| Background:Primary immune thrombocytopenia(ITP)is a hemorrhagic disease with a reduced platelet count in peripheral blood.It is mediated by a variety of mechanisms such as humoral immunity and cellular immunity.Rituximab(RTX)is one of the second-line treatments recommended by current guidelines,which mainly exerts its curative effect by deleting CD20+B cells.Currently,there are few studies of immune status changes before and after RTX treatment in ITP patients.In addition,some ITP patients are ineffective with RTX treatment,and few studies have explained the reasons for the heterogeneity of RTX efficacy.Besides,there is no clear marker that can predict the efficacy of RTX.Objective:To reveal the effect of RTX on the immune system of ITP.To explore the therapeutic mechanism of RTX.To screen for markers that can predict the efficacy of RTX.Methods:(1)The clinical data of ITP patients were collected.Peripheral blood samples were collected before and after RTX treatment.Flow cytometry was used to analyze the ratios of various immune cell populations.The CD19+B cell population includes B cell subsets(naive B cells,memory B cells,preplasma cells)and regulatory B cells(Bregs).The CD3+T cell population includes CD4+/CD8+T cells,helper T helper cells(Th),regulatory T cells(Tregs).and suppressor T cells(Ts).The monocyte Fcγ receptors(Fcγreceptors,Fcγ Rs)including FcyRⅠ/Ⅱa/Ⅱb/Ⅲ.Peripheral blood mononuclear cells(PBMCs)were isolated in vitro and were stimulated and cultured.The apoptosis level was detected by Annexin V/7-AAD staining.The proliferation level was detected by BrdU staining.The T cell activation level was detected by CD25/CD69 staining.Plasma cytokine levels were detected by enzyme-linked immunosorbent assay(ELISA).Plasma platelet membrane glycoprotein(GP)Ⅱb/Ⅲa,Ⅰb/Ⅸ,Ⅰa/Ⅱa levels were detected by the GTI platelet autoantibody detection kit.(2)The transcriptome and TCR/BCR full-length V(D)J sequences of PBMCs before and after RTX treatment were sequenced by single-cell immune library sequencing technology.The gene expression differential analysis,gene set enrichment analysis,and cellular heterogeneity analysis were performed.(3)Compare the differences in clinical and experimental indicators before RTX treatment between the response(R)and the no response(NR)patients.Analyze the changes in the immune system at various stages after RTX treatment,and screen for predictors of the efficacy of RTX.Results:(1)Before RTX treatment,the level of B cell activation,plasma cell antibody secretion,humoral immune response,related type Ⅰ IFN signaling pathway gene sets,and the proportion of naive B cells in the R group were higher than those in the NR group.The proportion of memory B cells in the R group was lower than that in the NR group.(2)Before RTX treatment,the T cell activation and differentiation level,T cell-mediated immune response,cytotoxicity score,the Th1/Th2 ratio,T cell apoptosis level,and proliferation level in the NR group were higher than those in the R group.The T cell naive score in the NR group was lower than that in the R group.(3)Before RTX treatment,the monocyte differentiation,antigen presentation level,and major histocompatibility complex(MHC)Ⅰ and MHC Ⅱ score in the NR group were significantly higher than those in the R group.There was activation of the IFN-γ signaling pathway in monocytes in the NR group.(4)After RTX treatment,the number of B cells decreased,the reconstitution of memory B cells was delayed,and the titer of platelet membrane glycoprotein antibody decreased;(5)After RTX treatment,the T cell cytotoxicity score,CD3+T cells ratio,Th1/Th2 ratio and Ts ratio increased,memory T cells naive score,plasma chemokine CX-C motif chemokine 13(CXCL13)and granzyme B concentrations decreased;The CD4+/CD8+T cells ratio and Tregs ratio in the R group were higher than those in the NR group,and the Ts ratio in the NR group was higher than that in the R group.(6)After RTX treatment,the MHC Ⅱ score of monocytes,the ratio of activating receptor to inhibitory receptor increased,and the expression of FcγRⅠ and FcγRⅡb decreased;(7)The naive B cells ratio and Th1/Th2 ratio in peripheral blood before RTX treatment were reliable markers for predicting efficacy.Conclusion:The main mechanism of RTX treatment of ITP is to reduce the humoral immune response by deleting B cells.T cell-mediated cellular immune response and monocyte/macrophage antigen presentation and phagocytosis were compensatively enhanced after RTX treatment.RTX is more effective in the treatment of ITP patients with the humoral immune response as the main pathogenic mechanism than in patients with the cellular immune response as the main pathogenic mechanism.Peripheral blood naive B cells ratio and Th1/Th2 ratio can be used as markers to predict the efficacy of RTX.Background:Thrombotic thrombocytopenic purpura(TTP)is a rare thrombotic microangiopathy.The main clinical manifestations are microangiopathic hemolytic anemia,thrombocytopenia,and neuropsychiatric symptoms.The main treatment methods include plasma therapy,glucocorticoids,rituximab,and immunosuppressants.Objective:To analyze the clinical characteristics,treatment and outcome of TTP patients.Methods:The clinical data of 83 patients with TTP from May 1998 to May 2019 were analyzed retrospectively.Results:Among the 83 patients,there were 27 males and 56 females,with a median age of 39(10-68)years.41 cases(49.4%)showed pentalogy syndrome and 79 cases(95.2%)showed triad syndrome.78.0%(46/59)of the patients had a PLASMIC score of 6 or higher.TTP gene mutations was detected in 5 of 10 patients.The activity of von Willebrand factorcleaving protease(ADAMTS13),which was detected in 10 patients before plasma exchange(PEX),was less than 10%in 9 patients.83 patients were treated with PEX/plasma infusion and glucocorticoid,35 of which were treated combined with rituximab and/or immunosuppressant.The median follow-up was 34(1-167)months,the effective rate was 81.9%,the remission rate was 63.9%,the relapse rate was(35.7 ±7.1)%,and the 3-year overall survival(OS)rate was(78.6±4.6)%.The effective rate(72.9%vs 94.3%,P=0.019)and OS rate[(63.8±7.5)%vs(94.3±3.9)%,χ2=8.450,P=0.004]in the group treated with PEX/PI and glucocorticoid alone were lower than those in the group treated combined with rituximab and/or immunosuppressant.Cox multivariate analysis showed that age(HR=1.111,95%CI 1.044-1.184,P=0.001)and alanine transaminase(ALT)/aspartate aminotransferase(AST)(HR=1.353,95%CI 1.072-1.708,P=0.011)were independent risk factors for OS.Conclusion:Most patients with TTP have triad syndrome,accompanied by a decrease in ADAMTS13 activity.Plasma infusion and glucocorticoid combined with rituximab,immunosuppressive therapy could improve overall survival.The prognosis of patients with older age and high ALT/AST ratio is poor. |
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