Jizhou Zhang

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

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.

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.

Anti-inflammatory effects of interleukin-35 in acquired aplastic anemia

Interleukin (IL)-35 is a novel regulatory cytokine primarily produced by regulatory T cells. Accumulating evidence has established that IL-35 plays an important role in the regulation of immune homeostasis, but little is known regarding the function of IL-35 in acquired aplastic anemia (AA). The aim of the present study was to investigate the expression of IL-35 and its effects on T cell response in AA. Our study demonstrated that significantly decreased plasma levels of IL-35 in AA were closely correlated with disease severity. In vitro stimulation experiment further confirmed the anti-inflammatory effects of IL-35, including suppressing the proliferation of CD4(+) and CD8(+) effector T cells, inhibiting the secretion of interferon-γ, tumor necrosis factor-α and IL-17 and promoting the production of transforming growth factor-β by peripheral blood mononuclear cells from patients with AA. Furthermore, we established that IL-35 inhibited the differentiation of type 1 T cells and T helper 17 cells but promoted the differentiation of type 2 T cells. Accordingly, the expression of T-bet and RORγt was inhibited while the expression of GATA3 was induced after IL-35 treatment. In summary, our findings suggested that decreased IL-35 might contribute to the loss of immune-tolerance and be critically involved in the pathogenesis of AA.

Impaired Autophagy in Adult Bone Marrow CD34+ Cells of Patients with Aplastic Anemia: Possible Pathogenic Significance.

Aplastic anemia (AA) is a bone marrow failure syndrome that is caused largely by profound quantitative and qualitative defects of hematopoietic stem and progenitor cells. However, the mechanisms underlying these defects remain unclear. Under conditions of stress, autophagy acts as a protective mechanism for cells. We therefore postulated that autophagy in CD34+ hematopoietic progenitor cells (HPCs) from AA patients might be impaired and play a role in the pathogenesis of AA. To test this hypothesis, we tested autophagy in CD34+ cells from AA samples and healthy controls and investigated the effect of autophagy on the survival of adult human bone marrow CD34+ cells. We found that the level of autophagy in CD34+ cells from AA patients was significantly lower than in age/sex-matched healthy controls, and lower in cases of severe AA than in those with non-severe AA. Autophagy in CD34+ cells improved upon amelioration of AA but, compared to healthy controls, was still significantly reduced even in AA patients who had achieved a complete, long-term response. We also showed that although the basal autophagy in CD34+ cells was low, the autophagic response of CD34+ cells to “adversity” was rapid. Finally, impaired autophagy resulted in reduced differentiation and proliferation of CD34+ cells and sensitized them to death and apoptosis. Thus, our results confirm that autophagy in CD34+ cells from AA patients is impaired, that autophagy is required for the survival of CD34+ cells, and that impaired autophagy in CD34+ HPCs may play an important role in the pathogenesis of AA.

Basal level of Th17 immune response is not enhanced in aplastic amemia

Acquired aplastic anemia (AA) is an immune mediated bone marrow failure syndrome which is associated with impaired T-cell immune responses. In this work we investigated the role of Th17 immune response in AA. Our results showed that the absolute numbers of circulating IL-17-producing CD4(+) T cells and CD4(+)CD161(+)CCR6(+) cells, the blood plasma level of IL-17, and the expression level of Th17 lineage-specifying transcription factor RORC in circulating CD4(+) T cells, were not increased in AA. These results suggest that Th17 immune response may not play an important role in the pathogenesis of AA.

Role of vitamin D receptor gene polymorphisms in aplastic anemia: a case–control study from China

Vitamin D receptor (VDR) gene and its polymorphisms are highlighted as candidate components for susceptibility to various autoimmune diseases. The aim of this study was to investigate the role of VDR polymorphisms (rs2228570, rs1544410, rs7975232, and rs731236) in aplastic anemia (AA).

ASXL1 single nucleotide polymorphisms rs62206933, rs117901891, and rs74638057 identify a subgroup of acquired aplastic anemia in Chinese Han patients

Sir, Acquired aplastic anemia (AA), a bone marrow failure syndrome, is characterized by pancytopenia in peripheral blood (PB) and bone marrow (BM) hypoplasia. AA shows genetic instability: Myelodysplastic syndrome (MDS)-related gene mutations were detected in 24 (17.4%) AA patients, including ASXL1 mutations in 14 (10.1%) patients who had a greater risk of transformation to MDS [1]. A total of 32 somatic mutations typically seen in MDS and other myeloid malignancies were detected in 29 (19%) patients with AA; 11 evolved to MDS, including 7 of 12 that were ASXL1-mutated [2]. Acquired somatic mutations of ASXL1 are common in AA and are relevant to myeloid transformation and prognosis; thus, we postulated that some single nucleotide polymorphisms (SNPs) in the ASXL1 gene may also associated with AA, which would be helpful to predict prognosis and risk of progression to MDS. In this study, we analyzed ASXL1 SNPs and biological features in 134 Han Chinese patients with AA. Our results showed that 8.2% (11 of 134) patients had the recurrent conjoined rs62206933, rs117901891, and rs74638057 genotype (WT1), which was closely associated with poor prognosis in patients with nonsevere AA (P = 0.025) and had a greater risk of transformation to MDS. Bone marrow aspirates were collected from patients with pancytopenia at a single institution (Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College). One hundred and thirty-four Han Chinese patients with AA (females, 57; males, 77; median age, 28.5 years; age range, 2 to 76 years) diagnosed according to standard criteria [3] had complete clinical data for this study. The date of the last follow-up was November 1, 2014 or progression to MDS/acute myeloid leukemia (AML), and the median follow-up was 37.3 months (range, 6–325 months). All patients were screened for paroxysmal nocturnal hemoglobinuria (PNH) clone, CD8 T cells and CD4 T cells by flow cytometry. The level of interleukin (IL)-1, IL-2, IL-4, IL-6, and tumor necrosis factor (TNF) in plasma was determined by enzyme-linked immunosorbent assay and chromosome analyses, and ASXL1 SNPs (reference sequence: NM_015338.5) were searched by direct sequencing after polymerase chain reaction amplification of genomic DNA (primers for sequencing are listed in Table S1). Annotated single nucleotide polymorphisms were based on the 1000 genomes database (http://www.ncbi.nlm.nih.gov/variation/tools/1000genomes). Of 134 patients with AA, five ASXL1 SNPs were identified in exon 12, including c.1954G>A, c.2985C>T, c.3519G>A, c.3692C>T, and c.3759U>C, which had been previously reported separately as rs3746609, rs62206933, rs117901891, rs74638057, and rs4911231 (http://www.ncbi.nlm.nih.gov/ projects/SNP/snp_ref.cgi); 8.2% (11 of 134) patients had the recurrent conjoined rs62206933, rs117901891, and rs74638057 genotypes; rs3746609 only appeared in one AA patient; and rs3746609 was common in both AA and MDS (Figure S1). Comparisons of pretreatment features between WT1 group and patients without WT1 genotypes (WT2 group) are shown in Table 1. WT1 was more frequent in patients aged <16 years (15.4%, 6 of 39) and in patients aged >50 years (16.7%, 3 of 18), compared to patients aged 16–50 years (2.6%, 2 of 77), and the difference was significant (P = 0.016). The median age of patients with WT1 was younger than those with WT2 (26.5 years vs 28.7 years), but this did not reach statistical significant (P = 0.690). The frequency of WT1 in very severe AA (VSAA), SAA, and non-SAA (NSAA) patients was 14.3% (2 of 14), 4.2% (2 of 48), and 9.7% (7 of 72), respectively (P = 0.253; not significant). The WT1 group had lower reticulocyte (31.8 9 10/L vs 36.5 9 10/L, P = 0.613), neutrophil (0.97 9 10/L vs 1.13 9 10/L, P = 0.495), and platelet (19.3 9 10/L vs 22.1 9 10/L, P = 0.601) counts, and significantly higher level of serum TNF than the WT2 group (2.04 ng/ml vs 1.53 ng/ml, P = 0.024). There was no significant difference between the WT1 and WT2 groups with regard to IL-1, IL-2, IL-4, and IL-6 levels; sex (P = 0.909); rate of PB lymphocytes (P = 0.385); positive rate of neutrophil-alkaline phosphatase (N-ALP) (P = 0.807); positive index of N-ALP (P = 0.503);

Involvement of interleukin-21 in the pathophysiology of aplastic anemia.

Recently enhanced T‐helper type 17 (Th17) immune responses and deficient CD4+CD25hiFoxP3+ regulatory T cells (Tregs) have been reported in acquired aplastic anemia (AA). Interleukin‐21 (IL‐21), a CD4+ T‐cell‐derived proinflammatory cytokine, modulates the balance between Th17 cells and Tregs. However, its role in AA remains unclear.

Persistent elevated bone marrow plasma levels of thrombopoietin in patients with aplastic anemia

Acquired aplastic anemia (AA) is an immune-mediated bone marrow (BM) failure syndrome. Plasma thrombopoietin (TPO) levels are elevated in AA patients with reduced blood counts. However, whether elevated TPO can recover to normal level in AA patients in complete remission (CR) after efficient immunosuppressive therapy (IST) is unknown. We measured TPO levels of BM plasma in a large cohort of AA patients with focusing on patients in CR after IST. Our data showed that BM plasma TPO levels were still high in patients who had reached normal blood counts levels for a long time. We speculate that downregulated expression of MPL (TPO receptor) in HSPC (hematopoietic stem and progenitor cells) may be related to the upregulated TPO in AA.