Polyreactive Antibodies In Kidney Transplant Recipient Contribute To HLA Reactivity And Influence Long-term Kidney Allograft Loss

Antibody mediated graft rejection has been a major cause of allograft loss and one ofcritical factors to kidndy graft long-term survival. Thus far, it is commonly accepted thatantibody mediated rejection and graft loss has been mainly attributed to the development ofDSA. The contribution of NDSA or non-HLA antibodies has been largely overlooked. Casesof antibody mediated rejection in the absence of DSA have also been reported. Additionalstudies support a contribution of non-HLA antibodies to pre-sensitization. More specifically,serum IgG reactivity to autoantigens such as vimentin, oxidized lipids has been associatedwith increased rejection rates and reduced graft survival. Besides DSA, non-donor specificantibodies and non-HLA antibodies have been important factors to affect graft survival.Sensitizing antibodies after transplantation are primarily the antibodies reactive to humanleukocyte antigen which expressed on donor cells. It is very common that NDSA aredetectable in kidney transplant recipient besides DSA. However, the exact causes of thedevelopment of NDSA without specific immune sensitizing is obscure. The most widelyacceptable explanation is the theory of cross reaction that antibodies cross-reactive to multipleHLA alleles recognize “public” epitopes shared among polymorphic HLA alleles. However,NDSA could be detected in the absence of DSA and when patients do not have history ofprior immunizing events. It has also been reported that the mean time of development ofNDSA is shorter than development of DSA. This phenomenon could not be merely explainedby the theory of cross reaction.Natural antibodies are distinct antibodies that develop without any evidence ofimmunization. Natural antibodies have the capacity to react different kinds of antigens whichdon’t share the related antigenic determinant, hence revealing a pattern of polyreactivity.Natural antibodies play an essential role in human innate immune. An important characteristicof natural antibodies is their capacity to react to apoptotic cells. Thus far, the studies about polyreactive antibodies focus on human antoimmune disease,whereas, the research about polyreactive antibodies in the context of organ transplantation hasbeen rarely reported. In our studies, we detected the correlation between polyreactiveantibodies and HLA antibodies at the level of monoclonal B cells, monoclonal antibodies andthe level of serum of patients who had underwent kidney transplantation, respectively. In themeantime, we also performed the studies about the correlation between polyreactiveantibodies in kidney transplant recipients and antibody mediated rejection as well aslong-term kidney graft survival.Results:1、The correlation between polyreactive antibodies and development of HLA antibodies.In our studies, we isolated a number of B cells from PBMC of a kidney recipient and anexplanted kidney graft, respectively and obtained monoclonal B cell clones though invitroimmortalization. All the B cell clones secreted antibodies and some of them had capacity toreact to multiple antigens such as LPS, dsDNA, insulin as well as cell lysates, hence showinga pattern of polyreactivity which is different from “lock-key” specific reactivity betweenantigen and antibody. Moreover, we found that all polyreactive antibodies had the capacity toreact to apoptotic cells. Noticeably, we found that some polyreactive monoclonal antibodiescould react to multiple HLA antigens, hence revealing the correlation between polyreactiveantibodies and HLA antibodies at the clone level. Then we extended our research to the levelof serum and found that compared with serum negative reactivity to HLA, the serumspecimens with positive reactivity to HLA showed higher IgG polyreactivity (P=0.009, P <0.001, P <0.001, respectively). Using Spearman correlation test, we observed a strong positivecorrelation between IgG polyreactivity and HLA reactivity (P=0.004, P<0.001, P=0.019).Among patients with positive HLA Class I antibodies, serum samples with higherpolyreactivity recognized more HLA class I molecules compared to serum samples with lowerpolyreactivity (P=0.030), hence revealing the correlation between polyreactive antibodies andHLA antibodies at the serum level.2、The correlation between pre-transplant serum polyreactive antibodies and antibody mediated rejection as well as long-term graft survivalThrough the studies about serum reactivity of300kidney transplant recipients, we foundthe reactivity of polyreactive IgG in pre-transplant patients was significantly higher than thatin healthy subjects (P=0.011). The difference observed in IgG polyreactivity between the twogroups was not solely due to serum immunoglobulin concentration.The mean duration of follow-up for300patients included in this retrospective study was81.2±35.3months.46patients lost their grafts, these patients had significantly higher IgGpolyreactivity compared to those with functioning graft (P<0.001). Remarkably, among the46patients who lost their grafts, IgG polyreactivity was significantly increased in those whosegraft loss was attributed to AMR compared to patients with other causes of graft loss(P=0.033). Kaplan-Meier survival curve showed that the long-term graft survival wassignificantly lower in pre-transplant patients with higher polyreactive IgG (P<0.001). FurtherCox proportional hazards analysis showed that polyreactive IgG was independent risk factorfor graft loss (Hazard ratio=2.271, P <0.001).Our studies showed that IgG reactivity to apoptotic cells was almost exclusivelymediated by IgG1and IgG3subclasses. Antibodies reactive to apoptotic cells could alsoactivate complement, leading to C4d deposition on target cells. Moreover, we observed astrong association between IgG1and IgG3reactivity to apoptotic cells and C4d deposition ontarget cells (P<0.001, P=0.005).Our retrospective studies found the significant association between polyreactiveantibodies and HLA antibodies not only at the level of monoclonal antibody but also at thelevel of serum of kidney transplant recipients. Besides the cross reactivity theory, we provideda new proof to explain the development of HLA antibodies especially NDSA. Polyreactiveantibodies correlated with antibody mediated rejection and they were independent risk factorsfor graft loss. Our findings have significant implications for the assessment of transplantcandidates. Taking polyreactive antibodies into consideration in addition to anti-HLAantibodies during pre-transplant evaluation would likely improve the risk assessment of thegraft outcome. Moreover, patients with high polyreactivity could also possibly benefit fromdesensitization treatments.