Figure 2. CCR7⁺ iNKT cells are enriched in the emigrating iNKT population and depend on Klf2.

(A) Experimental scheme to track recent thymic emigrants (RTE) through intra-thymic injection of NHS-biotin (left column) followed by enrichment of biotin⁺ splenic cells 24 hr after intra-thymic labeling, compared to ‘old’ cells (biotin^– cells from spleen) (right column). (B) Expression of CCR7 in thymic, RTE and biotin⁻ iNKT cells, the grey shade represents thymic iNKT cells, the solid black line represents RTE iNKT cells, the dashed black line represents biotin⁻ iNKT cells (top row); frequency of CCR7⁺ cells in thymic, RTE and biotin⁻ iNKT cells, the solid grey circle represents thymic iNKT cells, the open circle represents RTE iNKT cells, the open square represents biotin⁻ iNKT cells (bottom row). Data are pooled from 5 independent experiments with 3–6 mice in each. ^****p<0.0001 (one-way ANOVA) Each symbol represents an individual mouse; small horizontal lines indicate the mean. (C) Mixed bone marrow chimeras were generated with 25:75 ratio of donor bone marrow cells using CD45.2⁺ CD45.2⁺ B6 Klf2 cKO cells and CD45.1⁺ CD45.2⁺ B6 Wt cells, or with 50:50 ratio of donor bone marrow cells using CD45.2⁺ CD45.2⁺ B6 Wt cells and CD45.1⁺ CD45.2⁺ B6 Wt cells. Eight weeks later, chimeras received intra-thymic labeling with NHS-biotin to track RTE among CD4, CD8 and iNKT cells. Data are representative of 2 independent experiments with 4–8 mice in each. (D) Statistical analysis of thymic, RTE and biotin⁻ CD4 T cells, CD8 T cells and iNKT cells in 8-week-old BM chimeras reconstituted with Klf2 cKO and Wt cells (top row) or with Wt and Wt cells (bottom row). Data are pooled form 2 independent experiments with 4–8 mice in each. Numbers indicate the ratio between the cells derived from each donor source. ^****p<0.0001 (one-way ANOVA). ns, not significant, p>0.05 (one-way ANOVA). Each symbol represents an individual mouse; small horizontal lines indicate the mean. (E) Expression of Klf2^GFP in CCR7⁺ iNKT, stage 0 iNKT, CCR7^– iNKT cells and CD8 SP cells (top row). Normalized gMFI of Klf2^GFP in CCR7⁺ iNKT, stage 0 iNKT, CCR7^– iNKT cells (bottom row). Data are pooled from 2 independent experiments with 2 mice in each. ^****p<0.0001 (one-way ANOVA). Each symbol represents an individual mouse; small horizontal lines indicate the mean.

Figure 2—figure supplement 1. Intra-thymic labeling with NHS-biotin to identify RTEs in the periphery and Rag2^GFP+ splenic iNKT cells are CCR7⁺.

(A) Gating strategy to identify RTEs of CD4 and CD8 T cells in spleen. (B) Expression of Rag2^GFP in RTE CD4 or CD8 T cells, total splenic CD4 or CD8 T cells, and Wt total splenic CD4 or CD8 T cells. Data are representative of two independent experiments with three mice in each (C) Normalized Rag2^GFP gMFI of RTE iNKT cells and biotin⁻ iNKT cells in spleen. Data are pooled from two independent experiments with three mice in each. ^**p=0.0025 (unpaired two tailed t test). Each symbol represents an individual mouse; small horizontal lines indicate the mean. (D) Normalized Rag2^GFP gMFI of CCR7⁺ RTE iNKT cells and CCR7⁻ RTE iNKT cells in spleen. Data are pooled from two independent experiments with three mice in each ^***p=0.0006 (unpaired two tailed t test). Each symbol represents an individual mouse; small horizontal lines indicate the mean. (E) Rag2^GFP+ iNKT cells predominantly express CCR7 (right column) when identified in spleen from B6 Rag2^GFP mice (left column), green shade indicates Rag2GFP⁺ iNKT cells and black line indicates Rag2^GFP– iNKT cells.

Figure 2—figure supplement 2. RTE iNKT cells express high level of Klf2^GFP.

(A) Normalized Klf2^GFP gMFI of RTE iNKT cells and biotin⁻ iNKT cells. Data are pooled from two independent experiments with 2–4 mice in each. ^****p<0.0001 (unpaired two tailed t test). Each symbol represents an individual mouse; small horizontal lines indicate the mean.