In vitro ‐generated MART ‐1‐specific CD 8 T cells display a broader T‐cell receptor repertoire than ex vivo naïve and tumor‐infiltrating lymphocytes

The differentiation of human hematopoietic stem cells into CD 8 T cells can be achieved in vitro with the OP 9‐ DL 4 system. We considered that in the absence of medullary thymic epithelial cells, which serve to restrict the breath of the T‐cell receptor ( TCR ) repertoire by expressing tissue‐restricted antigens, a distinct repertoire would be generated in vitro . To test this notion, we compared the TCR ‐Vα/Vβ ( TRAV / TRBV ) gene usage of major histocompatibility complex‐restricted antigen ( MART ‐1)‐specific T cells generated in vitro to that from ex vivo naïve T cells and tumor‐infiltrating lymphocytes ( TIL s) using high‐throughput DNA sequencing. In contrast to naïve T cells and TIL s, which showed the expected narrow TRAV repertoire, in vitro ‐generated MART ‐1‐specific T cells used almost all TRAV gene families and displayed unique CDR 3 lengths. Our work demonstrates that the OP 9‐ DL 4 system supports the creation of a broad antigen‐specific TCR repertoire, suggesting that T cells generated in vitro may undergo a different set of selection events that otherwise constrains the TCR repertoire of thymus‐derived T cells.