Diversity Analysis Of The TCR ?-chain CDR3 Repertoire In Systemic Lupus Erythematosus

Background:Systemic lupus erythematosus(SLE)is a typical systemic autoimmune disease,which characterized by producing a variety of antibodies,immune complex deposition and complement activation,resulting in multiple system and organ damage,such as the skin,joints,kidneys and central nervous system,and even induce renal function failure,lupus encephalopathy and severe secondary infection,which can be life-threatening and serious harm to human health.The etiology and pathogenesis of SLE has not been clear,but thought to be related to genetic,environmental triggers and immune system disorders.In recent years,researchers paid more and more attention on immunological study,and it is more and more in-depth in immune related study.With the continuous progress of science and technology,the research approach is becoming more perfect and mature,from traditional techniques,such as immunohistochemistry,immunofluorescence and antibody dependent cell mediated cytotoxicity(ADCC)experiment and so on to the development of a new generation of sequencing technology in view of the immune cells.Nowadays,immunology research has entered a new period.How to use sequencing technology to solve the immune related problem,will be of more concern to the researchers.Because the immune system is the body covering defence network,large and complex.Before the use of immune repertoire related technology,such as semi quantitative,multiplex PCR and high throughput sequencing technology,it is difficult for people to analyze immune diversity at the cellular level,and difficult to see the changes of disease specific.However,immune repertoire can reflect the polymorphism of T cell receptor alpha,beta,gamma and delta chains,can deeply dig the relationship between immune repertoire and disease.Through the establishment of the immune repertoire of SLE patients,to further elucidate the etiology and pathogenesis of SLE,deepen the understanding of the disease etiology,then provides a new opportunity for drug development,disease prevention,diagnosis and treatment,lay a solid foundation for the future implementation of individualized diagnosis,optimal treatment.Objective:To study the diversity of T lymphocyte immune repertoire in systemic lupus erythematosus(SLE)patients,to provide a new basis for early diagnosis of SLE and the accurate assessment of prognosis and therapeutic effect.Methods:After rigorous screening,10 SLE patients and 10 healthy people were selected as study group and control group of our study,respectively.Extract venous blood 5ml(EDTA lmg:5ml anticoagulant)in each object,and record the SLEDAI score.Whole genome DNA were extracted from whole blood of each subject.Designing primers for the region of V family and J family,to amplify the whole CDR3 area of T cell receptor(TCR).Combined with high throughput sequencing platform,to analyze the expression frequency of each gene family,V-J pairing,base insertion/deletion,the region of CDR3 diversity and length distribution characteristics of T cell TCR beta chain in peripheral blood T lymphocytes of SLE and NC group.To screen the highly clonal amplification(frequency greater than 0.5%)DNA sequence,amino acid sequence(AA)and V-J combination in SLE group and NC group,to screen the common DNA sequence,amino acid sequence and V-J from the mass of data.In addition,we analyze the TCR diversity and differentially expressed clones according to the Distinct(uniq)clone number,Simpson and Shannon Weiner coefficient,to reveal the immunological characteristics of T lymphocytes in SLE group and NC group,to elucidate the pathophysiological mechanism of SLE and find new immune therapy target.Results:1.The frequency of length distribution:the distributions of CDR3,VD indel,and DJ indel lengths were comparable between the SLE and NC groups.The 5 most frequently observed CDR3 lengths were 42,45,39,36,and 48 nt,the 5 most frequently observed VD indel lengths were 0,2,3,1,and 4 nt,the 5 most common observed DJ indel lengths were 0,1,2,3,and 4 nt.2.Clonal amplification degree:Compared with the normal control group,the highly clonal amplification DNA sequences(frequency greater than 0.5%)increased in SLE group(0.019%vs 0.008%),and clonal amplification degree was general increased.In addition,the frequency distribution showed that the majority of the repertoire comprised a small number of HECs and also showed a right-skewed distribution in which the majority of the clones were low frequency.3.TCR diversity:Through calculating the diversity coefficient,Simpson and Shannon Weiner coefficient of SLE and NC group,statisticing the significant differences.We found that the immune diversity of SLE patients was significantly lower than that in normal control group.4.Highly expanded clones(HEC):After statistical analysis,we screened the HECs in SLE and NC group,at the resolutions of DNA sequence(106 vs 73),AA sequence(107 vs 72)and V-J combination(472 vs 398).5.Public sequences:After statistical analysis,we screened the public DNA sequences and amino acid sequences in SLE group and NC group.Three DNA sequences and four aa sequences were shared by all 10 SLE patients;these sequences may provide biomarkers for SLE disease risk,diagnosis or prognosis.6.Skewed usage of TCR?V/J:Through systematically analyzing the V,D and J segment usage frequencies in PBMCs from ten normal volunteers and ten SLE patients,we found the expression levels of 10 TR?V segments and 6 TR?J segments are significantly different in the SLE group compared to the NC group.Conclusion:1.Compared with the normal control group,the degree of T cell clonal expansions in the SLE group was higher,and the immune diversity of SLE patients was significantly lower.2.Immune repertoire sequencing can reflect the diversity of immune repertoire in SLE patients which calculated at different resolutions of distinct DNA sequence,amino acid sequence,and V-J combination,to develop biomarkers for diagnosis of SLE disease.