Extended Data Fig. 6. B and T cell development and homeostasis in NR4A-deficient mice.

A-C. Splenocytes from WT, Nr4a1^−/− and Nr4a3^−/− mice were stained to detect T cell subsets. A. Plots depict gating. CM=central memory. EM=effector memory. B, C. Graphs depict T cell subsets. D. Thymocytes and splenocytes from WT, Nr4a1^−/− and Nr4a3^−/− mice were permeabilized and stained to detect Foxp3. Graph depicts % Tregs in CD4+ gate. E-G. Splenocytes from WT, Nr4a1^−/− and Nr4a3^−/− mice were stained to detect B cell subsets. E. Plots of B220+ cells (top row) to identify marginal zone (MZ). Bottom row depicts B220+ subsets after exclusion of MZ. F, G. Graphs depict B cell subsets (F) and IgM/IgD MFI on B220+CD23+ splenocytes (G). H, I. Non-competitive chimeras were generated by reconstituting lethally irradiated CD45.1+ WT mice with BM from either CD45.2+ mb1-cre mice (cre chimera), or CD45.2+ cDKO chimera for 8-10 weeks. Graphs depict splenic T cell subsets as gated in A. N=5 chimeras / genotype. J, K. Splenocytes from from mb1-cre, Nr4a1cKO, and cDKO mice were stained to detect B cells. Graphs depict % B220+ cells (J) and IgM/IgD MFI on B220+ splenic B cells (K). L. Graph depicts splenic subsets from chimeras above (H, I) as gated in E. M, N. Irradiated CD45.1+ recipients were reconstituted with 1:1 BM mix from donor A (CD45.1/2 WT mice) and donor B (either CD45.2+ mb1-cre control or CD45.2+ cDKO mice) for 10-12 weeks. Graphs depict relative reconstitution of B cell developmental subsets in the BM and spleen. N=5 for chimeras each. Panels depict N=3 biological replicates except N=5 got chimeras (H, I, L, M, N). Mean +/− SEM for all graphs. Statistical significance was assessed with two-way ANOVA with Tukey’s (B-D, F, G, K); two-tailed unpaired student’s t-test with Holm-Sidak (H, I, L); one-way ANOVA with Tukey’s (J). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001