Bailey to VG; Glenn Foundation for Medical Research and AI114800 to CJO; TR000118, Roger L. gene expression. ACEL-14-0945-s001.pdf (2.5M) GUID:?B5582BC9-393B-48D9-B2A4-79BF56EC866E Video S1 Activity of RAG2\/\ mice on eRapa at age 62?weeks (14.5?months). ACEL-14-0945-s002.3gp (21M) GUID:?E0700378-B265-42F2-A4AE-ADD644F9A8E2 Video S2 Activity of RAG2\/\ mice on Eudragit control at age 58?weeks (13.5?months). ACEL-14-0945-s003.3gp (21M) GUID:?E7E88501-5A0C-439A-9593-618003D1D205 Summary The mammalian (mechanistic) target of rapamycin (mTOR) regulates critical immune processes that remain incompletely defined. Interest in mTOR inhibitor drugs is heightened by recent demonstrations that the mTOR inhibitor rapamycin extends lifespan and healthspan in mice. Rapamycin or related analogues (rapalogues) also mitigate age\related debilities including increasing antigen\specific immunity, improving vaccine responses in elderly humans, and treating Flucytosine cancers and autoimmunity, suggesting important new clinical applications. Nonetheless, immune toxicity concerns for long\term mTOR inhibition, particularly immunosuppression, persist. Although mTOR is pivotal to fundamental, important immune pathways, little is reported on immune effects of mTOR inhibition in lifespan or healthspan extension, or with chronic mTOR inhibitor use. We comprehensively analyzed immune effects of rapamycin as used in lifespan extension studies. Gene expression profiling found many and novel changes in genes affecting differentiation, function, homeostasis, exhaustion, cell death, and inflammation in distinct T\ and B\lymphocyte and myeloid cell subpopulations. Immune functions relevant to aging and inflammation, and to cancer and infections, and innate lymphoid cell effects were validated and YXil7Rccr7studies of rapamycin\treated CD8+ T cells (Sinclair and other genes associated with T\cell exhaustion (e.g., (Tim\3); Table?S1). Reduced LAG3 in aged T cells was confirmed by flow cytometry (Fig.?2B). The (Ki\67) proliferation marker was strongly reduced Flucytosine by eRapa in all T\cell subsets. The acute activation marker was slightly reduced in PD1? T cells and unchanged in PD\1+ T cells (Table?S1). To test for the effect of rapamycin on functional T\cell exhaustion, Flucytosine we sorted PD\1+ and PD\1? T cells from mice fed eRapa or control diet for 6?months (five mice per group), stimulated them for 4?days with anti\CD3/CD28 antibodies, and assessed proliferation. PD\1? T cells from eRapa\ and control\fed mice proliferated equally well, whereas the PD\1+ T cells Rabbit Polyclonal to p44/42 MAPK from control mice proliferated poorly, consistent with exhaustion. This proliferative defect was partially reversed in PD\1+ T cells from eRapa mice (Fig.?2C) consistent with reversal of exhaustion. Open in a separate window Figure 2 eRapa reduces T\cell exhaustion markers. (A) PD\1+ cell prevalence in CD4+ and CD8+ splenic T cells from 24\ to 25\month\old male and female C57BL/6 mice on Eudragit (CTRL) or eRapa (RAPA) chow for 6?months (with anti\CD3/anti\CD28 antibodies for 2?days plus 5?ng/mL rapamycin (RAPA) or DMSO control (CTRL). MFI, mean fluorescence intensity. (D) eRapa\fed, young mice challenged with subcutaneous B16F10 melanoma cells have reduced PD\1+ CD4+ T cells in spleen and tumor\draining lymph nodes (DLN). (Th1), (Th2), (Th17), and (Th22) and increased (Th9) and (TFH) (Fig.?3A). Expressions of these transcription factors and associated cytokines were also modulated in all other T\cell populations analyzed. eRapa increased Th17 and Th22 signature genes in CD8+PD\1? cells (Fig.?3A) and affected chemokine receptor genes distinguishing Th subsets (e.g., decreased CXCR3 and CCR5 in all T\cell populations, and increased CCR6, CCR4, and CCR10 in CD4+PD\1+ T cells; Table?S2). Open in a separate window Figure 3 eRapa alters T helper (Th) pathway differentiation. (A) Log2 fold\changes (ratio of eRapa over CTRL normalized gene expression) in genes characteristic of Th/cytotoxic subsets in.