Take Advantage Of GeXP Multiple Gene Expression Genetic Analysis System To Detect The Spectratyping And Length Of TCRVα And TCRVβ Chain

Objective: Establish two multiplex reverse transcription polymerase chainreaction (mRT-PCR) methods to detect third complementarity-determining region’s(CDR3) spectratyping and length of the T-cell receptor (TCR) α and β chains for theanalysis of the proliferation of T cell clones by taking advantage of the multi-geneexpression system. Analysis clonal proliferation of TCR Vβ and TCR Vα family ofthe tumor infiltrating lymphocytes (TIL) in patients with lung carcinoma, and to clonethe TCR Vβ and TCR Vα gene to construct recombinant adenovirus vectorpDC315-TCRVα-IRES-TCRVβ. To evaluate the killing effect of TCR Vβ and TCRVα gene modified PBMC on different cancer cell lines to lay the foundation for thefuture discovery of tumor-specific TCR gene specific TCR gene-modified T cells foradoptive immunotherapy.Methods:1.T cells were isolated from peripheral blood in3healthy individuals, and thenextracted the total RNA, established two multiplex reverse transcription polymerasechain reaction (mRT-PCR) methods to detect CDR3spectratyping and length of22TCR Vβ family family for the analysis of the proliferation of T cell clones by takingadvantage of the multi-gene expression system.2. T cells were isolated from peripheral blood in3healthy individuals, and thenextracted the total RNA, established two multiplex reverse transcription polymerasechain reaction (mRT-PCR) methods to detect CDR3spectratyping and length of32 TCR Vα family for the analysis of the proliferation of T cell clones by takingadvantage of the multi-gene expression system.3. Total RNA was extracted from monocytes which were isolate from pleuralfluid of patients with lung cancer; then analyze the TCR Vβ family and TCR Vαfamily clonal proliferation situation of TIL in patients with lung carcinoma by the twomulti RT-PCR methods.4. The total RNA was extracted from the T cell isolated from the pleural effusion.The monoclonal/oligoclonal TCR Vβ genes were cloned and one correct sequencewas subcloned into the vector to construct shuttle plasmidpDC315-TCRVα-IRES-TCRVβ.5. The adenovirus backbone plasmid and shuttle plasmidpDC315-TCRVα-IRES-TCRVβwere co-transfered into HEK293cells withLipofectamine2000, and recombinant TCR Vβ and TCR Vα adenovirus wasgenerated. The exogenous target gene was analyzed by PCR using viral genomicDNA as template and the expression of target protein in PBMC was detected by flowcytometry. The recombinant adenovirus was propagated in HEK293cells, the titter ofviruses were detected by TCID50method. PBMC was infected by recombinant TCRVβ and TCR Vα adenovirus and subject to coculture with different tumor cells(NCI-H1299-HLA-A2+, NCI-H1299-HLA-A2-, HepG2,7402)36h after infection.The killing effects on tumor cells were separately analyzed by MTT after12,24and36hours.Result: Successfully establish two multiplex reverse transcription polymerasechain reaction (mRT-PCR) methods to detect CDR3spectratyping and length of22TCR Vβ family and32TCR Vα family for the analysis of the proliferation of T cellclones by taking advantage of the multi-gene expression system. GeXP detected TCRVβ family and TCR Vα family CDR3spectratyping of tumor infiltrating lymphocytes(TIL) in five patients with lung carcinoma, the results show that the families in patient2were all polyclonal, the remaining four patients appeared monoclonal andoligoclonal proliferation phenomenon. Successfully cloned the TCR Vα5and TCRVβ17gene from patients5, and properly constructed recombinant adenovirus shuttleplasmid pDC315-TCRVα5-IRES-TCRVβ17. The recombinant adenovirus werepacked by HEK293cells. Extraction of the recombinant adenovirus genome, PCRamplification obtained the Fiber35TCR Vβ17gene, the target gene was integratedinto the viral genome. Virus infected PBMC, the target gene was detect by flow cytometry. The killing assay showed that the lysis efficiency of PBMC infected withrecombinant TCR adenovirus on different tumor cells was: NCI-H1299(HLA-A2+)>HepG2(HLA-A2+)> NCI-H1299(HLA-A2-)>7402(HLA-A2+)Conclusion: GEXP detected CDR3of TIL in five lung cancer patients, theTCRVα and TCRVβ family appeared oligoclonal or monoclonal proliferationphenomenon (except P2) which presumably due induced tumor specific antigen.Clone TRAV5(TRAV5-TRAJ27) and TRBV17(TRBV17-TRBD2S2-TRBJ2S1) genefrom the pleural fluid of patients with lung cancer and construct recombinantadenovirus vector. Killing experiments showed that recombinant TCR adenovirusinfection of PBMC has a lethal effect on two lung adenocarcinoma cell line, the lethaleffect on HLA-A2+lung adenocarcinoma cell was significantly higher than that ofHLA-A2-lung adenocarcinoma cells, but has no effect on7402cells. The oligoclonalproliferation TCRVα, and TCRVβ family could caused by lung cancer.