Primary Study Of αβ TCR Second Rearrangement In SLE

BackgroundBased of Burnet and Talmagefa's clone (or cell) selection theory, the "taboo" T/B cell clones with self-response receptor experienced negative selection in the process of development, which were removed or inactivation as clonal anergy. T/B cells with other antigen-specific receptor will be further mature, and entere the peripheral lymphoid tissues to exercise their functions of T cells. After migration out from thymus, the rearrangement mechanisms is closed, no longer express recombination activating genes(RAGs) and no longer occurrence receptor gene rearrangements. However, over the past ten years, some abroad research results posed a challenge to this doctrine, new studies found that there are not only cell clonal selection but also receptor molecules choice, T cell can change its antigen specificity through receptor V(D)J gene rearrangement before clone select, replace the existing receptor to a new receptor, so as to avoid removing or apoptosis. Receptor secondary rearrangements occurrence ont only in the early development stage of immature T/B lymphocytes but also in peripheral mature T/B lymphocytes, the former known as "receptor editing", the latter known as "receptor revised".Current studies found that, T cell receptor secondary rearrangement make a major role in two areas: first, in the process of the thymus selection, if there are lake of positive choice ligand (suitable MHC), a large number of cells in the thymus triggered a sustained rearrangement at TCR a loci until new TCR go through positive choices. At the process of negative selection, when the T lymphocytes encounter self-antigen, in order to avoid self-reaction, TCR secondary rearrangement be activated, editing their receptor before remove or apoptosis, rescued in negative selection process. Second, peripheral mature B lymphocytes and T lymphocytes encounter virus infection in the immune response, viral antigen induced T cell tolerance through two channels: one is clone apoptosis or eliminated, the other is receptor revise, in order to avoid a large number of antigen-specific lymphocyte apoptosis, T cell revised its receptor, expression non-specific receptor and rescued, that the receptor no longer identify the tolerance original, thus the body without response to pathogenic microbes. Currently, it has been recognized that in the thymus positive/negative selection mechanism,safeguard self-tolerance and shaping diversity lymphocyte repertoire, receptor editing play a important role. Although in the studies of receptor revising, there are much controversial, but it have more potential significance for understanding the immunology major problems, such as autoimmune,lymphocyte reaction affinity maturation and tumor and virus-infected immune escape mechanism.As a typical autoimmune disease, Systemic Lupus Erythematosus(SLE) are considered associated with receptor editing/revising. As we all known, in PBMC of patients with SLE, there are existence of a large number of self-reactive antibodies, and these pathogenicity antibodies are self-reactive CD4+T cell-dependent. However, how do these antibodies and CD4+T been produced is not yet known. Many studies found that, regardless of human or animal SLE model have more unusual rearrangement than normal control group, like D-D fusion, second D-J_H rearrangement and V_H replacement. These abnormal rearrangements often raise the binding affinity of antibody or antigen receptor to DNA or RNA, perhaps these self-reactive T cells or antibody in SLE patients' PBMC is come from excessive or deficiencies receptor editing/revising which caused the abnormal rearrangements. ObjectiveIn the basis of the work has been done, integrate the latest technology to improve receptor secondary rearrangement research methods and systems, provide reliable and effective monitoring technical and analysis tools for the detection of alpha beta T cell TCR second rearrangement in peripheral blood mononuclear cell (PBMC) of patients with SLE.Detect the mRNA expression of rearrangement major regulatory protein, such as RAG1,RAG2,terminal deoxynucleo transferase(TdT) and Ku70&Ku80 in PBMC of patients with SLE. Detect the TCR rearrangement product alpha beta T cell signal joint TCR BD-BJ gene recombination DNA excision circles(TCR BD-BJ sjTRECs) by nest-PCR method. Detection of alpha beta T cell TCR BD-BJ gene recombination signal sequences (RSS) fracture end by Ligation Mediated PCR (LM-PCR) method. Provide the basic data for the study of TCR second rearrangement in vitro and T cells immune response mechanism in autoimmune disease.Detect the mRNA expression of alpha beta T cell TCR 32 AV families and 24 BV families in PBMC of patients with SLE. Monitoring T cell TCR alpha and beta chain CDR3 length distribution and polymorphism. Dynamic monitoring the changes of CDR3 profiles in different course of disease by immunoscope spectratype analysis technique. Screening the mono/oligoclonal T cell proliferative family which related to diseases, sequencing its TCR CDR3 regions and simulated the TCR protein's three dimensional structure. Provide basis data for the study of diagnosis and treatment of SLE and development new vaccine.Methods(1) Select fetal thymus tissue, T lymphoblastic leukemia cell line Jurkat and normal human PBMC as study sample. Synthetic the upstream and downstream primers of rearrangement main regulating protein gene, such as RAG1,RAG2 and TdT, Ku70, Ku80. Isolate total RNA from all samples, amplified and detected their mRNA expression, positive PCR products were sequenced; Synthetic the special joint BW Linker which link with the double-stranded RSS fracture end and synthetic it's primers, isolate total DNA from all samples, then link DNA to the BW-Linker and identified the expression by nested PCR, the second round positive PCR products were sequenced; Synthesis three upstream primers of TCR BJ1 and B J2 (BJ1-1,BJ1-2 and BJ1-3 or BJ2-1,BJ2-2 and BJ2-3) and two downstream primers of 3' end of BD1 and BD2 (BD1-1,BD1-2 or BD2-1,BD2-2) the TCR BD-BJ signal joint TCR excision DNA circles(sjTRECs)which may hydroponically form by BD1 joint with six BJ1 fragment or BD2 joint with seven BJ2 was amplified by nested-PCR method.(2) Synthesis the upstream primers of 24 TCR BV (TCRβchain variab gene) families and 32 TCR AV (TCR a chain variable gene) families of human T cells and common downstream primers of BC (βchain constant gene) and AC (a chain constant gene), marked the downstream primers with FAM, Selected T lymphoblastic leukemia cell lines Jurkat and healthy donors' PBMC as study object. Extraction samples' total RNA. Amplify the BV and AV gene CDR3 segments by nested-PCR. Genescan technology have been used to detecte T cell TCR CDR3 spectratype of normal polyclonal groups PBMC and analysis the length and sequence of CDR3 regions of monoclonal Jurkat cells TCR a andβchain.(3) Study the PBMC samples of clinical diagnosis SLE patients. Isolate total RNA and DNA from all samples,the total RNA retrovirus into eDNA, then detect the expression of RAG and TdT, Ku70, Ku80 mRNA by RT-PCR method. Detect the sjTRECs by nested PCR method and detect the double-stranded RSS fracture end by LM-PCR method. Amplify the CDR3 segments of 24BV families and 32AV families. Detection TCR aβCDR3 length polymorphism and distribution by immunoscope spectratype analysis technology. RT-PCR products of mono/oligoclonal proliferation T cell were sequenced. Analyze the CDR3 sequence and structure by bioinformatics.Results(1) Both in thymus tissue and Jurkat cells have detected RAG1 and RAG2 mRNA, only RAG1 but no RAG2 mRNA was detected in healthy doner's PBMC. Sequencing of RT-PCR product of Jurkat cells RAG1 and RAG2 showed that the sequence consistent with GeneBank reported sequence. Thymus tissue and Jurkat cells both have the expression of TdT, Ku70, Ku80 mRNA. Apart from TdT, the rest are all detected in healthy human PBMC. In the thymus DNA, four breakpoints of RSS 5' and 3' end have been detected. In Jurkat DNA, two RSS 5' and 3' breakpoints have been detected. But no RSS be detected in healthy human PBMC. Sequencing the LM-PCR products, the sequence is consistent with the report of Gene Bankd. BD1-BJ1 sjTRECs S1, S2, S4, S5, S6 and BD2-BJ2 sjTRECs S1, S2, S4, S5 be detected in Thymus DNA. BD2-BJ2 sjTRECs S1, S4 and BD2-BJ2 sjTRECs S2 were detected in PBMC. BD2-BJ2 sjTRECs S1,S5 were detected in Jurkat cells.(2) Analysis RT-PCR amplification products of Jurkat cell TCR 24 BV and 32 AV families in 2% agarose gel electrophoresis shows that only BV8 and AV1.1 family has specific bands. Analysis Jurkat BV8 and AV1.1 family's RT-PCR product in 6% denaturing polyacrylamide gel electrophoresis showed that both have a single band. Further using GeneScan software 672 automatic scanning analysis Jurkat BV8 and AV1.1 family's RT-PCR product showed that there was only one single peak. Analysis healthy human PBMC RT-PCR product of AV and BV family in 2% agarose gel showed a fuzzy bands. Analysis the products of each family in 6% denaturing polyacrylamide gel electrophoresis have shown over eight bands. Automatic analysis each family by GeneScan found a bell-map Gaussian distribution peak photo with above eight peak of each family and the the expression frequency of every family is similar, but every family has a different CDR3 length distribution polymorphism;(3) Analysis 34 TCR AV and 24 BV families CDR3 RT-PCR products of 20 cases SLE patients, the majority of families showed a fuzzy band in the 1.5% agarose gel electrophoresis, part of family have no band. At 6% sequencing gel electrophoresis, most of TCR AV and BV families showed eight or more bands, part of families less than eight bands. Use GeneScan software analysis showed that the 20 patients TCR CDR3 spectratype, showing single peak, little-side peaks or partial-peaks. The expression of several families extremely low or absent. Some families showed high frequency mono/oligoclonal proliferation in 20 patients. Part of mono/oligoclonal expansion family in the SLE patients had been analyzed, some conservative sequence and high-frequency J gene fragments have been found. The expression of RAG1,RAG2,Ku70,Ku80 mRNA have detected in all of 20 patients. The expression of TdT mRNA have detected in 16 patients. The sjTRECs have detected in all of 20 patients, The Rss breakpoints have detected only in two patients.Conclusion(1) Improve the detection of RAG1, RAG2, Ku70, Ku80 and TdT mRNA expression by RT-PCR method. Sequencing identification certified the method is stable and reliable. Detection of these types of major rearrangements regulatory protein mRNA expression can indirectly reflects whether T cells occurred rearrangement, improve the detection of recombinant intermediates RSS breakpoint by LM-PCR methods and detection of sjTRECs by nested-PCR combining fluorescence quantitative PCR can provide research methods to quantitative analysis ongoing second peripheral TCR rearrangement which occurred in vitro,abnormal rearrangements in the tumor,autoimmune diseases and so on. Meanwhile, it provide the basis of the mechanisms study and potential clinical study for artificial induced TCR second rearrangement in vitro.(2) The Immunoscope Spectratyping analysis technology can dynamicly monitoring TCR CDR3 spectratype, which is a relatively simple,reliable,high sensitivity method to the research of specific T cell, it can not only monitoring monoclonal cells but also can analyze groups polyclonal samples, and it can reflect the dynamic changes of CDR3 spectratype, and can screen out mono/oligoclonal specific T cells to sequencing and bioinformatics analysis it's TCR.(3) The spectratype of TCR CDR3 of PBMC of SLE patients showed significantly monoclonal and oligoclonal amplifier. The CDR3 region of a chain andβchain of mono/oligoclonal T cell have different sequence and structure. Suggest there are correlation between the TCR molecular characteristics with the individual and their own antigen HLA diversity. Different patients with the same family mono/oligoclonal proliferation and use of conservative sequence and high-frequency J gene fragments. This restrictive use of the family and fragment suggest the same or similar structure of self-antigen. RAG2 and TdT can be found expression in SLE patients. This differs from normal PBMC. Initial speculation in patients with SLE may exist TCR second rearrangement. However, for many reasons, such as time,funding and experimental conditions, the study of rearrangements in this work is very dredging shallow, far from being able to answer the above proposition. Further research is necessary to establish self-response cell lines in vitro, to exclude the effection of T cells just initial migration from the first rearrangement not completely shut closed. And enforce the real-time monitoring the changes of TCR on the level of gene and protein. Meanwhile monitor the expression of mRNA and protein of rearrangement participate protein; fluorescence quantitative analysis SjTRECs; And expand the number of research samples, so that improve statistical significance. Our research will provide methods and means to study the immune pathogenesis mechanism,individualized treatment and autoantigen T cell epitope vaccines of SLE.