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This study examines whether 6-thioguanine resistant T cells (mutant) from metastatic melanoma patients are enriched for melanoma-associated T cells compared to T cells obtained analogously without thioguanine selection (wild-type). Melanoma-associated antigen pentamer staining was performed on 5 tumour and 9 peripheral blood samples from metastatic melanoma patients. T cell receptor beta chain repertoire was examined via Sanger sequencing of mutant and wild-type in blood and tumour from metastatic melanoma patients at times of tumour progression (n = 8) and via Illumina sequencing in tumour derived T cells and in uncultured T cells (uncultured), wild-type and mutant from blood before and after immune checkpoint blockade (n = 1). Mutant from tumour (3 of 5; P &lt; 0.001), but not blood (0 of 9), were enriched compared to wild-type for binding melanoma-associated antigen pentamers. T cell receptor beta analysis in patients with tumour progression (n = 8) detected increased melanoma associated T cells in mutant compared to wild-type from blood (Monte Carlo P = 10). Comparison of blood samples before and after immune checkpoint blockade with prior tumor from one metastatic melanoma patient detected increased T cell receptor beta sharing between tumour and mutant compared to tumour and wild-type or tumour and uncultured: 11.0% (72/656), 1.5% (206/13 639) and 1.3% (381/29 807), respectively (Monte Carlo P = 10 for mutant versus wild-type and mutant versus uncultured). These data demonstrate that mutant in metastatic melanoma patients are enriched for melanoma-associated T cells and are candidate probes to study in vivo melanoma-reactive T cells.

Enrichment of melanoma-associated T cells in 6-thioguanine-resistant T cells from metastatic melanoma patients