| Objective: 1)to explore the predictive value of the expression of CD39 and CD103 on T cells in peripheral blood of patients with nasopharyngeal carcinoma(NPC).2)to observe the phenotypic changes of CD39+T cells and T cells in peripheral blood of patients with nasopharyngeal carcinoma at different treatment time points,and to explore the role of CD39+CD8+T cells in the treatment of nasopharyngeal carcinoma.3)to evaluate the function of tumor-specific T cells in nasopharyngeal carcinoma in vitro and to explore the functional heterogeneity of tumor-specific CD8+T cells.Methods: 1)60 NPC and 25 healthy controls were collected in the affiliated Tumor Hospital of Xinjiang Medical University with the clinical data and followed up.15 ml PBMC was isolated from elbow vein blood of the patients and multicolor flow cytometry staining was performed to analyze the expression of CD39 and CD103 on T cells.2)24 groups of PBMC of patients were collected from the patients before treatment,1 month after treatment and 6 months after treatment.Multi-color flow cytometry was used to detect the changes of CD39,CD103 and differentiation phenotypes(CD27,CCR7,CD45RA)on T cells at different timepoint during treatment,as well as the co-expression with immune checkpoint PD-1and Tim-3.3)EBV peptide tetramer was used to isolate EBV specific T cells from NPC patients.T cell function experiments,such as T cell affinity assay,intracellular cytokine detection,flow cytometry staining,cytokine assay and TCR sequencing were performed.Results: 1)the distribution of CD39 and CD103 on the T cells in PBMC of NPC patients was different from that in healthy controls.The expression of CD39+CD8+T cells,CD39+CD4+T,coexpressed CD39+CD103+CD8+T cells and CD39+CD103+CD4+T cells in NPC group was significantly higher than that in healthy controls.Compared with the clinical features,it was found that the expression rate of CD39+CD103+CD8+T cells in patients with distant metastasis was significantly higher than that in patients without distant metastasis(P<0.05).Univariate analysis showed that clinical stage,N stage and CD39+CD103+CD8+T cell expression level were the factors affecting PFS in patients with nasopharyngeal carcinoma,and the difference was statistically significant.Multivariate analysis showed that CD39+CD103+CD8+T cells and N stage were independent prognostic factors of PFS(HR=0.147,95%CI ? 0.028-0.781,P=0.024;HR=12.187,95%CI ? 2.561-57.995,P=0.002).Survival curve analysis showed that the PFS of patients with high expression of CD39+CD103+CD8+T cells was better than that of patients with low expression of CD39+CD103+CD8+T cells.2)According to the expression of CD27,CCR7 and CD45 RA on T cell,memory T cells in peripheral blood can be naive type(CD27+CCR7+CD45RA+),early type(CD27+CCR7+CD45RA-),intermediate type(CD27+CCR7-CD45RA-),advanced type(CD27-CCR7-CD45RA-)and late type(CD27-CCR7-CD45RA+).compared with CD39-CD8+T cells,CD39+CD8+T cells showed more central memory T cells,early effector memory T cells and middle and late effector memory T cells,while fewer initial and terminal T cells,the difference was statistically significant(P<0.001).Compared with CD39-CD4+T cells,CD39+CD4+T cells showed more early effector memory T cells,but fewer initial T cells,the difference was statistically significant(P<0.001),CD39+CD8+T cells and CD39+CD4+T cells expressed only a few inhibitory receptors PD1 and Tim-3.In CD39+CD8+T cells,the co-expression of PD1+ accounted for 11.46% and the co-expression of Tim3+ accounted for 88.53% and 4.77%,respectively.In CD39+CD4+T cells,co-expression of PD1+accounted for 5.54%,Tim3+ accounted for 94.43%,and coexpression of Tim3+ accounted for 2.43%.The changes of CD39+CD8+T cells before and after treatment: the expression rate of CD39+CD8+T cells increased 1 month after treatment,and decreased 6 months after treatment,and the difference was statistically significant(P<0.05).The expression rate of CD39+ CD4+T cells increased 1 month after treatment,and remained at a high level 6 months after treatment.Compared with that before treatment,the difference was statistically significant(P<0.05).Changes of T cell differentiation phenotype before and after treatment: the expression ratio of initial CD8+T cells decreased significantly 1 month after treatment,and remained at a low level 6 months after treatment compared with that before treatment.The expression ratio of middle and late effect memory CD8+T cells and terminal CD8+T cells increased 1 month after treatment,and remained at a high level 6months after treatment,and the difference was statistically significant(P<0.05).The expression ratio of initial CD4+T cells decreased significantly 1 month after treatment,and it was still much lower than that before treatment 6 months after treatment.The expression ratio of middle and late effector CD4+T cells to terminal CD4+T cells increased 1 month after treatment,and it was still higher 6 months after treatment,and the difference was statistically significant(P < 0.05).The increased proportion of CD39+CD8+T cells in peripheral blood was positively correlated with the increased proportion of CD8+ memory T cells in early effector type(R=0.469,P=0.031)and middle and late effector type(R=0.508,P=0.019).The difference was statistically significant.The increased proportion of CD39+CD4+T cells in peripheral blood was not positively correlated with the increased proportion of early effector CD4+ memory T cells,and the difference was statistically significant(R=0.439,P=0.047).3)tumor specific T cells were screened from 4 patients.The surface CD39 and CD103 of tumor specific T cells were higher than that of total T cells,and tumor antigen specific T cells were early effect(CD27+CD45RA-CCR7-)memory T cells.Two HLA-A11-restricted EBVSSCSSCPLSK-specific T cell lines and clones were cultured.In vitro experiments showed that the specific T cell lines from the same source had different antigenic sensitivity.TCR antibody detection showed that all clones had the same TCR Vb,and selected 6 T cell clones with different antigen sensitivity.TCR sequencing showed that all the clones were TRAV22 TRAJ43 TRBV10-2 TRBJ2-5.Killing function test and intracellular cytokine staining showed that the killing function and IFN-? secretion ability of 6 T cell clones were consistent with antigen sensitivity,and all 6 clones could secrete IL-2,IL-10,IFN-?,TNF-? and GM-CSF.The results of chemokine staining showed that there was almost no expression of CCR3,CCR4,CCR5,XCR1 and CXCR4,and the expression of CCR2 was different among clones.it was found that the expression rate of CXCR4 increased after T cell activation and remained high 8 hours later,while the expression rate of PD-1increased after T cell activation,peaked at 4 hours and decreased after 8 hours,but still not decreased to the initial level.Conclusion: 1)the expression level of CD39+CD103+CD8+T cells PBMC of NPC patients is related to the PFS.High expression of CD39+CD103+CD8+T cells is correlated with the better prognosis of patients.2)CD39+CD8+T cells in peripheral blood are high quality effector cells.Radiotherapy and chemotherapy can increase the expression level of CD39+CD8+T cells in peripheral blood of patients with nasopharyngeal carcinoma,so it may be the main contributor to good control of metastasis in patients with nasopharyngeal carcinoma after radiotherapy and chemotherapy.3)tumor specific T cells with the same TCR have different antigen sensitivity,cytokine spectrum and killing ability in vitro.Therefore,further study on the heterogeneity of tumor specific CD8+T cells in vitro may be of potential significance for T cell immunotherapy of nasopharyngeal carcinoma. |
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