Immune-mediated Bone Marrow Failure Research Articles

The state of the art in the treatment of severe aplastic anemia: immunotherapy and hematopoietic cell transplantation in children and adults.

Acquired aplastic anemia (AA) is an immune-mediated bone marrow (BM) failure where marrow disruption is driven by a cytotoxic T-cell-mediated autoimmune attack against hematopoietic stem cells. The key diagnostic challenge in children, but also in adults, is to exclude the possible underlying congenital condition and myelodysplasia. The choice of treatment options, either allogeneic hematopoietic cell transplantation (alloHCT) or immunosuppressive therapy (IST), depends on the patient's age, comorbidities, and access to a suitable donor and effective therapeutic agents. Since 2022, horse antithymocyte globulin (hATG) has been available again in Europe and is recommended for IST as a more effective option than rabbit ATG. Therefore, an update on immunosuppressive strategies is warranted. Despite an improved response to the new immunosuppression protocols with hATG and eltrombopag, some patients are not cured or remain at risk of aplasia relapse or clonal evolution and require postponed alloHCT. The transplantation field has evolved, becoming safer and more accessible. Upfront alloHCT from unrelated donors is becoming a tempting option. With the use of posttransplant cyclophosphamide, haploidentical HCT offers promising outcomes also in AA. In this paper, we present the state of the art in the management of severe AA for pediatric and adult patients based on the available guidelines and recently published studies.

Quality of life after immune suppressive therapy in aplastic anemia.

Acquired aplastic anemia (AA) is a rare form of immune-mediated bone marrow failure, which can result in life-threatening infections or bleeding if left untreated. Treatment consists of either immune suppressive therapy (IST) or allogeneic stem cell transplantation (alloHSCT). While considerable research has been published regarding survival, response rate and toxicity of both treatments, knowledge on the impact on quality of life (QoL) is scarce. We used the recently developed AA-specific QoL questionnaire (QLQ-AA/PNH-54) to evaluate QoL in a single center cohort of AA patients who were successfully treated with IST. The 54 questions represent 12 different QoL domains. Results were analyzed for all patients and grouped based on hematologic response (complete response (CR) or partial response (PR)). Thirty-six successfully treated adult patients (15 in CR, 21 in PR) completed the questionnaire (median age 54years, range 21-71; median time since last IST 5years, range 0-41). Fatigue was experienced by 83% of patients. Even though total QoL scores did not significantly differ between patients with PR and CR (105 vs 92, p-value 0,17) there appeared to be a trend towards higher scores in patients with PR, especially in domains concerning psychological wellbeing. This trend was most clear in the domains fear of progression (2,12 in PR patients vs 1,73 in CR patients; p-value 0,08) and role functioning (2,22 vs 1,88; p-value 0,07). In conclusion, patients with AA continue to experience psychological and physical effects despite successful IST.

Formononetin reverses Treg/Th17 imbalance in immune-mediated bone marrow failure mice by regulating the PI3K/Akt signaling pathway

BackgroundSevere aplastic anemia (SAA) is a syndrome of bone marrow failure which is life-threatening. Recent studies have demonstrated that CD4 + T cell subsets, including T regulatory (Treg) and T helper 17 (Th17) cells, play a pivotal role in the pathogenesis of SAA. Formononetin (FMN) is a natural compound extracted from the traditional Chinese medicine Huangqi, which has the ability to regulate the imbalance of Treg/Th17 cells in some inflammatory diseases. Nevertheless, the therapeutic effect of FMN in SAA has yet to be definitively established. Therefore, the objective of this research was to investigate the effect of FMN on SAA and elucidate its underlying mechanism.MethodsIn vivo experiments, the mice were divided into the following five groups: control, model, low-dose FMN, high-dose FMN, and positive control cyclosporine A group. The immune-mediated bone marrow failure (BMF) mouse model was established by the total body X-ray radiation and lymphocyte infusion. After 10 days of continuous administration of FMN, the numbers of Treg/Th17 cells in the bone marrow and spleen were assessed by flow cytometry. The protein expressions of PI3K/Akt pathway in the bone marrow and spleen was assessed by immunohistochemistry and western blotting. In vitro, the impact of FMN on the differentiation of naive CD4 + T cells into Treg cells was investigated by flow cytometry and ELISA.ResultsIn comparison with the control group, the model group showed a reduction in bone marrow nucleated cells, a significant decrease in peripheral blood cells, and an altered CD8 + /CD4 + T cell ratio. These findings indicate the successful establishment of a mouse model of immune-mediated BMF. After FMN treatment, there were the increased levels of red blood cells and hemoglobin. In addition, FMN mitigated the bone marrow destruction and restored the CD8 + /CD4 + T cell ratio. Furthermore, in comparison with the control group, the model group showed the decreased levels of Treg cells and the increased levels of Th17 cells. After FMN treatment, there was a significantly increased number of Treg cells and a decreased number of Th17 cells. Additionally, FMN remarkably down-regulated the expression levels of PI3K and Akt proteins in immune-mediated BMF mice.ConclusionsFMN alleviates immune-mediated BMF by modulating the balance of Treg/Th17 cells through the PI3K/Akt signaling pathway.

Dioscin alleviates aplastic anemia through regulatory T cells promotion

ABSTRACT Objectives: Aplastic anemia (AA) is one of the immune-mediated bone marrow failure disorders caused by multiple factors, including the inability of CD4 + CD25 + regulatory T cells (Tregs) to negatively regulate cytotoxic T lymphocytes (CTLs). Dioscin is a natural steroid saponin that has a similar structure to steroid hormones. The purpose of this study is to look into the effect of Dioscin on the functions of CD4 + CD25+ Tregs in the AA mouse model and explore its underlying mechanism. Methods: To begin with, bone marrow failure was induced through total body irradiation and allogeneic lymphocyte infusion using male Balb/c mice. After 14 consecutive days of Dioscin orally administrated, the AA mouse model was tested for complete blood counts, HE Staining of the femur, Foxp3, IL-10 and TGF-β. Then CD4 + CD25+ Tregs were isolated from splenic lymphocytes of the AA mouse model, Tregs and the biomarkers and cytokines of Tregs were measured after 24 h of Dioscin intervention treatment in vitro. Results: Dioscin promotes the expression of Foxp3, IL-10, IL-35 and TGF-β, indicating its Tregs-promoting properties. Mechanistically, the administration of Dioscin resulted in the alteration of CD152, CD357, Perforin and CD73 on the surface of Tregs, and restored the expression of Foxp3. Conclusion: Dioscin markedly attenuated bone marrow failure, and promoted Tregs differentiation, suggesting the maintenance of theimmune balance effect of Dioscin. Dioscin attenuates pancytopenia and bone marrow failure via its Tregs promotion properties.

Diagnosis and treatment of aplastic anemia and lower-risk myelodysplastic neoplasms

Myelodysplastic neoplasms (MDS) are clonal hematological malignancies arising from gene mutations. Immunosuppressive therapies (IST) are effective in lower-risk MDS (LR-MDS) with characteristics such as hypoplastic marrow with low blasts or low ring sideroblasts, and with a small increase of PNH clones or decrease of megakaryocytes. Differential diagnosis of these LR-MDS cases from AA can be difficult, and precise diagnosis requires careful evaluation of bone marrow cellularity and dysplasia. To decide on an appropriate treatment strategy for LR-MDS, it is important to evaluate the underlying pathology, and preferentially select IST as first-line therapy in patients with features that indicate immune-mediated bone marrow failure.

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>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>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 >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.

The effectiveness of a novel treatment of TIM-3(-) NK cells infusion in murine models of immune-mediated bone marrow failure.

T-cell immunoglobulin and mucin-containing domain (TIM)-3 exerts its inhibitory effect on NK cells and participates in the immune pathogenesis of SAA. In this study, we aimed to explore a novel treatment method of TIM-3(+) NK or TIM-3(-) NK cell infusion in combination with immunosuppressive therapy for bone marrow failure (BMF)/aplastic anemia (AA) mice. BMF/AA mouse model was constructed. The TIM-3 expression and functional molecules on TIM-3(+) and TIM-3(-) NK cells of the BMF group, total body irradiation (TBI) group, and normal control (NC) group mice were detected by flow cytometry. After treatment, the general condition, whole blood cell and bone marrow cell (BMC) count, and immune condition of mice from each group were compared. TIM-3 expression in the peripheral blood NK cells of BMF mice was significantly lower than that of the TBI and NC group mice. TIM-3(-) NK cells expressed more NKG2D receptors than TIM-3(+) NK cells. The levels of P-Akt and PI3K in TIM-3(-) NK cells were higher than those in TIM-3(+) NK cells. On the 17th day after BMF induction, the weight, peripheral whole blood cell count, and BMC count of BMF mice decreased significantly compared with that of the NC group mice. The therapeutic effect in the TIM-3(-) NK cell treatment group was better than that in the TIM-3(+) NK cell treatment and CsA treatment groups. Concurrently, the ratio of CD4+ T and CD8+ T cells of BMF mice was significantly lower than that of the NC group mice. The therapeutic effect in CsA + TIM-3(-) NK group was more significant than that of the CsA treatment and the CsA + TIM-3(+) NK groups. In this study, we found that the general condition, peripheral whole blood cell and BMC count, and immune status of BMF mice improved significantly after CsA + TIM-3(-) NK cell treatment. These results may provide further insights into the immune pathogenesis of SAA and novel therapeutic ideas for improving SAA treatment.

Partial SAA patients benefit from delayed response of IST.

Severe aplastic anemia(SAA)is a severe disease characterized by immune-mediated bone marrow failure and pancytopenia. Immunosuppressive therapy (ATG plus CsA, IST) is the standard treatment for patients who are not suitable for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Some patients have a delayed response after 6 months of ATG, and unnecessary to be given secondary ATG or allo-HSCT. We attempted to distinguish patients who may get potential delayed response from those who were really not responsive to IST. We collected data from 45 SAA patients who were assessed no-response to IST at 6 months after rATG and failed to receive secondary ATG or allo-HSCT. CsA plus eltrombopag (EPAG) group has an extra 75% response rate while CsA maintenance group has an extra 44% response rate at 12 months. ATG was applied within 30 days after diagnosis, ATG dosage was suffificient (ATG/lymphocyte ≥2), and absolute reticulocyte count (ARC) was ≥30×109 /L at 6 months, indicated patients could get delayed response and benefifit from CsA maintenance. Addition of EPAG could give an even better response. Otherwise, secondary ATG or allo-HSCT treatment were recommended to be given immediately. https://www.chictr.org.cn/searchproj.aspx, identifier ChiCTR2300067615.

Effects of Quercetin-3-O-β-D-Glucuronide on Platelet Apoptosis and Activation in Mice with Immune-Mediated Bone Marrow Failure via PI3K/AKT Pathway

To investigate the effect of quercetin-3-O-β-D-glucuronide(QG) on platelet apoptosis and activation in mice with immune-mediated bone marrow failure via PI3K/AKT pathway. An immune-mediated bone marrow failure mice model was established and forty C57BL/6 mice were randomly assigned into 4 groups: normal group, model group,cyclosporine (CsA) group and QG group, with 10 mice in each group.The mice in CsA group were intragastrically administered with 0.027 g/kg CsA daily and the mice in QG group were intragastrically administered with 0.2 g/kg QG daily, while the mice in the normal group and model group were intragastrically administered with normal saline, respectively. After three days of modeling, the mice were euthanized, and the blood was collected through the tail vein. Part of the blood was used for blood routine examination, and the other part was used to prepare washed platelets. Some of the prepared washed platelets were used to detect the expressions of BAX, BAD, caspase-9, phosphatidylserine (PS), platelet activated complex-1 (PAC-1) and P-selectin by flow cytometry; some of washed platelets was used to determine the protein contents of PI3K, p-PI3k, AKT and p-AKT by Western blot; the other part of the washed platelets was used to measure the expressions of PI3K mRNA and AKT mRNA by real-time quantitative PCR (RT-qPCR). In the model group, the absolute platelet count of the mice was significantly decreased, and the levels of BAX, BAD, caspase-9, PS, PAC-1, and P-selectin in the washed platelets were significantly increased, compared to the normal group (P<0.05). At the same time, the expression levels of PI3K, p-PI3K, AKT, p-AKT proteins and PI3K mRNA, AKT mRNA in model group were significantly reduced, compared to the normal group (P<0.05). In the CsA group and QG group, the expression levels of BAX, BAD, caspase-9, PS, PAC-1, and P-selectin in the washed platelets were significantly reduced (P<0.05), while the levels of PI3K, p-PI3K, AKT, p-AKT and PI3K mRNA, AKT mRNA were significantly increased, compared to the model group (P<0.05). QG can reduce platelet apoptosis in mice with immune-mediated bone marrow failure, and activation of some platelets is involved, which may be related to the regulation of PI3K/AKT pathway.

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

Avatrombopag Added to Standard Immunosuppressive Therapy for Severe Aplastic Anemia

Acquired aplastic anemia is immune-mediated bone marrow failure disease. Antithymocyte globulin (ATG) and cyclosporine based immunosuppressive therapy (IST) is effective for severe aplastic anemia (SAA), but only 12-17% of patients achieve complete response at 6 months. Complete hematologic response predicts better survival of patients, so clinicians have been trying to further improve the complete response rate. Addition of eltrombopag improves both the overall response rate and complete response rate of SAA patients, but its hepatotoxicity is still worrying. Avatrombopag is a novel, oral, small-molecule thrombopoietin receptor agonist being developed to provide a predictable increase in platelet counts in immune thrombocytopenia and thrombocytopenia in patients with chronic liver disease. To date, there is no report on the effectiveness of avatrombopag in treating aplastic anemia. Here we enrolled 32 severe and very severe aplastic anemia (SAA/VSAA) patients who received ATG, cyclosporine and avatrombopag as first-line treatment between June 2021 and March 2022. Thirty-one patient received porcine ATG (20mg/kg.d for 5 consecutive days) and one patient received rabbit ATG (3mg/kg.d for 5 consecutive days). Avatrombopag was added within 3 months after ATG initiation at dose of 40-60mg per day and administered for at least 3 months. Cyclosporine was given orally at dose of 3.5-5mg/kg.d. There were 17 SAA patients and 15 VSAA patients. The median neutrophil count was 0.31 (range 0.01-1.01) ×109/L, median reticulocyte count was 8 (range 2-54) ×109/L, and median platelet count was 5 (range 1-16) ×109/L. All patients achieved the end-point of 3 months and 23 patients achieved end-point of 6 months. One patient with VSAA died at 1 month due to infections and was counted as no response at 3 months and 6 months. The rate of complete response was 15.6% and rate of overall response was 46.9% at 3 months. The rate of complete response at 6 months was 30.4% and rate of overall response was 65.2%. The complete response rate at 3 months and 6 months were both higher than historical results of standard IST (ATG and cyclosporine, 5.2% at 3 months and 14.6% at 6 months). Avatrombopag was safe, and there was no drug-related liver dysfunction. No patient discontinued avatrombopag due to adverse events. In conclusion, the addition of avatrombopag to IST was safe and associated with higher complete response rate among patients with SAA/VSAA.

Granulocytic myeloid-derived suppressor cells to prevent and treat murine immune-mediated bone marrow failure

AbstractMyeloid-derived suppressor cells (MDSCs) are immature myeloid cells that originate in the bone marrow (BM) and have immunoregulatory functions. MDSCs have been implicated in the pathogenesis of several autoimmune diseases but have not been investigated in immune aplastic anemia (AA). We examined the roles of granulocytic-MDSCs (G-MDSCs) in murine models of human AA and BM failure (BMF). As both prophylaxis and therapy, BM-derived G-MDSCs improved pancytopenia and BM cellularity and suppressed BM T-cell infiltration in major histocompatibility complex (MHC)-matched C.B10 BMF mice. These effects were not obtained in the MHC-mismatched CByB6F1 AA model, likely because of MHC disparity between G-MDSCs and donor T cells. Single-cell RNA sequencing demonstrated that G-MDSCs downregulated cell cycle–related genes in BM-infiltrated T cells, consistent with suppression of T-cell proliferation by G-MDSCs through reactive oxygen species pathways. Clearance of G-MDSCs in the MHC-mismatched CByB6F1 model using anti-Ly6G antibody facilitated T cell–mediated BM destruction, suggesting an intrinsic immunosuppressive property of G-MDSCs. However, the same anti-Ly6G antibody in the MHC-matched C.B10 AA model mildly mitigated BMF, associated with expansion of an intermediate Ly6G population. Our results demonstrate that G-MDSC eradication and therapeutic efficacy are immune context-dependent.

Levamisole Suppresses CD4+ T-Cell Proliferation and Antigen-Presenting Cell Activation in Aplastic Anemia by Regulating the JAK/STAT and TLR Signaling Pathways.

Aplastic anemia (AA) is a life-threatening disease primarily caused by a metabolic disorder and an altered immune response in the bone marrow (BM) microenvironment, where cytotoxic immune cells attack resident cells and lead to hematopoietic failure. We previously reported an efficient strategy by applying cyclosporin (CSA) combined with levamisole (CSA+LMS-based regimen) in the treatment of AA, but the immunoregulatory mechanism of LMS was still unclear. Here, the therapeutic effects of LMS were examined in vivo using the BM failure murine model. Meanwhile, the proportion and related function of T cells were measured by flow cytometry in vivo and in vitro. The involved signaling pathways were screened by RNA-seq and virtual binding analysis, which were further verified by interference experiments using the specific antagonists on the targeting cells by RT-PCR in vitro. In this study, the CSA+LMS-based regimen showed a superior immune-suppressive response and higher recession rate than standard CSA therapy in the clinical retrospective study. LMS improved pancytopenia and extended the survival in an immune-mediated BM failure murine model by suppressing effector T cells and promoting regulatory T-cell expansion, which were also confirmed by in vitro experiments. By screening of binding targets, we found that JAK1/2 and TLR7 showed the highest docking score as LMS targeting molecules. In terms of the underlying molecular mechanisms, LMS could inhibit JAK/STAT and TLR7 signaling activity and downstream involved molecules. In summary, LMS treatment could inhibit T-cell activation and downregulate related molecules by the JAK/STAT and TLR signaling pathways, supporting the valuable clinical utility of LMS in the treatment of AA.

Somatic mutations and clonal expansions in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder caused by a mutation of the X-linked PIGA gene, resulting in a deficient expression of glycosylphosphatidylinositol (GPI)-anchored proteins. While large clonal expansions of GPI(−) cells cause hemolytic symptoms, tiny GPI(−) cell populations can be found in healthy individuals and remain miniscule throughout life. The slight expansion of PNH clones often occurs in patients with acquired aplastic anemia (AA), an autoimmune bone marrow (BM) failure caused by autoreactive cytotoxic T lymphocyte attack on hematopoietic stem and progenitor cells (HSPCs). The presence of PNH clones is thought to represent the immune pathophysiology of BM failure and be derived from GPI(−) HSPCs that evaded immune attack against HSPCs. However, which mechanisms underlie the selection of GPI(−) HSPCs as well as their overwhelming clonal expansion remains unclear. Ancestral or secondary somatic mutations in GPI(−) HSPCs contribute to the clonal expansion of the aberrant HSPCs in certain patients with PNH; however, it remains unclear whether such driver mutations are responsible for clonal expansion of all patients. Increased sensitivity to TGF-β in GPI(−) HSPCs partly explains the predominance of GPI(−) erythrocytes in immune-mediated BM failure. CD4+ T cells specific to antigens presented by HLA-DR15 on HSPCs also contribute to the immune escape of GPI(–) HSPCs. Studying the evolution of HSPCs in AA and PNH will yield further information for understanding human autoimmunity and stem cell biology.

Abnormal Proteomics Profile of Plasma Reveals the Immunological Pathogenesis of Severe Aplastic Anemia.

Severe aplastic anemia (SAA) is an immune-mediated bone marrow failure characterized by pancytopenia. This study was aimed at uncovering proteins of plasma that were differentially expressed in SAA patients. 8 SAA patients and 8 health controls were enrolled and detected by data independent acquisition (DIA) technology. 154 differential expression proteins (DEPs) in plasma of SAA patients were identified. GO and KEGG analyses indicated DEPs were mainly involved in the immune system process. Specifically, C-C motif chemokine 18 (CCL18), matrix metalloproteinase-3 (MMP3), histidine-rich glycoprotein (HRG), and lactotransferrin (lactoferrin (Lf)) may play an important role in the immune pathogenesis of SAA. CCL18, MMP3, HRG, and Lf might be potential biomarkers for SAA.

Aplastic anemia: Pathophysiology

Bone marrow failure (BMF) syndromes are a heterogeneous group of benign hematological conditions characterized by uni- or multi-lineage marrow and/or peripheral blood cytopenia(s), and can be classified in constitutional or acquired syndromes based on pathophysiology. In inherited diseases, germline mutations occur in the hematopoietic stem and progenitor cell (HSPC) compartment causing a progressive loss of normal hematopoiesis, while in acquired syndromes, HPSC compartment disruption can be caused by an extrinsic direct damage by external cytotoxic agents on the stem cell pool or by an autoimmune attack against HSPCs. Aplastic anemia is an acquired immune-mediated BMF syndrome where marrow disruption is driven by a cytotoxic T cell-mediated autoimmune attack against HSPCs sustained by type I interferons that polarize the immune system toward T helper 1 responses in early phases and then toward T helper 17 and effector memory CD8+ T cell in late stage and severe disease. Cytokines and chemokines also play a crucial role in driving immune responses and HSPC growth inhibition and apoptosis, as described for interferon-γ and tumor necrosis factor α. In this review, we summarize current knowledge on acquired aplastic anemia pathophysiology.

Severe aplastic anemia after COVID-19 mRNA vaccination: Causality or coincidence?

The development of various autoimmune diseases has been reported after COVID-19 infections or vaccinations. However, no method for assessing the relationships between vaccines and the development of autoimmune diseases has been established. Aplastic anemia (AA) is an immune-mediated bone marrow failure syndrome. We report a case of severe AA that arose after the administration of a COVID-19 vaccine (the Pfizer-BioNTech mRNA vaccine), which was treated with allogeneic hematopoietic stem cell transplantation (HSCT). In this patient, antibodies against the SARS-CoV-2 spike protein were detected both before and after the HSCT. After the patient's hematopoietic stem cells were replaced through HSCT, his AA improved despite the presence of anti-SARS-CoV-2 antibodies. In this case, antibodies derived from the COVID-19 vaccine may not have been directly involved in the development of AA. This case suggests that the measurement of vaccine antibody titers before and after allogeneic HSCT may provide clues to the pathogenesis of vaccine-related autoimmune diseases. Although causality was not proven in this case, further evaluations are warranted to assess the associations between vaccines and AA.

The GPI-anchored protein CD109 protects hematopoietic progenitor cells from undergoing erythroid differentiation induced by TGF-β.

Although a glycosylphosphatidylinositol-anchored protein (GPI-AP) CD109 serves as a TGF-β co-receptor and inhibits TGF-β signaling in keratinocytes, the role of CD109 on hematopoietic stem progenitor cells (HSPCs) remains unknown. We studied the effect of CD109 knockout (KO) or knockdown (KD) on TF-1, a myeloid leukemia cell line that expresses CD109, and primary human HSPCs. CD109-KO or KD TF-1 cells underwent erythroid differentiation in the presence of TGF-β. CD109 was more abundantly expressed in hematopoietic stem cells (HSCs) than in multipotent progenitors and HSPCs of human bone marrow (BM) and cord blood but was not detected in mouse HSCs. Erythroid differentiation was induced by TGF-β to a greater extent in CD109-KD cord blood or iPS cell-derived megakaryocyte-erythrocyte progenitor cells (MEPs) than in wild-type MEPs. When we analyzed the phenotype of peripheral blood MEPs of patients with paroxysmal nocturnal hemoglobinuria who had both GPI(+) and GPI(-) CD34+ cells, the CD36 expression was more evident in CD109- MEPs than CD109+ MEPs. In summary, CD109 suppresses TGF-β signaling in HSPCs, and the lack of CD109 may increase the sensitivity of PIGA-mutated HSPCs to TGF-β, thus leading to the preferential commitment of erythroid progenitor cells to mature red blood cells in immune-mediated BM failure.

Granulocytic Myeloid-Derived Suppressor Cells in Murine Models of Immune-Mediated Bone Marrow Failure

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immature myeloid cells with immunoregulatory function. Limited published studies have reported conflicting data concerning the effects of MDSCs on autoimmune diseases and graft-versus-host disease. MDSCs can be divided into two major subsets, more abundant granulocytic (G-MDSCs) and monocytic (M-MDSCs). We examined G-MDSCs in murine models of human bone marrow failure (BMF).We first characterized bone marrow (BM) MDSCs from C.B10 mice. CD11b +Ly6G +Ly6C low G-MDSCs suppressed in vitro proliferation of both CD4 and CD8 T cells from C57BL/6 (B6) mice, while Ly6G +Ly6C - cells had no effect and Ly6G -Ly6C + cells increased T cell proliferation (Fig. 1A). We then tested G-MDSCs in vivo utilizing antibody-mediated cell depletion. Lymph node (LN) cells from B6 donor mice were injected into sub-lethally irradiated major histocompatibility-mismatched CByB6F1 mice to induce BMF. Anti-Ly6G antibody injection worsened cytopenias and BM hypoplasia, and they increased BM CD4 and CD8 T cell infiltration. In contrast, anti-Ly6G antibody injection in the minor histocompatibility-mismatched C.B10 BMF model improved platelet counts and reduced BM CD8 T cells. The pathogenic and protective effects in the two models correlated with differential anti-Ly6G antibody modulation of G-MDSCs: in the CByB6F1 model, anti-Ly6G antibody eradicated G-MDSCs in blood and BM while in the C.B10 model the same antibody generated a novel G-MDSC cell population, of identical Ly6C lowCD11b + phenotype but intermediate Ly6G expression, which was not present in the CByB6F1 animals after antibody injection. When we examined the efficacy of G-MDSCs in C.B10 BMF: Ly6G + cells were enriched from BM of normal C.B10 donors (94%-97% Ly6C lowLy6G +CD11b +), and injected at the time of marrow failure initiation. Mice infused with Ly6G + cells had significantly higher levels of WBC, RBC, platelets, and total BM cells, decreased BM CD4 and CD8 T cell infiltration, and improved BM cellularity. These results indicated a protective role of G-MDSCs. When G-MDSCs were injected at day 3 after LN cell infusion, treated mice again had higher levels of WBC, RBC, platelets, and total BM cells at day 14, alleviating BMF. As both prophylaxis and therapy, G-MDSCs decreased Fas expression and Annexin V binding of residual BM cells, suppressed intracellular levels of gamma interferon and tumor necrosis factor alpha, as well as cell proliferation protein Ki67 levels in BM CD4 and CD8 T cells, relative to BMF control mice. TotalSeq simultaneously detecting surface proteins and mRNA expression in whole BM mononuclear cells in the therapy model showed an increased proportion of myeloid cells and reduced proportion of T cells in marrow from G-MDSC-treated mice based on cell surface markers and marker gene expression (Fig. 1B). Gene pathway analysis revealed down-regulation of Fas expression and reduced program cell death in total BM cells and decreased expression of genes related to cell cycle in infiltrating T cells from Ly6G + cell-treated mice-both results consistent with suppression by G-MDSCs of T cell proliferation and protection of target BM cells from apoptosis. In vitro culture of T cells from B6 mice with G-MDSCs which had been isolated from C.B10 BM cells showed dose-dependent suppression of T cell proliferation.In conclusion, our results demonstrate an active role of G-MDSCs in protecting BM from immune-mediated destruction, by suppression of T cell proliferation in the BM. G-MDSCs might have clinical application as treatment in human aplastic anemia and other immune-mediated and autoimmune diseases. [Display omitted] DisclosuresYoung: Novartis: Research Funding.

Functional Characteristics of FAS-L Resistant and IL-2 Deprived Regulatory T Cells (Treg A) in Idiopathic Aplastic Anaemia

IntroductionAplastic anemia (AA) is an immune mediated bone marrow failure syndrome characterized by reduced numbers and dysfunctional regulatory T cells (Tregs). Using deep phenotyping, we have previously identified an immune signature that predicts clinical response to immunosuppression (IST) (Kordasti et al., Blood 2016). The so-called Treg A (CD4+CD25hiCD127loCD45RAhiCD95-CCR4lo) were higher in IST non-responders, whereas responder patients had a higher proportion of Treg B (CD4+CD25hiCD127loCD45RAloCD95+CCR4hi). The aims of the current study were to investigate the reasons for the reduction of specific Treg subset, the ex vivo expandability of the remaining Tregs in response to IL-2, their function and stability following expansion. The efficacy and longevity of these cells were tested in an animal model, with the aim of using autologous ex vivo expanded Tregs as a potential treatment for AA.Methods and ResultsFAS-L-induced apoptosis leads to reduction of Treg B, this effect can be reduced by low-dose IL-2Given the role of FAS-L positive T cells in the pathophysiology of AA pathophysiology and the fact that CD95 (FAS) is one of the significant non-redundant markers that defines Treg B, we tested the FAS-L sensitivity of these cells. After 5 hours culture with FAS-L (5 mg/ml), the number of apoptotic cells was significantly higher in Treg B compared to Treg A (3 replicates, p < 0.02). The effect was prevented by the addition of low-dose IL-2 (5 U/mL) to the culture.Treg expansion and their functionTotal Tregs from AA and healthy donors (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 polyclonally 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). Although Treg A had lower level of p-STAT5 in the presence of low-dose IL-2, this reached to a similar level to Treg B following culture with higher doses of IL-2. To investigate the potential differences between Treg A and B in terms of function and expandability, Treg A and B were sorted from 6 HDs and 4 AA patients, and then expanded for 4 weeks (as above). Treg A and B expanded in HDs (Treg A 45x, and B 33x fold increase, p = n.s.), whereas Treg A expanded at a higher rate compared to Treg B in AA (Treg A 2120x, and B 90x fold increase, p = 0.03). The in vitro expanded Treg A and B both suppress proliferation of conventional T cells (Tconv) (n = 6, p < 0.001, no difference between A and B, figure a). Treg stability was assessed by DNA methylation analysis using deep amplicon bisulfite sequencing of the Treg-specific demethylated region (TSDR) . TSDR CpG sites in the expanded Treg A and Treg B populations from HD and AA patients are 2% and 13% methylated respectively, confirming their stability.Treg function in vivoTo test the suppressive ability of these Tregs, we transplanted NSG-SGM3 mice with either HDs PBMCs alone (n = 3), or HDs ex vivo expanded Tregs alone (n = 3) or HDs PBMCs co-injected with HDs ex vivo expanded Tregs (n = 3). Mice injected with both PBMCs and Tregs received a further dose of Tregs at day 16 and then weekly injections until day 28. Mice injected with PBMCs only had worst overall survival compared to the mice with PBMCs + Tregs or Tregs only (13 versus 35 days, p < 0.0001) and developed acute GVHD during the first 10 days following the injection (day 13 PBMCs mice 17.36 g versus PBMCs + Tregs mice 22.7 g, p = 0.0052, figure b). There was no weight difference between PBCs + Tregs mice and Tregs only mice.ConclusionsIn summary, although the FAS-L sensitive Tregs, which are the most functional Tregs, are reduced in AA, the remaining Tregs in these patients are IL-2 responsive and can be expanded in vitro . We were able to sort Treg subpopulations (Treg A and B) from AA patients and show that they are expandable, stable and functional up to 8:1 ratio with Tconv. We have also tested the efficacy of ex vivo expanded Tregs in preventing GVHD in a mouse model. Overall, the current study suggests that in AA there may be an increased exposure to FAS-L and an insufficient level of IL-2, which selectively induce apoptosis of Treg B. Moreover, the ex vivo expansion of stable and functional AA Treg subsets is feasible. The expanded Tregs are likely to be able to control the inflammatory response in these patients, hence pave the way toward a novel cellular therapy for AA. [Display omitted] DisclosuresKordasti:Celgene: Research Funding.