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Regulatory T cells play a critical role in maintaining immune homeostasis and in preventing and controlling unwanted immune activation. These cells are often studied in the context of human peripheral blood, but can also be isolated from other biofluids. Here we describe methods for the isolation and functional characterization of human CD4

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.

Key Points Anti-TNF maintains IL10 expression in CD4+ T cells at the transcriptional level. IKZF3 is enriched in IL-10+ CD4+ T cells; degrading IKZF3 disrupts IL-10 production. Overexpression of IKZF3 does not drive IL10 or activate local enhancers., Visual Abstract, The expression of anti-inflammatory IL-10 by CD4+ T cells is indispensable for immune homeostasis, as it allows T cells to moderate their effector function. We previously showed that TNF-α blockade during T cell stimulation in CD4+ T cell/monocyte cocultures resulted in maintenance of IL-10–producing T cells and identified IKZF3 as a putative regulator of IL-10. In this study, we tested the hypothesis that IKZF3 is a transcriptional regulator of IL-10 using a human CD4+ T cell–only culture system. IL-10+ CD4+ T cells expressed the highest levels of IKZF3 both ex vivo and after activation compared with IL-10–CD4+ T cells. Pharmacological targeting of IKZF3 with the drug lenalidomide showed that IKZF3 is required for anti-CD3/CD28 mAb–mediated induction of IL-10 but is dispensable for ex vivo IL-10 expression. However, overexpression of IKZF3 was unable to upregulate IL-10 at the mRNA or protein level in CD4+ T cells and did not drive the transcription of the IL10 promoter or putative local enhancer constructs. Collectively, these data indicate that IKZF3 is associated with but not sufficient for IL-10 expression in CD4+ T cells.

TNF‐blockade has shown clear therapeutic value in rheumatoid arthritis and other immune‐mediated inflammatory diseases, however its mechanism of action is not fully elucidated. We investigated the effects of TNF‐blockade on CD4+ T cell activation, maturation, and proliferation, and assessed whether TNF‐inhibitors confer regulatory potential to CD4+ T cells. CyTOF and flow cytometry analysis revealed that in vitro treatment of human CD4+ T cells with the anti‐TNF monoclonal antibody adalimumab promoted IL‐10 expression in CD4+ T cells, whilst decreasing cellular activation. In line with this, analysis of gene expression profiling datasets of anti‐TNF‐treated IL‐17 or IFN‐γ‐producing CD4+ T cells revealed changes in multiple pathways associated with cell cycle and proliferation. Kinetics experiments showed that anti‐TNF treatment led to delayed, rather than impaired T‐cell activation and maturation. Whilst anti‐TNF‐treated CD4+ T cells displayed some hyporesponsiveness upon restimulation, they did not acquire enhanced capacity to suppress T‐cell responses or modulate monocyte phenotype. These cells however displayed a reduced ability to induce IL‐6 and IL‐8 production by synovial fibroblasts. Together, these data indicate that anti‐TNF treatment delays human CD4+ T‐cell activation, maturation, and proliferation, and this reduced activation state may impair their ability to activate stromal cells., Through deep phenotyping using CyTOF and functional assays we have shown that TNF inhibition using adalimumab altered the phenotype and function of multiple populations of human CD4+ T‐cells, leading to an overall decreased pro‐inflammatory potential. These results provide insight into the anti‐TNF mechanism of action in human CD4+ T‐cells.

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 compared to normal bone marrow samples. Transcriptional profiling revealed down-regulation of transcripts related to pro-inflammatory pathways in MDS-derived cells as compared to normal bone marrow. 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.

CD4+ CD25high CD127low/- FOXP3+ T regulatory cells are responsible for maintaining immune tolerance and controlling excessive immune responses. Treg cell use in pre-clinical animal models showed the huge therapeutic potential of these cells in immune-mediated diseases and laid the foundations for their applications in therapy in humans. Currently, there are several clinical trials utilizing the adoptive transfer of Treg cells to reduce the morbidity in autoimmune disorders, allogeneic HSC transplantation, and solid organ transplantation. However, a large part of them utilizes total Treg cells without distinction of their biological variability. Many studies on the heterogeneity of Treg cell population revealed distinct subsets with different functions in the control of the immune response and induction of peripheral tolerance. Some of these subsets also showed a role in controlling the general homeostasis of non-lymphoid tissues. All these Treg cell subsets and their peculiar properties can be therefore exploited to develop novel therapeutic approaches. This review describes these functionally distinct subsets, their phenotype, homing properties and functions in lymphoid and non-lymphoid tissues. In addition, we also discuss the limitations in using Treg cells as a cellular therapy and the strategies to enhance their efficacy.

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.

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.

SummaryExpansion and acquisition of Th1 cell effector function requires metabolic reprogramming; however, the signals instructing these adaptations remain poorly defined. Here we found that in activated human T cells, autocrine stimulation of the complement receptor CD46, and specifically its intracellular domain CYT-1, was required for induction of the amino acid (AA) transporter LAT1 and enhanced expression of the glucose transporter GLUT1. Furthermore, CD46 activation simultaneously drove expression of LAMTOR5, which mediated assembly of the AA-sensing Ragulator-Rag-mTORC1 complex and increased glycolysis and oxidative phosphorylation (OXPHOS), required for cytokine production. T cells from CD46-deficient patients, characterized by defective Th1 cell induction, failed to upregulate the molecular components of this metabolic program as well as glycolysis and OXPHOS, but IFN-γ production could be reinstated by retrovirus-mediated CD46-CYT-1 expression. These data establish a critical link between the complement system and immunometabolic adaptations driving human CD4+ T cell effector function.

Physiological health must balance immunological responsiveness against foreign pathogens with tolerance toward self-components and commensals. Disruption of this balance causes autoimmune diseases/chronic inflammation, in case of excessive immune responses, and persistent infection/immunodeficiency if regulatory components are overactive. This homeostasis occurs at two different levels: at a resting state to prevent autoimmune disease, as autoreactive effector T-cells (Teffs) are only partially deleted in the thymus, and during inflammation to prevent excessive tissue injury, contract the immune response, and enable tissue repair. Adaptive immune cells with regulatory function ("regulatory T-cells") are essential to control Teffs. Two sets of regulatory T cell are required to achieve the desired control: those emerging de novo from embryonic/neonatal thymus ("thymic" or tTregs), whose function is to control autoreactive Teffs to prevent autoimmune diseases, and those induced in the periphery ("peripheral" or pTregs) to acquire regulatory phenotype in response to pathogens/inflammation. The differentiation mechanisms of these cells determine their commitment to lineage and plasticity toward other phenotypes. tTregs, expressing high levels of IL-2 receptor alpha chain (CD25), and the transcription factor Foxp3, are the most important, since mutations or deletions in these genes cause fatal autoimmune diseases in both mice and men. In the periphery, instead, Foxp3(+) pTregs can be induced from naïve precursors in response to environmental signals. Here, we discuss molecular signatures and induction processes, mechanisms and sites of action, lineage stability, and differentiating characteristics of both Foxp3(+) and Foxp3(-) populations of regulatory T cells, derived from the thymus or induced peripherally. We relate these predicates to programs of cell-based therapy for the treatment of autoimmune diseases and induction of tolerance to transplants.

Introduction Severe aplastic anemia (SAA) is an immune mediated bone marrow failure syndrome characterized, among other features, by reduced numbers and dysfunctional regulatory T cells, as shown by our group. Using mass cytometry, we identified an immune signature that predicts clinical response toimmunosuppression (IST)consisting of two Treg subsets (A and B), differentially expressing CD45RA, CD95, CD7, CD28, and CCR4(Kordasti Blood 2016). Treg B are more suppressive and enriched in cell cycle related proteins, suggesting their proliferative potential. The aim of this study was to investigate the in vitro expandability of Treg subpopulations as well as their function, stability, plasticity, clonality and gene signature following expansion with the aim of using autologous in vitro expanded Tregs as a therapeutic option for AA patient. Methods and results Treg expansion, phenotype, and function Total Tregs from AA and HDs are sensitive to low dose IL-2 as shown by STAT5 phosphorylation (p < 0.001, n = 6 AA and 2 HDs). Moreover Tregs from AA and HDs can be expanded at a comparable rate (fold increase 33x vs 21x, p = n.s. n = 6 AA and 8 HDs) in a Treg promoting culture (stimulation with anti CD3/CD28 beads and IL-2 1,000 IU/ml for 4 weeks with all-trans retinoic acid and rapamycin). The expanded total Tregs have the phenotype of Treg B. To investigate the potential differences between Treg A and B in terms of function and expandability, Treg A (CD4+CD25hiCD127loCD45RAhiCD95-CCR4lo) and B (CD4+CD25hiCD127loCD45RAloCD95+CCR4hi) were sorted from 6 HDs and expanded for 4 weeks (as above). On average Treg A and B expanded equally (Treg A 45x, and B 33x fold increase, p = n.s.). After expansion Treg A up-regulate CD95 expression (p = 0.005) similar to Treg B phenotype. Expanded Treg A and B both suppress proliferation of autologous Tconv (n = 3, p < 0.001) as well as the IFN-g and TNF-a secretion. Only Treg B suppressed IL-17A secretion (n = 3, p < 0.05) (figure 1). Treg-specific demethylated region (TSDR) Treg stability was assessed by DNA methylation analysis by deep ampliconbisulphitesequencing of the TSDR. TSDR CpG sites in expanded HDs and AA Tregs are > 98% unmethylated, confirming their stability. Interestingly, TSDR sites were less methylated in Treg B compared to Treg A (5% v 35%), but it decreases to a similar level as Treg B after expansion. Treg plasticity after expansion To investigate the plasticity, total Tregs, Treg A, and Treg B were cultured for 5 days in a Th17 polarizing culture (containing IL-1b,IL-6, IL-2). After 5 days of culture, only total Tregs were secreting IL-17A (p = 0.013), but neither Treg A, nor Treg B. Despite IL-17A secretion, Tregs were still able to suppress autologous Tconv proliferation (n = 3, p= 0.001). T cell receptor diversity To track the clonal origin of Treg A and B, we performed amplification and sequencing of CDR3 using immunoSEQ Platform, as already described. Our results show that both AA and HDs expanded Tregs show a comparable level of TCR VbCDR3 diversity (average clonality score 0.12); moreover Tregs, Treg A, and Treg B are polyclonal, pre and post expansion (clonality score 0.01 for all the subsets). Finally, Treg A and B do not share any TCR sequence and do not have any overlap in their TCR repertoire, suggesting they originate from different clones. Gene expression Expanded Tregs (total, A and B) were compared to established human Treg gene signature (Ferraro PNAS 2014). Treg B aremore enriched in Treg genes compared Treg A (Tregs B vs A false discovery rate (FDR) < 0.0001). IL-7 and CD80 are among the top 5 expressed genes in all 3 subpopulations, which are important for Tregs' motility and homeostasis. Conclusions In summary, AA Tregs can be expanded in vitro and expanded Tregs are more similar to Treg B, which are lacking in SAA as shown before. Although we were not able to sort Treg subpopulations from AA patients due to very low numbers, the expanded Treg A and B from HDs demonstrate a functional Treg profile with minimal plasticity. Interestingly the main difference between Treg A and B is the ability of Treg B to suppress IL-17A secretion. This is important, as IL-17 plays a crucial role in AA pathophysiology (Peffault de Latour Blood 2010). Overall, the current study suggests that in vitro expansion of AA Tregs is feasible. The expanded Tregs show the phenotype of the most functional Treg subset and likely to control the inflammatory response inAA, hence pave the way toward a novel cellular therapy for SAA. Disclosures Marsh: Alexion pharmaceuticals: Honoraria.