| Background and Objection:Immunotherapy provides an attractive treatment of cancer, the advantage of which is that it can enhance or reconstruct the patients’ anti-tumor immunity with weak side effect. Currently, there are mainly two strategies for tumor immunotherapy, namely, therapeutic vaccination and adoptive cell therapy, and the latter normally refers to the transfusion of autologous or allogenic immune cells into tumor-bearing hosts. The immune cells include Cytotoxic Lymphocytes (CTLs) with tumor-reactive T cells as its major ingredient and tumor-infiltrating lymphocytes (TILs) etc. The transfusion of tumor-reactive CTLs has been employed in treatment of metastatic melanoma, Epstein-Barr virus Induced Lymphoma and other tumors.But the clinical application of adoptive cell therapy in most solid tumor is dependent on the solution of two major problems.1) How to obtain sufficient numbers of tumor-reactive T cells.2) The efficacy of adoptive immunotherapy in humans is often limited by the failure of cultured T cells, particularly cloned CD8+T cells, to persist in vivo.A major restriction of adoptive T cell therapy is that highly avid T cells can only be separated from some particular patients, because tumor lacks of antigenicity [4]. To solve this problem, reactive T cell clones were selected from TILs. Through this approach, researchers identified the genes of T Cell Receptors (TCRs), which can specifically recognize tumor antigen, and then transduced the genes encoding tumor-specific TCRs into mature T cells to acquire TCR gene engineered T cells that can specifically recognize antigen positive tumor cells in vitro and can mediate anti-tumor immunity after being re-injected into the patient. TCR gene engineered T cells were first employed in clinical trials treating melanoma, in which MART-1antigen specific TCR genes were transduced into melanoma patients’peripheral blood lymphocytes (PBLs). The transfused TCR gene engineered T cell then directly destroy antigen positive tumor in vivo. By this means, complete tumor regression were observed in some cases. Similar results have been achieved in clinical trials in which NY-ESO-1antigen-specific TCR gene engineered T cells were utilized to treat melanoma and synovial sarcoma patients.A critical point, which determines the antitumor efficacy of TCR gene engineered T cells, is to identify the TCRs that can recognize tumor antigens and select T cell subsets that are suitable for adoptive cell therapy.Based on differentiations, T cells fall into three categories, naive T cells, effector T cells (TE), and memory T cells. In order to obtain sufficient cell numbers for adoptive transfer, T cell usually need to be stimulated in vitro. A vital reason for the short-term survival of transfused T cells is that effector phenotypes are acquired after T cells being activated in vitro. Compared with effector T cell, there are reasons to believe that transfer of cells with memory properties, including enhanced recall response and the ability to undergo self-renewal, may be superior mediators of an antitumor response in vivo.According difference of phenotype, homing ability and function, memory T cells can be divided into effector memory T cells (TEM) and central memory T cells (TCM)-Tcm express CD62L and CCR7, which can promote the homing of them to lymph nodes and enable them to proliferate rapidly upon re-exposure to the same antigen. Berger’s landmark research modeled on macaques has proved that effector T cells (TE) derived from Tcm can persist long-term in vivo, and reacquired phenotypic and functional properties of memory T cells.We sought to obtain tumor-reactive T cells population with improved capacity to persist and heightened anti tumor reactivity by the following strategy.1) T cell modified by tumor antigen specific TCR gene.In previous research, we had screened AFP peptide binding specific TCR genes-TCRV α12.2-V β7.1, by gene preferential usage of TILs isolated from hepatocellular carcinoma patients. The antigen specificity of T cell is redirected by the modification of tumor specific TCRV α12.2-V β7.1, which enable T cell to recognize antigen-positive tumor cell.2) The activation of tumor specific TCR gene modified T cell induces the differentiation of memory T cell.To investigate the differentiation of memory T cell, after activation of TCR gene modified T cell stimulated by tumor antigen, and determine the phenotype and antitumor reactivity of TCR transferred T cell induced memory T cell. 3) TCR gene transferred central memory T cell (Tcm).Since memory T cell is superior to CD8+T cells in the persistence and antitumor efficiency, and intrinsic properties of T cells that are isolated for adoptive therapy determine their fate in vivo. We than attempt to obtain a superior tumor-reactive T cell population by isolating CD8+central memory T cells for transferring with tumor specific TCR gene.Chapter1Enhanced anti tumor reactivity by TCR gene transferred T cell induced memory T cellObjectiveTo investigate the differentiation of memory T cell after the activation of TCR gene transferred T cell stimulated by tumor antigen in vitro. And determine the phenotype and antitumor reactivity of TCR transferred T cell induced memory T cell.Methods and materials1. T cell culture, stimulation and transduction1) Human peripheral monocytes (PBMC) were isolated by Ficoll-plaque plus (GE Healthcare) density gradient centrifugation of buffy coats from healthy donors.2) Primary stimulation was accomplished by McAb to costimulatory molecule and cytokine on day13) Transduction was performed2days after initial stimulation using Ad5F35-TRAV-TRBV adenovirus supernatant produced in HEK293packaging cells. 4) The relative efficiency of transgene expression in T cells was detected by flow cytometry (3-14days after transduction).5) Restimulation was performed on day5, using T2cell pulsed with9-amino HLA-A2+epitope from AFP2i8-226(LLNQHACAV).2. Phenotype changes of TCR gene transferred T cell stimulated by tumor antigen.Expression of maker molecule CD45RO、CD62L、CD44on TCR gene transferred T cells was detected by FACS from3-14days after stimulation3. Anti tumor reactivity of TCR gene ttansferred T cell after stimulated by tumor antigen1) CTL activatity. After coculture24hours with different target cells:HepG-2(HLA-A2+AFP+), SMMC-7721(HLA-A2+AFP+), and MCF-7(HLA-A2+AFP-) at different E/T ranging from30:1to3:1, tumor specific lysis of TE from PBMC control, Stimulated PBMC, TCR gene transferred T cell and Stimulated TCR gene ttansferred T cell was evaluated by MTT assays.2) Induced apoptosis. HepG-2was coculture4hours with TE of each group (E/T30:1). The frequency of apoptosis target cell was detected by Annexin V/PI double-labeled FACS.The proportion of FasL+cell in TE of each group was detected by FACS.3)Cytokine secretion. The secretion of cytokine IFN-γ and IL-2of TE coculture with HepG-2(E/T30:1) were quantified by ELISA assaysStatistical analysisThe experimental data are expressed as mean (x)±standard deviation (s). Statistical analysis was carried out by SPSS v13.0software (SPSS Inc, Chicago, USA).After checking for normal distribution and homogeneity of variances, Factorial design variance analysis was used to detect the main effects and interaction of factors. Analysis of one fact was evaluated using Independent samples T test or One way Anova. Probabilities (P)<0.05was considered to be statistical difference and P<0.01was considered to be significantly statistical difference.Results1. Ad5F35chimeric adenoviral vector transduced T cell effectively. The highest tranduce efficiency was obtained at MOI100(17.120±0.983%, which was significantly higher than other groups(P<0.001) The frequency of exogenous TCR positive cell is about30%3days after transduction. The frequency of exogenous TCR decreased over the time.2. Tumor specific TCR gene modification redirected the antigen specificity of T cell effectively; TCR gene transferred T cells were activated by stimulation of tumor antigen AFP. The frequency of CD45RO+cell increased after activation, reached (39.150±4.005%)14days after stimulation in TCR gene transferred and stimulated with antigen group, which is significantly higher than other groups. Antigen stimulation initiated the differentiation process of memory T cell. As indicated by CD62L, CD44fluorescent McAb labeling, phenotype of most CD45RO+cells are CD62L-CD44-. The frequency of CD62L positive cell increased as time went on after stimulation. Cell clusters with CD62L+CD44+Tcm phenotype appeared in TE cells eventually.3. Tumor specific TCR gene modification enhanced the ability of T cells to lyse HLA-A2+AFP+target cells:HepG-2(TCR gene transferred group:25.479±8.574%, control group:6.107±1.047%), SMMC-7721(TCR gene transferred group: 24.368±8.411%, control group:6.024±1.269%).The difference was statistically significant (P<0.001)(n=5). Stimulating by tumor antigen could improve specific lysis of TCR gene transferred T cells (HepG-2:33.467±12.625%, SMMC-7721:32.027±12.309%), showing significant difference with TCR gene transferred group (P<0.001). The percent specific lysis of antigen stimulated TCR gene transferred T cell was significantly higher than that of TCR gene transferred T cell (P<0.001). TCR gene modification and antigen stimulation had no significant effect on the specific lysis of T cell against AFP-target cell MCF-7(P>0.05), which indicates antigen specificity of TCR gene modification.4. Tumor specific TCR gene modification enhanced the ability of T cells to induce HepG-2apoptosis (TCR gene transferred group:16.400±1.272%, control group:1.375±0.573%), showing significant difference(P<0.001)(n=4). The frequency of apoptosis target cell induced by antigen stimulated TCR gene transferred T cell (28.750±4.160%) was significantly higher than that of TCR gene transferred T cell (P=0.001). TCR gene modification and antigen stimulation promoted the expression of FasL on TE cells which mediate apoptosis in Fas-FasL pathway. The frequency of FasL+T cells in TCR gene transferred group (14.700±1.706%) was significantly higher than that of control group(1.725±0.427%)(P<0.001)(n=4). The frequency of FasL+T cells in antigen stimulated TCR gene transferred T cell (27.650±2.686%) was significantly higher than that of TCR gene transferred group(P<0.001).5. Tumor specific TCR gene modification enhanced T cells to secret IFN-γ which mediate anti tumor response((TCR gene transferred group:25.962±2.488ng/ml, control group:3.736±0.412ng/ml, P<0.001, n=5). The production of IFN-γ by antigen stimulated TCR gene transferred T cell (33.394±2.520ng/ml) was significantly higher than that of TCR gene transferred T cell (P=0.002).ConclusionTumor specific TCR gene modification redirected antigen specificity of T cell effectively. Antigen specific T cell stimulated by tumor antigen initiated the differentiation of memory T cell, which enabled TE acquire phenotypic character of Tcm and mediated enhanced response if re-exposure to tumor antigen.Chapter2Enhanced anti tumor reactivity of Tcm modified by TCR gene transferred.ObjectiveTo investigate phenotypic character and anti tumor reactivity of TE derived from sorted CD8+Tcm modified by tumor specific TCR gene.Methods and materials1. TCM sorting, stimulation and transduction1) CD8+T cells were isolated from PBLs of HLA-A2+healthy donors by magnetic bead negative selection, CD62L+CD44+cells were then separated by positive selection.2) Primary stimulation was accomplished by McAb to costimulatory molecule and cytokine on day13) Transduction was performed2days after initial stimulation using Ad5F35-TRAV-TRBV adenovirus supernatant produced in HEK293packaging cells. 4) Restimulation was performed on day5, using T2cell pulsed with9-amino HLA-A2+epitope from AFP218-226(LLNQHACAV).5) In vitro growth of TE derived from CD8+T cell and Tcm was measured by counting viable cells using trypan blue dye exclusion.6) Telomera length of CD8+T cell and TCM was measured by in situ hybridization and flow cytometry (Flow-FISH) on day14which is climax of cell numer after stimulation.2. Phenotype changes of TE derived from TCM stimulated by tumor antigen. Expression of maker molecule CD45RO, CD62L, CD44, CD28and CCR7on TE derived from Tcm and CD8+T cells were detected by FACS from7-28days after stimulation.3. Anti tumor reactivity of TE derived from TCR gene transferred TCM.1) The relative efficiency of transgene expression in Tcm and CD8+T cells was detected by FACS (3-14days after transduction).2) CTL activity,1days after restimulation, Cytotoxicity of TE (derived from TCR gene transferred or untransferred Tcm and CD8+T cell) against target cells (HepG-2, SMMC-7721and MCF-7) at different E/T ranging from30:1to3:1was evaluated by the calcein release assay.3) Antibody blocking experiments. Target cells (HepG2, SMMC-7721) were incubated with anti-human HLA-A2mAb before coculture. Cytotoxicity of TE derived from TCR gene transferred Tcm against HLA-A2+target cells (HepG-2, SMMC-7721) was evaluated by the calcein release assay.4) Cytolytic granule protein secretion, ldays after restimulation, TE from TCR gene transferred or untransferred Tcm and CD8+T cells were cocultured4h with target cells (HepG2, SMMC-7721) at30:1(E/T). The frequency of perforin+and Granzyme B+cells in TE was detected by intracellular staining with FACS.5) Cytokine secretion.1days after restimulation, TE from TCR gene transferred or untransferred Tcm and CD8+T cell were cocultured overnight with target cells (HepG2, SMMC-7721, and MCF-7) at30:1(E/T). IFN-γ and IL-2contents in the supernatants were determined by ELISA assays.Statistical analysisThe experimental data are expressed as mean (x)±standard deviation (s). Statistical analysis was carried out by SPSS v13.0software (SPSS Inc., Chicago, USA). After checking for normal distribution and homogeneity of variances, Factorial design variance analysis was used to detect the main effects and interaction of factors. Analysis of one fact was evaluated using Independent samples T test and One way Anova. Probabilities (P)<0.05was considered to be statistical difference and P<0.01was considered to be significantly statistical difference.Result1. The frequency of Tcm was13%-35%of CD8+T cells, which was variable among12different donors. CD62L+CD44+TCM were sorted from6HLA-A2+samples. Isolating efficiency reached97%or higher.2. TE from each subset displayed rapid expansion after stimulation, and significantly greater number of cells was obtained from TE derived from TcM(day14,19.421±3.003×106/ml) compared with that from CD8+T cells(day14,14.533±1.874×106/ml)(t=-5.349, P<0.001)(n=15). After reaching the peak at day14, number of TE from Tcm displayed slower decline than that from CD8+T cells. The number of TE derived from Tcm was significantly higher than that of TE derived from CD8+T cells on day21(t=-12.226, P<0.001), day28(t=-16.472, P<0.001) and day 35(t=-20.203, P<0.001). The relative telomere length of TE derived from TCM(1.005±0.053) was longer than that from CD8+T cells(0.968±0.017). However, the difference was not significant (P=0.228)(n=4).3. After being stimulated, the frequency of CD62L+cell in TE derived from CD8+T cells gradually decreased to the extent that cannot be detected. On the contrary, the frequency of CD62L+cell in TE derived from Tcm maintained certain level (>10%). The frequency of CD44+cells in TE derived from CD8+T cells gradually decreased, reaching20%at the end of culture. In contrast, the frequency of CD44+cells in TE derived from Tcm constantly keeps relatively high, accounting for85%during the whole culture period. Especially in mid-late period of culture (21days later), cell clusters with high CD44expression appeared in TE from TCM,all CD62L positive cells were CD44high cells, i.e., CD62L+CD44high cluster.4. The expression of CD45RO in TE cells derived from Tcm was higher than that in TE cells derived from CD8+T cells during the culture period. Similar to the trend of CD62L+T cells, the frequency of CD28+cells and CCR7+cells in TE cells derived from Tcm maintained certain level during the culture period (>19%), whereas the frequency of CD28+cells and CCR7+cells in TE cells derived from CD8+T cells gradually decreased to lower extent (<10%).5. TCM and CD8+T cell were efficiently transduced by Ad5F35chimeric adenoviral vector. The frequency of TCRa12+-TCRVβ7+cells is30-36%of T cells3days after transduction, the frequency of TCRa12+-TCRVβ7+cells declined gradually over time after transduction.6. TCR gene transfer enhanced the ability of T cells to lyse HLA-A2+AFP+target cells:HepG-2(TCR gene transferred TCM:32.382±14.311%, TCM:10.566±2.034%(n=6). SMMC-7721(TCR gene transferred TCM:29.512±12.246%, TCM:10.878±2.114%)(n=6). AFP antigen stimulation enhanced specific lysis of TCR gene transfer T cm, which was significantly higher than TCR gene transfer CD8+T cell (HepG-2:24.424±9.340%, SMMC-7721:21.368±6.948%) in10:1(P=0.04) and30:1(P=0.015) coculture with HepG-2, and in10:1(P=0.007) and30:1(P=0.037) coculture with SMMC-7721.7There are no statistical differences in level of specific lysis between TCR gene transferred T cells and untransferred control groups in the CTL assays using AFP-MCF-7as target cells, which indicates antigen specificity of TCR gene modification.8. After blocking the HLA-A2sites on the surface of HepG-2and SMMC-7721with HLA-A2mAb, the specific lysis effects of TCR gene transferred T cells could be significantly eliminated. The specific lysis was significantly lower after blocking by HLA-A2mAb in3:1,10:1and30:1using HepG-2(t=-3.406, t=-20.945, t=-15.188. P=0.003, P<0.001, P<0.001)(n=10) or SMMC-7721(t=-4.021, t=-15.640, t=-12.443. P=0.001, P<0.001,P<0.001)(n=10) as target cells. These results indicate that TCR gene transferred CTL recognized target tumor cells in an HLA-A2-restricted manner.9. After coculture with HLA-A2+AFP+target cells, the frequency of perforin+and granzyme B+cell in TE derived from TCR gene transferred TCM are higher than that in untransferred TCM-The frequency of perforin+and granzyme B+cell in TE derived from TCR gene transferred CD8+T cells and untransferred CD8+T cells are both lower than that of corresponding TCM cell groups.10. After coculture with HLA-A2+AFP+target cells, TCR gene transferred TCM produced greater quantities of IFN-y (coculture with HepG-2:35.761±7.311ng/ml, coculture with SMMC-7721:27.310±3.672ng/ml) than untransferred TCM (coculture with HepG-2:3.288±0.740ng/ml, coculture with SMMC-7721:3.211±1.181ng/ml), showing significant difference(P<0.001). Furthermore, Content of IFN-y secretion in TCR gene transferred Tcm was higher than that of TCR gene transferred CD8+T cell (coculture with HepG-2:27.920±3.722ng/ml, coculture with SMMC-7721:24.673±2.279ng/ml). The difference was statistically significant (HepG-2, donor1: P=0.003, donor2:P=0.017. SMMC-7721, donor1:P=0.005, donor2:P=0.001)ConclusionIsolating central memory T cells rather than CD8+T cells for insertion of gene encoding tumor-specific TCR may provide a superior tumor-reactive T cell population for adoptive transfer. |
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