However, RCDII IELs lack CD8 and surface CD3-TCR complex [21-24], and whether ACD IELs express CD8αα was not indicated [21]. Freshly isolated RCDII and ACD IELs express higher Bcl-XL but lower Bcl-2 compared with IELs from healthy donors [21]. Therefore, these IEL lines likely do not resemble normal primary CD8αα+ IELs, and the IL-15-mediated

survival signals in normal CD8αα+ iIELs remain elusive. Here, we delineated the IL-15-induced survival signals in primary murine CD8αα+ iIELs in vitro, and confirmed their role in vivo. IL-15 supports CD8αα+ iIEL survival through the activation of the Jak3-Jak1-PI3K-Akt-ERK pathway to upregulate Bcl-2 and Mcl-1. Furthermore, this signaling axis does not affect the level of Bim, but promotes the dissociation of Bim from the Bim-Bcl-2 complex and maintains the dissociated Bim in a phosphorylated state. These results ERK inhibitor suggest a new mechanism by which IL-15 ABT-888 cell line modulates the members of the Bcl-2 family to support cell survival. We previously found that IL-15Rα supports the survival of CD8αα+ iIELs in vivo, and that exogenous IL-15 maintains live CD8αα+ iIELs

in vitro in an IL-15Rβ-dependent manner [2]. To dissect the IL-15-mediated survival signals using the in vitro system, we cultured CD8αα+ iIELs in 50 ng/mL of IL-15, as this amount of IL-15 stably maintained the percentage of live cells up to 64 h (Fig. 1A, top panels). Although 50 ng/mL of IL-15 induces proliferation of murine NK cells in vitro [25], it had little mitogenic effect on CD8αα+ iIELs as few PFKL cell in G2/S/M phase appeared by 64 h of culturing in IL-15 (Fig. 1A, lower panels). On the other hand, 50 ng/mL of IL-15 supported cell survival as shown by the relatively low percentage of cells in sub-G1 phase (Fig. 1A, lower panels). We investigated IL-15-triggered survival signals in CD8αα+ iIELs in vitro first by using inhibitors. Cells were treated with individual inhibitor for 1 h before the addition of IL-15. The inhibitor treatment did not alter the level of IL-15Rβγ on CD8αα+ αβ and γδ iIELs (Supporting Information Fig. 1A and B). Inhibitors of Jak3, PI3K (LY294002), protein kinase B/Akt (Akt) (Akt IV) and MEK (U0126) abolished IL-15′s

prosurvival, whereas inhibitors of p38 mitogen-activated protein kinase (SB203580) and mammalian target of rapamycin inhibitor (rapamycin) had no effect (Fig. 1B, line graphs). The effective inhibitors diminished IL-15′s prosurvival effect in a dose-dependent manner (Supporting Information Fig. 1C). As the αβ and γδ cell composition of CD8αα+ iIELs remained the same before and after culturing in medium alone, in IL-15, or in IL-15 plus each inhibitor (Fig. 1B, bar graphs), the IL-15-triggered survival signals are similar in the two subsets at the level of Jak3, PI3K, and ERK1/2 activation. Consistent with the inhibitors’ effects on CD8αα+ iIEL survival (Fig. 1B), IL-15 induced phosphorylation of Jak1, Akt, and ERK1/2 (Fig. 1C) with delayed kinetics for ERK1/2 phosphorylation.