(A) Expression of Rag2GFP and CCR7 in thymic iNKT cells (TCR-β+ 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-γt+ 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-γt+ 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-γt, huCD2, PLZF, LEF1, Egr2 and CD4 with CCR7 in thymic iNKT cells (TCR-β+ 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-γt 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-γt+ 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.
Figure 1—source data 1. High proliferative and precursor potential of CCR7+ iNKT cells.
Figure 1—source data 2. Thymic CCR7+ iNKT cells are distinguished from stage 0 iNKT cells and give rise to iNKT subsets in periphery.
Figure 1—source data 3. Consistent, robust and unbiased labeling of thymocytes by intra-thymic injection of biotin.