Experimental Study Of Distribution, Phenotype, Differentiation, Recruitment, And Clinical Significance Of CD4~+IL-17~+cells In The Tumor Microenvironment Of Pancreatic Cancer Patients

Objective To investigate the percentage of CD4+IL-17+cells in the tumor tissuesand peripheral blood of pancreatic cancer patients, and analyze the distribution ofCD4+IL-17+cells in the tumor microenvironment and peripheral blood of pancreatic cancerpatients.Methods The percentage of CD4+IL-17+cells in the tumor tissues of26pancreaticcancer patients and peripheral blood of20pancreatic cancer patients was detected by flowcytometry analysis (FACS). The corresponding adjacent normal tissues in26cases and20healthy volunteers were used as control respectively. The percentage of CD8+IL-17+cellsin the tumor tissues was also detected by FACS. The percentage of CD4+IL-17+cells wascompared according to the different clinical stage and node metastasis group. Thepercentage of CD4+IL-17+cells in the tumor tissues, corresponding adjacent normal tissuesand peripheral blood of12same pancreatic cancer patients were analyzed.Results The percentage of CD4+IL-17+cells detected by FACS was significantlyhigher in tumor tissues (5.28±1.65%) compared with corresponding adjacent normaltissues (3.37±0.93%, P＜0.05). In addition, the percentage of CD4+IL-17+cells wassignificantly higher in stage III-IV tumors than stage I-II tumors (6.98±2.78%VS4.41±1.09%, P＜0.05). The percentage of CD8+IL-17+cells was higher in TIL than NIL (P＞0.05). The percentage of CD4+IL-17+cells in peripheral blood of patients (2.58±1.05%)was significantly higher than healthy volunteers (1.18±0.32%, P＜0.05). The percentage ofCD4+IL-17+cells was higher in stage III-IV tumors than stage I-II tumors (3.55±1.49% VS2.08±0.78%,P＜0.05). Moreover, the percentage of CD4~+IL-17~+cells was higher intumors with lymph node metastasis than tumors without lymph node metastasis(4.01±1.66%VS2.39±1.13%, P＜0.05). In the same patient, the percentage ofCD4~+IL-17~+cells in tumor tissues is highest (5.05±1.71%), which is lowest in peripheralblood (2.19±0.68%).Conclusions The percentage of CD4~+IL-17~+cells in pancreatic tumor tissues wassignificantly higher corresponding adjacent normal tissues. The percentage of CD4~+IL-17~+cells in peripheral blood of pancreatic cancer patients was significantly higher than healthyvolunteers. In the same patient, the percentage of CD4~+IL-17~+cells in tumor tissues ishighest, which is lowest in peripheral blood. Taken together, the data indicate that cells areinriched in the tumor microenvironment and peripheral blood of pancreatic cancer patients.Part Ⅱ The phenotype and function of CD4~+IL-17~+cells in tumormicroenvironment of pancreatic cancer patientsObjective To identify the phenotypic characteristics of CD4~+IL-17~+cells and theexpression profile of CD4~+IL-17~+cells-associated cytokines and transcription factor intumor microenvironment of pancreatic cancer patients.Methods The phenotype (HLA-DR,CD25,PD-1,Foxp3,CD45RO,CD45RA)and chemokine receptors (CCR1,CCR2,CCR4,CCR5,CCR6,CCR7,CXCR4) anddifferent immune cell subsets (Th1,Th2,Treg,tumor-associated macrophages) ofCD4~+IL-17~+cells in tumor tissues were detected by FACS. The relationship betweenCD4~+IL-17~+cells and different immune cell subsets was analyzed. The mRNA expressionlevels of CD4~+IL-17~+cells-associated cytokines (IL-17A,IL-23,IL-6,TGF-,IL-1) andtranscription factor in tumor tissues of pancreatic cancer patients were determined by Realtime-polymerase chain reaction (Real time-PCR). The serum concentration of IL-17andIL-23in patients were determined by enzyme linked immunosorbent assay (ELISA).Results Most of the tumor-infiltrating IL-17~+cells were CD4~+IL-17~+cells.CD4~+IL-17~+cells in tumor microenvironment of pancreatic cancer express little T-cellactivation function markers HLA-DR, CD25, and immune suppression markers PD-1,Foxp3. These cells also show the phenotype of memory T cells (CD45RO~+CD45RA-).CD4~+IL-17~+cells in tumor tissues express low levels of CCR1, CCR5, and CCR7, and express high levels of CCR2, CCR4, CCR6, and CXCR4. FACS reveals that CD4~+IL-17~+cells were inversely correlated with Th1cells, and positively correlated with Treg cells.However, there were no quantitative correlations between CD4~+IL-17~+cells and Th2cellsand tumor-associated macrophages (TAM). Real time-PCR shows high levels ofCD4~+IL-17~+cells-associated cytokines (IL-17A, IL-23, IL-6, TGF-, IL-1) andtranscription factor in tumor tissues of pancreatic cancer. Moreover, ELISA shows highserum concentration of IL-17(69.2±28.5pg/ml) and IL-23(266.5±98.1pg/ml) in patients.The serum concentration of IL-17(75.8±29.0pg/ml) and IL-23(279.9±102.1pg/ml) washigher in stage III-IV tumors than stage I-II tumors (P＜0.05).Conclusions CD4~+IL-17~+cells in tumor microenvironment of pancreatic cancerpatients have their own phenotype including the T cell function markers and chemokinereceptor expression profiles. CD4~+IL-17~+cells-associated cytokines and transcriptionfactor were highly expressed in tumor tissues and serum of pancreatic cancer patients.Part Ⅲ Themechanism of differentiation and recruitment ofCD4~+IL-17~+cells in tumor microenvironment ofpancreatic cancer patientsObjective To explore the possible mechanism of differentiation and recruitment ofCD4~+IL-17~+cells in tumor microenvironment of pancreatic cancer patients.Methods TAM from pancreatic cancer tissues were isolated by FACS, and thenormal macrophages (M) isolated from PBMC of pancreatic cancer patients were used ascontrol. The na ve CD4~+T cells were isolated from PBMC of pancreatic cancer patients bymagnetic cell sorting system (MACS). After TAM or normal M was cocultured withna ve CD4~+T cells and tumor cells for48hours, the percentage of CD4~+IL-17~+cells wasdetected by FACS, and the expression levels of the CD4~+IL-17~+cells-associated cytokineswere determined by Real time-PCR and ELISA. The cytokines secreted by TAM weredetected by Real time-PCR. The changes of percentage of CD4~+IL-17~+cells were detectedby FACS after cytokines blocking assay. Regulatory T (Treg) cells from pancreatic cancertissues were isolated by FACS. The changes of percentage of CD4~+IL-17~+cells wasdetected by FACS in the presence different concentrations of tumor-associated Treg cells. The chemokine receptors (CCR1,CCR2,CCR4,CCR5,CCR6,CCR7) of CD4~+IL-17~+cells in tumor microenvironment and PBMC of pancreatic cancer patients were detected byFACS. Migration assay was conducted between the chemokine (RANTES,MCP-1,CCL22,CCL20) and CD4~+IL-17~+cells in PBMC. The above chemokines of tumormicroenvironment in pancreatic cancer patients were detected by FACS.Results After TAM or normal M was cocultured with na ve CD4~+T cells andtumor cells, TAM was more efficient than normal M in eliciting CD4~+IL-17~+cells andtheir associated cytokine production (P＜0.05), and the induction was dose dependent.TAM expresses higher levels of IL-23, IL-6, and IL-1, compared with normal M (P＜0.05). Blockade of IL-23or IL-6can largely reduce TAM-mediated induction ofCD4~+IL-17~+cells. Tumor-associated Treg cells can suppressed CD4~+IL-17~+cellsdifferentiation in a dose-dependent manner. CD4~+IL-17~+cells in PBMC of patients expresshigh levels of CCR2and CCR6, median levels of CCR4, and low levels of CCR1.Migration assay shows that both MCP-1and CCL20can effectively induce the migrationCD4~+IL-17~+cells in PBMC. The expression of both MCP-1and CCL20in tumor tissueswas significantly higher than corresponding adjacent normal tissues (P＜0.05). Theexpression of CCL22and RANTES in tumor tissues was relatively low, and there was nosignificantly difference between corresponding adjacent normal tissues.Conclusions TAM secreted a series of cytokines, in which IL-23and IL-6play amajor role. It might be the major mechanism for differentiation of CD4~+IL-17~+cells in thetumor microenvironment of pancreatic cancer. MCP-1and CCL20expression in tumortissues could specially interacted with CCR2and CCR6of CD4~+IL-17~+cells in PBMC,which promotes the migration and trafficking of CD4~+IL-17~+cells into the tumormicroenvironment. It might be the major mechanism for the inrich of CD4~+IL-17~+cells inthe tumor microenvironment of pancreatic cancer.Part IV The clinical significance of CD4~+IL-17~+cells in tumormicroenvironment of pancreatic cancer patientsObjective To investigate the clinical significance of CD4~+IL-17~+cells in tumormicroenvironment of pancreatic cancer patients. Methods Immunohistochemistry (IHC) was performed to detect IL-17~+cells in51pancreatic tumor tissues, as well as expression of CD34and VEGF in20tumor tissues.The relationship between the expression of IL-17~+cells in tumor tissues and tumorangiogenesis, clinic pathological parameters, and survival time of pancreatic cancerpatients were analyzed.Results The percentage of CD4~+IL-17~+cells in tumor tissues was positively correlatedwith microvessel density (MVD) and the expression of VEGF in tumor tissues (P＜0.05).IL-17~+cells was shown to mainly locate in cytoplasm, and the frequency of positive cells intumor tissues was higher than corresponding adjacent normal tissues (P＜0.05). Thepresence of IL-17~+cells in tumor tissues was associated with tumor, node, and metastasis(TNM) stage and lymph node metastasis (P＜0.05), but not with patient sex, age, tumorsize, tumor location, histological grade, and local invasion (P＞0.05). Of the51pancreaticcancer patients, follow up was successful for50(98.0%). The follow up period was2–67months, and the mean survival time of these patients was16.6±4.8months. Patients withhigher levels of intratumoral IL-17~+cells had significantly shorter survival time thanpatients with lower levels of intratumoral IL-17~+cells (P＜0.05). The univarite analysisshow tumor size, TNM stage, lymph node metastasis, and levels of intratumoral IL-17~+cells were associated with survival, but multivariate analysis show only TNM stage wereindependent prognostic factor for survival.Conclusions The distribution of CD4~+IL-17~+cells in tumor tissues was positivelycorrelated with tumor angiogenesis. The expression of IL-17~+cells in tumor tissues wasrelated with the clinic pathological parameters (TNM stage and lymph node metastasis) andsurvival time of the patients. CD4~+IL-17~+cells may be served as one of the importantimmune indicators for predicting the prognosis of pancreatic cancer patients.