Fig 2: Thymic iNKT effector subsets are predominantly resident and may influence thymocytes maturation.(A) Experimental scheme of parabiosis surgery. (B) Representative flowcytometry profile of spleen and thymus from the CD45.2+ host of a parabiotic pair after 30 days of parabiosis surgery, the number indicates the frequency of CD45.2+ (host) and CD45.1+ (parabiotic counterpart) cells in CD8 T cells and iNKT cells. Numbers adjacent to outlined areas indicate percent cells in each throughout. Data are representative of 2 independent experiments with 3 parabiotic pairs in each. (C) The frequency of cells derived from the host parabiont in splenic CD4+ T, CD8+ T and iNKT cells as well as thymic iNKT cells. Data are pooled from 2 independent experiments with 3 parabiotic pairs in each. ns, not significant, p=0.8722 (one-way ANOVA). ***p=0.0009, ****p<0.0001 (one-way ANOVA). Each symbol represents an individual mouse; small horizontal lines indicate the mean. (D) CCR7+ Tbx21GFP– and CCR7– Tbx21GFP+ thymocytes were sorted from BALB/c Tbx21GFP mice and intra-thymically transferred to congenic BALB/c recipients (left column). After 5 days, the donor derived iNKT cells were recovered in thymus and spleen from mice received CCR7+ Tbx21GFP– thymocytes or CCR7– Tbx21GFP+ thymocytes (right column). Numbers adjacent to outlined areas indicate percent cells in each throughout. Data are representative of 5 independent experiments. (E) Cell number of donor derived iNKT cells recovered in thymus and spleen from mice that received CCR7+ Tbx21GFP– thymocytes or CCR7– Tbx21GFP+ thymocytes. Data are pooled from 5 independent experiments with 2 mice in each. ns, not significant, p=0.1508 (unpaired two tailed t test). **p=0.0079 (unpaired two tailed t test). Each symbol represents an individual mouse; small horizontal lines indicate the mean. (F). Expression of YFP in DP thymocytes (left column) or thymic NKT1 cells (NK1.1+ CD44+) (right column), the grey shade represents cells from Wt mice, the solid green line represents cells from Ifn-?YFP mice; Numbers adjacent to solid black line indicate percent of Ifn-?YFP+ cells from Ifn-?YFP mice; Data are representative of 4 independent experiments with 3–5 mice in each. (G) Frequency of Ifn-?YFP+ cells within thymic NKT1 cells. Data are pooled from 4 independent experiments with 3–5 mice in each. Each symbol represents an individual mouse; small horizontal lines indicate the mean. (H) Expression of Qa2 in CD4 SP or CD8 SP thymocytes from Wt or Cd1d KO mice. Numbers adjacent to outlined areas indicate percent cells in each throughout. Data are representative of 5 independent experiments. (I) Frequency of Qa2+ cells in CD4 SP or CD8 SP thymocytes from Wt, Cd1d KO and Ifn-?RKO mice. Data are pooled from 5 independent experiments, n = 18 (Wt), n = 11 (Cd1d KO), n = 6 (Ifn-?RKO). *p=0.0426, **p=0.0023, ****p<0.0001 (one-way ANOVA). ns, not significant, p=0.2676 (CD4 SP), p=0.8848 (CD8 SP) (one-way ANOVA). Each symbol represents an individual mouse; small horizontal lines indicate the mean. (J) Expression of RankL in thymic NKT1, NKT2 and NKT17 cells (upper panel). Normalized gMFI of RANKL in thymic NKT1, NKT2 and NKT17 cells (bottom panel). Data are representative of 3 independent experiments with 2–3 mice in each. **p=0.0027 (NKT1 vs NKT2), *p=0.0391 (NKT1 vs NKT17) (one-way ANOVA). (K) Expression of Aire in TEC from Wt, Cd1d KO or Aire KO mice. Numbers adjacent to outlined areas indicate percent cells in each throughout. Data are representative of 4 independent experiments. (L) Frequency and number of Aire+ mTEC (left and middle column), number of total TEC (right column) from Wt and Cd1d KO mice. Data are representative of 4 independent experiments, n = 5 (Wt), n = 6 (Cd1d KO). ****p<0.0001, **p=0.0032, ns, not significant, p=0.5209 (unpaired two tailed t test). Each symbol represents an individual mouse; small horizontal lines indicate the mean.10.7554/eLife.34793.025Figure 5—source data 1.Thymic iNKT cells are resident and influence thymic microenvironment.10.7554/eLife.34793.026Figure 5—source data 2.CD69 and CD103 are dispensable for thymic retention of NKT1 and NKT17 cells.

Fig 3: T-cell development in Gli3+/+, Gli3+/- and Gli3-/- E17.5 plus 4 days in FTOC and E18.5 thymus. Flow cytometry analysis. Scatter plots show mean±s.e.m. Each point represents thymus from an individual mouse embryo. (A-H) E17.5 FTOC for 4 days. Gli3+/+ (n=14), Gli3+/- (n=14) and Gli3-/- (n=11). (I,J) E18.5 Gli3+/+ (n=3) and Gli3-/- (n=3) thymus. (A) CD8 against CD4. (B) Percentage of populations (relative to mean of WT) giving significance by Student's t-test compared with WT for SP4 (Gli3+/-, P<0.03; Gli3-/-, P<0.001), SP8 (Gli3-/-, P<0.03) and SP4:SP8 ratio (Gli3+/-, P<0.02; Gli3-/-, P<0.0006). (C) CD3 staining on Gli3+/+, Gli3+/- and Gli3-/- thymocytes, giving the percentage of CD3hi cells. (D) Percentage of CD3hi thymocytes in Gli3+/+, Gli3+/- and Gli3-/- (relative to mean of WT littermate). (E) CD8 against CD4, gated on CD3hi. (F) Percentage of populations gated on CD3hi, giving significance by Student's t-test compared with WT for CD3hi SP4 (Gli3+/-, P<0.002; Gli3-/-, P<0.0004), CD3hi SP4:SP8 ratio (Gli3-/-, P<0.002) and CD3hi DP (Gli3+/-, P<0.05; Gli3-/-, P<0.02). (G) HSA against Qa2 expression, gated on SP4 cells from Gli3+/+ and Gli3-/-. (H) Relative percentage of HSA- Qa2+ cells in the SP4 population, giving significance by Student's t-test compared with WT (P<0.002). (I) Percentage of CD69+ cells in SP4 and DP populations, giving significance by Student's t-test for SP4 (P<0.07) and DP (P<0.05). (J) CD69 expression on SP4 and DP cells in a representative experiment.