Figure 11. Mtor hypomorphic mutation corrects Rasgrp1Anaef–induced increase in naive T-cell CD44 expression and accumulation of CD44+Helios+PD-1+CD4+ cells and autoantibodies.
(A) B6.Rasgrp1Anaef mice were intercrossed with B6.Mtorchino mice to generate the single and double-mutant mice. Representative CD44 histograms of CD62L+FOXP3−CD4+ splenocytes from these chino, Anaef, and Anaef.chino mutants were overlaid against wild-type cells and plotted. Below, CD44 MFI of mice was normalized against average CD44 MFI of wild-type mice across two independent experiments and graphed, with columns showing mean ± SEM. Significance indicated using a 1-way ANOVA and Tukey’s post-test at n = 13 WT, 5 chino, 15 Anaef, and 4 Anaef.chino. *p<0.05, **p<0.01, ***p<0.001. (B) Representative CD44 vs FOXP3 plots for these four genotypes from (A), which display CD4+ splenocytes gated into naïve (CD44lo FOXP3−), activated/memory (CD44hi FOXP3−), and T-reg (FOXP3+) populations. Absolute numbers of these three subsets across all four genotypes is graphed below, with columns showing mean ± SEM. Significance indicated using a 1-way ANOVA and Tukey’s post-test at n = 13 WT, 5 chino, 15 Anaef, and 4 Anaef.chino. *p<0.05, **p<0.01. (C) Bone marrow cells from sibling mice described in (A) and (B) were used to reconstitute irradiated B6.SJL CD451/1 mice, which were analyzed 28 weeks after irradiation. Plots show HELIOS vs PD-1 expression on FOXP3− CD4+ splenocytes, representative of both nonchimeric and chimeric mice. Absolute number of HELIOS+ PD-1+ FOXP3− CD4+ splenocytes per mouse is graphed below, with columns showing mean ± SEM. Significance indicated using a 1-way ANOVA and Tukey’s post-test at n = 11 WT, 10 chino, 10 Anaef, 11 Anaef.chino. ***p<0.001. (D) Chimeric mice from (C) were bled 27 weeks after irradiation and the presence of homogeneous nuclear ANA in blood plasma was measured by immunofluorescence on HEp-2 cells and scored in a blinded manner.