gene expression and surface-protein expression from tens of thousands of single cells. Using human polyclonal CD8+ T cells with known antigen specificity and TCR sequences, we demonstrate over 98% precision for detecting the correct antigen specificity. We also evaluate gene expression and phenotypic differences among antigen-specific CD8+ T cells and characterize phenotype signatures of influenza- and Epstein-Barr virus-specific CD8+ T cells that are unique to their pathogen targets. Moreover, with the high-throughput capacity of profiling hundreds of antigens simultaneously, we apply TetTCR-SeqHD to identify antigens that preferentially enrich cognate CD8+ T cells in patients with type 1 diabetes compared to healthy controls and discover a TCR that cross-reacts with diabetes-related and microbiome antigens. TetTCR-SeqHD is a powerful approach for profiling T cell responses in humans and mice.
Subject(s)
Antigens/immunology , CD8-Positive T-Lymphocytes/immunology , High-Throughput Nucleotide Sequencing , Receptors, Antigen, T-Cell/genetics , Single-Cell Analysis , Antigens/metabolism , Antigens, Viral/immunology , Antigens, Viral/metabolism , Autoantigens/immunology , Autoantigens/metabolism , Autoimmunity , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/virology , Case-Control Studies , Cell Separation , Cells, Cultured , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/metabolism , Herpesvirus 4, Human/immunology , Herpesvirus 4, Human/pathogenicity , Humans , Orthomyxoviridae/immunology , Orthomyxoviridae/pathogenicity , Phenotype , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolismABSTRACT
Betaherpesvirus dUTPase homologs are core herpesvirus proteins, but little is known about their role during infection. Human cytomegalovirus (HCMV) UL72 and murine cytomegalovirus (MCMV) M72 have been designated dUTPase homologs, and previous studies indicate UL72 is dispensable for replication and enzymatically inactive. Here, we report the initial characterization of MCMV M72. M72 does not possess dUTPase activity, and is expressed as a leaky-late gene product with multiple protein isoforms. Importantly, M72 augments MCMV replication in vitro and during the early stage of acute infection in vivo. We identify and confirm interaction of M72 with the eukaryotic chaperonin tailless complex protein -1 (TCP-1) ring complex (TRiC) or chaperonin containing tailless complex polypeptide 1 (CCT). Accumulating biochemical evidence indicates M72 forms homo-oligomers and is a substrate of TRiC/CCT. Taken together, we provide the first evidence of M72's contribution to viral pathogenesis, and identify a novel interaction with the TRiC/CCT complex.
