(B) WT and TSC1DC-KO BMDCs were either NT or treated with 100 ng/ml of LPS overnight (LPS). and TSC1DC-KO pLNs (= 4) were analyzed by flow cytometry. (E) The percentages of cDCs (CD11c+MHC-II+, pregated as F4/80?CD64?) and cDC subsets (XCR1+ and SIRP+ cDCs) in kidneys of WT and TSC1DC-KO mice (= 6) were analyzed by circulation cytometry. The total cell figures were counted by a hemocytometer HLM006474 under a microscope. (F) The percentages of total T cells (CD3+) and T-cell subsets (CD8+ and CD4+ T cells) of pLNs from WT and TSC1DC-KO mice were analyzed by circulation cytometry. (G) The percentages of total T cells (CD3+) and T-cell subsets (CD8+ and CD4+ T cells) and B cells (CD19+B220+) of mLNs from WT and TSC1DC-KO mice were analyzed by circulation cytometry. (H) Na?ve and memoryCphenotype CD4+ T cells of WT and TSC1DC-KO spleens (= 4) were analyzed by circulation cytometry, and the percentages were calculated. The data are offered as means SEM (** 0.01; analyzed by Students test). These experiments were repeated at least once with similar results. Underlying data are available in S1 Data and S1 Uncooked Images. CCR7, chemokine (C-C motif) receptor 7; CD, cluster of differentiation; cDC, classical DC; DC, dendritic cell; Mac pc, macrophage; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; Mig DC, migratory DC; mLN, mesenteric lymph node; NK, natural killer cell; pLN, peripheral lymph node; SIRP, transmission regulatory protein ; SSC, part scatter; TCM, central memory space T cell; TEM, effector memory space T cell; TN, na?ve T cell; Tsc1, tuberous sclerosis complex subunit 1; TSC1DC-KO, specific ablation of in the DC compartment; WT, wild-type; XCR1, chemokine (C motif) receptor 1.(TIF) pbio.3000420.s001.TIF (1.4M) GUID:?B836D17B-F6B3-487A-AA91-CD794EA660FD S2 Fig: TSC1-mTORC1 in DCs, rather than macrophages, affects CD8 T-cell homeostasis. (A and B) The percentages of total T cells (CD3+) and T-cell subsets (CD8+ and CD4+ T cells) of spleens and pLNs from WT, TSC1DC-KO, and TSC1/mTORDC-DKO mice (A) or TSC1/RaptorDC-DKO mice (B) were analyzed by circulation cytometry. (C) Spleens from WT and TSC1DC-KO mice (= 6) were isolated and immediately stained with annexin V and PI; after cell surface marker staining, early apoptotic CD4+ T cells (annexin V+PI?) were calculated. The data are offered as means SEM (*** 0.001, analyzed by College students test). (D) The percentages of total T cells (CD3+) and T-cell subsets (CD8+ and CD4+ T cells) and B cells (CD19+B220+) of spleens, pLNs, and mLNs and percentages of different T-cell populations in thymuses from WT and TSC1M/N-KO mice were analyzed by circulation cytometry. The data are offered as means SEM. These experiments were repeated at least once, HLM006474 and similar results were obtained. Underlying data are available in S1 Data. CD, cluster of differentiation; DC, dendritic cell; DN, double negative; DP, double positive; mLN, mesenteric lymph node; mTor, mechanistic target of rapamycin; mTORC1, mTOR complex 1; PI, propidium iodide; pLN, peripheral lymph node; Rptor, regulatory connected protein of MTORc1; SP, solitary positive; SSC, part scatter; Tsc1, tuberous sclerosis complex subunit 1; TSC1DC-KO, specific ablation of in the DC compartment; WT, wild-type.(TIF) pbio.3000420.s002.TIF (1.6M) GUID:?3799D93E-6879-4AAA-BA10-D6B67F41051E S3 Fig: mTOR ablation restores CD8 T-cell responses in TSC1DC-KO mice. (A) The 6C8-week-old WT and TSC1DC-KO littermates (= 4) were i.v. infected with 104 CFU of L.M-OVA. After 7 days, the spleens were isolated, and KLRG1+ and CD44+ CD8+ T cells were analyzed by circulation cytometry, and the percentages of different type of cells among CD8+ T cells and cell figures were determined; the data are offered as means SEM (** 0.01, *** 0.001; analyzed by Students test). (B) In total, Pik3r1 5 106 splenocytes from infected mice were restimulated with 10 ng/ml OVA257-264 for 5 hours in the presence of brefeldin A. The percentages of IFN- and TNF-producing CD8+ T cells were analyzed by intracellular staining adopted with circulation cytometry. The data are offered as means SEM. These experiments were conducted three times with similar results. (C) The 6C8-week-old WT, TSC1DC-KO, and TSC1/mTORDC-DKO mice (= 4) were infected with L.M.-OVA as with (A). After 7 days, the spleens were isolated and 5 106 splenocytes from infected mice were restimulated with 10 ng/ml OVA257-264 for 5 hours in the presence of brefeldin A. The percentages of IFN-producing CD8+ T cells HLM006474 were analyzed by intracellular staining followed by circulation cytometry, and cell figures were calculated accordingly (left panel). The percentages and numbers of the OVA-specific CD8+ T cells were also HLM006474 analyzed by circulation cytometry (right panel). The data are offered as means SEM (* 0.05, ** 0.01, *** 0.001; analyzed by Students test). This experiment was performed twice with HLM006474 related results. (D) WT, TSC1DC-KO, and TSC1/mTORDC-DKO.
(B) WT and TSC1DC-KO BMDCs were either NT or treated with 100 ng/ml of LPS overnight (LPS)