Deep-phenotyping of Tregs identifies an immune signature for idiopathic aplastic anemia and predicts response to treatment

Idiopathic aplastic anemia (AA) is an immune-mediated and serious form of bone marrow failure. Akin to other autoimmune diseases, we have previously shown that in AA regulatory T-cells (Tregs) are reduced in number and function. The aim of this study was to further characterize Treg subpopulations in AA and investigate the potential correlation between specific Treg subsets and response to immunosuppressive therapy (IST) as well as their in-vitro expandability for potential clinical use. Using mass cytometry (CyTOF) and an unbiased multidimensional analytical approach, we identified two specific human Treg subpopulations (Treg A and Treg B) with distinct phenotypes, gene-expression, expandability and function. Treg subpopulation B, predominates in IST responder patients, has a memory/activated phenotype (with higher expression of CD95, CCR4 and CD45RO within FOXP3hi, CD127lo Tregs), expresses the IL- 2/STAT5 pathway and cell-cycle commitment genes. Furthermore, in-vitro expanded Tregs become functional and with the characteristics of Treg subpopulation B. Collectively, this study identifies human Treg subpopulations that can be used as predictive biomarkers for response to IST in AA and potentially other autoimmune diseases. We also show that Tregs from AA patients are IL-2 sensitive and expandable in-vitro, suggesting novel therapeutic approaches such as low dose IL-2 therapy and/or expanded autologous Tregs and meriting further exploration

Reduced frequencies and functional impairment of dendritic cell subsets and non-classical monocytes in myelodysplastic syndromes

In myelodysplastic syndromes (MDS) the immune system is involved in pathogenesis as well as in disease progression. Dendritic cells (DC) are key players of the immune system by serving as regulators of immune responses. Their function has been scarcely studied in MDS and most of the reported studies didn't investigate naturally occurring DC subsets. Therefore, we here examined the frequency and function of DC subsets and slan+ non-classical monocytes in various MDS risk groups. Frequencies of DC as well as of slan+ monocytes were decreased in MDS bone marrow (BM) compared to normal bone marrow (NBM) samples. Transcriptional profiling revealed down-regulation of transcripts related to pro-inflammatory pathways in MDS-derived cells as compared to NBM. Additionally, their capacity to induce T cell proliferation was impaired. Multidimensional mass cytometry showed that whereas healthy donor-derived slan+ monocytes supported Th1/Th17/Treg differentiation/expansion their MDS-derived counterparts also mediated substantial Th2 expansion. Our findings point to a role for an impaired ability of DC subsets to adequately respond to cellular stress and DNA damage in the immune escape and progression of MDS. As such, it paves the way toward potential novel immunotherapeutic interventions

Treg sensitivity to FasL and relative IL-2 deprivation drive Idiopathic Aplastic Anemia immune dysfunction:Mechanism of Treg depletion in AA

Idiopathic aplastic anemia (AA) has 2 key characteristics: an autoimmune response against hematopoietic stem/progenitor cells and regulatory T-cells (Tregs) deficiency. We have previously demonstrated reduction in a specific subpopulation of Treg in AA, which predicts response to immunosuppression. The aims of the present study were to define mechanisms of Treg subpopulation imbalance and identify potential for therapeutic intervention. We have identified 2 mechanisms that lead to skewed Treg composition in AA: first, FasL-mediated apoptosis on ligand interaction; and, second, relative interleukin-2 (IL-2) deprivation. We have shown that IL-2 augmentation can overcome these mechanisms. Interestingly, when high concentrations of IL-2 were used for in vitro Treg expansion cultures, AA Tregs were able to expand. The expanded populations expressed a high level of p-BCL-2, which makes them resistant to apoptosis. Using a xenograft mouse model, the function and stability of expanded AA Tregs were tested. We have shown that these Tregs were able to suppress the macroscopic clinical features and tissue manifestations of T-cell–mediated graft-versus-host disease. These Tregs maintained their suppressive properties as well as their phenotype in a highly inflammatory environment. Our findings provide an insight into the mechanisms of Treg reduction in AA. We have identified novel targets with potential for therapeutic interventions. Supplementation of ex vivo expansion cultures of Tregs with high concentrations of IL-2 or delivery of IL-2 directly to patients could improve clinical outcomes in addition to standard immunosuppressive therapy

CD161 expression characterizes a sub-population of human regulatory T cells that produces IL-17 in a STAT3 dependent manner

Treg cells are critical for the prevention of autoimmune diseases and are thus prime candidates for cell-based clinical therapy. However, human Treg cells are “plastic”, and are able to produce IL-17 under inflammatory conditions. Here, we identify and characterize the human Treg subpopulation that can be induced to produce IL-17 and identify its mechanisms. We confirm that a subpopulation of human Treg cells produces IL-17 in vitro when activated in the presence of IL-1β, but not IL-6. “IL-17 potential” is restricted to population III (CD4(+)CD25(hi)CD127(lo)CD45RA(−)) Treg cells expressing the natural killer cell marker CD161. We show that these cells are functionally as suppressive and have similar phenotypic/molecular characteristics to other subpopulations of Treg cells and retain their suppressive function following IL-17 induction. Importantly, we find that IL-17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL-17. Finally, we show that CD161(+) population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL-17-producing Treg-cell population at these sites. As IL-17 production from this Treg-cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary

Human retinoic acid-regulated CD161+ regulatory T cells support wound repair in intestinal mucosa.

Repair of tissue damaged during inflammatory processes is key to the return of local homeostasis and restoration of epithelial integrity. Here we describe CD161+ regulatory T (Treg) cells as a distinct, highly suppressive population of Treg cells that mediate wound healing. These Treg cells were enriched in intestinal lamina propria, particularly in Crohn's disease. CD161+ Treg cells had an all-trans retinoic acid (ATRA)-regulated gene signature, and CD161 expression on Treg cells was induced by ATRA, which directly regulated the CD161 gene. CD161 was co-stimulatory, and ligation with the T cell antigen receptor induced cytokines that accelerated the wound healing of intestinal epithelial cells. We identified a transcription-factor network, including BACH2, RORγt, FOSL2, AP-1 and RUNX1, that controlled expression of the wound-healing program, and found a CD161+ Treg cell signature in Crohn's disease mucosa associated with reduced inflammation. These findings identify CD161+ Treg cells as a population involved in controlling the balance between inflammation and epithelial barrier healing in the gut