| Objective: In human leukocyte antigen(HLA)mismatched allogeneic hematopoietic stem cell transplantation(Allo-HSCT),the presence of donor-specific antibody(DSA),especially the complement C1q-binding DSA,is an important risk factor for inducing graft rejection(GR)and graft failure(GF),but the mechanism has not been fully elucidated.Additionally,there is still a lack of unified and effective treatment for highrisk sensitized transplant recipients.By analyzing the typical clinical cases,extracting scientific questions and furtherly using the peripheral blood of included patients and synthetic HLA peptides to establish an in vitro immune response model,the present study aimed to clarify the mechanism of synergistic effect of DSA-C1 q in regulating B cell immune and mediating GR,explore the prevention and treatment effect on GR by inhibiting complement activity at different levels in the complement classic pathway,so as to provide new ideas for immunotherapy of this kind of clinical problems.Methods: Clinical samples of DSA-positive patients who underwent allo-HSCT in our hospital from September 2020 to October 2021 were collected,the DSA level during HSCT was dynamically monitored,and the desensitization effect and transplant outcome were followed up,at the same time,the HLA-negative patients transplanted were collected as the negative control.By detecting the clinical samples and establishing the HLA-DSA-C1 q response model in vitro,the regulatory role of DSA and C1 q in the occurrence and development of GR was analyzed in depth:(1)The exploration of the association between DSA-mediated GR and complement classic pathway(CP)activation.To evaluate the actual occurrence of complement activation in patients during the posttransplant period,the dynamic expression levels of plasma complement proteins C1 q,C2,C4,C3 a and C5 a in plasma of patients were detected dynamically after transplantation by western blotting(WB)or enzyme linked immunosorbent assay(Elisa).(2)To investigate the synergistic effect of C1 q and DSA on mediating complementdependent cytotoxicity(CDC).Donor lymphocytes were incubated with patient serum containing DSA,and then flow cytometry was applied to the detection of immunoglobulin(Ig)isotypes and C1 q binding.Lactate dehydrogenase(LDH)assay was used to evaluate the CDC effect of DSA-positive serum on donor cells and the intervention effect of complement C1 inhibitor(C1-INH).The HLA antigen-antibody reaction model was further constructed in vitro,the serum of the sensitized patient(P1)suffered graft failure was used as the direct source of DSA and complement proteins,the specific HLA antigens were synthesized according to the mismatched HLA loci sequence between the patient P1 and the donor,then the synthesized HLA antigens were reacted with the serum of the patient P1 by Elisa,the levels of Ig G,Ig M,Ig A type DSA and related complement proteins C1 q,C3b,and membrane attack complex(MAC)were detected to further verify the DSA antibody isotype and the whole process of DSA-C1 q induced complement CP at the molecular level.(3)To explore the potential molecular mechanism of DSA-C1 q co-regulating the activity and function of B cells and mediating GR progression.The constructed HLADSA-C1 q reaction system was used to co-culture human peripheral blood B cells for 3days,the expression of CD38 on the cell surface of different groups was detected by flow cytometry to assess the cell maturity,cell viability was detected by CCK8 assay,the intracellular phosphorylation of t-AKT and p-AKT in cells as well as the secretion of Ig G DSA and total Ig G in culture supernatant were detected by WB and Elisa.The effects of exogenous C3 d or C3-INH(compstatin)on the activity of B cells and antibody secretion in the co-culture system were observed to determine whether DSA-C1 q promote the activation of B cells and the secretion of antibodies by inducing the generation of complement activation product C3 d.Results:(1)A total of seven DSA positive patients undergoing primary allo-HSCT were collected in the present study,one(P1)of them with high levels of HLA-I and II DSA suffered GF,the patient was insensitive to plasma exchange treatment and experienced elevated DSA rebound at HLA-B*40:01 and HLA-DRB1*09:01 locus,bone marrow chimerism detection at 3 weeks after transplantation showed that the patient suffered GR.The results of plasma complement protein detection in this patient showed persistent consumption of C1 q,C2 and C4 and up-regulated expression of active complement fragments C3 a and C5 a after HSCT,suggesting that the development of GR in the patient was accompanied by continuous complement CP activation.The transplantation engraftment was succeeded in other six DSA-positive patients,among them,one case(P2)had HLA-I and II DSA,and three cases(P3-P5)had HLA-I DSA,and two cases(P5-P6)had HLA-II DSA,two patients(P2,P6)were temporarily detected to have C1 q,C2,C4 consumption and C3 a,C5a production in the early stage after HSCT,but no longer occurred in the post cell engraftment and recovery period.The immune reconstitution of the included three HLA antibody-negative patients(P8-P10)recovered to normal after allo-HSCT,the results of plasma complement protein showed that there was no complement activation in these three patients after allo-HSCT.(2)Consistently high levels of Ig G and C1 q binding were detected after HSCT of DSApositive patient(P1)who occurred GR.Among the DSA positive patients with successful engraftment,low levels of Ig G,C1 q were detected in only one patient(P2)during the early post-transplant period,no significant levels of Ig G and C1 q was detected in the other five patients(P3-P7)with DSA positive as well as the HLA antibodies negative patients(P8-P10).The patient’s serum of DSA positive patient who suffered GR mediated significantly stronger CDC on donor cells,and the strength of cytotoxicity was dependent on the concentration of DSA.While the serums of DSA negative or positive patients who achieved successful engraftment showed lower CDC effect on the donor cells,C1 inhibitor can effectively interfere with DSA-mediated CDC.Significant level of Ig G DSA was detected by incubating the specific HLA antigens with the serum containing DSA of matched HLA loci,while neither Ig M nor Ig A was observed to participate in the reaction,meanwhile,significantly high levels of C1 q,C3and MAC were fatherly detected indicating that Ig G DSA can initiate the whole process of complement CP by recruiting C1 q after forming immune complexes with HLA antigens.(3)Compared with B cells cultured alone,the B cells co-cultured with HLA-DSA-C1 q reaction system showed significant up-regulated of CD19+CD38+ expression on B cells,higher cell viability,increased p-Akt/t-AKT ratio and higher Ig G production in the cell supernatant,which indicated that the HLA-DSA immune complex combined with C1 q could effectively activate B cells and induce the secretion of antibodies against specific antigens by activating the complement CP.Moreover,C3-INH and exogenous C3 d could completely inhibit and further promote the effect of DSA-dependent complement pathway on B cell activity and function,respectively,which confirmed that DSA mediated B cell activation by inducing the activation of complement classic pathway and the production of C3 d.Conclusions: DSA cooperated with C1 q to regulate the B-cell immunity of patients by activating complement CP,which mediates the occurrence and development of GR in allo-HSCT.On the one hand,Ig G DSA can directly destroy the hematopoietic stem cells from the donor by mediating CDC in an antibody concentration dependent manner.On the other hand,the active fragment of complement can aggravate GR by affecting the activity and function of recipient B cells,and promoting the secretion of de novo antibodies.Regulation of the complement CP by C1 or C3 inhibitor might be a key breakthrough in the precise prevention and treatment of GR in high-risk sensitized recipients of allo-HSCT. |
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