Xingxin Li

Intrinsic impairment of CD4+CD25+ regulatory T cells in acquired aplastic anemia

Acquired aplastic anemia (AA) is an immune-mediated bone marrow (BM) failure attacked by autoreactive effector T cells and BM is the main target organ. CD4(+)CD25(+) regulatory T cells (Tregs) were believed to control development and progression of autoimmunity by suppressing autoreactive effector T cells, but little was known regarding the function of Tregs in AA. Our study demonstrated that both peripheral blood (PB) and BM had decreased frequencies of Tregs, accompanied with a reversed lower ratio of Treg frequencies between BM and PB in AA. PB Tregs in AA had impaired migratory ability because of lower CXCR4 (but not for CXCR7) expression. Interestingly, we first showed that impairment of Treg-mediated immunosuppression was intrinsic to Tregs, rather than resistance of effector T cells to suppression in AA by coculture assays and criss-cross experiments in vitro. Furthermore, Tregs in AA were less able to inhibit interferon-γ production by effector T cells. Defective immunosuppression by Tregs could contribute to impaired hematopoiesis conducted by effector T cells in vitro. Our study provided powerful evidence that impairment of Tregs played a critical role in the pathophysiology of AA. Thus, patients with AA might greatly benefit from a Treg-oriented immunosuppressive strategy.

Interleukin-27 enhances the production of tumour necrosis factor-α and interferon-γ by bone marrow T lymphocytes in aplastic anaemia

Aplastic anaemia (AA) is considered as an immune‐mediated bone marrow failure syndrome. The mechanism is involved with a variety of immune molecules including interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α) and interleukins (ILs). IL‐27 is a novel member of the IL‐12 family, which mediates T cell response and enhances the production of IFN‐γ. However, little is known about the role of IL‐27 in the development of AA. This study investigated the role of IL‐27 and its receptor IL‐27R in the pathogenesis of AA. Both the mRNA expression of IL‐27/IL‐27R subunits in the bone marrow mononuclear cells (BMMNCs) and the levels of IL‐27 in the marrow plasma in AA were found to be higher than in controls. Increased IL‐27 correlated with the disease severity of AA. Subsequently, we stimulated marrow T lymphocytes with recombinant human (rh)IL‐27 and found that rhIL‐27 enhanced the production of TNF‐α and IFN‐γ in both CD4+ and CD8+ T lymphocytes from AA patients. We also detected increased TNF‐α and IFN‐γ in the supernatants of BMMNCs from AA patients after IL‐27 stimulation. In conclusion, our data suggest that elevated IL‐27 and IL‐27‐induced TNF‐α and IFN‐γ overproduction might be involved in the pathogenesis of AA.

Outcomes of optimized over standard protocol of rabbit antithymocyte globulin for severe aplastic anemia: a single-center experience.

Background Previous reports showed that outcome of rabbit antithymocyte globulin (rATG) was not satisfactory as the first-line therapy for severe aplastic anemia (SAA). We explored a modifying schedule of administration of rATG. Design and Methods Outcomes of a cohort of 175 SAA patients, including 51 patients administered with standard protocol (3.55 mg/kg/d for 5 days) and 124 cases with optimized protocol (1.97 mg/kg/d for 9 days) of rATG plus cyclosporine (CSA), were analyzed retrospectively. Results Of all 175 patients, response rates at 3 and 6 months were 36.6% and 56.0%, respectively. 51 cases received standard protocol had poor responses at 3 (25.5%) and 6 months (41.2%). However, 124 patients received optimized protocol had better responses at 3 (41.1%, P = 0.14) and 6 (62.1%, P = 0.01). Higher incidences of infection (57.1% versus 37.9%, P = 0.02) and early mortality (17.9% versus 0.8%, P<0.001) occurred in patients received standard protocol compared with optimized protocol. The 5-year overall survival in favor of the optimized over standard rATG protocol (76.0% versus. 50.3%, P<0.001) was observed. By multivariate analysis, optimized protocol (RR = 2.21, P = 0.04), response at 3 months (RR = 10.31, P = 0.03) and shorter interval (<23 days) between diagnosis and initial dose of rATG (RR = 5.35, P = 0.002) were independent favorable predictors of overall survival. Conclusions Optimized instead of standard rATG protocol in combination with CSA remained efficacious as a first-line immunosuppressive regimen for SAA.

Mutations of ASXL1 and TET2 in aplastic anemia

Acquired aplastic anemia (AA), characterized by pancytopenia in peripheral blood (PB) and bone marrow (BM) hypoplasia, is a bone marrow failure syndrome. The late evolution to myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML) is the most common clonal complication in refractory patients

The polymorphisms of human leukocyte antigen loci may contribute to the susceptibility and severity of severe aplastic anemia in Chinese patients.

The human leukocyte antigen (HLA) system has been reported to be involved in the development of aplastic anemia (AA). We compared and analyzed HLA-A, B, C, DRB1 and DQB1 alleles in 96 Chinese severe AA (SAA) patients to those in 600 healthy people chosen randomly from the China Marrow Donor Program to investigate the association of HLA class I and II allele polymorphisms with disposition of SAA and its severity degree in Chinese population. The DNA of patients was extracted and HLA high-resolution genotyping was conducted using polymerase chain reaction-sequence based typing technique. The gene frequencies of A(∗)02:01, A(∗)02:06, B(∗)13:01, DRB1(∗)07:01, DRB1(∗)09:01, DRB1(∗)15:01 and DQB1(∗)06:02 in SAA patients were significantly higher than in controls (all P<0.05), while the allelic frequencies of A(∗)02:07, A(∗)11:01 and B(∗)40:01 were notably lower in SAA patients than those in the controls (P = 0.001, 0.002, 0.005, respectively). Comparison among different severity of SAA groups exhibited significant increases of DRB1(∗)15:01 (P = 0.027) and DQB1(∗)06:02 (P = 0.013), but obviously lower frequencies of B(∗)46:01 (P = 0.023) and DRB1(∗)09:01 (P = 0.020) in non-VSAA patients than in VSAA patients. Thus, our results identified several risk and protective HLA alleles for Chinese SAA patients. Moreover, DRB1(∗)15:01, DQB1(∗)06:02, B(∗)46:01 and DRB1(∗)09:01 may be associated with severity of SAA.

The polymorphisms of T cell–specific TBX21 and STAT4 genes may contribute to the susceptibility of Chinese individuals to aplastic anemia

T cell-specific T-box transcription factor gene (TBX21) and signal transducer and activator of transcription 4 (STAT4) have been suggested as 2 candidate genes for conferring susceptibility to autoimmunity. We herein hypothesized that the polymorphisms of TBX21 and STAT4 genes might contribute to the susceptibility of Chinese individuals to aplastic anemia (AA) as T cell-mediated immune disease characterized by hypoplasia and pancytopenia. We investigated the distributions of TBX21 (T-1993C and T-1514C) and STAT4 (rs7574862) polymorphisms in 202 adult patients with AA and 195 healthy controls by polymerase chain reaction-restriction fragment length polymorphism. The frequencies of T-1993C (but not T-1514C) genotype and allele distribution were significantly higher in AA patients than in controls. The T allele (TT + TG genotypes) of STAT4 variant rs7574865 was associated with increased susceptibility of Chinese people to AA. Our results indicated that single nucleotide polymorphisms in TBX21 and STAT4 might contribute to susceptibility to AA in the Chinese population.

Increased Bone Marrow (BM) Plasma Level of Soluble CD30 and Correlations with BM Plasma Level of Interferon (IFN)-γ, CD4/CD8 T-Cell Ratio and Disease Severity in Aplastic Anemia

Idiopathic aplastic anemia (AA) is an immune-mediated bone marrow failure syndrome. Immune abnormalities such as decreased lymphocyte counts, inverted CD4/CD8 T-cell ratio and increased IFN-γ-producing T cells have been found in AA. CD30, a surface protein belonging to the tumor necrosis factor receptor family and releasing from cell surface as a soluble form (sCD30) after activation, marks a subset of activated T cells secreting IFN-γ when exposed to allogeneic antigens. Our study found elevated BM plasma levels of sCD30 in patients with SAA, which were closely correlated with disease severity, including absolute lymphocyte count (ALC) and absolute netrophil count (ANC). We also noted that sCD30 levels were positively correlated with plasma IFN-γ levels and CD4/CD8 T-cell ratio in patients with SAA. In order to explain these phenomena, we stimulated T cells with alloantigen in vitro and found that CD30+ T cells were the major source of IFN-γ, and induced CD30+ T cells from patients with SAA produced significantly more IFN-γ than that from healthy individuals. In addition, increased proportion of CD8+ T cells in AA showed enhanced allogeneic response by the fact that they expressed more CD30 during allogeneic stimulation. sCD30 levels decreased in patients responded to immunosuppressive therapy. In conclusion, elevated BM plasma levels of sCD30 reflected the enhanced CD30+ T cell-mediated immune response in SAA. CD30 as a molecular marker that transiently expresses on IFN-γ-producing T cells, may participate in mediating bone marrow failure in AA, which also can facilitate our understanding of AA pathogenesis to identify new therapeutic targets.

Differential expression profile of Th1/Th17/Th2-related chemokines and their receptors in patients with acquired bone marrow failure syndromes.

Th1/Th17/Th2-related chemokines (CXCL10/CCL20/CCL2) and their receptors (CXCR3/CCR6/CCR2) have rarely been studied in acquired bone marrow failure syndromes (BMFs). We evaluated the concentrations of CXCL10, CCL20 and CCL2 in plasma and BM fluid from aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH), and myelodysplastic syndromes (MDS) patients by enzyme-linked immunosorbent assay (ELISA). Real-time quantitative polymerase chain reaction (RT-PCR) was performed to determine mRNA expressions of those chemokines and their receptors. CCL20 levels in both plasma and BM fluid from AA, PNH and MDS patients were significantly higher than those in the corresponding samples from healthy controls; there were no differences in terms of CXCL10 and CCL2 levels. Significantly higher expressions of CXCR3 and CCL20 mRNA, meanwhile significantly lower expression of CCR2 mRNA in both peripheral blood mononuclear cells (PBMNCs) and bone marrow MNCs (BMMNCs) from AA and PNH patients were observed, with no differences in terms of CXCL10, CCL2 and CCR6 mRNA expressions. CCR6 mRNA expressions in both PBMNCs and BMMNCs from MDS patients were significantly higher than of the corresponding samples from controls. Our study implicated that CXCL10-CXCR3, CCL20-CCR6 and CCL2-CCR2 interaction might play important roles in Th1 and Th17 (but not for Th2) cells trafficking toward BM in acquired bone marrow failure syndromes.

Decreased expression of vitamin D receptor may contribute to the hyperimmune status of patients with acquired aplastic anemia

Acquired aplastic anemia (AA) is an immune‐mediated bone marrow failure syndrome. 1α,25‐Dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D, is a critical modulator of immune response via binding with vitamin D receptor (VDR). Previous studies have established that 1,25(OH)2D3 and VDR were involved in the pathogenesis of some autoimmune diseases. In this study, we evaluated the involvement of 1,25(OH)2D3 and VDR on T‐cell responses in AA. Plasma 25(OH)D3 levels were comparable between patients with AA and healthy controls. Surprisingly, VDR mRNA was significantly lower in untreated patients with AA than in healthy controls. Subsequent in vitro experiments revealed that 1,25(OH)2D3 treatment suppressed the proliferation of lymphocytes and inhibited the secretion of interferon‐γ, tumor necrosis factor‐α, and interleukin‐17A, meanwhile promoting the production of transforming growth factor‐β1 in patients with AA. Moreover, 1,25(OH)2D3 inhibited the differentiation of type 1 and Th17 cells but induced the differentiation of type 2 and regulatory T cells. Interestingly, VDR mRNA was elevated in healthy controls after 1,25(OH)2D3 treatment, but not in patients with AA. In conclusion, decreased expression of VDR might contribute to the hyperimmune status of AA and appropriate vitamin D supplementation could partly correct the immune dysfunction by strengthening signal transduction through VDR in patients with AA.

A novel tri-allelic mutation of TMPRSS6 in iron-refractory iron deficiency anaemia with response to glucocorticoid.

Benjamin Chaigne Caroline Dartigeas Lotfi Benboubker Franc ois Chaumier Marjan Ertault S everine Lissandre Marion Stacoffe Franc ois Maillot H el ene Blasco Patrick Vourc’h Philippe Colombat Emmanuel Gyan Service d’h ematologie et th erapie cellulaire, Centre hospitalier universitaire de Tours, Service de m edecine interne, Centre hospitalier universitaire de Tours, UMR CNRS 7292, Universit e Franc ois Rabelais, and Service de biochimie et de biologie mol eculaire, Centre hospitalier universitaire de Tours, Tours, France E-mail: emmanuel.gyan@univ-tours.fr