Research On The Effect Of CD137 Signaling On CIK Cells' Proliferation And Function

CD137 is a TNFR superfamily member that is expressed in activated natural killer (NK) cells, T cells and dendritic cells (DCs). It is believed that CD137L binding to CD137 mediates costimulatory signal that results in proliferation and production of cytokines of T cells, NK cells. In addition, CD137 signaling can protect cells from activation-induced cell death. Cytokine-induced killer cells (CIK cells) have cytotoxic activity in a broad spectrum of tumor cell targets in a non-MHC-restricted manner. But the expansion efficiency and cytotoxicity of CIK cells remain to be improved presently. In CIK cell cultures the greatest lytic activity is found in the population of CD3⁺CD56⁺ cells. So it has been generally accepted that increase of proliferation rate and anti-tumor activity of CD3⁺CD56⁺ effector cells may be an efficient approach to enhance the anti-tumor capacities of CIK cells. However, no data to date have been available as to CD137 signaling modulating function of CD3⁺CD56⁺ cell subpopulation. In this paper, CD137 costimulatory signaling was mediated by CD137 mAb which enhance the capacities of CIK cells proliferation and cytotoxicity. We further investigated the mechanisms of the CD137 signaling on regulation the function of CIK cells. That was provided a new efficient approach to elevate the therapeutic effect of CIK cells adoptive immunotherapy in solid tumors.Objective: To investigate the effect of CD137 signaling on regulation the function of CIK cells from healthy human and lung cancer patients' PBMCs.Methods: CIK cells were induced from healthy human or lung cancer patients' PBMCs in the presence of CD3mAb, IL-2, IFN-γ. CD137mAb or mouse IgG1 isotype control was added to experimental group (CD137-CIK group) or control group (IgG1-CIK group), respectively.Study of healthy human CIK cells: (1) Cell proliferation was determined by cell counting with trypan blue exclusion test. (2) Apoptosis and necrosis of CIK cells was detected by fluorescence- activated cell sorter (FACS) using AnnexinV- FITC/PI staining. (3) Cytotoxicity was measured by LDH Cytotoxicity assay. (4) Phenotypes and cytokines production of CD3⁺CD56⁺cells were analyzed by FACS. (5) To explore the impact of CIK cells on CD4⁺Th0 cells differentiation, they were co-cultured and then IFN-γand IL-4 production in CD4⁺T cells were analyzed by FACS. Study of lung cancer patients' CIK cells: (1) Phenotypes of CIK cells were analyzed by FACS. (2) Cytotoxicity was measured by LDH Cytotoxicity assay.Results: Healthy human: (1) CD137mAb could dramatically promote proliferation of CIK cells. The concentration of CD137-CIK group was arrived at (9.87±0.57)×10⁶/ml and IgG1-CIK group was (7.02±0.68)×10⁶/ml (p<0.05); (2) At the 28 days of culture, the percentages of CD3⁺CD56⁺cells in CD137-CIK group and IgG1-CIK group were respectively achieved in (39.86±4.69)% and (29.14±5.12)%(p<0.05); (3) At the 14 days, the rate of apoptosis and necrosis of CD137-CIK cells was lower than IgG1-CIK cells; (4) The CD137-CIK cells possessed greatly higher ability to kill tumor cell line A549 than IgG1-CIK cells(20:1, P>0.05; 10:1 and 5:1, P<0.05); (5)The expression of IFN-γ, IL-2 and TNF-αin CD3⁺CD56⁺cells in experimental group increased significantly compared with control group; However, the production of TGF-β₁, IL-4, IL-10 in CD3⁺CD56⁺ cells decreased significantly in CD137mAb treated CIK cells; (6) The expression of NKG2D was up regulated and NKG2A was down regulated on CD3⁺CD56⁺cells in CD137-CIK group compared with control group. (7) In addition, the result showed that CD137 mAb could enhance the abilities of CIK cells to increase the production of IFN-γand reduce the production of IL-4 in CD4⁺T cells. Lung cancer patients: (l)At the 28 days of culture, the percentages of CD3⁺CD56⁺cells in CD137-CIK group and IgG1-CIK group were respectively achieved in (30.1±7.3)% and (21.7±5.7)%(p<0.05); (2) The CD137-CIK cells have higher ability to kill tumor cell line A549 than IgG1-CIK cells (10:1 and 5:1, P<0.01; 20:1, P<0.05).Conclusion: This study suggested that CD137 signaling could significantly elevate the expansion efficiency and cytotoxicity of CIK cells from PBMCs of healthy human and lung cancer patients. That could enhance the abilities of CIK cells to kill tumor cells in a non-MHC restricted manner. Furthermore, CD137 signaling can enhance Th1 type cytokines production by CD3⁺CD56⁺ cells which could activate the CTL immune response to mediate the MHC restricted anti-tumor effect; It can also promote the capability of CIK cells inducing CD4⁺Th0 cells to Th1-type immunity which could balance the immunologic derangement of cancer patients. These rusults could offer enough feasibility evidences to enhance the therapeutic efficacy of CIK cells adoptive immunotherapy in solid tumor.