| Objective:1.To analyze the level of tissue-infiltrating CD8+CD103+ Tissue-resident memory(Trm)cells and its clinical relevance in gastric cancer patients.2.To study the phenotype and functional characteristics of CD8+CD103+Trm cells in gastric cancer patients.3.Explore the mechanism of CD8+CD103+Trm cell function decline in gastric cancer tissue.Methods:1.105 cases of gastric cancer patients’ postoperative pathological sections(tumor sections and matched non-tumor normal sections)were collected for immunohistochemical double staining,and the number of CD8+CD103+Trm cells in tissues was detected;And analyze the correlation with clinical stage,lymph,nerve invasion,pathological,differentiation,vascular invasion,the size of tumor and tumor invasion depth.2.Fresh tissues from 54 patients with gastric cancer were collected and digested by enzyme,and then stained and detected by flow cytometry,and the proportion,number,phenotype and function of CD8+CD103+Trm cells were analyzed.3.The level of TGF-β1protein in gastric cancer tissues was detected by enzyme linked immunosorbent assay(ELISA),and the correlation between TGF-β1 concentration and granzyme B-expressing CD8+CD103+Trm cell levelas well as perforin-expressing CD8+CD103+Trm cells level was analyzed.Purified CD8+T cells was stimulated by rhTGF-β1 in vitro,and the expression of granzyme B and perforin in CD8+CD103+Trm cells was analyzed.Results:1.To analyze the level of tissue-infiltrating CD8+CD103+Tissue-resident memory(Trm)cells and its clinical relevance in gastric cancer patients.1.1 Immunohistochemical double staining was used for gastric cancer patient samples,and 3-5 visual fields were randomly counted under high magnification.The number of CD8+CD103+Trm cells was counted and the differences between groups were compared.The results showed that there was no significant difference in the number of CD8 and CD 103 double positive cells between tumor and normal tissues(37/HPF vs.33/HPF,P>0.05).According to flow cytometry analysis,the results showed that the frequency of CD8+CD103+Trm cells in tumor tissues of gastric cancer patients were significantly lowerthan thatin normal tissues(67.6%vs.78.2%,P<0.05).Whereas every 106 cells were homogenized,the number of CD8+CD103+Trm cells had no statistical difference between tumor tissues and normal tissues(9332/106 vs.7698/106,P>0.05).1.2 The number of CD8+CD103+Trm cells in tumor tissues of gastric cancer patients with tumor diameter greater than 5 cm,undifferentiated tissue,which invaded the whole stomach wall,was significantly lower than that of gastric cancer patients with tumor diameter less than 5 cm,tissue differentiation and shallow invasion depth.The number of CD8+CD103+Trm cells in tumors of patients with nerve,vascular invasion and regional lymph node metastasis was significantly lower than that of patients without nerve,vascular invasion and regional lymph node metastasis.According to TNM staging analysis,the results showed that with the progression of gastric cancer,the number of CD8+CD103+Trm cells in tumor tissues of gastric cancer patients gradually decreased,and the decreased number of CD8+CD103+Trm cells was negatively correlated with the progression degree of gastric cancer.According to the median number of CD8+CD103+Trm cells in high magnification,gastric cancer patients were divided into two groups:low CD8+CD103+Trm cells and high CD8+CD103+Trm cells,the follow-up time was 60 months.The survival rate of patients was counted and analyzed by Log-rank.The results showed that the survival rate of high CD8+CD103+Trm cell array was significantly higher than that of low CD8+CD103+Trm cell array.2.Phenotypic and functional characteristics of CD8+CD103+Trm cells in gastric cancer patients2.1 Compared with CD8+CD103-T cells,the infiltrating percentages of CD45RA+CCR7+subsets and CD45RA+CCR7-subsets in CD8+CD103+Trm cells in gastric cancer tissues decreased significantly(1.0%vs.10.5%,2.0%vs.14.3%,P<.05).While the proportion of CD45RA-CR7-subgroup increased significantly(94.1%vs.70.9%,P<0.05),but there was no significant difference for the percentage of CD45RA-CCR7+subset in CD8+CD103-T cells and CD8+CD103+Trm cells(3.0%vs.4.3%).Compared with CD8+CD103-T cells,CD8+CD103+Trm cells expressed higher levels of CD69,PD-1 and CD39(73.5%vs.61.4%,79.3%vs.47.8%,23.3%vs.6.1%,P<0.05),but expressed lower level of KLRG1(12.2%vs.58.1%,P<0.05).2.2 Compared with their CD8+CD103-T cell counterparts,the expression levels of granzyme B and perforin in tumor-infiltrating CD8+CD103+Trm cells were significantly decreased(granzyme B:37.5%vs.47.9%,P<0.05,perforin:4.0%vs.8.5%,P<0.05).The expression levels of granzyme B and perforin in tumor-infiltrating CD8+CD103+Trm cells were significantly decreased after stimulation with lymphocyte stimulator(granzyme B:36.1%vs.47.5%,P<0.05,perforin:3.5%vs.7.8%,P<0.05).Inaddition,compared with normal tissues,the expression levels of granzyme B and perforin of CD8+CD103+Trm cells in tumor tissues were decreased(granzyme B:35.7%vs.41.0%,P<0.05,perforin:2.7%vs.4.3%,P<0.05).3.The dysfuntional mechanism of CD8+CD103+Trm cells in gastric cancer patients.3.1 The results of ELISA showed that TGF-β1 protein concentration in gastric cancer was negatively correlated with levels of granzyme B and perforin in CD8+CD103+Trm cells(P<0.05).3.2 Furthermore,recombinant human rhTGF-β1 was used to stimulate cells in vitro,which showed that compared with those without rhTGF-β1 stimulation,the expression of granzyme B and perforin in CD8+CD103+Trm cells stimulated by rhTGF-β1 decreased significantly(P<0.05).Conclusion:1.The infiltration level of CD8+CD103+Trm cells in gastric cancer is closely associated with disease progression.2.Compared with normal tissues,the frequency of CD8+CD103+Trm cells in gastric cancer decreased,and its anti-tumor function also decreased.3.TGF-β1 stimulation could down-regulate the expression of granzyme B and perforin in CD8+CD103+Trm cells to reduce their anti-tumor function. |
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