with anti-Bcl-2 antibody, we found that BimEL was coimmunoprecipitated from freshly purified CD8αα+ iIELs (data not shown) as well as from cells cultured in IL-15 for 40 h (Fig. 4D). The MEK inhibitor diminished IL-15-induced https://www.selleckchem.com/products/bay-57-1293.html BimEL phosphorylation, while inducing an increase of BimEL that coimmunoprecipitated with Bcl-2 (Fig. 4D). This result implies that the phosphorylation of BimEL by ERK1/2 prevents its association with Bcl-2. Taken together, these results demonstrate that Bcl-2 and Bim participated in the survival and death of CD8αα+ iIELs under the influence of IL-15. IL-15 modulated the balance between Bcl-2 and Bim via upregulation of Bcl-2 and reduction of the association between Bcl-2 and Bim. As the maintenance of CD8αα+ iIELs in the intestine requires IL-15Rα of IEC , we next investigated the Rapamycin ic50 role of Bcl-2, Mcl-1, and Bim in IL-15-mediated CD8αα+ iIEL survival in vivo by adoptive transfer of huBCL-2 tg, huMCl-1 tg,
double tg, or Bim−/− CD8αα+ iIELs into non-tg WT and Il15ra−/− recipient mice . The number of recovered donor cells was normalized to the number of input donor cells as a percentage of input cells for comparison among different recipients and different experiments. All types of donor cells showed a lower recovery in KO than in WT recipients, indicating the prosurvival effect of the IL-15 system in vivo (Fig. 5A). The recovery of huBCL-2 tg, double tg, and Bim−/− cells were better than that of non-tg cells in the iIEL compartment of WT cAMP and Il15ra−/− recipients (Fig. 5A) and in the spleen of Il15ra−/− recipients (Supporting Information Fig. 5). The result of WT recipients indicates a positive and a negative role for Bcl-2 and Bim, respectively, in CD8αα+ iIEL survival in the presence of IL-15. The result of KO recipients indicates that overexpression of Bcl-2 or removal of Bim from CD8αα+ iIEL enhanced cell survival in Il15ra−/− recipient, which implies that Bcl-2 and Bim are
downstream effectors in the IL-15-mediated survival pathway in vivo. This interpretation is supported by the in vitro signaling study (Fig. 2A, D and 3). As the intravenously transferred iIELs migrate from the blood system, including the spleen, to the intestine , the increased donor cell recovery in the iIEL compartment likely reflected a cumulative maintenance benefit in the spleen and intestine. Moreover, the composition of αβ and γδ subsets in all types of donor cells recovered from the iIEL compartment was similar to that before transfer (Fig. 5B). This result implies that the involvement of Bcl-2 and Bim in the IL-15-mediated survival in vivo was similar in the two iIEL subsets. It is noted that the percentage of the αβ subset increased in Bim−/− CD8αα+ iIELs, which is due to a sevenfold increase of αβ cell number in comparison with that in B6 mice (Supporting Information Fig. 4B).