Modulating CD8+ T Cell Responses

Purified CD8⁺ T cells were stimulated with recombinant human IL-7 (10 ng/mL; R&D Systems, Minneapolis, Minnesota, USA) [22 (link)] for 48 h in the presence of anti-CD3/CD28, along with either JAK inhibitor (10 µmol/L; Sigma-Aldrich, Temecula, California, USA), STAT5 inhibitor (250 µmol/L; Merck Millipore), or PI3K inhibitor (LY294002) (25 µmol/L; Sigma-Aldrich) as previously described [20 (link)]. Control cells were only stimulated with anti-CD3/CD28 for maintenance of CD8⁺ T cell survival. In certain experiments, stimulated CD8⁺ T cells were washed twice. 10⁴ of CD8⁺ T cells were co-cultured with 10⁵ of melanoma cell line SK-MEL-5 cells for 48 h.