Single-cell RNA-seq of human CD4+ and CD8+ T cells transduced with a TAA-specific low-avidity TCR and the CD8αβ co-receptor

Most naturally occurring major histocompatibility class I restricted T cell receptors (TCRs) that target over-expressed tumor-associated self-antigens (TAAs) are of low avidity due to selection and tolerance in the host and are CD8 co-receptor dependent. Adoptive T cell transfer with TCR-engineered T cells thus rely on the function of CD8+ T cells and do not exploit beneficial CD4+ T cell functions. Hence, we developed a novel strategy that combines expression of a TAA-specific low-avidity TCR with the CD8ab co-receptor and explored the properties of purified transgenic CD8+ and CD4+ T cells separately, in vitro and in vivo. We found that CD8ab co-transfer enhanced TCR+ CD8+ T cell function by increasing their serial tumor killing capacity, indicating that limited availability of endogenous CD8 co-receptors impedes full deployment of their functional potential. Engineered CD4+ T cells were efficiently reprogrammed into hybrid multifunctional cytotoxic and helper T cells at the single-cell level: they recognized and killed cells expressing the cognate class I restricted tumor antigen, became serial killers, produced mostly TH1 and preserved some TH2 cytokines, and showed superior anti-tumor function in vivo in a leukemia xenograft model. CD8ab co-transfer restored the TCR-pMHC interaction in CD4+ T cells and enhanced early TCR signaling events. Single-cell RNA-seq profiling suggests that co-transferred CD4+ T cells acquired a cytotoxic gene expression program and at the same time retained CD4 lineage specific features. In conclusion, we present a novel approach that allows us to (1) enhance the function of TCR-transgenic CD8+ T cells and (2) to manufacture class I pMHC targeted hybrid cytotoxic and helper T cells with both CD8+ and CD4+ T cell functions readily available at the single-cell level. 9 samples were analyzed from three different timepoints of the experiment: freshly isolated (frozen on Day 0 and thawed on Day 21), expanded (Day 14), and cocultured (Day 21). Single-cell isolation (10XGenomics) and library preparation (10XGenomics) were performed at two timepoints (Day 14 and Day 21). Two samples per lane were loaded for samples with 3000 single cells, and 1 sample per lane was loaded for samples with 5000 single cells. The sequencing was performed in a paired-end PE100-100 configuration. No reads data provided due to human data policies