(a) Contribution of WT and Egr1^-/- (Egr1KO) cells to the iNKT compartment in thymus (T), spleen (S) and liver (L) of mixed bone marrow chimeras generated by injecting Egr1^-/- (CD45.2) and F1(C57BL/6xB6-LY5.2/Cr) (CD45.1;CD45.2) bone marrow cells into lethally irradiated B6-LY5.2/Cr recipient mice (CD45.1). Mean and SEM is shown on the right (n = 5). Significance was assessed using a paired t-test. **<0.01, *<0.05. This is one out of two independent experiments. (b) Percentages and absolute numbers of iNKT cells in the thymus (T), spleen (S) and liver mononuclear cells (L) of WT and Egr-2^f/f-lck-Cre (EGR2KO) mice stained with CD4, CD8, PBS57-loaded CD1d tetramer and TCRβ. (c) Percentages and absolute numbers of iNKT cell subpopulations in gated TCR-β^hiPBS57-CD1dtet⁺ thymocytes from WT and EGR2KO mice. Results representative of five independent pairs in three independent experiments. The bar graphs show the average and SEM of all the experiments. Significance was assessed using an unpaired t-test. ***<0.001, **<0.01.

(a) Thymic profile of littermate wild-type (WT), or Egr1^-/-;Egr2^f/flckcre (DKO) mice (b) Percentages and absolute numbers of iNKT cells in the thymus (T), spleen (S) and liver mononuclear cells (L) of WT and EgrDKO mice stained with CD4, CD8, PBS57-loaded CD1d tetramer and TCRβ. (c) Percentages and absolute numbers of iNKT cell subpopulations in gated thymic TCR-βhiPBS57-CD1dtet⁺ of WT and EgrDKO mice. Results representative of five independent pairs in three independent experiments. The bar graphs show the average and SEM of all the experiments. Significance as assessed using the unpaired t-test. ***<0.001, **<0.01

(a) Thymic profile of WT and dnRas mice (b) Percentages and absolute numbers of iNKT cells in the thymus (T), spleen (S) and liver mononuclear cells (L) of normal littermate controls (WT) and dnRas mice stained with CD4, CD8, PBS57-loaded CD1d tetramer and TCRβ. (c) Percentages and absolute numbers of iNKT cell subpopulations in gated TCR^hiPBS57-CD1dtet⁺ thymocytes from WT and dnRas mice. Results representative of nine independent dnRas and WT pairs analyzed in five experiments, except for the CD44/NK1.1 histograms (n = 2). The bar graphs show the average and SEM of all the experiments. Significance as assessed using a two-tailed unpaired t-test. ***<0.001, **<0.01. (d) Gating strategy used to sort the different populations. (e) Expression of Egr-1, Egr-2 and Id3 in sorted Tet⁺ HSA^hi and Tet⁺ HSA^lo from normal littermate control (WT) and dnRas mice. Bar graphs show relative expression of dnRas compared to WT for three independent experiments. Each experiment was an independent sort of a WT and dnRas pair. Expression in each experiment was normalized to the expression levelis in Tet⁺ HSA^hi WT cells. Significance as assessed using a two-tailed unpaired t-test. **<0.01.

(a) Donor contributions in bone marrow chimeras generated by injecting MiG-Bcl2 transduced Egr2^f/flck-Cre (Egr2KO) (CD45.2) and wild-type (CD45.1;CD45.2) cells into lethally irradiated CD45.1 recipients, analyzed at 8–12 weeks (left), and a representative histogram of GFP expression in MiG-Bcl2 transduced Egr2KO thymocytes (right). (b) TcR thymic profiles (upper panel) and iNKT cells frequency (lower panels) in wild-type, Egr2KO and Egr2KO-Bcl2 donor cells in the thymus (T) and Liver (L) of chimeras described in (a). (c) Percentages of different cell subpopulations from WT Egr2KO and Egr2KO;Bcl-2 in the mixed bone marrow chimeras mice. Results representative of eight independent chimeras in two independent experiments. The bar graphs show the average and SEM of all the experiments. Significance as assessed using the unpaired t-test. ***<0.001.

Slamf1, Slamf3, Slamf6, Slamf5 and CD1d expression levels in DP thymocytes mice were assessed by flow cytometry. Shown are representative histograms, and the mean and SEM of the normalized MFI of DP populations. In (A) WT vs. dnRas (n = 5). In (B) WT vs. Egr1^-/-;Egr2^f/f-lck-Cre (Egr-DKO) (n = 3). (C) SAP and Bcl_xL expression levels in DP thymocytes from WT or dnRas mice were assessed by intracellular flow cytometry. Shown are representative histograms, and the mean and SEM of the normalized MFI of DP populations (n = 5). To normalize the MFI, we averaged the MFI for the WT mice in each experiment and considered that value 1. The bar graphs show the average and SEM of all the experiments. Significance was assessed using the unpaired t-test ***<0.001, **<0.01 *<0.05.