Shahram Kordasti

Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group

Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as ‘normal’, ‘suggestive of’, or ‘diagnostic of’ MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique.

IL-17-producing CD4(+) T cells, pro-inflammatory cytokines and apoptosis are increased in low risk myelodysplastic syndrome.

Immunological responses are increasingly recognised as being important in the initiation and progression of myelodysplastic syndrome (MDS). Indeed, autoimmune diseases commonly occur in association with MDS, particularly in subtypes with a low risk of leukaemic transformation. This study showed for the first time that the numbers of CD3+ CD4+ IL‐17 producing T cells (Th17) were markedly increased in low risk MDS compared with high risk MDS (P < 0·01). An inverse relationship between the numbers of Th17 cells and naturally occurring CD4+CD25high FoxP3+ regulatory T cells (Tregs) were also described. The Th17:Tregs ratio was significantly higher in low risk disease (P < 0·005) compared with high risk MDS and was correlated with increased bone marrow (BM) apoptosis (P < 0·01). Tregs from MDS patients suppressed interferon‐γ (IFN‐γ) secretion by effector CD4+ T cells but had no effect on interleukin (IL)‐17 production. In addition, the serum levels of IL‐7, IL‐12, RANTES and IFN‐γ are significantly elevated in low risk MDS, while inhibitory factors, such as IL‐10 and soluble IL‐2 receptor, are significantly higher in high risk disease. The ‘unfavourable’ Th17:Tregs ratio in low risk MDS may explain the higher risk of autoimmunity and the improved response to immune suppression in patients with low risk MDS compared to those with high risk disease.

Prospective study of rabbit antithymocyte globulin and cyclosporine for aplastic anemia from the EBMT Severe Aplastic Anaemia Working Party

Rabbit antithymocyte globulin (rATG; thymoglobulin, Genzyme) in combination with cyclosporine, as first-line immunosuppressive therapy, was evaluated prospectively in a multicenter, European, phase 2 pilot study, in 35 patients with aplastic anemia. Results were compared with 105 age- and disease severity-matched patients from the European Blood and Marrow Transplant registry, treated with horse ATG (hATG; lymphoglobulin) and cyclosporine. The primary end point was response at 6 months. At 3 months, no patients had achieved a complete response to rATG. Partial response occurred in 11 (34%). At 6 months, complete response rate was 3% and partial response rate 37%. There were 10 deaths after rATG (28.5%) and 1 after subsequent HSCT. Infections were the main cause of death in 9 of 10 patients. The best response rate was 60% for rATG and 67% for hATG. For rATG, overall survival at 2 years was 68%, compared with 86% for hATG (P = .009). Transplant-free survival was 52% for rATG and 76% for hATG (P = .002). On multivariate analysis, rATG (hazard ratio = 3.9, P = .003) and age more than 37 years (hazard ratio = 4.7, P = .0008) were independent adverse risk factors for survival. This study was registered at www.clinicaltrials.gov as NCT00471848.

Functional characterization of CD4+ T cells in aplastic anemia

The role of CD4(+) T cells in the pathogenesis of aplastic anemia (AA) is not well characterized. We investigate CD4(+) T-cell subsets in AA. Sixty-three patients with acquired AA were studied. Th1 and Th2 cells were significantly higher in AA patients than in healthy donors (HDs; P = .03 and P = .006). Tregs were significantly lower in patients with severe AA than in HDs (P < .001) and patients with non-severe AA (P = .01). Th17 cells were increased in severe AA (P = .02) but normal in non-severe AA. Activated and resting Tregs were reduced in AA (P = .004; P = .01), whereas cytokine-secreting non-Tregs were increased (P = .003). Tregs from AA patients were unable to suppress normal effector T cells. In contrast, AA effector T cells were suppressible by Tregs from HDs. Th1 clonality in AA, investigated by high-throughput sequencing, was greater than in HDs (P = .03). Our results confirm that Th1 and Th2 cells are expanded and Tregs are functionally abnormal in AA. The clonally restricted expansion of Th1 cells is most likely to be antigen-driven, and induces an inflammatory environment, that exacerbate the functional impairment of Tregs, which are reduced in number.

CD161 expression characterizes a subpopulation 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+CD25hiCD127loCD45RA−) 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.

Rationale for the clinical application of flow cytometry in patients with myelodysplastic syndromes: position paper of an International Consortium and the European LeukemiaNet Working Group

Abstract An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.

The effects of 5-azacytidine on the function and number of regulatory T-cells and T-effectors in myelodysplastic syndrome

Expansion of regulatory T cells occurs in high-risk myelodysplastic syndrome and correlates with a poor prognosis. DNA methyltransferase inhibitors, particularly 5-azacytidine, have been shown to increase the survival of patients with high-risk myelodysplastic syndrome. It is not entirely clear whether this improvement in patients’ survival is related to the effects of DNA methyltransferase inhibitors on the immune system and/or the direct effect of these drugs on the dysplastic clone. In this study we investigated the effect of 5-azacytidine on the function and proliferation capability of regulatory T cells and T-helper cells. The number and function of CD4+ T-cell subsets in 68 patients with intermediate-2/high-risk myelodysplastic syndrome were serially assessed at diagnosis and following treatment. The in-vitro effects of 5-azacytidine on CD4+ T-cell subsets isolated from both healthy donors and patients with myelodysplastic syndrome were also investigated. The number of peripheral blood regulatory T cells was significantly higher in myelodysplastic syndrome patients than in healthy donors and responders to treatment (P=0.01). The absolute numbers of T-helper 1 and T-helper 2, but not T-helper 17, cells were significantly reduced following 12 months of treatment (P=0.03, P=0.03). The in vitro addition of 5-azacytidine to CD4+ T cells reduced the proliferative capacity of regulatory T cells (P=0.03). In addition, the 5-azacytidine-treated regulatory T cells had reduced suppressive function and produced larger amounts of interleukin-17. The FOXP3 expression in 5-azacyti-dine-treated T-effectors was also increased. Interestingly, these FOXP3+/interleukin-17+ cells originated mainly from effector T cells rather than regulatory T cells. Our data suggest that 5-azacytidine has profound effects on CD4+ T cells, which correlate with disease status after treatment. Furthermore, despite the demethylation of the FOXP3 promoter and increased FOXP3 expression following 5-azacytidine treatment, these phenotypic regulatory T cell-like cells lack the regulatory function and cytokine profile of regulatory T cells. These findings are important in correlating the clinically relevant immunomodulatory effects of 5-azacytidine.

Comparison of Regulatory T Cells in Hemodialysis Patients and Healthy Controls: Implications for Cell Therapy in Transplantation

BACKGROUND AND OBJECTIVES Cell-based therapy with natural (CD4(+)CD25(hi)CD127(lo)) regulatory T cells to induce transplant tolerance is now technically feasible. However, regulatory T cells from hemodialysis patients awaiting transplantation may be functionally/numerically defective. Human regulatory T cells are also heterogeneous, and some are able to convert to proinflammatory Th17 cells. This study addresses the suitability of regulatory T cells from hemodialysis patients for cell-based therapy in preparation for the first clinical trials in renal transplant recipients (the ONE Study). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Healthy controls and age- and sex-matched hemodialysis patients without recent illness/autoimmune disease on established, complication-free hemodialysis for a minimum of 6 months were recruited. Circulating regulatory T cells were studied by flow cytometry to compare the regulatory T cell subpopulations. Regulatory T cells from members of each group were compared for suppressive function and plasticity (IL-17-producing capacity) before and after in vitro expansion with and without Rapamycin, using standard assays. RESULTS Both groups had similar total regulatory T cells and subpopulations I and III. In each subpopulation, regulatory T cells expressed similar levels of the function-associated markers CD27, CD39, HLA-DR, and FOXP3. Hemodialysis regulatory T cells were less suppressive, expanded poorly compared with healthy control regulatory T cells, and produced IL-17 in the absence of Rapamycin. However, Rapamycin efficiently expanded hemodialysis regulatory T cells to a functional and stable cell product. CONCLUSIONS Rapamycin-based expansion protocols should enable clinical trials of cell-based immunotherapy for the induction of tolerance to renal allografts using hemodialysis regulatory T cells.

Rationale for the clinical application of flow cytometry in patients with myelodysplastic syndromes: position paper of an International Consortium and the European LeukemiaNet Working Group.

Abstract An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.

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 and an unbiased multidimensional analytical approach, we identified 2 specific human Treg subpopulations (Treg A and Treg B) with distinct phenotypes, gene expression, expandability, and function. Treg B predominates in IST responder patients, has a memory/activated phenotype (with higher expression of CD95, CCR4, and CD45RO within FOXP3(hi), CD127(lo) Tregs), expresses the interleukin-2 (IL-2)/STAT5 pathway and cell-cycle commitment genes. Furthermore, in vitro-expanded Tregs become functional and take on the characteristics of Treg 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.