Immune-mediated Aplastic Anemia Research Articles

Umbilical Cord Blood-Derived Cells Can Reconstruct Hematopoiesis in an Aplastic Anemia Animal Model.

To explore the efficacy and the mechanism of the umbilical cord-derived cells combined with cyclosporine A (CsA) in treating aplastic anemia (AA) in mice. Immune-mediated AA model mice were treated with CsA + UC mesenchymal stem cells (UC-MSC), CsA + umbilical cord blood regulatory T cells (UCB-Treg), UC-MSC, UCB-Treg, CsA alone, or blank control, respectively (n = 9 mice/group). CsA and the cell infusion was administered on d0. Routine peripheral blood testing was performed once weekly; bone marrow colony culture, bone marrow cell flow cytometry, peripheral blood T cell subsets, and serum inflammatory cytokines tests were performed on d14. Transcriptome sequencing was performed for cells from CsA + UC-MSC, CsA + UCB-Treg, and CsA groups to detect the possible related genes. Gene function cluster and signal pathway enrichment analysis were also performed. Blank control mice died due to pancytopenia within 21 days, whereas mice in other groups survived for >28 days. On d14, the CsA + UC-MSC and CsA + UCB-Treg groups had higher white blood cell (WBC) counts than the other groups (p < 0.05), along with higher burst-forming unit (BFU) and colony-forming unit-granulocyte, macrophage (CFU-GM) counts (p < 0.01). The CsA + UC-MSC group had the highest BFU count (p < 0.01). The CsA + UC-MSC and CsA + UCB-Treg groups exhibited the highest bone marrow CD34+ cell proportion (9.68% ± 1.35% and 8.17% ± 0.53%, respectively; p < 0.01). Tumor necrosis factor (TNF)-α and interleukin (IL)-2 levels in the CsA + UC-MSC group (p < 0.05) and TNF-α, interleukin-2, and interferon (INF)-γ levels in the CsA + UC-Treg group (p < 0.01) were lower than those in the CsA group. Compared with CsA treatment, CsA + UC-MSC significantly downregulated the histone methylation pathway (p < 0.05), whereas CsA + UCB-Treg significantly upregulated energy metabolism processes (p < 0.05). Treatment with CsA + UC-MSC upregulated superoxide dismutase activity compared with CsA + UCB-Treg treatment. Adding UC-MSC or UCB-Treg to CsA markedly enhanced the reconstruction of hematopoiesis in AA mice, with UC-MSC eliciting greater efficiency than UCB-Treg. Accordingly, the addition of these cells could further improve immune abnormalities.

Clonal Hematopoiesis in Cartilage-Hair Hypoplasia

Background: Cartilage-hair hypoplasia (CHH) is an autosomal recessive disorder characterized by short stature, hypotrichosis, combined immunodeficiency, and macrocytic anemia. CHH is caused by germline mutations in the RMRP gene encoding the RNA component of the mitochondrial RNA processing endoribonuclease. CHH is heavily enriched in the Finnish (1 in 23000 births) and the Amish (1 in 1340 births) populations. Transient macrocytic anemia is common in children with CHH, whereas severe hypoplastic macrocytic anemia is only present in 10% of pediatric CHH patients and can be treated with allogeneic hematopoietic stem cell transplantation (HSCT). The mechanism behind hypoplastic anemia in CHH is unknown: while erythroid differentiation from hematopoietic stem cells is impaired in vitro in CHH, immune dysregulation may also play a role in this phenotype. Clonal hematopoiesis is a common phenomenon of aging. Patients with inherited and immune-mediated bone marrow failure (BMF) show distinct spectra of clonal hematopoiesis mutations, reflecting context-specific selection of hematopoietic stem cell clones. However, the spectrum of clonal hematopoiesis is yet to be characterized in CHH. Methods: To assess the landscape of somatic alterations in hematopoietic cells in CHH, we performed whole exome sequencing (WES) on twelve bone marrow samples of four pediatric (age 4 to 13 years) and eight adult (age 20 to 52 years) unrelated subjects with CHH. Three children with CHH had severe hypoplastic anemia at the time of DNA sampling and three patients underwent HSCT during follow-up. Seven adults with CHH had normal blood counts and no severe immune phenotypes despite other clinical manifestations of CHH. Matched skin fibroblast samples were available for five CHH adults in the cohort for whom we performed tumor-normal variant calling. For the remaining cases, variant calling was based on tumor-only samples and stringent filtering to exclude germline variants. Additionally, we queried the FINRISK dataset, a Finnish cohort comprising WES and SNP microarray data from 10 129 participants, for the presence of CHH associated RMRP variants and evaluated their association with clonal hematopoiesis of indeterminate potential (CHIP) and mosaic chromosomal alterations (mCAs). Results: The median WES coverage of the bone marrow samples was 445x (347-505). Eleven out of 12 patients were homozygous for the RMRP founder variant n.71A&amp;gt;G and one patient was compound heterozygous for two pathogenic RMRP variants. We observed no somatic reversion of the germline RMRP variants. Two pediatric patients had variants in the CHIP genes: CHH1 (age 13 years) with TP53 hotspot variant R175C (variant allele frequency, VAF 0.006) and CHH2 (age 4 years) with BCORL1 P1681Qfs*20 (VAF 0.025). One fifty-year-old patient had a DNMT3A splicing variant c.1555-1G&amp;gt;C (VAF 0.022), likely consistent with CHIP observed in older general population. To identify clonal hematopoiesis outside the classical CHIP genes, we performed conservative filtering of the remaining somatic variant calls, resulting in a list of 36 high-confidence coding variants (VAF &amp;gt;0.02). These variants were present in 10 out of 12 patients; no recurrent genetic alterations were observed. Finally, the analysis of the FINRISK dataset revealed eleven participants with RMRP disease-associated variants; each participant had one heterozygous RMRP variant. No enrichment of CHIP variants or mCAs were observed in carriers of RMRP variants. Conclusion: We observed TP53 and BCORL1 mutations in the bone marrow samples of pediatric patients with CHH. In comparison to other CHIP-associated conditions, BCORL1 variants are disproportionately represented in immune-mediated aplastic anemia, potentially suggestive of similar immune-mediated origin of hypoplastic anemia in CHH. TP53 mutations are abundant in inherited BMF syndromes; however and in contrast to classical inherited BMF syndromes, CHH is not associated with increased risk of myeloid malignancies. No enrichment of classical or non-classical clonal hematopoiesis mutations were observed in adults with clinical CHH diagnosis or in RMRP variant carriers in the population-level FINRISK cohort. This is consistent with transient selection pressure on hematopoietic stem cells in childhood CHH. To our knowledge, our study is the first attempt to characterize the spectrum of clonal hematopoiesis in CHH.

Familial immune-mediated aplastic anaemia in six different families.

We studied the pathophysiology of aplastic anaemia (AA) in six different pairs of relatives without a family history of hematologic disorders or congenital AA. Five and four of the six pairs shared the HLA-DRB1*15:01 and B*40:02 alleles, respectively. Glycosylphosphatidylinositol-anchored protein-deficient blood cells were detected in eight of the 10 patients evaluated. In a mother-daughter pair from one family, flow cytometry detected leukocytes lacking HLA-A2 due to loss of heterogeneity in chromosome 6p. Whole-exome sequencing of the family pair revealed a missense mutation in MYSM1. These results suggest that genetic inheritance of immune traits might underlie familial AA in some patients.

Trisomy-6: The Ying and Yang of Chromosome 6 Aberrations

Karyotypic aberrations of chromosome 6 (chr.6), include duplications (trisomy 6; +6), deletion of its short arm (del6p), uniparental disomy 6 (UPD6p), as well as various microduplications and microdeletions. Among many important genes, the HLA locus is located on 6p, and loss of heterozygosity and allelic loss have been described as means of immune escape in immune-mediated aplastic anemia (AA) and in relapse of myeloid neoplasia following allogeneic bone marrow transplant. The pathogenesis of duplication of chr.6 appears more complex and might involve deletion of a putative tumor suppressor gene (TSG) causing duplication of the HLA locus with increased HLA expression. We have encountered several patients with +6 who inspired us to investigate the clinical and molecular phenotypes of +6. A total of 29 +6 MDS patients were identified, of whom 16 had isolated +6 (Iso +6). When compared to normal karyotype (NK) MDS (n=420), median age was younger in Iso +6 (70 vs 42 years, P<.0001). While no differences were noted for white blood counts, Iso +6 patients had lower platelets (median 27 vs. 112 x 103/uL, P=.19), were more frequently anemic (median hemoglobin 8.4 vs. 9.5 g/dL, P=.04) and showed higher frequency of hypocellularity at marrow evaluation (62.5% vs. 9.5%, P=0.0016) as opposed to NK MDS. Indeed, 67% of Iso +6 patients had a clinical picture reminiscent of AA or MDS phenotype which might have evolved from an antecedent AA phase. However, 67% of Iso +6 MDS had high risk MDS (IPSS-R ≥3.5) and 37% transformed to AML. Thus, our cohort indicated a phenotypic dichotomy which include hypocellular MDS/AA and high-risk disease with a poor prognosis. Of all +6 MDS, mutations most commonly affected TP53 (69%) followed by EZH2 (19%). Recurrent copy number variations (CNV) such as micro-duplications and microdeletions may be used to identify culprit genes. Indeed, microdeletions involving HLA have previously hinted towards the pathogenic role of HLA deletions. We obtained CNV data and compared them to conventional germline chromosome 6 CNV gains publicly available in the Database of Genomic Variants (DGV). Genes pertaining to significantly duplicated segments were then identified using the UCSC Genome Browser (https://genome.ucsc.edu/cgi-bin/hgGateway). In our cohort, in addition to 833 cases with del6p, we have identified microduplications involving 6p locus in 115 (11.5%) NK MDS cases. Overall, a total of 1198 6p copy gains were found in these patients, ranging in size 0.09-7.8Mb and with a median of 0.12Mb. The genomic architecture of each segment was investigated using the UCSC Genome Browser, from which only complete gene duplications that mirror an extra chromosomal copy were considered. Complete duplications were most commonly seen in loci containing genes of DUSP22 (12.2%) and most importantly HLA-DRB1 (10.4%), HLA-DRB5 (10.4%), HLA-DQA1 (9.6%) and MICA (9.6%). All of these five genes were significantly enriched in NK MDS as compared to healthy controls (DUSP22 3% P<0.0001, HLA-DRB1 0.4% P<0.0001, HLA-DRB5 0.5% P<0.0001, HLA-DQA1 0.5% P<0.0001 and MICA 0.2% P<0.0001) (Fig 1). Interestingly, while checking the Cosmic Cancer Browser database (https://cancer.sanger.ac.uk), we noticed that copy gains involving HLA-DRB1 and HLA-DRB5 were also more common in primary AML than in healthy controls from DGV (3.6 vs 0.4%, P<0.0001 and 3.6 vs 0.5%, P<0.0001, respectively). While the involvement of the HLA genes hints towards an immune-mediated mechanism underlying the pathogenesis of +6 MDS, further involvement in primary AML may suggest a common mechanism through which CN gains of the HLA locus promote myelo-neoplasia. We stipulate that while clonal deletion or UPD of HLA locus can be a result of immune pressure as seen in AA and during GvL, clonal duplication may lead to increased autoantigens presentation beyond the TCR activation threshold, triggering immune reactions ultimately resulting in hypocellular marrow and AA-like presentation. Alternatively, +6 and related lesions may lead to risk allele duplication and consequential more efficient presentation of a specific antigenic peptide. In more advanced diseases, deletion of TSG may also play a role, with progression reflecting a somatic pressure to overcome immune inhibition. HLA constellation and mutational studies will be presented in the context of the findings of 6p duplications and microdeletion in MDS and AA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Significance of a Miniscule GPI(-) Granulocyte Population in Patients with Bone Marrow Failure

[Background] Small (0.003-1.0%) populations of glycosylphosphatidylinositol (GPI)-deficient granulocytes (Grs) (sPNH-GPs) are often detected in patients with acquired aplastic anemia (AA) and low-risk myelodysplastic syndromes, and are thought to represent a marker of immune-mediated bone marrow (BM) failure. On the other hand, our recent studies revealed that miniscule (<0.003%) GPI(-) Gr populations (mPNH-GPs), which are derived from mono- or oligo-clonal PIGA-mutated hematopoietic stem cells (HSCs), are detectable in the peripheral blood (PB) of 80% of healthy individuals (HIs) and some cord blood (CB) samples (ASH 2021, #2181). The high prevalence of mPNH-GPs in HIs and the similarly high (60%) prevalence of PNH-phenotype Grs in AA patients suggest that the proliferation of PIGA-mutated HSCs may inevitably occur in immune-mediated AA. However, our earlier study failed to detect mPNH-GPs in some AA patients in long-term remission who possessed HLA-class I allele-lacking (HLA[-]) Grs after immunosuppressive therapy (IST) (unpublished data). In patients with long-standing AA, the prevalence of mPNH-GPs may be lower than that in HIs due to the replacement of mPNH-GPs by other aberrant Grs. [Methods] To test our hypothesis, we examined PB samples from 40 patients who developed AA 1-43 (median 11) years previously and were all negative for sPNH-GPs for the presence of mPNH-GPs. At the time of sampling, 28 including 6 patients possessing HLA(-) Grs were in remission after IST (n=8), thrombopoietin-receptor agonists (TPO-RAs, n=14), anabolic steroids (AS) (n=3) after failing IST, or spontaneous resolution (n=3). Eight patients were refractory to all therapies, 3 of which were transfusion dependent. The remaining 4 with non-severe AA had not been treated. Four patients with pure red cell aplasia (PRCA) who had a 12-32 year history of anemia responding to cyclosporine (CsA) were included in this analysis. PB samples from 93 HIs and 22 CB served as controls, and samples from a patient with chronic myeloid leukemia (CML) were used as a positive control for clonal hematopoiesis. mPNH-GPs were deemed positive when 4 or more CD11bhigh dots that formed tight clusters were present in the FLAER- fraction of Grs that were enriched using microbeads coated with PE-labeled anti-CD55 and anti-CD59 antibodies. [Results] We first determined the prevalence of mPNH-GPs in HIs (n=93) at different ages. The prevalence was approximately 80% in all different age groups with the exception of the CB, which showed a prevalence of 40.9% (Figure 1). In contrast, mPNH-GPs (0.101-1.998x10-3%) were detected only in 7 (17.5%) of the 40 patients with a long history of AA (Figure 1). These 7 patients were refractory to IST but were in remission at the time of sampling after responding to TPO-RAs (n=3, Figure 2) and AS (n=1); while 3 experienced spontaneous resolution of AA after cessation of treatment. Of the 33 AA patients who were negative for mPNH-GPs, only 8 were in remission, and 5 of the 8 possessed HLA(-) Grs that accounted for 75%-99% (median 96%) of the total Grs (Figure 2). All 4 patients with PRCA who were negative for sPNH-GPs possessed mPNH-GPs, despite the fact that they responded to CsA. The lack of mPNH-GPs in AA patients with complete clonal hematopoiesis with HLA(-) Grs prompted us to examine a CML patient whose Grs were 100% BCR-ABL-positive. mPNH-GPs were undetectable at the diagnosis, but became detectable 2 weeks after the initiation of nilotinib therapy, and increased after 6 months when the patient achieved cytogenetic remission (Figure 2). [Discussion] The persistence of mPNH-GPs in AA patients who failed to respond to IST suggests that immune mechanisms may have been not involved in the patients in whom GPI-deficient Grs remained miniscule. Given that mPNH-GPs were undetectable in AA patients possessing HLA(-) Grs and in many patients with long-standing AA refractory to IST, the presence of mPNH-GPs may be a marker of oligo- or poly-clonal hematopoiesis, which can be eliminated by the clonal expansion of HLA(-) HSCs or some other HSCs with intrinsic abnormalities in patients with long-standing AA, findings that are compatible with the absence of mPNH-GPs at the diagnosis in a CML patient. Lastly, the presence of mPNH-GPs without expansion in PRCA patients responding to CsA suggests that immunopathogenesis of PRCA is distinct from that of CsA-responsive AA, which turns mPNH-GPs into sPNH-GPs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Aplastic anemia following the severe acute respiratory syndrome coronavirus-2 vaccine.

Aplastic anemia following the severe acute respiratory syndrome coronavirus-2 vaccine.

The role of inflammation in hematopoiesis and bone marrow failure: What can we learn from mouse models?

Hematopoiesis is a remarkable system that plays an important role in not only immune cell function, but also in nutrient transport, hemostasis and wound healing among other functions. Under inflammatory conditions, steady-state hematopoiesis switches to emergency myelopoiesis to give rise to the effector cell types necessary to fight the acute insult. Sustained or aberrant exposure to inflammatory signals has detrimental effects on the hematopoietic system, leading to increased proliferation, DNA damage, different forms of cell death (i.e., apoptosis, pyroptosis and necroptosis) and bone marrow microenvironment modifications. Together, all these changes can cause premature loss of hematopoiesis function. Especially in individuals with inherited bone marrow failure syndromes or immune-mediated aplastic anemia, chronic inflammatory signals may thus aggravate cytopenias and accelerate disease progression. However, the understanding of the inflammation roles in bone marrow failure remains limited. In this review, we summarize the different mechanisms found in mouse models regarding to inflammatory bone marrow failure and discuss implications for future research and clinical practice.

The characterization analysis of pathogenic T cells in immune-mediated aplastic anemia mouse model

目的通过DsRed小鼠淋巴细胞输注加全身辐照（TBI）建立免疫介导的再生障碍性贫血（AA）C.B10-H2b/LilMcd (C.B10)小鼠模型，分析C.B10 AA小鼠模型中病理性T细胞在不同器官的动态归巢过程及特征。方法通过异体淋巴细胞输注加TBI建立AA小鼠模型，以TBI组为对照，分析外周血细胞和骨髓单个核细胞数，验证造模效果。在造模的第3、6、9、12天，处死小鼠并收集骨髓、脾脏、淋巴结和胸腺单个核细胞，流式细胞术分析DsRed+ T细胞的动态变化及效应记忆T细胞（TEM）、中央记忆T细胞（TCM）、初始T细胞（naïve T）和效应T细胞（TEFF）的比例。PCR Array分析AA小鼠不同组织内DsRed+病理性T细胞活化相关基因的表达谱。结果与TBI组相比，AA组小鼠在造模第9、12天出现严重骨髓衰竭，第12天时骨髓有核细胞数、外周血细胞数均显著减少（P值均<0.05）。AA组小鼠骨髓、脾脏、淋巴结内DsRed+ T细胞比例随时间增加而增加。骨髓中的DsRed+ T细胞比例在第3、6天低于脾脏与淋巴结，但在第12天时高于脾脏与淋巴结（P值均<0.05）。整个骨髓衰竭形成过程中，胸腺中DsRed+ T细胞比例均最低。在第12天时，AA小鼠骨髓、淋巴结和脾脏单个核细胞中，DsRed+CD3+CD4+ T细胞比例分别为（91.38±2.10）％、（39.78±6.98）％、（67.87±12.77）％，三者之间两两比较均有统计学意义（P值均<0.05）；DsRed+CD3+CD8+ T细胞比例分别为（98.21±1.49）％、（94.06±4.20）％、（96.29±1.23）％，差异均无统计学意义（P值均>0.05）。第9、12天，AA组小鼠骨髓中几乎所有的DsRed+CD4+ T或DsRed+CD8+ T细胞均为TEM，而淋巴结中包括TEM、TCM以及naïve T。PCR Array结果显示：骨髓中DsRed+CD4+或DsRed+CD8+ T细胞中CD38、IFN-γ、LAG3、CSF1、SPP1及TNFSF13B表达增高，但 DsRed+CD4+ T细胞中FOXP3、CTLA4表达降低。结论DsRed小鼠淋巴细胞输注可导致C.B10小鼠发生骨髓衰竭，并可以在该AA模型小鼠中追踪DsRed+病理性T细胞。在C.B10 AA模型小鼠体内，DsRed+CD8+和DsRed+CD4+ T细胞先归巢到脾脏与淋巴结，在其中扩增分化，最终转运至骨髓并转化为TEM。胸腺对异体T细胞的归巢过程更具抵抗性，提示胸腺在免疫介导AA模型的骨髓衰竭过程中可能是一个免疫豁免部位。在C.B10 AA模型小鼠的骨髓衰竭形成过程中，归巢到骨髓的DsRed+病理性T细胞较迁移到脾脏与淋巴结的细胞具有更强的免疫活性，而免疫抑制活性则减弱。

Kinetics of immune activated T cells in aplastic anemia mouse model

目的探索再生障碍性贫血（AA）小鼠模型中供者来源T细胞不同时间点动力学变化。方法构建AA小鼠模型，分别于不同时间点采用流式细胞术测定模型小鼠脾脏与骨髓内供者T细胞比例、活化分子表达、细胞周期及功能亚群，评估不同时期T细胞功能状态。结果①半致死剂量照射联合主要组织相容性抗原（MHC）半相合的淋巴结细胞输注成功构建T细胞免疫介导的AA小鼠模型。②AA小鼠脾脏中供者T细胞从移植第3天后开始明显浸润，并逐渐出现CD4+/CD8+比例倒置，第5天开始进入骨髓，以CD8+细胞浸润为主。③CD69在供者CD4+细胞中表达高峰晚于CD8+细胞，CD25在CD4+细胞与CD8+细胞中表达水平的变化趋势相同，但在CD8+细胞中的表达高于CD4+细胞。④脾脏内供者CD4+细胞S/G2/M期比例在移植后第3天即达高峰，约12％，而CD8+细胞中S/G2/M期比例在移植后第5天达高峰，约20％，且两者在进入骨髓后S/G2/M期比例均再次升高，但移植第3天后其比例在脾脏与骨髓CD8+细胞中持续高于CD4+细胞。⑤脾脏内免疫活化的T细胞经历短暂的中枢记忆T细胞（TCM）阶段后迅速分化为效应记忆T细胞（TEM），进入骨髓后部分TEM分化为效应细胞进一步发挥效应功能。结论AA小鼠模型中供者T细胞进入异体后迅速活化，5天内达增殖高峰，并完成向TEM细胞的分化，5天后开始进入骨髓进一步增殖损伤造血。

Bushen Jianpi Quyu Formula Alleviates Myelosuppression of an Immune-Mediated Aplastic Anemia Mouse Model via Inhibiting Expression of the PI3K/AKT/NF-κB Signaling Pathway.

Bushen Jianpi Quyu Formula (BSJPQYF), an experienced formula, has been used to treat aplastic anemia (AA) more than three decades. To determinate the effect of BSJPQYF on AA, we constructed an immune-mediated AA mouse model. All mice were divided into four groups: control, model, low dose (0.85 g/mL), and high dose (1.7 g/mL BSJPQYF) group. They were administered with different concentrations of BSJPQYF or normal saline for 14 days. Besides, components of BSJPQYF were analyzed by electrospray ionization and mass spectrometry (ESI-MS). Subsequently, mouse peripheral blood and femurs were collected, and bone marrow mesenchymal stem cells (BMSCs) were isolated by fluorescence-activated cell sorting (FACS). Among them, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), and interferon-γ (IFN-γ) were measured by ELISA assay, PI3K, AKT, p-AKT, NF-κB, p-NF-κB, TNF-α, and cleaved caspase-3 proteins were detected by western blot. Compared with standard compounds, we identified three compounds of BSJPQYF, namely, icariin, kaempferol and tanshinone iia, as potentially effective compounds for the treatment of AA. Through an in vivo study, we found the administration of BSJPQYF in high dose for 14 days could significantly increase peripheral blood count and bone marrow (BM) cells, meanwhile decrease TNF-α, TGF-β, and IFN-γ levels. Besides, it could suppress the protein expression of PI3K and the phosphorylation of AKT and NF-κB to restrict the protein expression of TNF-α, eventually reduce the protein expression of cleaved caspase-3. This study demonstrated the therapeutic effects of BSJPQYF in AA, which could alleviate myelosuppression through inhibiting the expression of the PI3K/AKT/NF-κB signaling pathway.

Role of imbalance of M1/M2 subsets of bone marrow macrophages in the pathogenesis of immune-mediated aplastic anemia in mice

目的探讨巨噬细胞对新型免疫介导的再生障碍性贫血（AA）模型小鼠发病的影响。方法以正常雌性B6D2F1（F1）小鼠作为空白对照（对照组，3只），应用双磷酸盐（Clodronate）脂质体尾静脉注射的方法去除小鼠的巨噬细胞（CLO组，4只），以PBS脂质体处理小鼠为阴性对照（PBS组，4只）。并在注射脂质体后以全身辐照（TBI）联合异基因淋巴细胞输注的方法制备F1 AA小鼠模型，分别记为CLO+AA组和PBS+AA组。骨髓活检病理学检查、外周血细胞计数观察各组小鼠AA的程度；流式细胞术检测小鼠骨髓CD4+/CD8+ T淋巴细胞亚群和巨噬细胞亚群；ELISA法检测各组小鼠外周血血浆中IFN-γ、TNF-α、G-CSF、GM-CSF、TPO、EPO的水平变化。结果与PBS+AA组相比，去除巨噬细胞的CLO+AA组小鼠骨髓脂肪化明显改善。PBS+AA组和CLO+AA组的HGB分别为（91.50±31.63）、（110.65±24.15）g/L，PLT分别为（90.85±121.90）×106/L、（461.13±483.45）×106/L，差异均有统计学意义（P值均<0.05）。PBS+AA组和CLO+AA组CD4+ T淋巴细胞占骨髓细胞的比例分别为（18.50±10.17）％、（7.58±8.00）％；CD8+ T淋巴细胞占骨髓细胞的比例分别为（36.23±6.40）％、（6.67±5.78）％。去除巨噬细胞后，CD4+和CD8+ T淋巴细胞比值均下降，但CD8+ T细胞减少程度更大，差异均有统计学意义（P值均<0.05）。和PBS+AA组相比，CLO+AA组小鼠的脾脏和骨髓巨噬细胞比例均明显减少，M1型巨噬细胞减少程度明显高于M2型，差异均有统计学意义（P值均<0.05）。PBS+AA组和CLO+AA组外周血中IFN-γ水平分别为（602.37±104.62）、（303.01±87.22）ng/L；TNF-α水平分别为（34.46±1.42）、（23.25±4.21）ng/L；GM-CSF水平分别为（9.32±2.00）、（64.85±12.25）ng/L；G-CSF水平分别为（5 891.78±2 632.39）、（17 784.16±488.36）ng/L；EPO水平分别为（9 667.31±4 501.95）、（2 078.02±897.56）ng/L；TPO的浓度分别为（6.36±2.09）、（11.67±2.86）ng/L，差异均有统计学意义（P值均<0.05）。结论该实验证实在新型F1 AA小鼠模型中巨噬细胞参与了AA的发病。提前去除巨噬细胞，F1 AA模型小鼠的血象和骨髓损伤程度得以改善。正负造血调控因子的改变和巨噬细胞M1/M2亚群失衡可能是造成AA微环境损伤、最终引起AA发病的重要机制。

How we('ll) treat paroxysmal nocturnal haemoglobinuria: diving into the future.

SummaryParoxysmal nocturnal haemoglobinuria (PNH) is characterized by complement‐mediated intravascular haemolysis, severe thrombophilia and bone marrow failure. While for patients with bone marrow failure the treatment follows that of immune‐mediated aplastic anaemia, that of classic, haemolytic PNH is based on anti‐complement medication. The anti‐C5 monoclonal antibody eculizumab has revolutionized treatment, resulting in control of intravascular haemolysis and thromboembolic risk, with improved long‐term survival. Novel strategies of complement inhibition are emerging. New anti‐C5 agents reproduce the safety and efficacy of eculizumab, with improved patient convenience. Proximal complement inhibitors have been developed to address C3‐mediated extra‐vascular haemolysis and seem to improve haematological response.

Somatic mutations in lymphocytes in patients with immune-mediated aplastic anemia

The prevalence and functional impact of somatic mutations in nonleukemic T cells is not well characterized, although clonal T-cell expansions are common. In immune-mediated aplastic anemia (AA), cytotoxic T-cell expansions are shown to participate in disease pathogenesis. We investigated the mutation profiles of T cells in AA by a custom panel of 2533 genes. We sequenced CD4+ and CD8+ T cells of 24 AA patients and compared the results to 20 healthy controls and whole-exome sequencing of 37 patients with AA. Somatic variants were common both in patients and healthy controls but enriched to AA patients’ CD8+ T cells, which accumulated most mutations on JAK-STAT and MAPK pathways. Mutation burden was associated with CD8+ T-cell clonality, assessed by T-cell receptor beta sequencing. To understand the effect of mutations, we performed single-cell sequencing of AA patients carrying STAT3 or other mutations in CD8+ T cells. STAT3 mutated clone was cytotoxic, clearly distinguishable from other CD8+ T cells, and attenuated by successful immunosuppressive treatment. Our results suggest that somatic mutations in T cells are common, associate with clonality, and can alter T-cell phenotype, warranting further investigation of their role in the pathogenesis of AA.

Sphingosine 1-Phosphate Receptor Modulator ONO-4641 Regulates Trafficking of T Lymphocytes and Hematopoietic Stem Cells and Alleviates Immune-Mediated Aplastic Anemia in a Mouse Model.

ONO-4641 is a second-generation sphingosine 1-phosphate (S1P) receptor modulator that exhibits selectivity for S1P receptors 1 and 5. Treatment with ONO-4641 leads to a reduction in magnetic resonance imaging disease measures in patients with relapsing-remitting multiple sclerosis. The objective of this study was to explore the potential impact of ONO-4641 treatment based on its immunomodulatory effects. Severe aplastic anemia is a bone marrow (BM) failure disease typically caused by aberrant immune destruction of blood progenitors. Although the T helper type 1-mediated pathology is well described for aplastic anemia, the molecular mechanisms driving disease progression remain undefined. We evaluated the efficacy of ONO-4641 in a mouse model of aplastic anemia. ONO-4641 reduced the severity of BM failure in a dose-dependent manner, resulting in higher blood and BM cell counts. By evaluating the mode of action, we found that ONO-4641 inhibited the infiltration of donor-derived T lymphocytes to the BM. ONO-4641 also induced the accumulation of hematopoietic stem cells in the BM of model mice. These observations indicate, for the first time, that S1P receptor modulators demonstrate efficacy in the mouse model of aplastic anemia and suggest that treatment with ONO-4641 might delay the progression of aplastic anemia. SIGNIFICANCE STATEMENT: ONO-4641 is a second-generation sphingosine 1-phosphate (S1P) receptor modulator selective for S1P receptors 1 and 5. In this study, we demonstrated that ONO-4641 regulates the trafficking of T lymphocytes along with hematopoietic stem and progenitor cells, leading to alleviation of pancytopenia and destruction of bone marrow in a bone marrow failure-induced mouse model mimicking human aplastic anemia.

Panaxdiol Saponins Component Promotes Hematopoiesis and Modulates T Lymphocyte Dysregulation in Aplastic Anemia Model Mice.

To investigate the potential efficacy of panaxadiol saponins component (PDS-C) in the treatment of aplastic anemia (AA) model mice. Totally 70 mice were divided into 7 groups as follows: normal, model, low-, medium-, high-dose PDS-C (20, 40, 80 mg/kg, namely L-, M-, H-PDS-C), cyclosporine (40 mg/kg), and andriol (25 mg/kg) groups, respectively. An immune-mediated AA mouse model was established in BALB/c mice by exposing to 5.0 Gy total body irradiation at 1.0 Gy/min, and injecting with lymphocytes from DBA mice. On day 4 after establishment of AA model, all drugs were intragastrically administered daily for 15 days, respectively, while the mice in the normal and model groups were administered with saline solution. After treatment, the peripheral blood counts, bone marrow pathological examination, colony forming assay of bone marrow culture, T lymphocyte subpopulation analysis, as well as T-bet, GATA-3 and FoxP3 proteins were detected by flow cytometry and Western blot. The peripheral blood of white blood cell (WBC), platelet, neutrophil counts and hemoglobin (Hb) concentration were significantly decreased in the model group compared with the normal group (all P<0.01). In response to 3 dose PDS-C treatment, the WBC, platelet, neutrophil counts were significantly increased at a dose-dependent manner compared with the model group (all P<0.01). The myelosuppression status of AA was significantly reduced in M-, H-PDS-C groups, and hematopoietic cell quantity of bone marrow was more abundant than the model group. The colony numbers of myeloid, erythroid and megakaryocytic progenitor cells in the model group were less than those of the normal mice in bone marrow culture, while, PDS-C therapy enhanced proliferation of hematopoietic progenitor cells by significantly increasing colony numbers (all P<0.01). Furthermore, PDS-C therapy increased peripheral blood CD3+ and CD3+CD4+ cells and reduced CD3+CD8+ cells (P<0.05 or P<0.01). Meanwhile, PDS-C treatment at medium- and high doses groups also increased CD4+CD25+FoxP3+ cells, downregulated T-bet protein expression, and upregulated GATA-3 and FoxP3 protein expressions in spleen cells (P<0.05). PDS-C possesses dual activities, promoting proliferation hematopoietic progenitor cells and modulating T lymphocyte immune functions in the treatment of AA model mice.

Rapamycin ameliorates immune-mediated aplastic anemia by inhibiting the proliferation and metabolism of T cells

Aplastic anemia (AA) is a serious blood system disease that threatens human health. At present, the main cause of this disease is believed to be immune hyperfunction. However, the specific metabolic mode involved in the occurrence of lymphocytes in AA is still unknown. In addition, whether rapamycin, a specific blocker of the mTOR signaling pathway, plays a therapeutic role by inhibiting lymphocyte metabolism remains unclear. We induced an AA mouse model through the classical immune-mediated pathway and simultaneously administered rapamycin intervention therapy. First, the AA-associated phenotypic changes and the efficacy of rapamycin in the treatment of AA were discussed. Second, the proliferation and metabolic pathway of bone marrow (BM) lymphocytes in AA and the effect of rapamycin on this process were determined. Finally, the expression levels of mTOR pathway-related proteins were analyzed. By inhibiting the mTOR signaling pathway, rapamycin could ameliorate the phenotype of the immune-mediated AA model and inhibit the proliferation of T cells by preventing cell cycle transition from G0 to G1 phase. Moreover, we found that mitochondrial oxidative phosphorylation is involved in the metabolic reprogramming of T cells in AA and that rapamycin can inhibit this process. We confirmed that mitochondrial oxidative phosphorylation is involved in the metabolic reprogramming of T cells in AA and further extended the mechanism of rapamycin in treating AA by inhibiting the mTOR signaling pathway. This viewpoint may provide a new therapeutic idea for clinical applications.

Development and application of the immune-mediated aplastic anemia mouse model

再生障碍性贫血（AA）的发病机制主要是免疫异常，免疫介导的AA小鼠模型的建立有利于AA发病机制的深入研究。制备免疫介导的AA小鼠模型有病毒感染、异基因淋巴细胞输注、全身辐照（TBI）加异基因淋巴细胞输注等方法。我们分别以干扰素-γ（IFN-γ）与白消安合用及TBI加异基因淋巴结细胞输注方法成功制备了两种AA小鼠模型，检测结果显示这两种模型更接近人类AA的特点，其中改良的杂交一代（F1）AA小鼠的制备方法在国内实验条件下易于操作，更适合用于推广，有望成为新的AA基础研究平台。.

Altered expression of leptin and leptin receptor in the development of immune-mediated aplastic anemia in mice.

The current study aimed to explore the levels of leptin (LEP) and LEP receptor (LEP-R) on the progression of aplastic anemia (AA) with bone marrow fat conversion. An AA model was developed by infusing C57BL/6 lymph node cells into BALB/c mice. At 0, 3, 6, 9, 12, 15 and 18 days after modeling, routine blood counts, bone marrow biopsy slides, lymphocyte subsets (CD4+ and CD8+ T cells) and cytokine levels [including interleukin (IL)-2, IL-4, IL-5 and interferon-γ] were assessed. LEP and LEP-R levels in peripheral blood serum, mesenchymal stem cells (MSCs) and bone marrow were also analyzed by enzyme-linked immunosorbent assay, polymerase chain reaction and immunohistochemistry. The relevance of LEP, LEP-R and other factors was analyzed by Pearson's correlation analysis. Peripheral pancytopenia (reduced count of white blood cells, red blood cells, hemoglobin and platelets), abnormal immune factor levels and histological changes in bone marrow sections were detected in the AA model mice, suggesting that these mice mimicked the pathological changes commonly observed in AA. In addition, following the establishment of AA, the LEP level was gradually increased and the LEP-R level was reduced in the mice over time (P<0.05). The expression of adipogenic genes, including CCAAT/enhancer-binding protein (C/EBP)α, C/EBPβ and peroxisome proliferator-activated receptor γ, was markedly increased, while the expression of the osteogenic gene runt-related transcription factor 2 was reduced compared with the levels in the control group (P<0.05). Taken together, damage to LEP-R may lead to dysregulation of LEP and the enhancement of MSCs to differentiate into adipocytes, resulting in excessive fat in bone marrow of AA patients.

Chinese Herbal Formula, Modified Danggui Buxue Tang, Attenuates Apoptosis of Hematopoietic Stem Cells in Immune-Mediated Aplastic Anemia Mouse Model.

A derivative formula, DGBX, which is composed of three herbs (Radix astragali, Radix Angelicae sinensis, and Coptis chinensis Franch), is derived from a famous Chinese herbal formula, Danggui Buxue Tang (DBT) (Radix astragali and Radix Angelicae sinensis). We aimed to investigate the effects of DGBX on the regulation of the balance between proliferation and apoptosis of hematopoietic stem cells (HSCs) due to the aberrant immune response in a mouse model of aplastic anemia (AA). Cyclosporine (CsA), an immunosuppressor, was used as the positive control. Our results indicated that DGBX could downregulate the production of IFNγ in bone marrow cells by interfering with the binding between SLAM and SAP and the expressions of Fyn and T-bet. This herbal formula can also inhibit the activation of Fas-mediated apoptosis, interferon regulatory factor-1-induced JAK/Stat, and eukaryotic initiation factor 2 signaling pathways and thereby induce proliferation and attenuate apoptosis of HSCs. In conclusion, DGBX can relieve the immune-mediated destruction of HSCs, repair hematopoietic failure, and recover the hematopoietic function of HSCs in hematogenesis. Therefore, DGBX can be used in traditional medicine against AA as a complementary and alternative immunosuppressive therapeutic formula.

Research on Xijiao Dihuang Decoction suppressing platelet apoptosis in immune-mediated aplastic anemia based on mitochondrial mediated pathway

Research on Xijiao Dihuang Decoction suppressing platelet apoptosis in immune-mediated aplastic anemia based on mitochondrial mediated pathway