Fig 2: Thymic CCR7+ iNKT and MAIT cells are at an early developmental stage and give rise to distinct effector subsets in the thymus.(A) Expression of Rag2GFP and CCR7 in thymic iNKT cells (TCR-ß+ CD1d-PBS57+) (left column), and of CD24 (middle column) and CD69 (right column) on cells with different levels of Rag2GFP. Data are representative of 3 independent experiments with 3–4 mice in each. (B) Expression of NK1.1 and CD44 in thymic CCR7+ iNKT (black dots), IL-4+ (human CD2+) NKT2 (red dots), ROR-?t+ NKT17 (blue dots), T-bet+ NKT1 (green dots) cells together with total thymic iNKT cells (grey dots). Numbers in quadrants indicate percent cells in each for CCR7+ iNKT (black dots), IL-4+ (human CD2+) NKT2 (red dots), ROR-?t+ NKT17 (blue dots) and T-bet+ NKT1 (green dots) cells. Data are representative of 4 independent experiments with 2–3 mice in each. (C) Expression of T-bet, ROR-?t, huCD2, PLZF, LEF1, Egr2 and CD4 with CCR7 in thymic iNKT cells (TCR-ß+ CD1d-PBS57+ CD24–). Data are representative of 3 independent experiments with 2–3 mice in each. Numbers in quadrants indicate percent cells in each (throughout). (D) Frequency of Ki67+ cells in each population of thymic iNKT cells. Data are pooled from three independent experiments with 2–3 mice in each. ****p<0.0001 (one-way ANOVA, Tukey’s multiple comparisons test) Each symbol represents an individual mouse; small horizontal lines indicate the mean. (E) Expression of Tbx21GFP, ROR-?t and human CD2 in CCR7+ iNKT cells sorted from BALB/c Tbx21GFP KN2 mice before intra-thymic transfer (left column) or 5 days after transfer in the thymus of congenic BALB/c recipient mice (right column). (F) Frequency of Tbx21GFP+, ROR-?t+ or human CD2+ cells in donor cells before or 5 days after intra-thymic transfer into the thymus of congenic BALB/c recipient mice. Each symbol represents an individual recipient mouse; small horizontal lines indicate the mean. (G) SPADE analysis of thymic iNKT cells from B6 KN2 mice supports that CCR7+ NKTp are a distinct lineage from the effector subsets, NKT1, NKT2 and NKT17. Representative figure shows differential expression of human CD2 in each population of iNKT cells. (H) CCR7 and PD-1 distinguish two cell populations (top row, right column) within PLZFhi iNKT cells (top row, left column), and expression of human CD2 and CD44 in CCR7+ NKTp (grey), NKT1 (green), NKT2 (red) and NKT17 (blue) are shown as overlays (bottom row). Data are representative of 3 independent experiments with 3 mice in each. (I) Expression of Rag2GFP together with CCR7 (middle column) in thymic MAIT cells (far left column), and expression of CD24 and CD69 in CCR7+ and CCR7– MAIT cells with different level of Rag2GFP (far right two columns). Data are representative of 2 independent experiments with 3 mice in each.10.7554/eLife.34793.006Figure 1—source data 1.High proliferative and precursor potential of CCR7+ iNKT cells.10.7554/eLife.34793.007Figure 1—source data 2.Thymic CCR7+ iNKT cells are distinguished from stage 0 iNKT cells and give rise to iNKT subsets in periphery.10.7554/eLife.34793.008Figure 1—source data 3.Consistent, robust and unbiased labeling of thymocytes by intra-thymic injection of biotin.

Fig 3: PG enhances antitumor immunity via reducing PD-1 on the surface of CD8+ T. (A) The heat map of Pearson correlation coefficients (PCCs) evaluated by the four methods between the gene expression level of PG targets and immune phenotypes; Benjamini–Hochberg (BH) adjusted p-values of PCCs <0.05 are shown in white. (B) LLC tumor infiltrating CD8+ T cells in PG-treated versus WT mice analyzed by flow cytometry. Flow cytometry analysis of single-cell suspensions obtained from LLC tumors and stained for detecting CD8+ T cells (CD3+CD8+). Representative dot plots with indicated percentages of CD8+ T cells populations within CD45+ cells in LLC tumors from the WT and PG-treated groups, respectively. (C) Protein level of IFN-? and granzyme B in WT and PG-treated groups by means of western blotting. (D) Transcription levels of IFN-? and granzyme B in WT and PG-treated groups by means of RT-PCR. (E) Protein level of PD-1 in WT and PG-treated groups. (F) Flow cytometry analysis of single-cell suspensions obtained from LLC tumors and stained for detecting PD-1+ CD8+ T cells. Representative dot plots with indicated percentages of PD-1+ CD8+ T-cell populations within CD8+ T cells in LLC tumors from WT and PG-treated groups, respectively. All plots of flow cytometry analysis were gated on total live cells. Data are from distinct samples and presented as the mean ± SEM. **p < 0.01, ***p < 0.001 compared with WT by unpaired t-test; n > = 3.

Fig 4: T cell exhaustion in the KPC tumor microenvironment. (a) Percentage of CD8+ CD45+ T cells in live cells in tumor and spleen. (b) Percentage and (c) flow cytometry gating of PD-1+ T cells in CD8+ T cell populations in tumor and spleen. Statistical analysis of %CD8+ CD45+ T cells in live cells (a) and %PD-1+ cells in T cells (b) between indicated groups was performed by Student T test, *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001. Mean ± SD for n = 4 mice per group.

Fig 5: Srsf1-deficient Treg cells exhibit impaired phenotype and suppressive function(A) Spleen cells were isolated from WT and T cell Srsf1-KO mice, and expression levels of Treg-associated molecules in CD4+CD25+Foxp3+ Treg cells were analyzed by flow cytometry. Dot plots (left) and histograms (right) show representative data of frequencies and mean fluorescence intensity (MFI) of markers in gated Treg cells.(B) Graphs show the percentage of CTLA4+, PD1+, and CD39+ cells gated on Treg cells and quantification of MFI of CD103, ICOS, GITR, LAG3, and Helios (n = 4–10 each).(C) Spleen cells from WT or T cell Srsf1-KO mice were isolated and conventional CD4 T (Tconv) cells and Treg cells (CD4+CD25+CD127lo) were sorted by flow cytometry. Tconv cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and co-cultured with Treg cells at increasing ratios for 7 days, and proliferation of Tconv cells was analyzed by flow cytometry. Representative plots are shown.(D) Graph shows proliferation of Tconv cells (n = 8 WT, n = 10 KO).(E) B6 mice were given 2.5% dextran sodium sulfate (DSS) in water to induce colitis for 8–10 days. One day before the initiation of DSS administration, PBS or flow-cytometry-sorted Treg cells (CD4+CD25+CD127lo) from WT or T cell Srsf1-KO mice were injected into B6 mice. Graph shows body weight of mice from five independent experiments (n = 6 each).(F) Representative image of colon after DSS administration for 8–10 days. Graph shows colon length of WT Treg and KO Treg group (n = 6 WT, n = 5 KO).(G) Naive CD4 T cells were adoptive transferred into Rag1-/- mice to induce colitis together with PBS or sorted Treg cells (CD4+CD25+CD127lo) from WT or T cell Srsf1-KO mice. Graph shows time course of body weight of Rag1-/- recipient mice (n = 4–5 each).*p < 0.05; **p < 0.005; and n.s., no significant difference, two-way ANOVA test (D, E, and G) and unpaired t test (B and F); mean ± SEM.