Generating stem-like memory T cells with antioxidants for adoptive cell transfer immunotherapy of cancer

Among the multiple factors that are responsible for the success of adoptive cell transfer (ACT) immunotherapy for cancer, the differentiation status of the in vitro expanded T cell product at the time of transfer seems to play a major role. In particular, less differentiated memory CD8⁺ T cells endowed with self-renewing capacity and multipotency exert the most potent antitumor activity. To this aim, expansion protocols that generate sufficient numbers of tumor-specific CD8⁺ T cells with superior capacity to persist in vivo following ACT are needed. We describe a procedure for the differentiation of TCF-1⁺ stem-like CD8⁺ memory T cells from peripheral blood naïve precursors that takes advantage of the use of antioxidants, in particular N-acetylcysteine (NAC), in combination with T cell receptor stimulation and proinflammatory cytokines. We additionally describe how to conduct in vitro assays to test the stem-like features of the generated cells at the phenotypic, functional and metabolic level. Balancing the oxidative metabolism by the addition of antioxidants during in vitro manipulation of CD8⁺ T cells results in the generation of cell products with potent antitumor characteristics following ACT.