| Objectives: Gastric cancer(GC)is one of the leading cause of cancer related death in the world,and regulatory T cells(Tregs)are considered as one of central factors to facilitate tumor tolerance by inhibiting antitumor immunity.Recently,Tregs have been proposed as the heterogeneous T population with distinct phenotype and function,which are recognized as a main factor to result in the confusing consequences with regard to the researches involving Tregs and GC.So far,there is little known about the relationship between these subpopulations of Tregs and GC.So we conduct this research to determine the expression of these Treg subtypes in both the peripheral circulation and tumor tissue of patients with GC and investigate the relationship between the Treg subgroups and the different clinicopathological characteristics of GC.Methods: Peripheral blood in patients with GC and healthy donor,and stomach tissues from the tumor and adjacent nontumor in a same patient were collected to gain the mononuclear cells.These fresh mononuclear cells were then stained with the surface markers anti-CD3,anti-CD4,anti-CD45 RA,anti-CD25 and intracellular anti-Foxp3.Further dividing Treg cells into three subtypes CD45RA+Foxp3lo(rTreg),CD45RA-Foxp3hi(aTreg),CD45RA-Foxp3lo(non-Treg)based on the CD45 RA and Foxp3 expression.The percentage of Tregs and subtypes were analyzed by FACSAria flow cytometry.Statistical analyses were performed in Graph Pad Prism 5.0 software.The significantly differences between groupswere determined using the Mann-Whitney U test(between two groups)and Kruskal-wallis test(between multi-groups).Results: The percentages of CD4+CD25+ Foxp3+Tregs were significantly increased in both peripheral blood of GC(GC-PBMC)and Tumor Infiltrating lymphocyte(TIL)compared to those in peripheral blood of healthy donor(HD-PBMC)and the nontumorous stomach tissue(NIL)in the same patients,respectively.(P<0.0001,P<0.0001,respectively).The percentages of aTreg and non-Treg cells in GC-PBMC were dramatically higher than those in HD-PBMC(P < 0.0001,P < 0.0001,respectively),the difference was significant.The percentages of aTreg and non-Treg cells in TIL were significantly higher than those in NIL(P<0.0001,P< 0.001,respectively).The clinical impact of Tregs and subtypes on clinicopathological features of GC was examined.We found that there was no obvious correlation between the mean percentage of Treg subtypes and tumor site,size,differentiation,stage,serum concentration of CEA,but there was significant correlation between the mean percentage of Treg subtypes and the state of metastases(lymph node and/or distant metastases).The mean percentage of CD4+CD25+Foxp3+Tregs was significant higher in PBMC and TIL with tummor metastases than those without metastases(P<0.05,P<0.01,respectively).In the analyses of subtypes,only the mean percentage of aTreg cells in TIL with metastases was found significantly increased in comparison to those without metastases(P<0.01).Conclusions: We demonstrated that the percentage of Tregs in both peripheral circulation and tumor tissue of GC was greatly raised compared to the controls.Importantly,the most significantlyelevated subtype were aTreg cells and only those in TIL were correlated with tumor metastases.These results imply that aTreg cells are the major subtype of Tregs in tumor microenvironment responsible for the suppression of antitumor immunity to facilitate the tumor progression.Therefor in the future targeted immunotherapy,local and selective depletion of these tumor infiltrating Treg subtype without depleting other cell types and/or systemic Tregs is expected to be an effective therapeutic approach for patients with GC.Meanwhile,we found that non-Treg cells were also dramatically increased in both peripheral blood and tumor tissue of GC,but not obviously correlated with the GC.Hence,more accurate data will be obtained by identifying distinct Tregs subtypes rather than the whole population of Tregs in the future study of GC. |
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