Effects Of Rituximab On T Lymphocyte Subsets In The Treatment Of Idiopathic Membranous Nephropathy And Its Clinical Significances

BackgroundIdiopathic membranous nephropathy(IMN)is a common pathological type in adult nephrotic syndrome with a peak age of 40 years.At present,IMN is considered to be an antibody-mediated autoimmune disease,and the main target antigen is M-type phospholipase A2 receptor(PLA2R).With the progress of research,more auto-antigens,such as thrombospondin type 1 domain-containing 7A(THSD7A)and neural tissue encoding protein with EGF-like repeats(NELL1),have also been discovered.Rituximab(RTX)is a type of human/mouse chimeric monoclonal antibody which targeted CD20+B lymphocytes,RTX can specifically identify the surface antigen on B lymphocyte and selectively inhibit B lymphocyte proliferation,which will give rise to the diesease of antibody and inhibit the formation of immune complex,and than the level of proteinuria has sustained remission.RTX has become the first-line treatment of IMN.In addition to the inhibition of B cells,some research also showed that RTX in the treatment of IMN patients can achieve continuous clinical remission when the B cells are not completely exhausted at early stage or the number of B cells begins to recover at late period.Moreover,minimal change disease(MCD)and focal segmental glomerular sclerosis(FSGS)are podocytic disease,most of research thought the pathogenesis mainly related to T lymphocyte dysfunction,Rituximab has achieved significant efficacy in the treatment of such diseases,These all suggest that the effects of rituximab are not limited to B cells.T lymphocytes are an important part of human immunity,complementary to the role of B lymphocytes.T lymphocytes can protect our body from foreign microbial invasion by auxiliary B cell response and secretion of various inflammatory factors.Cellular immunity is mainly through activation of T cell subsets to finish immune response.According to different phenotype,T cells are classified into three groups including CD3+CD4+,CD3+CD8+and CD3+CD4-CD8-T cells.Different subsets of T lymphocytes play different roles in the immune and inflammatory responses.Regulatory T cells(Treg)is a special type of T lymphocytes in CD4+T cell,in addition to effectively inhibiting autoimmune responses,Treg may also participate in the induction of graft tolerance and regulation of tumor immunity.Thus,Treg plays a key role in maintaining the stability of the internal environment.A large number of studies have confirmed that the deregulation of Treg and function can lead to common autoimmune diseases,such as systemic lupus erythematosus(SLE),immune thrombocytopenia(ITP),multiple sclerosis(MS),etc.And some studies have found that Treg is also reduced in IMN patients.The proliferative differentiation of Treg in the thymus and make functions in the periphery mainly depends on the activation of the IL-2/IL-2R singnal pathway,and IL-2/IL-2R signal also helps to maintain the homeostasis of Treg in the periphery.IL-2Rα chain is also known as T cell activation antigen or CD25.When the antigen promotes the activation of receptor(TCR)on the surface of T cells,T lymphocytes secrete more IL-2,and then positive feedback increases the expression of CD25 on the surface of T lymphocytes.Few researches on the changes of T lymphocyte subsets in peripheral blood and the relationship between IL-2/IL-2R and Treg in IMN.CD4+T can also secrete a variety of inflammatory cytokines,which can be divided into Thl-related factors such as IFN(interferon)-γ to mediate cellular immunity,Th2-related factors such as interleukin-4(IL-4)and interleukin-10(IL-10)to mediate humoral immunity,Th17-related factors such as interleukin-17a(IL-17a)are involved in promoting pathological inflammation.In this study,we used the flow analysis to detect the counts of T lymphocyte subsets and simultaneously evaluated the level of cytokines and hypersensitive C-reactive protein(Hs-CRP),which will more comprehensively assess the inflammatory status and severity of the disease in IMN patients.In the first part of this research,we use a cross-sectional study of 58 IMN patients to analyzed the changes of peripheral blood T lymphocytes subsets and related cytokines,and then exploring the correlation between T lymphocytes subsets and clinical indicators to reveal their clinical significance.In the second part,we selected 33 patients with complete 6-month follow-up data after a course of rituximab from the first part to further analyze the changes of the abnormal subsets at baseline after rituximab treatment and the relationship between T cells and prognosis,which aim to provide a basis for multi-target evaluation of the role of rituximab in membranous nephropathy.Chapter 1 Changes in peripheral blood T lymphocytes in patients with idiopathic membranous nephropathy and its correlation with clinical indicatorsAbstract:Objective:By examining the level of T lymphocyte subsets in the peripheral blood of patients with idiopathic membranous nephropathy,the abnormal T cell subsets were selected,and exploring their role in the pathogenesis of idiopathic membranous nephropathy,so as to provide a basis for treatment and prognosis.Methods:A total of 58 patients with idiopathic membranous nephropathy were selected from the Department of Nephrology in the First Affiliated Hospital of Soochow University from January 2020 to December 2022,and 25 healthy donors were selected as the control group in the same period.Distribution of peripheral T and B lymphocyte subsets and mature natural killer(NK)cells were analysed by flow cytometry.The level of T lymphocyte-associated cytokines(IL-2,IL-10,IL-17a,and IFN-γ)were measured by ELISA.Results:1.Compared with healthy donors,the level of peripheral hemoglobin,albumin,IgG and IgA were lower in the IMN patients and the differences were statistically significant(P<0.05).The level of cholesterol,triglyceride,peripheral blood creatinine,uric acid and low density cholesterol were higher in the IMN patients(P<0.05).2.Serum cytokines:IL-10 and IL-17a were higher in the IMN patients than the healthy donors(P<0.05),while IL-2 and IFN-y were not have significant difference between the two groups.3.Peripheral lymphocyte subsets:3.1 The expression level of T lymphocytes and B lymphocytes were significantly higher in the IMN patients(P<0.05);3.2 CD3+CD4+T cells and its subpopulations including CD4+CCR7+CD45RAcentral memory T cells,CD4+CCR7-CD45RA-effector memory T cells,CD4+CD25+T lymphocytes(IL-2Ra)were increased in the IMN patients;while CD4+CD25+CD127lo regulatory T cells were lower in the IMN patients(P<0.05).3.3 CD3+CD8+T cells and their subsets CD8+CCR7+CD45RA-central memory T cells and CD8+CCR7-CD45RA-effector memory T cells were lower in the IMN patients compared with healthy donors(P<0.05).3.4 There were no significant difference in CD3+CD4-CD8-double-negative T cells and CD 16+CD56+NK cells between the two groups.4.Correlation analysis:there was no obvious correlation between abnormal T lymphocyte subsets and PLA2R,Hs-CRP and other clinical indicators.Conclusions:1.Severe cellular immune abnormalities are found in IMN patients,which presented as rising B lymphocytes,suggesting autoimmune activation.On the other hand,it shows the activation of some T lymphocyte subsets,which aggravates the immune inflammatory response in IMN patients.2.CD25 is an activation marker of T cell,and this study found that CD4+CD25+T(IL-2a)lymphocytes were significantly increased in IMN patients,while CD8+CD25+T showed no obvious abnormalities,suggesting that CD4+T cell activation plays a major role in T cell activation.3.Abnormalities of CD4+T lymphocyte subsets in IMN patients are mainly manifested in CD4+central memory T cells,rising CD4+central effector memory T cells,and downregulation of CD4+CD25+CD127lo regulatory T cells,indicating that there is an imbalance of T lymphocyte subsets in IMN patients and patients all have decreased ability to suppress autoimmune response,and impaired self-regulation mechanism will lead to further aggravation of glomerular damage.4.The changes in T lymphocyte subsets were not associated with PLA2R and Hs-CRP,suggesting that the abnormal T lymphocyte subsets were independent of elevated B cells and their antibody changes,and that the activation of T lymphocytes was also not associated with inflammatory infection.Chapter 2 Effects of rituximab on T lymphocyte subsets in the treatment of idiopathic membranous nephropathy and its clinical significanceObjective:According to the abnormal T cell subsets selected in the first part of the article,the changes of T lymphocyte subsets before and after rituximab therapy were further analyzed,we observed the clinical efficacy and incidence of adverse effects after rituximab and explored the clinical significance of rituximab on T lymphocyte subsets were evaluated.Methods:A total of 33 patients diagnosed with idiopathic membranous nephropathy admitted in the Department of Nephrology of Soochow University from January,2020 to June,2022.The Patients were completed a standard course of rituximab and followed up for at least 6 months.Clinical data at baseline(before renal puncture),laboratory indicators and immune-related indexes were collected.The correlation of T lymphocyte subsets with clinical indicators was analyzed.The patients were divided into initial treatment group and non-initial treatment group.Then according to pretreatment clinical index before rituximab we also divided our patients into low-medium-risk group and high-risk group.It was divided into infected group and uninfected group according to whether infection occurred after medication.Clinical outcome defined clinical remission,no remission or relapse after 6 months of RTX treatment.the predictive value of relevant indicators for RTX treatment was assessed by binary logistic regression analysis.Results:1.The complete remission rate after 6 months of RTX treatment was 12.12%,the partial remission rate was 69.70%,and the incidence of infection was 12.12%.2.Anti-PLA2R antibody titer,24-hour urine protein quantification,total cholesterol,HDL-C,and LDL-C were significantly decreased compared with those before treatment(P<0.05).Hemoglobin and peripheral blood albumin levels increased and IgG levels increased than those before treatment(P<0.05).3.Treg increased after RTX treatment(P<0.05);total B lymphocytes and T lymphocyte subsets including central memory T cells,CD4+/CD8+,CD4+CD25+T cells decreased(P<0.05),but CD3+T cells,CD4+T cells and NK cell counts were no statistically significant difference.4.In this study,there was no obvious difference in T lymphocyte subsets before treatment in different risk stage;anti-PLA2R titer,urinary protein quantification and CD4+CD25+T(IL-2Rα)were decreased in low-medium-risk group and high-risk group,peripheral blood albumin and Treg in low-medium-risk group groups were increased(P<0.05).However,the changes of Treg in the high-risk group didn’t find significant difference.5.The study found that patients used RTX as the first therapy had lower urine protein quantification,creatinine levels(P<0.05),and higher hemoglobin,albumin,and Treg levels(P<0.05)compared with patients who had other immunosuppression before RTX treatment.6.We also observed that baseline anti-PLA2R antibody titers in the remission group were lower(P<0.05),however,T lymphocyte subsets showed no significant changes in partial remission and not remission group.7.We didn’t observed any difference before RTX treatment in the infected and uninfected groups.CD4+T lymphocyte subsets decreased in both groups after RTX treatment,but Hs-CRP was higher in infected patients compared with uninfected patients(P<0.05).8.Correlation analysis of IMN group data:CD8+effector T cells and B lymphocyte count were positively correlated with PLA2R after RTX treatment.However,no correlation between Hs-CRP and T lymphocyte subsets was found before and after treatment.9.In order to clarify the relationship between T lymphocyte subsets and prognosis,clinical outcome(remission,non-remission)was taken as the dependent variable,and clinical indicators were included in the binary logistic analysis,we observed Anti-PLA2R antibody titers were inversely correlated with remission.Then we took T and B lymphocytes and their subsets into our analysis,we find no correlation between T lymphocyte subsets and remission.Conclusion:1.rituximab for IMN achieved a significant clinical remission within 6 months.2.There was no significant change in the total number of T cells after rituximab treatment,but the increased CD4+/CD8+,CD4+central memory T cells,CD4+CD25+T(IL-2Rα)cells decreased compared with those before treatment,and the decreased Treg increased compared with that before treatment.All these results suggests that RTX can correct the unbalanced T lymphocyte subsets.3.Anti-PLA2R antibody titer and classical inflammatory index Hs-CRP showed no correlation with Treg and CD4+T lymphocyte subset,suggesting that changes in CD4+T lymphocyte subset after rituximab treatment were independent of B cell depletion and inflammatory response,considering that rituximab directly acts on CD4+T lymphocyte subset,but whether its specific target is in CD25 needs to be further investigated.4.Lower CD8+ T cell levels and higher B lymphocyte levels in IMN patients were associated with the remission rate of rituximab treatment,which can be the indicators of IMN patients to observe clinical response after RTX.