CD8 T cell phenotype in IL-15 TG/MT, IL-15 KO/MT and MT tumors. (A) CD8 T cell immunohistochemistry on representative IL-15 KO/MT, MT and IL-15 TG/MT tumors. Levels of CD8 T cells were highest in IL-15 TG/MT tumors and lowest in IL-15 KO/MT mice (brown staining = CD8+, 20X) (B) Quantitation of CD8 T cells in tumors (n = 5 mice/group) (C) Quantitation of CD4 T cells in tumors assessed by immunohistochemistry (n = 5 mice/group) (D) Tumors from MT, IL-15 KO/MT and IL-15 TG/MT mice were digested and stained for flow cytometry. The CD8+/CD4+ T cell ratio is altered drastically in the presence or absence of IL-15 (n = 5 mice/group). (E) CD3 + CD8+ cells from tumors of IL-15 TG/MT mice have a higher percentage of CD62L + CD44+ central memory T cells (Representative of n = 3/group) (F/G) CD8 T cells were isolated by positive selection from tumors of mice and stimulated non-specifically. (F) After 48 hours, supernatant IFNγ/TNFα levels were highest in CD8 T cells from IL-15 TG/MT mice (n = 2/group) (Representative of 3 experiments). (G) In another set of experiments, after 12 hours of stimulation, Golgi Stop was added to determine via flow cytometry which cells were capable of producing IFNγ. A high proportion of IL-15 TG/MT CD8 + CD3+ cells produce IFNγ in comparison to MT CD8 T cells (n = 3/group). (H) Fewer CD8 + CD3+ T cells in IL-15 TG/MT mice express the exhaustion marker PD-1 (n = 3/group). *p < 0.05, p < 0.01.