Figure 5.

Mekk1 Signaling Controls p27^Kip1 Expression to Regulate iNKT Cell Proliferation

(A) WT and Map3k1^ΔKD mice were i.p. injected with α-GalCer for 3 days. RNA was isolated from WT and Map3k1^ΔKD splenic iNKT cells, processed, and hybridized onto Affymetrix arrays. Bioinformatics analysis was performed, and a heatmap comparing gene hits between WT and Map3k1^ΔKD iNKT cell microarray screens was constructed. The data are from three independent experiments (four mice per experiment).

(B) Splenic and liver iNKT cells were isolated from 3-day α-GalCer-immunized Lck^Cre/+Map3k1^f/f, WT mice stained with anti-phospho c-Jun antibody, and flow cytometry performed as indicated (red line, WT; blue line, Lck^Cre/+Map3k1^f/f). Data were representative of three independent experiments. Histograms show the phospho c-Jun present in the gated iNKT cell population.

(C) Splenic iNKT cells from 3-day α-GalCer-immunized Lck^Cre/+Map3k1^f/f and WT mice were isolated and stained with anti-p27^Kip1 antibody and flow cytometry performed as indicated (red line, WT; blue line, Lck^Cre/+Map3k1^f/f). Data were representative of three independent experiments. Histogram shows the p27^Kip1 present in the gated iNKT cell population.

(D) iNKT cells from the spleen and liver of WT and Lck^Cre/+Map3k1^f/f mice were isolated 3 days post-i.p. injection with α-GalCer and their RNA analyzed by real-time PCR as indicated (gray square, WT; black square, Lck^Cre/+Map3k1^f/f). The average relative expression (±SEM) of genes from three independent experiments was statistically analyzed, where appropriate, by two-tailed Student’s t test (^∗p ≤ 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001).

(E) WT, Map3k1^ΔKD, Cdkn1b^−/−, or Map3k1^ΔKD/Cdkn1b^−/− (DKO) mice were treated with water containing BrdU and i.p. immunized with α-GalCer (day 3) or left unstimulated (day 0). Splenocytes were extracted and analyzed as indicated. Representative results (±SEM) from three quantitated iNKT proliferation experiments were statistically analyzed, where appropriate, by two-tailed Student’s t test (^∗p ≤ 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001).

(F) WT, Cdkn1b^−/−, Map3k1^ΔKD, or Map3k1^ΔKD/Cdkn1b^−/− (DKO) iNKT cells were isolated and incubated in [³H] thymidine containing media for 24 hr in the presence of DMSO (control), SP600125, PD98059, SB203580, NSC697923, or SU9516. The average cpm (±SEM) from three independent experiments was statistically analyzed, where appropriate, by two-tailed Student’s t test (^∗p ≤ 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001).

(G) WT, Map3k1^ΔKD, and Lck^Cre/+Map3k1^f/f mice were immunized with α-GalCer for 3 days, splenic or liver iNKT cells isolated, and Cdkn1b ChIP performed with anti-phospho c-Jun antibody as indicated. The average relative signal (±SEM) from three independent experiments was statistically analyzed, where appropriate, by two-tailed Student’s t test (^∗p ≤ 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001).