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A strategy for uncovering germline variants altering anti-tumour CD8 T cell response

Ulaganathan, VK;Vasileva, M;

Among many factors known to alter the outcomes of T cell receptor (TCR)-induced proximal signalling, the role played by human germline variants in dictating the individuality of the anti-tumour CD8 T cell response has remained challenging to address. Here, we describe a convenient strategy for molecular and functional characterization of phosphotyrosine-altering non-synonymous single nucleotide variations (pTyr-SNVs) that directly impact TCR-induced proximal phosphotyrosine motif-based signalling pathways. We devise an experimental co-cultivation set-up comprising a C57BL/6 mouse-derived metastatic melanoma cell line engineered to constitutively present ovalbumin (OVA) antigens and retrovirally engineer syngeneic MHC Class I restricted OVA TCR-transgenic CD8 T cells (OT-I). Using the synthetic version of pTyr-SNV rs1178800678-G/T encoding ITGA4 p.S1027I variant as a prototype, which generates a membrane-proximal immunoreceptor tyrosine activation motif (ITAM), we show that under identical TCR stimulation conditions, genetically determined membrane-proximal ITAM elevates tyrosine phosphorylation of proximal signalling molecule 70 kDa zeta-chain associated protein (ZAP70) and the levels of cytotoxic effector molecule granzyme B (GZMB), thereby contributing to enhanced cytotoxic activity against metastatic melanoma cell line. This strategy paves the way for rapid molecular and functional characterization of anti-tumor immune response-linked germline nsSNVs and uncover the genetic determinants of individual-to-individual differences in anti-tumour CD8 T cell response.