Along this line, it was interesting that inflammatory Th17 differentiation was intact, if not enhanced, in the absence of γc which, however, can be explained by the negative effect of IL-2 signaling on IL-17 expression. Of note, because Pim1TgγcKO mice lack FoxP3+ Treg cells and since Pim1TgγcKO CD4+ T cells could be induced to differentiate into inflammatory T cells, it was surprising that we did not find any signs of autoimmunity in Pim1TgγcKO mice. The in vivo immune response of these mice is currently under
investigation. Collectively, the present study establishes prosurvival effects as the only factor downstream PI3K Inhibitor Library chemical structure of γc signaling that is required for CD4+ T-cell development. Such characteristics set these cells apart from other T-lineage cells that presumably also require lineage specification signals downstream of γc signaling. We expect that further functional studies of γc-deficient CD4+ T cells, together with genetic reconstitution of other select γc downstream
pathways, such as constitutively active Akt or STAT5, will help decipher the detailed molecular pathways in T-lineage cell development and maintenance. CD45.1+ or CD45.2+ C57BL/6 and γc-deficient mice were obtained from the Jackson Laboratory. Human Bcl-2 transgenic mice were provided by Dr. Alfred Singer (National Cancer Institute, Bethesda, MD, USA) . Pim1 transgenic mice have been described , and were provided by Dr. Anton Berns (The Netherlands Cancer Institute, Amsterdam, The Netherlands). Animal experiments phosphatase inhibitor library were approved by the National Cancer Institute Animal Care and Use Committee, and all mice were cared for in accordance with National Institutes of Health guidelines. Cells were stained and analyzed on LSRII, ARIAII, or FACSCalibur flow cytometers (Becton Dickinson). Dead cells were excluded by forward
light scatter gating and propidium iodide staining. Antibodies with the following specificities were used for staining: CD8β, CD44, HSA, IL-7Rα, FoxP3, Ki-67 (eBioscience); CD4, CD8α, TCR-β, CD103, γc, human CD3, IL-4, IL-17 (Becton Dickinson); γδ TCR, IFN-γ (Biolegend). For intracellular cytokine staining, in vitro differentiated cells were restimulated for 3 h with PMA and ionomycin with the addition of brefeldin A (eBioscience). Cells N-acetylglucosamine-1-phosphate transferase were fixed and permeabilized with IC fixation buffer (eBioscience). For nuclear FoxP3 staining, cells were first surface stained and then fixed and permeabilized using FoxP3 intracellular staining buffer set according to the manufacturer’s instructions (eBioscience). Active caspase-3 was assayed using a CaspGLOW active caspase-3 kit following the manufacturer’s instructions (eBioscience). Intestines were harvested and washed using 2% FBS in HBSS. After slicing into smaller pieces, intestines were washed using 2% FBS in HBSS and stirred for 20 min at 37°C in 10% FBS in HBSS with 1 mM DTT.