| Background:At present,the activated T cell-mediated destruction of hematopoietic stem/progenitor cells(HSPCs)is the widely-recognized pathogenesis of aplastic anemia(AA).As the most important antigen-presenting cells in humans,dendritic cells(DCs)can not only activate and expand naive T cells but also affect the differentiation of T cells into different subtypes.DCs play an important role in the upstream link of immune pathogenesis in AA patients,so it may be the reason for the breakdown of immune tolerance in AA patients.However,the role of DCs in the pathogenesis of AA has not been fully elucidated.Mesenchymal stem cells(MSCs)can regulate the immune function of DC,and the enhanced function of DC in AA patients may be related to the functional deficiency of AA-MSC.In addition,Abatacept.a cytotoxic T lymphocyte-associated antigen-4(CTLA-4)fusion protein,can block T cell activation and function by binding CD80 and CD86 and blocking their interaction with CD28 to exert immunosuppressive effects.Thus may be a potential direction for targeting DCs for the treatment of AA,but relevant reports have not been seen so far.Objectives:This study aims to investigate the potential role of DC in the pathogenesis of AA,and to explore the association between hyperreactivity of DC and immunosuppressive dysfunction of MSC.Hoping to investigate the feasibility of abatacept targeting DC in the treatment of AA.So that to lay the foundation for further elucidation of the pathophysiological mechanisms of AA and provide new directions for the treatment of AA.Methods:Peripheral blood(PB)and bone marrow(BM)samples from patients with healthy donors(HD)and AA patients of initial diagnosis,complete remission(CR)and partial remission(PR)were collected.(1)The proportion of DC subgroups in PB and BM was detected by flow cytometry;(2)The expression levels of CD80 and CD86 on DC,CD28 and CTLA-4 on T cells and regulatory T cells(Treg)cells were detected;(3)AA and HD peripheral blood CD14+ monocytes were separated and cultured in vitro to detect the expression of DC surface molecules,apoptosis and endocytosis;(4)CD3+ T cells were sorted and co-cultured with the two groups of DC induced culture in vitro to detect the ability of DC to stimulate the proliferation,activation and differentiation of T cells;(5)AA and HD bone marrow-derived MSCs were cultured in vitro and co-cultured with DC,respectively,to detect the phenotype of DC and the ability to stimulate the activation.proliferation and differentiation of T cells after co-culture;(6)Abatacept was added into the co-culture system of DC and CD3+ T cells.The expression of CD80 and CD86 on the DC surface and their effects on T cell proliferation,activation and differentiation were detected.Results:(1)Compared with HD,the proportion of myeloid DC(mDC)in PB and BM in newly diagnosed AA patients was significantly higher(PB:P<0.0001;BM:P=0.023),the proportion of plasmacytoid DC(pDC)decreased significantly(PB:P<0.0001;BM:P=0.001),the ratio of mDC to pDC increased significantly(PB:P<0.0001;BM:P=0.001);(2)The proportion of mDC and mDC/pDC in PB of newly diagnosed AA patients were positively correlated with the proportion of CD3+T lymphocytes,while the proportion of pDC was negatively correlated with CD4+ T lymphocytes.In addition,the pDC ratio in PB of newly diagnosed AA patients was positively correlated with neutrophil ratio,monocyte absolute value,reticulocyte(RET)ratio,RET absolute value and the proportion of myeloid leukemia fusion gene WT1,while negatively correlated with the proportion of lymphocyte.mDC/pDC ratio was negatively correlated with neutrophil ratio and RET absolute value,but positively correlated with lymphocyte proportion;(3)The expression of CD80 and CD86 on peripheral blood DC surface of newly diagnosed AA patients was higher than that of HD patients(P=0.00012;P<0.0001),DC surface CD80 in patients with CR and PR after treatment and CD86 was significantly down-regulated;(4)Compared with HD,the expression of CTLA-4 on CD4+T cells and Treg cells in PB of newly diagnosed AA patients was significantly decreased(P<0.0001;P=0.00016).In patients with CR and PR after treatment,CD4+ T cells and Treg cells surface CTLA-4 expression recovered from initial diagnosis;(5)Compared with HD,the expression rates of the co-stimulatory molecule CD80 and mature molecule CD83 of DC cultured in vitro in AA patients were significantly increased(P=0.00025;P=0.037).the apoptosis level was significantly decreased(P=0.015),and the ability to promote proliferation of CD3+,CD4+ and CD8+T lymphocytes was enhanced(P=0.003;P=0.012;P=0.007).At the same time,AA-DC significantly enhanced the ability of CD4+ T and CD8+ T cells to express CD25 and CD69(CD4+ T:P=0.033;P=0.045;CD8+ T:P=0.001;P<0.0001).In addition,the ability of AA-DC to promote the differentiation of Th0 cells to Th1 and Th17 cells was stronger than HD(P=0.031;P=0.032).The ability to promote the differentiation of Th0 to Treg cells was weaker than HD(P=0.002);(6)Both AA-MSC and HD-MSC inhibited the conversion of CD 14+monocytes into CD1a+ DCs,down-regulated the expressions of CD83 and CD86 on the surface of DC,and inhibited the immune function of DC.But there was no significant difference in the immunosuppressive effect of AA-MSC and HD-MSC on DC;(7)In vitro,abatacept can bind to CD80 on the surface of DC and significantly down-regulate the expression of CD80.Adding abatacept into the co-culture system of DC and CD3+ T lymphocytes significantly inhibited the proliferation of CD3+,CD4+and CD8+ T lymphocytes,and significantly inhibited the expression of CD25 and CD69 in CD4+ T and CD8+T cells.In addition,the ability of DC to promote the secretion of interferon-γ(IFN-γ)by CD4+and CD8+ T cells in AA patients was significantly weakened after the addition of abatacept,while the ability to promote the secretion of interleukin-4(IL-4)by CD4+ and CD8+ T cells was significantly enhanced.Conclusions:The increased number and hyperfunction of DC in AA patients may be involved in the immune pathogenesis of AA.The hyperfunction of DC in AA patients may not be related to the weakened immunosuppressive ability of MSC.Further studied are needed to explore the underlying mechanisms of the change in DC function in AA patients.Abatacept can block T cell activation by blocking the DC surface co-stimulatory molecule CD80 and is a promising strategy for AA treatment.Background:Activated cytotoxic T lymphocytes(CTLs)recognize the auto-antigens presented on hematopoietic stem/progenitor cells(HSPCs)through class I human leukocyte antigen(HLA)molecules and play an important role in the immune pathogenesis of aplastic anemia(AA).Previous reports demonstrated that HLA was related to the disease susceptibility and response to immunosuppressive therapy(IST)in AA patients.Recent studies have indicated that specific HLA allele deletions,which helped AA patients to evade CTL-driven autoimmune responses and escape from immune surveillance,may lead to high-risk clonal evolution.Moreover,regardless of the status of HLA mutation,patients who inherited specific HLA alleles had a more severe course of disease and more frequent clonal evolution.Therefore,HLA genotyping has a particular predictive value for the response to IST and the risk of clonal evolution.However,there are limited studies on this topic in the Chinese population.Objectives:To explore the relationship between HLA allele and the response to IST and the risk of high-risk clone evolution in Chinese patients with AA,and to determine the value of HLA genotyping in Chinese patients with AA.Methods:A total of 202 patients with HLA genotyping in our hospital from January 2012 to December 2020 were analyzed retrospectively.Among them,95 patients with AA were treated with IST.The relationships between different genotypes and baseline data,short-term,long-term response to IST and the high-risk clonal evolution were compared,and subgroup analysis was carried out according to age and disease severity.Then,the next-generation sequencing technique was used to compare the relationship between different genotypes and somatic mutation.Results:The alleles HLA-B*15:18 and HLA-C*04:01 were associated with a superior long-term response to IST(P=0.025;P=0.027,respectively),while the allele HLA-B*40:01 indicated an inferior result(P=0.02).The patients with HLA-A*02:07 allele had a poor reaction in the early stage after IST(P=0.019 at 3 months and P=0.027 at 6 months),but there was no significant difference in long-term response than the patients without those alleles(P=0.129).The allele HLA-A*01:01 and HLA-B*54:01 were associated with high-risk clonal evolution(P=0.032;P=0.01,respectively),and the former had a higher frequency in very severe AA(VSAA)patients than that in severe AA(SAA)patients(12.7%vs 0%,P=0.02).The HLA-DQ*03:03 and HLA-DR*09:01 alleles were associated with high-risk clonal evolution(P=0.015;P=0.015)and poor long-term survival(P=0.045;P=0.045)in patients aged≥40 years.Such patients may be recommended for early allogeneic hematopoietic stem cell transplantation rather than the routine IST treatment.Conclusions:Specific HLA alleles are associated with the response of AA patients to IST and the occurrence of high-risk clonal evolution.And HLA genotype has crucial value in predicting the outcome of IST and long-term survival in AA patients,and thus may assist an individualized treatment strategy. |
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