Preliminary Study Of Crosstalk Between Dendritic Cells And Natural Killers Mediated By MHC Class I Chain-related Protein A

Natural killer cells are the first defense line against virus-infected cells and tumor cells. Meanwhile, activated NK cells regulate adaptive immune responses by releasing a variety of cytokines. Dendritic cells (DCs) are highly specialized APCs that can activate naive T cells and initiate immune responses. Previous studies have shown that interaction between DCs and NK cells is likely to influence both innate and adaptive immune responses. DCs can activate resting NK cells and augment anti-tumor immunity by triggering NK cell effector functions. Reciprocally, NK cells can promote DCs maturation and polarize Th0 to Th1, thus enhancing adaptive immunity of immune defense and immune surveillance.Major histocompatibility complex class I chain-related protein A (MICA) is a ligand for the NKG2D receptor, which transduces positive intracellular signals in NK cells. The ligation of NKG2D with MICA results in activation of NK cells, increasing NK cells cytolytic activity and IFN-γproduction. Although MICA is not present on the surface of monocytes, but it exists in cytoplasm. Therefore, we attempted to study how MICA mediates the crosstalk between DCs and NK cells, and whether this crosstalk mediated by MICA is involved with immune escape of colon cancer.This study was divided into three parts as follows.Part I: NK cells activities up-regulated by MICA expression on dendritic cellsObjective: To investigate whether MICA is present on the surface of dendritic cells under different stimulations, and regulation of NK cell activities with enhanced expression level of MICA on DCs.Methods: Firstly peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals or colon cancer patients donors were developed into immature DCs in culture with GM-CSF and IL-4 for 5 days. Immature DCs were further stimulated with LPS,TNF-α,CD40L,IL-15 or IFN-αfor 24 h. MICA expression on the surface of monocytes, immature DCs (iDCs) and mature DCs (mDCs) was detected by flow cytometry (FCM). Next CD69 expression, cytotoxicity and IFN-γproduction of NK cells were measured when DCs with high level of MICA expression were cultured with NK cells. Lastly we observed activation of NK cells which were incubated with DCs in the presence of NKG2D-Ig protein and IL-12 antibody, respectively.Results: MICA was poorly expressed on the surface of monocytes, and increased expression level on iDCs was detected in both healthy individuals and colon cancer patients. Compared with iDCs, LPS, TNF-α, and CD40L had no effects on MICA expression on mature DCs. In contrast, IL-15 and IFN-αcan up-regulate MICA expression on mDCs from healthy individuals, but have no effects on mDCs from colon cancer patients. IFN-α-stimulated DCs promoted CD69 expression, IFN-γproduction and cytotoxicity of NK cells. IFN-γproduction and cytotoxicity of NK cells were decreased when recombinant NKG2D-Ig protein was added into the culture medium. However, only IFN-γproduction was decreased when anti-IL-12 neutralizing antibody was added.Conclusions: MICA was expressed on the surface of both iDCs and mDCs in healthy individuals and colon cancer patients. IL-15 and IFN-αcan up-regulate MICA expression on mDCs. DCs with high level of MICA expression enhanced NK cell function. The increased cytotoxicity of NK cells was dependent on the ligation of ligands on DCs and NKG2D receptor on NK cells. In addition, low expression level of MICA on mDCs may be associated with immune escape in colon cancer patients.PartⅡ: Effects of NK cells stimulated with MICA on dendritic cells activationObjective: To observe whether immobilized MICA (iMICA) could synergize NK cells to activate iDCs, and to study whether apoptotic MICA positive targets attacked by NK cells were liable to be phagocytosed by iDC.Methods: Firstly iDCs were co-cultured with fresh, or iMICA-incubated allogeneic NK cells, and IL-2-stimulated, or IL-2 and iMICA co-incubated autologous NK cells at 1:5 ratio for 24 hours. Frequency of CD86, or HLA-DR positive cells were analyzed by flow cytometry. Then iDCs were co-cultured with IL-2-stimulated or IL-2 and iMICA co-incubated autologous NK cells at 5:1 ratio for 24 hours. Expression of HLA-DR on DCs was analyzed by flow cytometry. Next anti-IFN-γneutralizing antibody was added into the culture medium to observe whether DC maturation promoted by NK cells was dependent on IFN-γ. At last iDCs were co-cultured with CFSE-labeled K562, or K562-MICA cells, which were previously attacked by LAK cells or treated by mitomycin C. After 24 hours, frequencies of HLA-DR and CFSE double positive cells were analyzed by flow cytometry.Results: When iDCs were co-cultured with allogeneic or autologous NK cells respectively at the ratio of 1:5, each group of NK cells killed iDCs, and iMICA was redundant for NK cells killing. When iDCs were co-cultured with autologous iMICA-stimulated NK cells at the ratio of 5:1, NK cells promoted HLA-DR expression on DCs. Addition of anti-IFN-γneutralizing antibody to the NK-DC culture medium reduced HLA-DR expression. Apoptotic debris from K562-MICA cells attacked by LAK cells or treated by mitomycin C were susceptible to be phagocytosed by iDC.Conclusions: IL-2 activated NK cells killed iDCs independent of iMICA. However, NK cells stimulated with iMICA promoted maturation of DCs via increasing IFN-γproduction. MICA positive tumor cells attacked by LAK cells were susceptible to phagocytosis of iDC.PartⅢ: Correlation study of MICA expression on monocytes with NK cell phenotype in colon cancer patientsObjective: To compare frequencies of MICA positive monocytes, and of NKG2D positive, CD16 positive, CD69 positive NK cells in healthy individuals and colon cancer patients. And to analysis of correlation between MICA expression on monocytes and function-associated receptors expression on NK cells.Methods: Peripheral blood mononuclear cells isolated from healthy individuals or colon cancer patients were double-labeled with antibodies in different color. The combinational antibodies were anti-CD14/anti-MICA , anti-CD56/anti-NKG2D ,anti-CD56/anti-CD69,anti-CD56/anti-CD16. Frequencies of double positive cells were measured by flow cytometry. Correlation of MICA expression on monocytes with NKG2D, or CD69 expression on NK cells was calculated with the linear correlation and regression analysis.Results: There was no significant difference of frequency of CD14/MICA double positive monocytes between healthy individuals and colon cancer patients. Percentages of CD56/NKG2D, CD56/CD69 double positive NK cells were decreased in colon cancer patients than in healthy donors, but percentage of CD56/CD16 positive cells didn't altered significantly in two groups. In addition, there was no correlation of MICA expression on monocytes with NKG2D, or CD69 expression on NK cells. Conclusions: Frequencies of MICA-positive monocytes were very low in both healthy individuals and colon cancer patients. Activities of NK cells mediated by NKG2D receptor were decreased in colon cancer patients, but MICA expression on peripheral blood monocytes was not associated with function of NK cells.In summary, MICA mediated the crosstalk between DCs and NK cells directly. In addition, MICA on the surface of tumor cells primed NK cells, and promoted DC to uptake apoptotic debris and to induce DCs cross-presenting antigen, and resulted in initiation of specific immune response. Therefore, biological therapies based on enhancing the crosstalk between DCs and NK cells will have strong effects against tumor.