Analysis Of TCR Vβ CDR3 And Cytotoxicity Of Allogenetic T Cells Induced By Nasopharyngeal Carcinoma Cell Line CNE2

BACKGROUNDTumor generation and development is the fighting results between tumor cells andhost immune system. In the usual condition, host immune system can surveil andclean mutant cells in the body. However, the fact is tumor cells have developedmultiple mechanisms to restrain and escape this surveillance including the lack ofcostimulatory molecules, MHC abnormality, tumor antigens modulation and secretionof apoptosis-inducing molecules. All these ways get a common result, i.e., tumorantigens can't be presented to immune effector cells efficiently, so effector cells haveno means to fully execute surveillance and cleaning functions. The T-cell-mediatedcellular immune response plays a critical role against tumors mainly depend oncytotoxic T lymphocyte (CTL). This effect can kill survival tumor cells in early stageof tumor generating, relieasing phase and after resecting operation. When tumor is inadvance stage, this approach is impractical for patients, particularly forimmunodeficient patients. For the past few years, there are some new developmentsin the study of MHC restriction. So, the allogenetic immunotherapy is payed moreand more importance in recent years.TCRβand TCR a compose a different origin double chain, express on T cells that is the molecule to recognize the special antigen and mediate immune response. Thecomplementarity-determining region (CDR) is the important part to recognize antigen.The CDR3 of BV and AV combine to the center of peptide, however, CDR1 andCDR2 combine to the Junction of peptide and MHC. CDR3 beta chain sizeheterogeneity arises during developmental DNA rearrangement when a BC segmentis joined with 1 of 24 BV segments via a diversity (D) and a joining (J) segment togive rise to a complete BV gene. The immunoscope spectratyping is a powerful toolto analyze CDR3 length and sequence of TCR diversity in health and disease.The T-cell-mediated immune response plays a critical role against tumors. Aneffective T-cell response is initiated by two distinct signals: signals 1 and 2. Signal 1is antigen specific and transduced by T-cell receptor interaction with the peptide/MHC on the surface of APCs. Signal 2 is delivered by a series of costimulatorymolecules in an antigen-nonspecific fashion. The combined effects of these twosignals lead to T-cell activation, proliferation, and differentiation into effector cells.In the absence of signal 2, T-cell activation is abortive and cannot proceed to thestage of efficient effector function. Their interaction also include maintain andinhance the juncture between APC and T lymphocyte. There are many costimulatorymolecules and their ligands, for example, B7/CD28,LFA-1/ICAM-1 and ICAM-2,CD2/LFA-3, but the most important is B7-1(CD80) and B7-2 (CD86).Biotin-avidin system (BAS) is basis on the special characters of biotin and avidinthat they can bind to antigens, antibodies and other molecule. The technology isefficient, durable, stabile and multiple levels amplified.Accompany by the development of molecular immunology and molecularbiology, for example, the immunoscope spectratyping technique has been used moreextensively, the study of generation, differentiation, regulation and responce/toleranceof T lymphocyte has more and more methord.Objective1. Establishing the immunoscope spectratyping technique for monitoring CDR3polymorphism and length distribution of human aβT cells. Analyzing the CDR3 size and distribution of TCR aβT cells in the peripheral blood of healthy volunteers. It isoffer new thinking and techniques for the purpose of study of the diagnose, andmechanisms of T cells immune response or tolerancd, and individualizedimmunotherapy of the infection disease, tumor and auto-immune disease.2. Interleukin 2 (IL-2) has the effect to promote all kind of T cells proliferation andsecrete cytokine in vitro. It is usually be used in the culture of T cells. Our study is toanalyze the drift of the complementarity-determining region 3 of T cell receptor betachain variable region in of healthy volunteers cultured with IL-2 and detect thenon-special killing effect by the way of LDH releasing. The aim is to exclud the IL-2side effcet for next study.3. The allogenetic T cells were activated by dendritic cell (DC) loaded nasopharyngealcarcinoma cells line CNE2 in vitro. The study is to explore the specific cytotoxicity toCNE2 and primary analysis TCR BV CDR3 of cytotoxic T lymphocyte (CTL) and toprovide fundational theory for allogenetic immunotherapy.4. Malignant cells often elude the immune system by lacking costimulatory signalsrequired for the generation of effective antitumor immunity. This technology involvesmodification of the cell membrane with a biotin derivative and decoration ofbiotinylated cells with a chimeric molecule composed of the extracellular domains ofthe human CD80 costimulatory molecule and core streptavidin (CD80-SA). To studythe possibility that tumor cells can be manipulated to express costimulate moleculeand converted into potent APCs to induce anti-tumor effect.Methods1. Design and synthesis of primers of 24 TCRβchain variable gene (TCR BV), TCRβchain constant gene (TCR BC) and the marked anti-sense TCR BV with FAM.Selectde 5 health volunteers, analysis of 24 TCR BV family CDR3 length byfollowing research flow-process. (1) Isolating total RNA from PBMC of all samples.(2) Synthesis of the first cDNA by the total RNA temple of all samples. (3) PCRamplification of CDR3 cDNA. (4) 1.5% agarose gel electrophoresis of the PCRproducts. (5) Analysis of CDR3 length by Immunoscope spectratyping. 2. Isolating the T cells from health volunteers and culturing the T cell in vitro withIL-2. Detecting the non-special killing effect of the T cells by the way of LDHreleasing. Analysis the drift of TCR BV gene CDR3 in healthy volunteers by theimmunoscope spectratypes to evaluate the clonal proliferaion of T cells.3. Monocytes were isolated from peripheral blood mononuclear cells of healthyvolunteers and cultured with GM-CSF, IL-4 and TNF-a, then the T cell activated bymature DC loaded by CNE2. The phenotype of monocytes and mature DC wasdetected by flow cytometry. Detecting the specific cytotoxicity by the way of LDHreleasing. Analysis the distribution of TCRβchain CDR3 in PBMC and CTL ofhealthy volunteers by immunoscope spectratyping, to evaluate the clonal proliferationof T cells.4. This technology involves modification of the CNE2 and CNE2/DDP with a biotinderivative and decoration of biotinylated cells with a chimeric molecule composed ofthe extracellular domains of the human CD80 costimulatory molecule and corestreptavidin (CD80-SA). PBLs from ealthy volunteers were isolated by centrifugationon Ficoll-Paque Plus followed by plastic adherence for 2 h. Effector PBLs (5×10~6)were stimulated in culture for 5-6 days with the deactivate tumor cells (1×10~6) leftundecorated or decorated with CD80-SA. Effector cells were then harvested andtested for cytotoxic activity against various tumor targets at different E:T ratios usingthe LDH releasing assay.Results:1. The PCR product of 24 TCR BV families of 5 health volunteers showed a blurband on a 1.5% agarose gel, showed more than eight bands on 6% acrylamidesequencing gel and showed Gaussian distribution by GeneScan except BV19. TheCDR3 showed all the CDR3 family has the similarity RI, but has the different lengthprofiles.2. It was showed Gaussian distribution of 24 TCR BV CDR3 families except BV19 inhealthy volunteers. The T cell cultured with IL-2, however, displayed someanomalous and oligoclonal expansion in different TCR BV families according to the individual. The drift of CDR3 has not obviously regularity. The T cells have notkilling effect to nasophargngal carcinoma cell line CNE2.3. The DC cultured with GM-CSF, IL-4 and TNF-a displayed typically characteristicsof DC phenotype. The immunoscope spectratypes of TCR BV gene CDR3 showedGaussian distribution in PBMC of healthy volunteers. The CTL activated by DCloaded with CNE2, however, displayed some anomalous and oligoclonal expansion indifferent TCR BV families and showed special cytotoxic ability against CNE2.Conclusion: The allogenetic CTL activated by DC loaded CNE2 showed specialcytotoxic ability to CNE2. The expansion of TCR BV families has some potentialvalue in Nasopharyngeal carcinoma therapy.4. The use of binding method as an alternative to conventional gene transferapproaches for immunotherapy depends upon efficient cell surface display ofexogenous proteins on the surface for periods of time necessary for the elicitation ofthe antitumor response. The decoration of CNE2 and CNE2/DDP with CD80-SAresulted in effective display of this chimeric molecule on the surface by FCM. Tumorcells displaying the chimeric molecule generated potent proliferative responses inPBLs from healthy volunteers. T cells primed with CD80-SA-decorated tumor cells,but not with unmanipulated cells, effectively lysed tumor targets in an antigen-specific manner detected by the way of LDH releasing.Conclusion1. We established the immunoscope spectratyping technique for analysis of the CDR3profiles and distribution of TCR aβT cells, it was a simple, stable and sensibletechnique for monitoring the special T cells, it was not only offer the information ofall the T cells in the samples, but also coule detect the monoclonal expansion oroligoclonal expansion T cells, furthermore, it did not resemble other T cells assay,immunoscope spectratyping technique needn't per-stimulate the T cells in thesamples in vitro, it would be applied into the diagnosis, mechanisms of T cellsresponse, individual therapy of the infection disease, tumor and auto-immune disease.2. It was showed Gaussian distribution of TCR BV CDR3 except BV19 in healthy volunteers. The T cell cultured with IL-2, however, displayed some anomalous andoligoclonal expansion in different TCR BV families and there are not killing effect tonasophargngal carcinoma cell line CNE2. The change has obviously individualdifference. The high RI BV fimily may be the result of memory T cell clonelproliferation that used to contact with some antigen. In conclusion, IL-2 has someinfluence of TCRβchain CDR3 in the T cell cultured in vitro. It is necessary toexlude the side effect of IL-2 in the study about TCR BV CDR3, otherwise we willjump to a incorrect result.3. Our former study shows that the T cells isolated from peripheral bloodmononuclear cells of healthy volunteers have no cytotoxicity to CNE2. However, theT cells activated by DC loaded by CNE2 have the specific cytotoxicity and clonalproliferation. The immunoscope spectratypes of TCR BV CDR3 showed Gaussiandistribution in PBMC of healthy volunteers. The CTL activated by DC loaded withCNE2 displayed some anomalous and oligoclonal expansion in different TCR Vβsubfamilies and showed special cytotoxic ability against CNE2. In conclusion: Theallogenetic CTL activated by DC loaded CNE2 showed special cytotoxic ability toCNE2. The expansion of TCRβsubfamilies has association with CTL and somepotential value in Nasopharyngeal carcinoma therapy.4. The study showed that it is possible that CNE2 and CNE2/DDP can bemanipulated to express costimulate molecule and converted into potent APCs toinduce anti-tumor effect. These results indicate that the CD80-SA molecule deliverseffective costimulatory signals that lead to not only T-cell proliferation but alsodifferentiation into killer cells. In conclusion, rapid and durable cell surface display ofcostimulatory proteins possesses the simplicity, safety, and efficacy required to makeit a clinically relevant alternative that will accomplish the same as gene transferapproaches in the treatment of a broad spectrum of immune-based disorders.