Eomes is required for the lymphoproliferative, but not autoimmune, component of murine ALPS.
A, Flow cytometry of freshly isolated, TCRβ⁺ lymph node cells from wild-type, lpr/lpr (with homozygous floxed alleles of Eomes but no Cre recombinase transgene), and lpr/lpr mice with T cell-specific deletion of Eomes (homozygous floxed alleles of Eomes and transgenic Cre recombinase driven by the Cd4 promoter). Results are representative of more than ten independent experiments. Of note, the percentage of CD4+ T cells, CD8+ T cells, B cells, and non-B/non-T populations was not reproducibly different between wild-type and Eomes-deficient lpr/lpr mice.
B, Photograph of spleens and lymph nodes from mice with the indicated genotypes. Results are representative of eight independent experiments.
C, Mass of spleens and pooled lymph nodes and D, total cell number contained therein from mice of the indicated genotypes. Bar graphs indicate mean values, and error bars represent standard error of the mean, n=8 mice per group. A one-way ANOVA with Tukey's post-comparison test was performed using Prism software. Labels of n.s. denote not significant (p>0.05); * denotes p<0.05; *** denotes p<0.001.
E, F Autoantibody production in 12- to 16-month-old female mice of the indicated genotypes. Results are representative of three independent experiments. E, Sera were isolated and analyzed for anti-double-stranded DNA antibodies by enzyme-linked immunosorbent assay (ELISA). Lupus-prone MRL-lpr/lpr mice serve as positive control for severe autoantibody titers. F, Anti-nuclear antibodies were detected by exposing HEp-2 cells to sera of indicated mice, followed by staining with a secondary, FITC-conjugated antibody reactive to mouse IgG.

Dysregulated expression of Eomesodermin is a hallmark of double-negative (DN) T cells in both human and murine ALPS.
A, CD4⁺, CD8⁺, and CD4⁻CD8⁻ (DN) T cells were sorted from wild-type and lpr/lpr mice (Fas^WT and Fas^lpr, respectively) and murine Eomes mRNA levels were analyzed by quantitative real-time RT-PCR (qRT-PCR). Values represent the mean ± S.E.M. of triplicate determinations normalized to hypoxanthine-guanine phosphoribosyltransferase. Results are representative of at least three independent experiments.
B, Intranuclear Eomes protein expression assessed by monoclonal antibody and flow cytometry of indicated subpopulations within the freshly isolated splenocytes of wild-type and lpr/lpr mice. Results are representative of three independent experiments.
C, qRT-PCR analysis of human Eomes (hEomes) mRNA in the sorted DN T cells of five ALPS patients (Pt1–Pt5) that have been diagnosed clinically and confirmed with genetic testing for Fas mutation (see Table 1 for additional clinical information). Comparison is made to bulk peripheral blood mononuclear cells (PBMCs) from two normal controls (NC1, NC2), sorted memory- (CD45RA⁻CD122^hi) and naïve- (CD45RA⁺CD122^lo) phenotype CD8⁺ T cells from normal controls, and sorted human natural killer (hNK) cells.
D, CD4⁺, CD8⁺, and DN T cells were fractionated from the PBMCs of three ALPS patients (Pt1, Pt4, Pt5) and hEomes mRNA levels were compared to bulk PBMCs from healthy donors. Values represent the mean ± S.E.M. Results are representative of two independent experiments.