TetTCRII-SeqHD: High-dimensional analysis of antigen-specific ex vivo CD4+T cells

Abstract Objective: We present a method for high-throughput and inexpensive generation of DRB1 tetramers to assess CD4+ T cell specificity with special applications in oncoimmunology. Methods: Peptides were generated by in vitro transcription and translation (IVTT) of dsDNA. These peptides were then loaded onto DRB1 MHC molecules via chemical exchange and tetramerized using DNA barcoded streptavidin as previously described. As a proof of concept, we generated 250 DNA-tetramers bearing peptide segments from a variety of naïve and experienced antigens. These tetramers were pooled and used to stain primary CD4+ T cells from healthy donors. These cells were then labeled with Abseq antibodies (BD Biosciences), sorted and loaded onto the BD Rhapsody. Results: We analyzed ~5000 CD4+ T cells from healthy peripheral blood and assessed their phenotype, specificity and TCR sequence. We demonstrated that this technique robustly identifies and characterizes antigen-specific CD4+ T cells. It replicates previous findings in low-throughput systems in terms of clonality and phenotype distribution across memory and naïve T cell phenotypes. Given these results, we then analyzed tumor infiltrating lymphocytes and identified foreign specific cells within this compartment. We also found explicit differences in the phenotypes of antigen specific cells within the tumor and a paired healthy tissue sample. Conclusions: In conclusion, we validate the use of a high-throughput technology for assessing the specificity, phenotype, TCR, and gene expression patterns in antigen-specific CD4+ T cells in peripheral blood and tumor infiltrating lymphocytes.