| Background and Objectives:T-cell large granular lymphocytic leukemia(T-LGLL)is a rare lymphoproliferative disorder,characterized by monoclonal T-cell large granular lymphocytosis,often clinically manifest as bone marrow failure and accompany with autoimmune symptoms.Immunosuppressive therapies are effective,but the dominant clone may persist even in responders.The pathogenesis of T-LGLL has not been fully elucidated.In this study,single-cell RNA sequencing(sc-RNA seq)and V(D)J profiling were performed to discern clonotypes and gene expression patterns of T lymphocytes from T-LGLL patients who were sampled before and after treatment.Methods:Peripheral blood samples were collected from five T-LGLL patients prior and post immunotherapy treatment.Surface proteins were specifically labeled for epitopes sequencing,Single-cell RNA seq was performed on the 10x Genomics Chromium Single Cell V(D)J+5’ Gene Expression platform,and sequencing obtained on the Illumina Platform.Barcode assignment,alignment,unique molecular index counting and T cell receptor sequence assembly were performed using Cell Ranger 6.1.0.Immune cells were clustered and annotated,then transcriptome characteristics of each group of cells were analyzed,T cell receptor(TCR)clonetype and clonesize were identified.Furthermore,differential expression genes were identified and pathway enrichment was conducted.Results:A total of 67,237 immune cells,including 22137 of T cells with intact TCRa and β chains were detected.By analyzing the characteristics of transcriptome and immune library,it was found that:(1)The function of antigen presenting cells(APC)in patients was enhanced:1)Antigen process and presentation pathway was enriched in monocytes and dendritic cells,the signal pathways related to innate immunity were also up-regulated.2)For B lymphocyte,both the binding ability to antigen and the regulatory of T cell activation and differentiation were enhanced.(2)During disease condition,the expression of cytotoxicity related genes in NK cluster was decreased,and the pathways related to cytolysis and cytotoxicity were down-regulated,but the regulation of T cell receptor signaling pathway,T cell proliferation,activation and differentiation was enhanced.(3)The immune cell composition of patients before treatment and in non-response condition was similar,with increased proportion of CD 8 T cell population.There was a more significant similarity on transriptome between CD8 T cells and NK cells.(4)The proportion of effector CD8 T cells(CD8_TE)in G2/M phase was significantly higher in patients than in healthy individuals.Moreover,in patients prior treatment,cytotoxic genes of CD8 TE cluster was increased,exhaustion related genes were also upregulated.Cell killing,interferon y signaling pathway and cytokine production were enhanced in CD8 TE from patients.But cell proliferation and toxicity of CD8 TE cluster in responders were decreased.(5)The clonal diversity of CD8 TE cells was significantly reduced and the dominant clones were amplified.The dominant clones were individually specific and drifted with disease status.Increased cytotoxicity,up-regulated T cell activation and cytokine secretion were observed in monoclonal T cells.The proportion of dominant clones decreased after treatment,but the dominant clones still existed in remission state,with a decreased toxic function.(6)In addition,for patient prior treatment,the proportion of Treg was decreased significantly and apoptosis pathways was enriched.After response,the proportion of Treg was restored,metabolism-related pathway was up-regulated and apoptosis-related pathways were down-regulated.Conclusion:The immune homeostasis was unbalanced in T-LGLL.APCs demonstrated with increased antigen presentation effect and enhanced regulation on T cell activation,differentiation and proliferation.Stronger cytotoxicity and proliferation ability were observed in CD8 TE cluster.They also showed decreased diversity of TCR,and dominant clones was observed in individuals.NK cells was confirmed with less toxic killing function,but enhanced regulation ability to T cells.Decreased number and impaired function of Tregs may contribute pathogenesis of T-LGLL.IST can partially remove the pathogenic LGL cells,reduce their toxicity and proliferation ability,and enrich the clone diversity.The function of Treg was restored after IST,and the number of Treg was up-regulated.Background and Objectives:Primary acquired aplastic anemia(AA)is a kind of bone marrow failure syndrome mediated by auto-activated T cells.The incidence of AA is relatively high in Asian countries(0.6/million/year).According to the severity of the disease,it can be divided into severe aplastic anemia(SAA)and non-severe aplastic anemia(NSAA).NSAA is characterized by high-proportion(the incidence is four times of SAA),heterogeneous condition and high risk of progression to SAA.Cyclosporine is often used to treat with NSAA,but the overall response rate to cyclosporine is only 50%.Furthermore,it’s difficult to identify who will be responsive to cyclosporine.In order to better reveal the immune characteristics of NSAA and refine the subgroup,explore the factors affecting cyclosporine response and provide new methods for diagnosis and treatment.We designed a prospective cohort of NSAA,and analyzed the characteristics of T-cell immunity by mass spectrometry flow cytometry.Methods:Based on diagnosis and treatment guidelines of AA and literature review,the inclusion criteria were defined and patients with AA were enrolled.All patients were treated by cyclosporine with a dose of 3-5mg/kg/d,and the efficacy was evaluated after 6 months.Bone marrow samples from 30 AA patients and 4 healthy controls were collected to analyze the immune characteristics of T cells by mass spectrometry flow cytometry.Bone marrow samples were also collected from 7 patients before and after treatment,and the changes of T-cell immune characteristics were analyzed by mass spectrometry flow cytometry.Results:We established a clinical cohort of 92 initially treated NSAA patients,all of whom received cyclosporine.We also defined the criteria for classification of transfusion-dependence and transfusion-independence.Seventy-nine patients received cyclosporine for 6 months or more.Thirty-three(41.8%)of them achieved response.Clinical indicators such as peripheral blood count and biochemistry tests prior treatment were insufficient to predict the response to cyclosporine.By analyzing data from mass spectrometry flow cytometry,we found that there were significant differences in immune characteristics on T cells between AA and healthy individuals.The expression of cytotoxic T lymphocyte associate protein-4(CTLA-4)on CD8+T cell subsets,Sphingosine-l-Phosphaphate Receptor 1(S1P1)on naive T cells and Interleukin 9(IL-9)in CD4+T cell subsets in AA were higher than that in healthy individuals.By analyzing the immune characteristics of T cells in the cyclosporine-treated responsive group and non-responsive group,we found that the expression level of CTLA-4 in CD8+T cells at diagnosis in responders was lower than that in nonresponders(16.59 ± 1.83%vs 28.67±4.22%,p<0.05),and the level of CTLA-4 on CD8+T cells in responders was significantly increased after treatment(33.94±6.38%vs 11.28±2.73%,p<0.05).The expression levels of S1P1(32.18 ±3.46%vs 21.86 ± 2.67%,p<0.05)and IL-9(24.52±2.97%vs 40.42 ± 4.80%,p<0.05)on naive CD4 in responsive group were lower as compared with nonresponders.Conclusion:As compared to healthy individuals,T cells demonstrated a signature of abnormal function in AA.The expression of CTLA-4,S1P1 and IL-9 on T cells in responders were different with non-responders,and the expression of CTLA-4 in responder patients was restored after treatment.These results suggest that T cell immune checkpoint molecules,T cell migration and differentiation are expected to be a marker for predicting the efficacy of immunosuppressive therapy,and provide a novel treatment direction for patients unresponsive to cyclosporine. |
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