FOXP3-Positive Regulatory T-Cells in Acquired Aplastic Anemia.
Authors: Solomou Elena E. , Rezvani Katayoun , Mielke Stephan , Malide Daniela , Visconte Valeria , Keyvanfar Keyvan , Bruno Tullia C. , Barrett A. John , Young Neal S. 
Published: October 2019

Abstract
CD4+CD25+ regulatory T cells (Treg) have been hypothesized to control the development and progression of autoimmunity by suppressing autoreactive T cells. Treg are characterized by constitutive expression of membrane CD25 and intracellular expression of the transcription factor forkhead box (FOX) P3. FOXP3 and NF-AT1 have key roles in regulatory T-cell development and function: induction of FOXP3 in naïve T cells by gene transfer results in gain of a regulatory phenotype, and NF-AT1 modulates transcription of FOXP3. Treg display their suppressive properties on CD4+ CD25- T cells when activated via the T cell receptor or Toll-like receptor-2 (TLR2). Decreased numbers of FOXP3-positive regulatory Tregs have been associated with impaired immune homeostasis. Treg numbers are deficient in patients with active systemic lupus erythematosus, GVHD, and autoimmune hepatitis; in multiple sclerosis, decreased FOXP3 impairs Treg function. Acquired aplastic anemia (AA), the paradigm of immune mediated bone marrow failure syndromes, is characterized by immune-mediated destruction of hematopoietic stem cells. To examine expression of CD4+CD25+ T-cells in this disease, peripheral blood mononuclear cells from AA patients, sampled at diagnosis and prior to immunosuppressive therapy (n=18), were examined by flow cytometry: CD4+CD25hi+ T-cells were markedly reduced or absent in patients compared to healthy controls (n = 12, 0.08± 0.01 vs 0.4± 0.1%, p=0.001). CD4+CD25hi+FOXP3+ T-cells were also significantly lower in patients (0.05± 0.01 vs 0.32± 0.1, p=0.001). By immunoblot, we observed significantly decreased and often undetectable FOXP3 protein levels in CD4+CD25+ T cells from patients (n=8) compared to controls (n=6). Low FOXP3 protein levels correlated with decreased FOXP3 mRNA levels, as measured by RT-PCR and quantitative real-time PCR experiments (n=5, p=0.03). There were no differences in TLR2 in immunoblots between patients and healthy controls despite differences in FOXP3 expression. Patients (n=8) with low FOXP3 levels also showed decreased or absent NF-AT1 protein levels. These data collectively implicate a transcriptional mechanism for FOXP3 down-regulation. In confocal microscopy, purified CD4+CD25+ T cells from patients (n=5) showed undetectable NF-AT1 and FOXP3 levels as compared to controls (n=3). Four patients studied 3–6 months after first sampling and post-immunosuppressive treatment showed increased Tregs and FOXP3 expression; four further patients in complete remission after immunosuppressive treatment had increased CD4+CD25hi+ T-cells as compared to treatment-naïve patients. CD4+CD25+FOXP3+ regulatory T-cells are decreased in most patients with AA, probably through transcriptional regulation; decreased NFAT1 could explain low FOXP3 expression and Treg frequency. Treg employed as cellular therapy in a murine model of immune-mediated AA can prevent T cell-mediated marrow destruction (Chen J and Young NS, unpublished data). Treg defects are now implicated in autoimmune marrow failure in aplastic anemia.