PRR enhances anti-tumor immunity and suppresses colitis by promoting the development and survival of naive T and iNKT cells

Figures

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  Figure 1. T cell development and survival after positive selection are impaired in PRR-deficient mice. (A) Representative FCM plots of CD4 and CD8 expression in the thymocytes of control (CD4Cre(-) Atp6ap2+/Y) and CD4-Cre PRRcKO (CD4Cre(+) Atp6ap2flox/Y) mice. (B) Numbers of DP (CD4+CD8+) (n = 5), CD4 SP (CD4+CD8−TCRβ+), and CD8 SP (CD8+CD4−TCRβ+) (n = 3-4) thymocytes in control and CD4-Cre PRRcKO mice. (C) Representative FCM plots of CD69 and CCR7 expression in thymocytes of control and CD4-Cre PRRcKO mice. Thymocytes mature through stage 1 (CCR7−CD69−), 2 (CCR7−CD69+), 3 (CCR7lowCD69+), 4 (CCR7highCD69+), and 5 (CCR7highCD69−). (D) Number of thymocytes at maturation stages in (C) (n = 5). (E) Representative FCM plots of CD4 and CD25 expression in CD4+TCRβ+ thymocytes of control and CD4-Cre PRRcKO mice. (F) Number of CD25+CD4+TCRβ+ cells and frequency of Tregs relative to CD4 SP cells in the thymus of control and CD4-Cre PRRcKO mice (n = 3-4). (G) Representative FCM plots of CD1d-tetramer and TCRβ staining in thymocytes of control and CD4-Cre PRRcKO mice. (H) Number of iNKT (CD1d-tetramer+TCRβ+) cells in the thymus of control and CD4-Cre PRRcKO mice (n = 4-5). (I) Lethally irradiated WT…

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  Figure 2. The survival of T cell subsets is impaired in the periphery of PRR-deficient mice. (A) Numbers of CD4 T (CD4+TCRβ+) (n = 3-4) and CD8 T (CD8+TCRβ+) (n = 4-5) cells in the spleens of control and CD4-Cre PRRcKO mice. (B) Number of CD25+CD4+TCRβ+ cells and frequency of Tregs relative to CD4 SP cells in the thymus of control and CD4-Cre PRRcKO mice (n = 3-4). (C) Representative FCM plots of CD1d-tetramer and TCRβ expression in splenocytes of control and CD4-Cre PRRcKO mice. (D) Number of iNKT (CD1d-tetramer+TCRβ+) cells in the spleens of control and CD4-Cre PRRcKO mice (n = 4-5). (E) Representative FCM histogram and MFI of Bcl-2 in naïve (CD44low) CD4 T cells (n = 7-8) and naïve CD8 T cells (n = 4) in the spleens of control and CD4-Cre PRRcKO mice. (F) Isolated naive CD4 T cells from control and CD4-Cre PRRcKO mice were cultured in medium without stimulation for 20 (h) Viable cell data were expressed as a percentage of the total cell number (n = 4). (G) Number of YFP+ cells in CD4 T, Treg, and iNKT cells in the spleen of CD4-CreERT2 PRRcKO mice (CD4-CreERT2(+) Atp6ap2+/YRosa26YFP/+) treated with tamoxifen (n = 4-5). Data are the mean ± SD with Student’s t-test and pooled from at…

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  Figure 3. Mitochondrial metabolism is impaired in the T cells of PRR-deficient mice. (A) Representative FCM histogram and MFI of intracellular TCF1 staining in CD4 T and CD8 T cells in the spleen of control and CD4-Cre PRRcKO mice (n = 3). (B) Representative FCM histogram and MFI of LysoSensor staining of naive (CD44low) and effector (CD44highCD62L−) CD4 T cells in the spleens of control and CD4-Cre PRRcKO mice (n = 3). (C) Representative FCM histogram and MFI of MitoTracker staining of naive and effector CD4 T cells in the spleen of control and CD4-Cre PRRcKO mice (n = 3). (D) Representative FCM plots of JC-1 Red/Green expression and the Red/Green JC-1 ratio in naive and effector CD4 T cells in the spleen of control and CD4-Cre PRRcKO mice (n = 3). (E) Translation levels assessed by puromycin MFI in naive CD4 T cells of control and CD4-Cre PRRcKO mice were analyzed using the SCENITH method without 2-DG and oligomycin (n = 3). (F) Graphs showing the percentages of glucose dependence, mitochondrial dependence, glycolytic capacity, and fatty acid and amino acid oxidation (FAAO) capacity in naive CD4 T cells, calculated using the SCENITH method (n = 3). Data are the mean ± SD with Student’s…

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  Figure 4. Activated and cytokine-producing T cells are increased in PRR-deficient mice. (A) Representative FCM plots of CD44 and CD62L expression in CD4 and CD8 T cells in the spleens of control and CD4-Cre PRRcKO mice. (B) Numbers of naive (CD44low), effector (CD44highCD62L−), and central memory (CD44highCD62L+) populations in CD4 and CD8 T cells in the spleens of control and CD4-Cre PRRcKO mice (n = 5). (C) Representative FCM plots of IFN-γ and IL-17A expression in splenic CD4 T cells of control and CD4-Cre PRRcKO mice. (D) Frequency of IFN-γ+ and IL-17A+ CD4 T cells in the spleens of control and CD4-Cre PRRcKO mice (n = 3-4). (E) Number of IFN-γ+ and IL-17A+ CD4 T cells in the spleens of control and CD4-Cre PRRcKO mice (n = 3-4). (F) Frequency of IFN-γ+ (n = 9), IL-17A+ (n = 11), and Foxp3+ (n = 6) CD4 T cells among the siLP CD45+ cells of the control and CD4-Cre PRRcKO mice. (G) Number of IFN-γ+ (n = 9), IL-17A+ (n = 11), and Foxp3+ (n = 6) CD4 T cells among siLP CD45+ cells of control and CD4-Cre PRRcKO mice. (H) Isolated naive CD4 T cells from control and CD4-Cre PRRcKO mice were labeled with CPD eFluor 450 and stimulated with anti-CD3 and anti-CD28 antibodies in vitro for 3 days.…

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  Figure 5. Colitis is exacerbated in PRR-deficient mice via a reduction in iNKT cells. (A–C) Sorted naive CD4 T cells from control and CD4-Cre PRRcKO mice were transferred into RAG2-deficient mice (n = 4). Frequency and number of IFN-γ+, IL-17A+, and Foxp3+ populations in CD4 T cells, which were derived from control and CD4-Cre PRRcKO mice, of colon LP CD45+ cells in the host mice (A). Changes in the body weight of the host mice after naive T cell transfer (B). Data are expressed as the percentage of the basal body weight. Weight-to-length ratio of the colon (C). (D–I) iNKT cells were transferred or not into DSS-treated control and CD4-Cre PRRcKO mice. (D) Changes in the body weights of control and CD4-Cre PRRcKO mice after DSS treatment. Data are expressed as the percentage of basal body weight (n = 10). (E) Colon length of DSS-treated control and CD4-Cre PRRcKO mice (n = 10). (F, G) Frequency of IFN-γ+, IL-17A+(F), Foxp3+, and IL-10+(G) CD4 T cells in the colon LP CD45+ cells of control and CD4-Cre PRRcKO mice (n = 9). (G) Frequency of IFN-γ+ ILC3 cells (CD3−B220−RORγt+) in the colon LP CD45+ cells of control and CD4-Cre PRRcKO mice (n = 5). (I) Number of neutrophils (Ly-6G+CD11b+) and M1…

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  Figure 6. Anti-tumor immune responses are impaired in PRR-deficient mice. (A) Changes in B16F10 tumor volume after implantation into control and CD4-Cre PRRcKO mice (n = 5-7). (B) Representative FCM plots of CD1d-tetramer and TCRβ expression on tumor-infiltrating CD45+ cells from control and CD4-Cre PRRcKO mice. (C) Cell density of iNKT cells (cells/g) among tumor-infiltrating CD45+ cells from control and CD4-Cre PRRcKO mice (n = 5-7). (D) Density of CD8 T cells (cells/g) and frequency of IFN-γ+ populations in CD8 T cells from tumor tissue of control and CD4-Cre PRRcKO mice (n = 5-7). (E) Cell density of CD4 T cells (cells/g) and frequency of IFN-γ+ population in CD4 T cells from tumor tissue of control and CD4-Cre PRRcKO mice (n = 5-7). (F) Cell density of granulocyte-like MDSCs (G-MDSCs, Ly-6G+CD11b+), monocyte-like MDSCs (M-MDSCs, Ly-6G−CD11b+Ly-6C+F4/80−), and NK (NK1.1+CD3−) cells (cells/g) in CD45+ cells of tumor tissue from control and CD4-Cre PRRcKO mice (n = 5-7). (H-J) iNKT cells were transferred into B16F10-inoculated control and CD4-Cre PRRcKO mice. (H) B16F10 tumor volume after implantation into control and CD4-Cre PRRcKO mice (n = 5). (I) Frequency of IFN-γ+ populations in…