The Diversity And Oligoclonality Of αβTCR In CD4~+ T Cells Of Porcine Immunized By CSFV-C Strain Vaccine

T cell receptor(TCR) which especially exists in heterodimer structure on the surface of T lymphocytes is the surface marker of T cells, and αβTCR recognizes antigenic peptide(derived from degraded antigens) presented by major histocompatibility complex(MHC) molecules. There are three hypervariable regions(complementary determining regions, CDR 1, 2 and 3) both in TCR-α and β chains. CDR3 is responsible for recognizing and interacting with various antigenic peptides. Since different CDR3 sequence represents different T cell clone, one type of CDR3 sequence refers to a specific T cell clone, thus the T cell clonality can be detected through monitoring the CDR3 spectratype. So far, due to the features of fast, sensitive and efficient, GeneScan Spectratyping method has been known as a common technique in determining TCR CDR3 spectratype in the researches of tumor, virus infection, autoimmunity disease and transplant rejection, and has successfully illuminated the αβTCR change patterns no matter in immune homeostasis or clinical disease status. Those antigenic specific TCR gene has been used in immunotherapy in clinical trials and have made some achievements. Till now, little relative research has been achieved on porcine TCR under pathogenic infection condition, however, the study of porcine immunology especially T cell mediated cellular immune response can not only benefit to porcine diseases prevention, but is also helpful to apply miniature pigs into human xenogenic organ transplantation. Therefore, in this study, we analyzed the αβTCR diversity and clonal expansion of porcine CD4~+ T cells under healthy and classical swine fever virus-C strain(CSFV-C strain) vaccine immuned conditions, to better understand the cellular immune mechanism against swine fever virus and will provide theoretical basis for the development of new anti-CSFV vaccines. Progress in this study are as follows:1. We analyzed the CDR3 length repertoire and diversity of TCR α and β chains in swine CD4~+ and CD8~+ T lymphocytes by immunoscope spectratyping technique. RT-PCR was used to clone swine 19 TCR AV and 20 TCR BV gene families in separated CD4~+ and CD8~+ T cells. Spectratying results showed that 19 TCR AV and 20 TCR BV gene families can expressed both in CD4~+ and CD8~+ T cells; the CDR3 spectratyping of all TCR AV and BV gene families in two sorted T cells presented a standard Gaussian distribution with more than eight peaks by Genescan; the expression frequency of CDR3 in these cells showed different expression patterns. Except for some gene families, most CDR3 recombined in frame. The αβTCR diversity showed both in CD4~+ and CD8~+ T cells under healthy state is responsible for the efficient and sufficient immune coverage. The CDR3 polymorphism and length diversity showed different expression and utilisation patterns in CD4~+ and CD8~+ T cells, which may be helpful to further study the porcine TCR CDR3 gene repertoire, functional complexity and specificity of TCR molecule.2. αβTCR usage pattern in CD4~+ T cells was observed after CSFV-C strain vaccine immunization. Four Hezuo mini-pigs were used as experimental animals immunized with CSFV-C strain vaccine, then the TCR lineage was monitored by using GeneScan-Spectratyping method. Results showed that most of the clonal expanded TCR gene families appeared prior to the detection of anti-CSFV antibody, and this kind of cloning trend gradually let down after reaching at the peak. The TCR patterns returned to normal Gaussian distribution 15 days post immunization. It can be showed that TCR AV14, AV38 and TCR BV6 S in #884 swine, TCR AV8-3S and TCR BV30 in #888 swine, TCR AV5 S, AV8-3S, AV8-4S, AV14 and TCR BV4 S, BV7 S, BV15 in #892 swine presented clonal expansion. Among these gene families, TCR AV14 both in #884 and #892 pigs, TCR AV8-3S both in #888 and #892 pigs showed clonal expression; furthermore, TCR AV5 S, AV38 and TCR BV6 S were also observed in peripheral blood mononuclear cells(PBMCs), which implied that PBMCs and CD4~+ T lymphocytes may react to the same antigenic peptide from classical swine fever.3. The conserved CDR3 amino acid motif of those clonal expanded αβTCR gene families in CSFV-C strain vaccine immunized CD4~+ T cells were cloned and analyzed. The CDR3 region of those monoclonal/oligoclonal expanded TCR gene families were acquired by RT-PCR. After clone sequencing and bioinformatics analysis, we found that although the CDR3 sequences were not identical in the same gene family, however, conserved amino acid can be observed. A conserved “LX” motif in all TCR AV gene families and a conserved “GGA” amino acid motif in all TCR BV gene families can be detected. Those conserved CDR3 amino acid may be related to recognition and interaction with antigenic peptide derived from classical swine fever virus, which will lay basis on the high throughput screening and development of polypeptide vaccines against classical swine fever.4. The full length of classical swine fever-C strain specific TCR AV5 S and TCR BV6 S gene were cloned, prior to the construction of two eukaryotic expression vectors TCR AV5S-pIRES2-EGFP and TCR BV6S-pIRES2-EGFP to observe the expression level in vitro through co-transfection, the sequences were analysed. Targeted gene sequence analysis showed that the full gene sequence of TCR AV5 S was 816 bp, encoding 272 amino acids; the full gene sequence of TCR BV6 S was 945 bp, encoding 315 amino acids. Co-expression can be observed both in separated and co-transfection. This will provide method basis for the follow-up study of the function of antigen specific TCR gene.