Functional Immunoregulation And Clinical Relevance Of NK Cells In Patients With Gastric Cancer

Gastric cancer (GC) is one of the most common malignancies. The morbidity and mortality of GC is very high and accounts for about8%total cases and10%total deaths from cancer worldwide. The clinical outcome of GC is influenced by the cross-talks among different types of cells which create an immunosuppressive environment that is tightly associated with tumor progression and poor prognosis.Besides tumor cells and tissue-resident cells, a great amount of immune cells are migrated in the GC environment. In the human tumors, the longer survival time of patients will be obtained while activated-immune cells and the secretion of their effector moleclues are increased. In addition, lymphocyte-deficient Rag2-/-I12rg"-/-mouse (lack T cells, B cells and NK cells) can spontaneous form tumors and display a highly susceptibility to tumor-induction after methylcholanthrene exposure. Therefore, infiltrated immune cells in the tumor microenvironment are associated with the development of tumors, and boosting host anti-tumor immune response can be used as treatment strategies to improve the efficacy of tumor immunotherapy.Generally, CD8+cytotoxic T cells (CD8+CTL)-mediated acquired immune response play a critical role in the anti-tumor immunity. Increased CD8+CTL in the tumor microenvironment predict good prognosis. However, our previous study indicated that the function of CD8+CTL was inhibited by myeloid-derived suppressor cells (MDSCs) and denoted a failure of CD8+CTL to kill tumor cells. In the recent years, the anti-tumor capacity of NK cells is noticeable. As a type of innate immune cells, NK cells have the natural ability to distinguish normal cells from "modified" malignant cells and eliminate their targets as well as CD8+CTL through the release of cytotoxic enzymes (granzymes and perforin) and/or soluble factors such as interferon-γ (IFN-y). NK cells can also induce a powerful reaction called antibody-dependent cellular cytotoxicity (ADCC) by expressing the Fc immunoglobulin fragment low-affinity receptor CD16. However, clinical and experimental studies demonstrate that the dysfunction of NK cells is often occured inhuman solid tumors and correlate with advanced disease progression. However, the nature,regulation, functional and clinical predictive values of NK cells in GC remains unknown.Monocytes/macrophages markly outnumber other antigen presenting cells in solidtumors and can influence anti-tumor immunity. In the tumor environment, tumor-derivedsignals polarize monocytes to M2macrophages and then these cells inhibit the anti-tumorimmune response of CD8+PCPTL and NK cells. Thus, whether the dysfunction of NK cells ismodulated by monocytes/macrophages in GC milieu, and what mechanism involved in theprocess needs to be elucidated.【Objectives】1. To analyze the distribution, phenotype and functional characteristics of NK cells inGC.2. To investigate the regulation and clinical relevance of NK cells in GC.【Methods】1. The distribution, phenotype and functional characteristics of NK cells in GCThe tissue specimens and fresh blood were obtained from65patients with GC in theDepartment of General Surgery and Center of Minimal Invasive Gastrointestinal Surgery,Southwest Hospital, Chongqing. Normal blood from30healthy individuals was enrolled asthe control. The gastric pathologic examination was assayed by H&E staining. Thedistribution of NK cells was detected by FCM and immunohistochemical staining. Theexpression of receptors on NK cells and its functional relevance molecules were analyzedby FCM.2. The regulation of NK cells in GCThe phenotype of tumor-infiltrating monocytes/macrophages was analyzed by FCM.The correlations between monocytes/macrophages and NK cells in tumors were evaluatedby spearman’s correlation assays. The co-localization of monocytes/macrophages and NKcells in GC tissues was examined by immunohistochemical staining.Fresh peripheral blood from normal individuals was purchased from the Department ofBlood Transfusion, Southwest Hospital. Monocytes were purified by CD14-positiveselection. The conditioned blood monocytes were obtained by culturing purified monocyteswith25%TSN, TTSN and NTSN for12hours. In the co-culture system, peripheral NK cells were isolated and cocultured withautologous blood monocytes or autologous TSN-, TTSN-, and NTSN-conditionedmonocytes at4:1ratio and rhIL-2was added at100U/ml. For blocking experiments, NKcells were pre-incubated with anti-2B4Ab, or a control IgG for30min, and thenco-cultured with monocytes. Alternatively, anti-TGF-β1Ab was directly added to theco-culture system. After7days, the phenotype and intracellular cytokine production wereanalyzed by FCM. The apoptosis of NK cells was detected by using7-AAD/annexinⅤapoptosis detection kit. Additionally, NK cells alone were cultured with rhIL-2for5days, and rhTGF-β1was added or not, then the expression of Ki-67and IFN-γ wasdetected by FCM.3. The clinical relevance of NK cells in GCThe baseline clinical characteristics of65patients with GC were analyzed and theclinical TNM stages of tumors were determined according to the international TNMclassification system. Overall patient survival time was defined from the date of surgery tothe date of death or last follow-up. Differences between two groups were measured byMann Whitney nonparametric test. Cumulative survival time was calculated by theKaplan-Meier method and two groups were compared by using the log-rank test. Statisticalanalysis was performed with the GraphPad Prism5.0Software. P<0.05was consideredstatistically significant.【Results】1. The distribution, phenotype and functional characteristics of NK cells in GC1.1Using FCM, the prevalence of NK cells, NKT cells and T cells was detected innormal blood、GC blood and tissues. The percentages of NK cells, NKT cells and T cells innormal blood were18.03%±1.82%,3.25%±0.38%and52.47%±2.56%; and in GC bloodwere18.00%±1.67%,2.92%±0.40%and53.00%±2.08%；and in nontumor tissues were4.41%±0.38%,0.93%±0.14%and16.24%±2.03%; and in peritumor tissues were3.55%±0.25%,1.06%±0.15%and22.27%±2.09%; and in tumor tissues were2.38%±0.17%,1.29%±0.19%and29.82%±2.02%。The percentages of these cells in blood were nostatistically difference between healthy individuals and GC patients, but significantlyhigher than those in the tissues of GC patients. Within the GC patient cohort, blood alsocontained significantly higher percentages of these cells than tissues. In addition, the percentage of NK cells in tumor tissues was significantly lower than those in peritumor ornontumor tissues, but the percentage of T cells was significantly increased in tumor tissues.Immunohistochemical staining showed that the infiltration of NK cells in tumors was alsoless than that in peritumor or nontumor tissues.1.2Compared with normal blood, the expression of NKG2D, NKp30and DNAM-1onNK cells of GC blood was significantly decreased. Compared with peritumor or nontumortissues, CD16expression on NK cells was down-regulated but NKp44and2B4on NK cellswas up-regulated in tumor tissues. However, the expression of activating receptors NKp30,NKp46, NKG2D, DNAM-1and CD94and inhibitory receptors NKG2A, CD158a/h,CD158b and CD158e1on NK cells had no significant difference among them.1.3Compared with peritumor or nontumor tissues, NK cells in tumor tissues expressedhigher CD69but lower Ki-67, TRAIL and IFN-γ. Although there was no difference on theexpression of perforin between tumor and nontumor tissues, the expression of CD107a wassignificantly decreased in tumor tissues.2. The regulation of NK cells in GC2.1The phenotype analysis showed that tumor-infiltrating monocytes (TAMs)co-expressed CD68, and expressed higher levels of co-stimulator molecules CD80, CD86,HLA-DR, and up-regulated CD16, Tie-2and CD163, suggesting that TAMs were a type ofactivated macrophages and had potential inhibitory phenotype. In addition, the frequency ofTAMs was negatively correlated with the percentage of NK cells in tumor tissues, anddouble immunohistochemical staining indicated that TAMs were co-localizated with NKcells within tumors.2.2Compared with normal blood monocytes, TSN-conditioned blood monocytes couldinduce NK cells to up-regulate CD69, but down-regulate Ki-67, perforin and TRAIL aswell as to inhibit the production of intracellular cytokines INF-γ and TNF-α. The survivalof NK cells in the co-cultured system showed that there was no difference in the apoptosisof NK cells co-cultured with TSN-monocytes or normal monocytes. These results indicatedthat tumor-derived signals-treated monocytes promote NK cells activation but significantlysuppress the proliferation and function of NK cells.2.3The expression of CD48, PD-L1, PD-L2, CD155and CD112on monocytes intumor tissues and nontumor tissues was not significantly different, and TSN-conditioned monocytes did not up-regulate the expression of these molecules. In the co-culture system,pre-treating with anti-2B4Ab did not attenuate the dysfunction of NK cells induced byTSN-conditioned monocytes. However, the capacity of NK cells for producing INF-γ andTNF-α was markedly restored while adding anti-TGF-β1Ab to neutralize TGF-β1signal.In addition, compared with NTSN-conditioned monocytes, TTSN-conditioned monocytesalso inhibited the production of INF-γ and TNF-α from NK cells, but neutralization withanti-TGF-β1Ab did also attenuate the dysfunction of NK cells induced by TTSN-conditioned monocytes. In vitro rhTGF-β1could directly inhibted the expression of Ki-67and INF-γ in NK cells. These results indicated that TSN-, TTSN-conditioned monocytesinduced the inhibition of NK cells by TGF-β1.3. The clinical relevance of NK cells in GCThe percentage of NK cells in tumor tissues obtained from patients with≥5cmtumor size, T3and T4invasion, neural invasion, lymphoid nodal and distance metastasiswas significant lower than that obtained from patient with＜5cm tumor size, T1and T2invasion, and without neural invasion or lymphoid nodal and distance metastasis. TNMstages analysis showed that the percentage of NK cells in tissues was gradually decreasedwith GC progression. However, only the percentage of NK cells in tumor tissues wassignificantly different according to the TNM stages. Comparing the patients with high(≥2.36%median level) vs low (<2.36%) level of NK cells in tumor tissues, the20-monthsurvival time was significantly shorter for those with low NK cell percentage.【Conclusion】1. In contrast with increased the percentage of T cells in tumor tissues, the percentageof NK cells was significantly decreased in tumor tissues compared with that in peritumorand nontumor tissues. Although there was no difference on the expression of receptors fromNK cells in tissues, the dysfunction of NK cells in GC environment was observed.2. TSN-, TTSN-conditioned monocytes inhibited the function of NK cells by TGF-β1but not CD48/2B4signal pathway.3. The percentage of NK cells in tumor tissues was decreased with tumor progressionand the patients with high NK percentage had longer overall survival time.