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: HFD-fed IL22ra1-/-female mice, but not males, develop an increase in the absolute number of adaptive immune cells in their livers and spleen compared with their WT littermates at 30 weeks. IHLs and splenocytes were extracted from fatty livers and spleen of IL22ra1-/- and WT female or male mice, respectively, and analyzed by flow cytometry. (A) Representative FACS plot showing an outline for the gating strategy of B cells (CD45+ CD19+ CD3-), T cells (CD45+ CD3+ CD19-), NK cells (CD45+ CD19- CD3- NK1.1+ NKp46-), CD4+ T cells (CD3+ CD19- CD4+), CD8+ T cells (CD3+ CD19- CD8+), T cell receptor?d T cells (CD3+ CD19- T cell receptor?d+), and NK–T cells (CD3+ CD19- NK1.1+). The indicated numbers of cell subsets of (B and C) IHLs and (D and E) splenocytes represent cell number/g of liver and splenocyte number/106 cells for (B and D) female and (C and E) male mice, respectively. Data are expressed as means ± SD for 10–13 mice per group/sex (data were pooled from 3 independent experiments). Mann–Whitney test. *P < .05 and **P < .01. SSC, side scatter characteristics.

Fig 4: Formation of gastric lymphoid follicles after Helicobacter suis infectionC57BL/6J WT, Myd88 KO, Trif KO, and Myd88/Trif DKO mice (n = 5 each) were analyzed 6 months after H. suis infection.(A) Histological examination and mean number of gastric lymphoid follicles. Original magnification, 200×.(B) Immunohistology of immunocompetent cells in the stomach (n = 5 each group). B220-positive cells (B cells), CD4-positive cells (helper T cells), CD11c-positive cells (dendritic cells; DCs), follicular dendritic cells (FDCs), NK1.1-positive cells (natural killer cells), and phalloidin (F-actin) were analyzed with confocal microscopy. Magnification, 100×.Ifng (C) and Cxcl13 (D) mRNA levels determined with qRT-PCR.(E) Immunohistology of H. suis distribution (n = 5 each) via confocal microscopy. Magnification, 100×.(F) H. suis 16S rRNA levels determined with qRT-PCR. (C, D, and F) Quantitative values were normalized to mouse ß-actin levels (n = 5).Data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, N.S., no significant difference (ANOVA). DKO, double knockout; N.D., not detected. Scale bar, 100 µm.