Distribution, Phenotype, Generation, And Functional And Clinical Relevance Of Tc17Cells In Human Gastric Cancer

Gastric cancer (GC) is the second most frequent cause of oncological death worldwideand carries an especially poor prognosis. Clinical relevance of GC is influenced bycross-talks between different cell types which create and maintain an immunosuppressiveenvironment that promotes GC tumor progression and attenuates therapeutic efficacy.Solid GC comprise not only malignant cells, but also many other non-malignant celltypes that contains many resident and migratory non-malignant cell types. Recent studies,particularly of oncogene-driven tumors in transgenic mice, have shown that the outcome ofprimary oncogenic events in epithelial cells can be significantly modified by the nature ofthe surrounding non-malignant cells, especially immune cells. So, it is becoming apparentthat the immune cell-regulated microenvironment can modify the neoplastic properties ofthe tumor cells and that immune cells can be used as therapeutic targets.T cells constitute a major component of immune cells within the tumor microenvironment. A subset of T cells that produce IL-17and exhibit potent pro-inflammatoryproperties has recently been detected in human tumors. This population can be furtherdivided into CD4+T cells (Th17cells) and CD8+T cells (Tc17cells). In mice, Tc17cellscan be generated through in vitro priming with Th17cell-polarizing cytokines such asIL-23and-despite exhibiting reduced cytotoxic activity-they afford increased protectionagainst influenza infection and tumors. By contrast, in rhesus macaques and sootymangabeys, Tc17cells promote disease progression during simian immunodeficiency virusinfection. Notably, in humans, virtually nothing is known about the distribution, phenotype,generation, and functional and clinical relevance of Tc17cells in GC.Monocytes/macrophages and myeloid-derived suppressor cells (MDSCs) are other twokey immune cell types within the tumor milieu that influence anti-tumor immunity.Monocytes respond to tumor-derived signals, such as CSF-1and IL-6, and release Th17 cell-polarizing cytokines and in turn polarize T cell responses. Whether such a mechanismexisted and, if it did, was also responsible for the polarization of Tc17cells induced bymonocytes in GC remains unclear. MDSCs suppress anti-tumor immunity and thereforepromote tumor progression. Interestingly, pro-inflammatory cytokine IL-17has been shownto promote MDSC development and regulate MDSC in mice, but the mechanisms by whichthis occurs and by which cell types and also its relevance to human malignancies especiallyGC are yet to be elucidated.【Objectives】1. To characterize the distribution and phenotype of Tc17cells in GC.2. To investigate the generation and regulation of Tc17cells in GC.3. To elucidate the functional and clinical relevance of Tc17cells in GC.【Methods】1. The distribution and phenotype of Tc17cells in GCThe fresh blood and GC specimens were collected in Department of General Surgeryand Center of Minimal Invasive Gastrointestinal Surgery, Southwest Hospital, ThirdMilitary Medical University. Tc17cells distribution was examined by FCM, doubleimmunofluorescence staining and double immunohistochemistry staining. The gastricpathologic examination was assayed by H&E staining. Tc17cell phenotype was examinedby FCM. The correlations between Tc17cells and other immune cells in tumors wereevaluated by correlation assays.2. The generation and regulation of Tc17cells in GCFresh peripheral blood, non-tumor tissue or tumor-infiltrating monocytes were selected.The supernatant-conditioned blood monocytes were generated by culturing bloodmonocytes with50%TCCS, NCCS, TTCS or NTCS. The monocyte culture supernatantswere collected for ELISA, and the cells were collected for surface marker staining.In a5-d incubation, bead-purified peripheral CD8+T cells were labeled with CFSE andco-cultured with autologous blood, non-tumor tissue-derived or tumor-infiltratingmonocytes; or TCCS-, NCCS-, TTCS-or NTCS-conditioned blood monocytes at2:1ratio.Alternatively, CD8+T cells were co-cultured with autologous blood monocytes in thepresence or absence of hr IL-6, IL-23, and/or IL-1β; or were co-cultured withtumor-infiltrating monocytes in the presence of neutralizing Ab against IL-6, IL-23, and/or IL-1β, or a control IgG. After5-d incubation, the supernatants were harvested for ELISA,and the cells for intracellular cytokine staining.3. The functional and clinical relevance of Tc17cells in GCPrimary tumor cells from GC patients with TNM stage Ⅰ were purified.Primarytumor cells were stimulated with hr IL-17, IL-22, IFN-γ, IL-17plus IL-22, or100%Tc17cell-polarizing culture supernatants. In some cases, neutralizing Abs for IL-17and/or IL-22was added into the culture. The culture supernatants were harvested for ELISA andchemotaxis assays.The secondary tumor cell culture supernatants were collected as chemoattractants.FACS-sorted tumor-infiltrating MDSCs were transferred into the upper chambers oftranswells. Chemokine CXCL12and chemoattractants were placed in the lower chambers.Migration was quantified by counting cells in the lower chamber and cells adhering to thebottom of the membrane. In some cases, blocking Ab for CXCR4was added into MDSCsuspensions and incubated before chemotaxis assay.Purified peripheral CD8+T cells were CFSE-labeled and co-cultured with samenumber of autologous, CD8+-depleted PBMCs that were pretreated with mitomycin C. Then,autologous FACS-sorted tumor-infiltrating MDSCs were added at1:1ratio. In transwellexperiments, CFSE-labeled CD8+T cells and autologous mitomycin C-treated feeders wereseeded at1:1ratio in the lower chamber; autologous tumor-infiltrating MDSCs were thenadded either in the lower or upper chamber. After5-d incubation, the supernatants wereharvested for ELISA, and the cells were analyzed by FCM for CFSE dilution.Overall patient survival was defined as the interval between date of surgery and date ofdeath or last follow-up, whichever occurred earlier. Cumulative survival time wascalculated by the Kaplan-Meier method. Multivariate analysis of prognostic factors foroverall patient survival was performed using the Cox proportional hazards model.【Results】1. The distribution and phenotype of Tc17cells in GC1.1Patients with GC showed a higher Tc17cell percentage in peripheral blood thanhealthy donors. Within the patient cohort, tumors contained a significantly higher Tc17cellpercentage than other tissues. Moreover, as the cancer progressed, we found that thepercentage of Tc17cells significantly increased in each of the tested tissues with the exception of TDLN. In tumors, this accumulation of Tc17cells was most notable from stageⅡ onwards. Similar observations were made when analyzing the total number of Tc17cellsper million total cells in each tissue.1.2Double immunofluorescence staining and double immunohistochemistry stainingof Tc17cells showed that Tc17cells were abundant in tumor tissues. These data abovesuggest that Tc17cells are enriched in GC microenvironment.1.3Intratumoral Tc17cells expressed less IFN-γ, IL-4, IL-10, and IL-9, butco-expressed IL-22and IL-17F, and expressed little HLA-DR, CD25, perforin, GrB, FoxP3,and PD-1. Intratumoral, peritumoral, and non-tumor tissue-derived Tc17cells all displayeda CD27-CD45RA-effector/memory phenotype, whereas peripheral or TDLN-derived Tc17cells displayed a CD27+CD45RA-memory or CD27+CD45RA+naive phenotype.Tumor-infiltrating Tc17cells were positively correlated with IL-22-producing T-cellsubsets, Th17cells, and CD14+TAMs, and relatively poorly correlated with other T-cellsubsets and CD56+NK cells.2. The generation and regulation of Tc17cells in GC2.1T cell-monocyte co-cultures showed that TAMs were superior to blood ornon-tumor-derived monocytes in inducing CD8+T cell proliferation and Tc17cellpolarization and that the resultant cells, rather than the monocytes themselves, readilyproduced IL-17. TAMs expressed higher levels of CD80, CD86, and HLA-DR than othermonocytes.2.2TTCS-and TCCS-conditioned blood monocytes also expressed higher levels ofCD80, CD86, and HLA-DR and induced significantly more IL-17production and Tc17cellpolarization. These suggest that tumor cells release soluble factors which activate and/ormodify monocytes within the tumor microenvironment to promote Tc17cell polarization.2.3IL-6, IL-1β, and IL-23were all significant up-regulated in TAM culturesupernatants when compared to the supernatants of monocytes isolated from autologousnon-tumor tissues or peripheral blood. Similar results were obtained from TTCS-or TCCS-but not NTCS-or NCCS-conditioned blood monocyte culture supernatants. Individual Abblockade of IL-6, IL-1β, or IL-23efficiently inhibited the generation of Tc17cells by40-60%, while a combination of all three Abs almost completely abrogated the induction ofTc17cells and the concomitant production of IL-17. Provision of exogenous IL-6, IL-1β, or IL-23significantly increased the frequency of Tc17cells either alone or in combination.These demonstrate that IL-6, IL-1β, and IL-23play an essential role in monocyte-andTAM-mediated Tc17cell induction in vitro, and suggest that a similar process mightoperate in vivo.3. The functional and clinical relevance of Tc17cells in GC3.1We found that the percentage of MDSCs among total CD45+cells was higher intumor tissues than that in peritumoral or non-tumor tissues, and was positively correlatedwith the frequency of Tc17cells. We also examined IL-17R expression on the MDSCs andfound that few tumor-infiltrating MDSCs expressed IL-17R but the majority oftumor-infiltrating MDSCs expressed CXCR4but not CCR4, CCR6, or CXCR3. By contrast,MDSCs in peritumoral or non-tumor tissues had little CXCR4expression. These suggestthat MDSCs may be induced to migrate into the tumor microenvironment throughCXCR4-mediated chemotaxis.3.2We found the positive correlation between Tc17cells and CXCL12, the chemokineligand for CXCR4, within tumors, and the high expression of IL-17R on GC cells, and thatIL-17induced CXCL12production by primary tumor cells in a dose-dependent manner.TAM-derived (but not blood or non-tumor tissue monocyte-derived) Tc17cell culturesupernatants induced CXCL12production, which was almost totally blocked by anti-IL-17neutralizing Ab. These suggest that Tc17cell-derived IL-17induces chemokine CXCL12production by tumor cells.3.3MDSC chemotaxis assay showed that culture supernatants from tumor cells treatedwith TAM-derived Tc17cell-polarizing culture supernatants induced significantly moreMDSC migration than those supernatants from tumor cells treated with non-tumor tissue orblood monocyte-derived culture supernatants, and this effect was lost upon pre-treatmentwith neutralizing Abs against IL-17and CXCR4. These data support a model wherein Tc17cells secrete IL-17to induce tumor cells to produce chemokine CXCL12which in turnrecruits MDSCs into the tumor microenvironment.3.4We found that MDSCs suppressed proliferation, IFN-γ production andperforin/GrB expression by CD8+T cells. Transwell assay showed that cell contact wasnecessary for the suppression of T-cell proliferation, IFN-γ production, and perforin/GrBexpression, indicating that tumor-infiltrating MDSCs are likely to suppress anti-tumor CD8+CTLs through a cell contact-dependent mechanism.3.5An increased Tc17cell percentage was correlated with increased CEA expressionand tumor size, advanced lymphatic invasion and vascular invasion. Comparing patientswith high (≥2.75%median level) vs low (<2.75%) Tc17cell percentage level, the21-monthsurvival rate was significantly lower for those with in higher Tc17cell percentage group.Similar results were obtained when the patient cohort was stratified based upon Tc17cellnumber. Importantly, the finding that intratumoral Tc17cell percentage independentlypredicted survival was verified by multivariate analyses using a Cox proportional hazardmodel.【Conclusion】1. Tc17cells, with a distinct cytokine and functional profile, accumulated in tumors ofGC patients.2. Tumor-activated monocytes secreted IL-6, IL-1β, and IL-23, which together favoredTc17cell polarization.3. Supernatants from Tc17cell-polarizing cultures induced CXCL12chemokineproduction by tumor cells, which in turn promoted CXCR4-dependent migration of MDSCsand subsequent impairment of anti-tumor CD8+CTLs function via a cell contact-dependentmechanism.4. Tc17cell percentage increased with tumor progression and independently predictedreduced overall survival.【Significance】These results highlight the practical importance of Tc17cells in GC, and indicate thatfurther study of the regulation mechanism and function of Tc17cells in human GCmicroenvironment may help to elucidate the characteristics of immune response and theimmunopathogenic mechanism of Tc17cells in GC microenvironment and targeting Tc17cells may be a novel therapy for GC.