Single cell RNA seq profiling of (auto-)antigen-specific CD8+ T cells in response to their target antigen

CD8⁺ T cells are key player of the adaptive immune response. Improving our understanding of antigen-specific T cell responses on the single cell level will be crucial for the development of highly effective future vaccines and a necessity to resolve or minimize errant immunity in autoimmune diseases and after transplantation. Currently, the profiling of peptide-specific CD8⁺ T cells is compromised by a low efficiency of multimer-based isolation following peptide stimulation.

We established an assay that circumvents the isolation of antigen-responsive CD8⁺ T cells via MHC class I multimers and applied it to the analysis of CD8⁺ T cells responding to their cognate viral antigen (Influenza M1_58-66, CMV pp65_495-503), beta cell autoantigen (IGRP_265-273) or control stimuli. Targeted single cell gene expression analysis and single cell RNA sequencing on more than 600 isolated cells was conducted to identify genes differentially expressed in response to the cognate antigen.

We identified sets of transcripts that clearly mark the antiviral response on a single cell level, across donors and that largely overlap for cells stimulated via PBMC or cell line-based antigen presentation. The approach also identified paired TCR alpha and beta chain sequences with the expected TCR gene usage for flu and CMV responses. Fewer differentially expressed genes were identified for IGRP-responsive cells and these differed from those seen in antiviral response. Again, paired TCR alpha and beta chain sequences could be obtained from the IGRP-responsive cells.

A valid method for the isolation of peptide responsive CD8⁺ T cells was developed. Cells isolated using this method provide detailed information on the response profile and TCR, and suggest that circulating beta cell antigen peptide-stimulated CD8⁺ T cells have a different gene expression profile than viral peptide-responsive CD8⁺ T cells.