The Clinical Significances Of Peripheral Regulatory T Cells And Nkg2D+Nk Cells In Patients With Colorectal Cancer

Background:Recent statistics show that colorectal cancer (CRC) is both the third most commonly diagnosed cancer and the third most fatal cancer worldwide with millions of new cancer cases being reported annually, causing approximately600,000deaths annually. With the changes of diet customs and diet structures, the morbidity and mortality of CRC are increasing in China. Because the early clinical symptoms of CRC are not obvious, most patients were diagnosed as the middle and advanced stages of CRC. At present the precise mechanisms of pathogenesis and immune escape of CRC are not yet completely understood. For the CRC patients having lost the chance of surgery, there are short of more efficient treatments. Therefore, it is so necessary to look for ideal indicators for early diagnosis, prognostic evaluation and therapeutic targets of CRC.Natural killer (NK) cells are a type of important innate immune effector cells in organisms, which can eliminate tumor cells and infectious cells invaded by virus or bacteria in organisms and exert capability of immunological surveillance. There are many kinds of activating and inhibitory receptors on the surface of NK cells. It is depending on the synthesis of all kinds of activating and inhibitory signals that NK cells exert their cytotoxicity. Natural-killer group2, member A (NKG2A) is an important inhibitory receptor in NK cells, which specially recognizes and binds its ligand HLA-E and transfers inhibitory signals to NK cells. Natural-killer group2, member D is a major activating receptor in NK cells, which specially recognizes and binds its ligands MICA/B, ULBPs and transfers activating signals to NK cells. Several studies have shown that the decline of NK cytotoxicity mediated by NKG2D might be one of reasons that resulted in tumor immune escape.Regulatory T cells (Tregs) are a type of important immune suppressive cells in organisms and may suppress the functions of some immunologic effector cells such as CD8+CTL cells, natural killer (NK) cells and dendritic cells. Tregs are one of the key elements of tumor immune escape. At present natural CD4+CD25+Foxp3+Tregs are studied most deeply among Tregs. Forkhead box protein3(Foxp3) is the key intracellular marker of Tregs and is important for Tregs’developments and functions. Studies have shown an increase in populations of Tregs in peripheral blood as well as tumor microenvironment in patients with different cancers such as colorectal cancer, hepatocellular cancer, ovarian cancer and prostate cancer. Moreover, the populations of Tregs were negatively correlated with the tumor developments and prognosis of patients. The population of Tregs recovered to normal levels after tumor resection, while it increased when tumor recurrence.Studies have shown that Tregs may suppress the activation of NK cells by cell-contact-dependent mechanism and secreting immunosuppressive cytokines such as TGF-β, IL-10, which consequently make tumor cells evade immunological surveillance. However, the relationships and clinical significances of Tregs and NK cells’ receptors NKG2A and NKG2D expressions in CRC remain obscure. On the basis of above situations as well as in order to further understand the relationships and clinical significances of Tregs, NK cells and NK receptors NKG2A and NKG2D expressions in CRC, we formulate the following objectives and methods so as to supply new thoughts and experimental basis for CRC dignosis and therapy.Objectives:1. To observe both the mRNA transcription and protein expression levels of inhibitory receptor NKG2A and activating receptor NKG2D in peripheral NK cells in CRC patients.2. To observe how blocking NKG2D receptor in purified NK cells affect NK cytotoxicity and CD107a degranulation.3. To observe the level of peripheral Tregs in CRC patients and the relationships between Tregs and lymph node transfer or clinical stages of CRC.4. To assay peripheral Tregs, NKG2D+NK cells and serum carcino-embryonic antigen (CEA) levels in CRC patients parallelly and evaluate their relations.Methods:1. Patients and controls Ninety-seven patients (52men and45women) with primary CRC were recruited from the gastrointestinal surgery ward of Shandong Provincial Hospital affiliated to Shandong University. These patients were diagnosed with CRC on the basis of colorectal cancer diagnosis standard (2010) issued by Ministry of Health. The patients had no history of other diseases such as diabetes, kidney disease, hepatic disease, or autoimmune disease. All patients were not received with radiotherapy or chemotherapy before sample collection. Forty-eight healthy subjects (26men and22women) from the physical examination centre of Shandong Provincial Hospital. The study was approved by Shandong Provincial Hospital affiliated to Shandong University Ethics Committee, and informed consent was acquired from each individual.2. Cells and cell culture Peripheral blood mononuclear cells (PBMCs) were isolated from15mL venous blood obtained from study subjects by using Ficoll-Hypaque density gradient centrifugation. NK cells were isolated from PBMCs by negative selection using magnetic cell separation. Flow cytometry revealed the purity of CD3-CD56+NK cells to be greater than95%. The obtained NK cells were cultured in RPMI1640medium with10%heat-inactivated fetal calf serum (FCS),1%penicillin-streptomycin and100U/mL recombinant human interleukin2(rIL-2) in37℃and5%CO2incubator.Human colon carcinoma cell line HT29cells were cultured in RPMI1640supplemented with10%FCS and were used as target cells. The medium was regularly changed, and the cells were always washed twice before use.In antibody-blocking experiments, purified NK cells were pre-incubated with different concentrations of anti-NKG2D neutralizing antibodies for30min before they were cultured with the target cells. Then, the cytotoxicity and CD107a degranulation assays were performed.3. Real-time PCR Total cellular RNA was extracted from PBMCs by using TRIzol reagent. Concentration and quality of the extracted total RNA were determined by measuring its light absorbance at260nm (A260) and the ratio of (A260/A280). A1μg of total RNA was reverse transcribed in a20-μl reaction mixture containing2μl of Maxima enzyme mix and4μl of5×PCR mix. The procedure for reverse transcription was performed as follows:10min at25℃,30min at55℃, and then5min at85℃.The obtained cDNA was diluted1:10before PCR analysis.The primers included CD94, NKG2A, NKG2D, and β-actin. Reactions were performed in a total volume of20μl, which included5μl of cDNA sample,5μl Of0.8μM primer pair, and10μl of2×PCR mix. PCR was performed as follows:5min at95℃and45cycles of15s at95℃,30s at60℃, and15s at72℃. Incubation and on-line detection of the PCR products were carried out using optical96-well plates and the LightCycler480sequence detection system (Roche, Germany). Finally, the PCR products were subjected to electrophoresis to determine whether the required products were formed.4. Flow cytometry PBMCs were stained with10μl anti-human CD3,5μl anti-human CD56,10μl anti-human NKG2A, and10μl anti-human NKG2D antibodies to assay NKG2A and NKG2D expression on the surface of NK cells. Isotype controls were used meanwhile.Tregs were assayed with regulatory T cells kit according to the manufacturer’s instructions. For surface antigens staining, added10μl anti-human CD4,10μl anti-human CD25antibodies to100μl of the whole blood and incubated for30min in the dark at4℃. Then1×RBC Lysis Buffer was added to the whole blood in the room temperature in order to lyse RBCs. For intracellular antigen staining, fixation/permeabilization working solution was added to the cells and incubated for60min in the dark before the cells were stained with anti-human Foxp3antibody. Isotype controls were used at the same time. For CD107a degranulation assays, NK cells and HT29cells were co-cultured at37℃. The volume of per well was200μl, then10μl anti-human CD107a antibodies were added into the well. After incubating for1h at37℃,1.7μl monensin (0.1mg/ml) was added into the well and the plate was incubated for another3h. Control wells were used meanwhile. After collecting the cells, they were stained with10ul anti-human CD3,5μl anti-human CD56antibodies to assay the expression of CD107a on NK cells.At least10,000cells were analyzed using a3-color EPICS XL flow cytometer (Beckman coulter, USA) or BD FACSⅡ flow cytometer (BD Bioscences, USA).5. Cytotoxicity assays HT29cells were used as target cells, and the purified NK cells were used as effector cells in the51Cr release assay. The HT29cells were labeled by incubating them with the51Cr isotope for1h at37℃. The labeled targets were then co-incubated with NK cells at different ratios for4h. The supernatant was harvested and analyzed using a y-counter (Packard Cobra Ⅱ5002, USA). The percentage of51Cr released was calculated. Spontaneous release was less than15%of the maximum release.6. Serum CEA assay Serum CEA protein in CRC patients was assayed using electrochemiluminescence method with Roche cobas e601(Roche, Germany) at the Department of Medical Biochemistry, Shandong Provincial Hospital. In healthy subjects, CEA<10ng/ml was considered as normal.7. Statistical analysis Data were analyzed using the GraphPad Prism (GraphPad Software, USA). A t test was used for comparing quantitative variables of two different groups. A one-way Anova was used for comparing the data of three or four groups. Spearman correlation analysis was performed to determine the association between two variables. Probability values were considered statistically significant if p <0.05.Results:1. The levels of peripheral NKG2A mRNA trscription, NKG2A protein in PBMCs and NKG2A expression in NK cells were similar between from patients with CRC and from healthy controls. However, the levels of peripheral NKG2D mRNA trscription, NKG2D protein in PBMCs and NKG2D expression in NK cells from CRC patients were significantly downregulated compared with healthy controls.2. After blocking NKG2D signaling with anti-NKG2D antibodies ex vivo, cytotoxicity and CD107a degranulation were gradually decreased along with the increasing concentration of anti-NKG2D neutralizing antibodies.3. NKG2A and NKG2D were also expressed on the membranes of CD3+CD56+NKT cells and CD3+CD56-T cells. However, NKG2A expression levels decreased successively in NK cells, NKT cells, and T cells. The NKG2D expression levels in T cells were lower than the corresponding levels in NK cells and NKT cells.4. CD4+CD25+Foxp3+and CD4+CD25highFoxp3+Tregs were significantly up-regulated in peripheral blood in CRC patients compared to healthy controls, while the numbers of Tregs were not related with lymph node transfer or clinical stages.5. The levels of CD4+CD25highFoxp3+cells were much higher than those of CD4+CD25lowFoxp3+cells in both CRC patients and healthy controls. 6. Increased CD4+CD25+Foxp3+as well as CD4+CD25highFoxp3+Tregs were not statistically correlated with decreased NKG2D expression in NK cells in peripheral blood from CRC patients.7. Peripheral CD4+CD25+Foxp3+as well as CD4+CD25high Foxp3+Tregs were not correlated with serum CEA protein in CRC patients although there were elevated levels of both Treg and CEA in severe advanced CRC patients.Conclusions:The imbalance of inhibitory receptor NKG2A and activating receptor NKG2D expresions in transcription and translation level in CRC patients may be associated with suppression of NK cell activity. We inferred that tumor cells may escape NK cell surveillance via the NKG2pathway in CRC patients; however, the underlying mechanism needs to be investigated further in detail. Immunosuppressive Tregs were significantly up-regulated in peripheral blood in CRC patients; however, peripheral NKG2D+NK cells were significantly down-regulated. Yet both Tregs and NKG2D+NK cells were not correlated with lymph node metastasis or clinical stages of CRC. Limited by the small sample size, we did not find significant correlations between increased Tregs and decreased NKG2D+NK cells. Similarly, no correlation was observed between Tregs and serum CEA protein. More studies are needed to explore the mechnism that Tregs and NKG2D+NK cells participate in immune escape of CRC.