B, Intranuclear Eomes protein expression assessed by mAb and flow cytometry of indicated subpopulations within the freshly isolated splenocytes of wild-type and lpr/lpr mice. Results are representative of at least three independent experiments. Values represent the mean ± SEM of triplicate determinations normalized to hypoxanthine-guanine phosphoribosyltransferase. 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 qRT-PCR. Results are representative of two independent experiments.ĭysregulated expression of Eomes is a hallmark of DN T cells in both human and murine ALPS. 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 with bulk PBMCs from healthy donors. Comparison is made to bulk PBMCs from two normal controls (NC1, NC2), sorted memory (CD45RA −CD122 hi), and naive (CD45RA +CD122 lo) phenotype CD8 + T cells from normal controls, and sorted human NK (hNK) cells. C, qRT-PCR analysis of human Eomes (hEomes) mRNA in the sorted DN T cells of five patients with ALPS (Pt1–Pt5) that have been diagnosed clinically and confirmed with genetic testing for Fas mutation (see Table I for additional clinical information). Results are representative of three independent experiments.

1B, 1C).ĭysregulated expression of Eomes is a hallmark of DN T cells in both human and murine ALPS. Levels of Eomes protein and mRNA in murine and human Fas-mutant DN T cells appeared comparable to or greater than levels found in NK cells and effector/memory CD8 + T cells ( Fig. 1A, 1B), expression of Eomes in ALPS patients was most dysregulated in the DN T cell subset ( Fig. The patient with highest levels of Eomes in DN T cells (Pt5) had notably early and aggressive onset of disease ( Fig. Each patient exhibited elevated levels of Eomes in DN T cells ( Fig. Comparable results were obtained from the PBMCs of ALPS-FAS patients, who harbor confirmed mutations in the Fas gene ( Fig. Expression of Eomes was most dysregulated in the DN T cell subset. 1B) levels were substantially higher in Fas-mutant T cells compared with cells of wild-type mice. We examined the expression of Eomes in the T cell subsets of Fas-mutant lpr/lpr mice (from the nonautoimmune-prone C57BL/6 background) and found that Eomes mRNA ( Fig. We sought to investigate the effects of T cell-specific deletion of Eomes on the abnormal T cells of Fas deficiency, and our results suggest that Eomes is essential for the development or maintenance of this population.

In this study, we report that Eomes dysregulation defines the DN T cells in lpr/lpr animals and in humans with ALPS.

Responsiveness to IL-15 and T-bet–independent induction of IFN-γ are both characteristics controlled by the transcription factor Eomes ( 11, 18). Additionally, Fas-mutant T cells were found to produce IFN-γ independently of the T-box transcription factor T-bet ( 17). Previous studies suggested that ALPS DN T cells exhibit heightened sensitivity to the cytokine IL-15 ( 7). Eomes expression is also a hallmark of noncanonical, IL-15–responsive, innate-like CD8 + T cells that acquire functions, such as IFN-γ production and cytolytic potential, during their development in the thymus ( 12– 16). Eomesodermin (Eomes), a paralog of T-bet, is expressed in effector/memory CD8 + T cells and NK cells and plays redundant roles with T-bet in the induction of cytokine secretion and cytotoxic capacity of CD8 + T lymphocytes ( 9– 11).

Shotty lymph nodes genetic