Analysis Of The TCR Immune Repertoire In Patients With Diffuse Large B-cell Lymphoma Before And After Chemotherapy

Background and PurposeDiffuse large B-cell lymphoma(DLBCL)is the most common type of non-Hodgkin’s lymphoma(NHL).It is characterized by strong heterogeneity,high invasiveness,and a typical clinical manifestation of rapidly growing painless lump.DLBCL patients have obvious heterogeneity in cytogenetics,molecular biology,clinical manifestations and there are obvious differences in response to treatment and prognosis.The National Comprehensive Cancer Network(NCCN)guidelines recommend the use of rituximab,cyclophosphamide,doxorubicin,vincristine and prednisone(R-CHOP)as a first-line treatment regimen,which can cure 50-70%of DLBCL patients.The 5-year overall survival rate is 60-70%.Due to the damage and suppression of the immune function,many DLBCL patients are accompanied with autoimmune hemolytic anemia,immune thrombocytopenia,Sjogren’s syndrome,hypogammaglobulinemia and other immune system diseases when the disease is diagnosed.Lymphoma patients often suffer from low immunity and secondary infections after chemotherapy,which also indicates that the therapy has an impact on the body’s immune function.Therefore,the immune function of DLBCL patients before and after chemotherapy may play a role in guiding clinical treatment and predicting clinical benefit.Immune repertoire refers to the diverse collection of all immune cells in an individual.At present,it mainly targets at T cell receptor(TCR)and B cell receptor(BCR).As an important component and effector cell in the immune system,T cells play an important role in inducing anti-tumor response.In-depth analysis of T cells can enable us to understand the immune function and the process of anti-tumor immune response.T cell-mediated antigen recognition depends on the interaction between TCR and major histocompatibility complex(MHC)molecules,and the highly variable complementarity determining region 3(CDR3)is the key region for TCR to recognize foreign antigens.Therefore,analyzing the diversity of the CDR3 region can directly reflect the function of T cells and the state of immune response,which is essential for evaluating the response of patients to treatment.In the past,CDR3 spectral analysis technology or Sanger sequencing technology was used to detect the high diversity of TCR CDR3 sequences,and quantitative analysis results of TCR can be obtained,but only a limited number of sequences can be analyzed.With the emergence of large-scale parallel sequencing,high-throughput sequencing of immune repertoire(HTS-IR)technology has been developed,which allows simultaneous analysis of the CDR3 sequence of all T cell receptors,quantifies the use of V and J genes,and fully reflects the diversity and differences of TCR CDR3.In addition,the read depth available for sequencing is increased,and even low-abundance TCR sequences can be detected.Therefore,the purpose of this study aims to determine the changes of immune function of newly treated DLBCL patients before and after chemotherapy at the level of TCR CDR3 through high-throughput sequencing.In terms of CDR3,the frequency of V gene and J gene usage,T cell clonotype and diversity,the differences of TCR immune repetoire between DLBCL patients and healthy controls will be compared,and the relationship between TCR diversity and clinical characteristics will be further evaluated,and the changes of TCR diversity in DLBCL patients before and after chemotherapy will be explored.Materials and Methods1.30 cases of newly diagnosed DLBCL patients from the First Affiliated Hospital of Zhengzhou University were included in this study.All patients received rituximab combined with CHOP treatment,and peripheral blood samples were collected at baseline and after four cycles of chemotherapy.Clinical information was collected from the hospital case system and clinical responses were evaluated,including complete response(CR),partial response(PR),stable disease(SD)and progressive disease(PD).30 healthy and age-matched volunteers served as controls.They had no recent infections,fevers,and no history of autoimmune disease or malignant tumor.2.Peripheral blood mononuclear cells(PBMC)were isolated from 10 mL peripheral blood samples by Ficoll,and RNA was extracted by reverse transcription PCR(RT-PCR)kit for reverse transcription.The sequence of TCR variable region was obtained based on specific primers,and then the sequence information was identified by high-throughput sequencing.The highly variable CDR3 sequence was identified and translated by data analysis.3.The diversity and similarity of TCR immune repertoire among samples were detected.The diversity of TCR was described and quantified by three indicators:Shannon index,clonotype and D50 value.Shannon index was calculated according to the clonal abundance of all unique CDR3 sequences.The Shannon index was positively correlated with the TCR diversity of the samples.Clonal type referred to the unique CDR3 sequence that identified a T cell clone,and clonotype referred to the number of unique CDR3 sequences in each sample.The D50 value was determined based on the number of dominant CDR3,and positively correlated with clonotype and Shannon index,which could simplify the description of diversity.Bhattacharyya coefficient was used to determine the similarity of TCR between the two samples,which was based on the frequency and homogeneity of the shared TCR sequencing reads in the two samples.The value ranged from 0 to 1,where 0 meant that there was no overlap,and 1 meant that the TCR repertoire were the same between the two samples.4.Statistical analysisUsing Mann Whitney U test to compare the differences between the two groups.Spearman rank test was used to analyze the correlation between variables.ROC curve showed the diagnostic ability of binary variables.SPSS 23.0 was used to calculate all statistical analyses,P<0.05 was considered statistically significant.Results1.Comparing the sequencing data of TCR immune repertoire between DLBCL patients and healthy controls,the CDR3,V gene and V-J pairing of patients were lower than those of controls.Further analysis showed that there were significant differences in the frequency of 27 V genes and 3 J genes between the two groups.Compared with the control group,the number of clonotypes and the D50 value of the TCR immune repertoire in the DLBCL patients were significantly reduced(P<0.0001;P<0.0001),while the Shannon index was not significantly different(P=0.0676).2.This study evaluated the relationship between TCR diversity and clinical features in DLBCL patients.TCR diversity was lower in patients with clinical features such as age greater than or equal to median age 53.5 years,stage Ⅲ-Ⅳ,IPI score and ECOG score greater than 1.In addition,the levels of lactate dehydrogenase(LDH)and 3-2 microglobulin were negatively correlated with TCR diversity.3.The baseline TCR diversity of patients with complete remission(CR)after four cycles of chemotherapy was higher than that of patients without complete remission(Non-CR).The frequency of V gene and J gene usage in CR group and Non-CR group was compared.It was found that TRBV7-7,TRBV11-1 and their combined prediction model could classify clinical reactions.4.The standard first-line chemotherapy regimens may lead to the change of T cell clone frequency in peripheral blood of DLBCL patients.By evaluating the proportion of TCR clonal changes before and after treatment,it was found that the percentage of clonotype reduction after treatment was similar to the percentage of clonotype increased.Further comparison of TCR similarity before and after treatment showed that the similarity index of CR group was lower than that of Non-CR group,indicating that CR patients had more clonal changes or new clones.Conclusions1.DLBCL patients had unique immunological characteristics and immune dysfunction.The diversity of their TCR immune repertoire was lower than that of normal people.2.After chemotherapy in DLBCL patients,the CR group had a higher baseline TCR diversity than the Non-CR group,and the TRBV7-7,TRBV11-1 and their combined prediction model could classify the CR group and the Non-CR group.3.Chemotherapy could induce the reconstitution of the TCR immune repertoire in DLBCL patients,and dynamic monitoring of TCR similarity before and after treatment could be used as one of the indicators to predict the clinical response.