Chemical Modification IGb3 Analogues Induce More IFN-γ-producing NKT Cells

Objective Natural killer T (NKT) cells are a unique subset of the mature T lymphocytes and play important role in immunoregulation. They are characterized by co-expression of the surface markers of NK cell and T cell:α/βTCR, NK1.1, CD16, LY49A and LY49C. The T cell receptors (TCRs) of invariant natural killer T (iNKT) cells are highly restricted. Different with other conventional T cells, they recognize glycolipids presented by the nonclassical MHC class I molecule CD1d. After activation via TCR engagement, they promptly secrete large amounts of both Th1 cytokines (IFN-γ), and Th2 cytokines (interleukin (IL)-4 and interleukin (IL)-10), thereby resulting in activation of a variety of other cell types, including dendritic cells (DCs), NK cells, B cells and conventional T cells. NKT cells are powerful immunoregulatory cells and act as an attractive target for immunotherapy on the basis of their ability to rapidly secrete cytokines and modulate immune responses. The secretion of specific Th1 and Th2 cytokines by NKT cells is critical to these immunomodulatory roles.Theα-galactosylceramide (α-GalCer), which is derived from a marine sponge, is well defined as an exogenous ligand of NKT cells, and directly contributes to activate NKT cells. Most animal experiments have demonstrated thatα-GalCer exhibits multiple immunotherapy roles in autoimmune diseases (for example, multiplesclerosis, autoimmunediabetes and experimental autoimmune encephalomyelitis), cancer, atherosclerosis and infectious diseases. However, the different cytokines produced by NKT cells afterα-GalCer treatment might have opposite effect, as Th1 cytokines often antagonize the action of Th2 cytokines and vice versa, which largely limits the clinic usage ofα-GalCer. Moreover, NKT cells have also been reported to aggravate liver injury and promote both atherosclerosis and animal abortion. Recently, Isoglobotrihexosylceramide(iGb3) has been identified as an endogenous ligand recognized by NKT cells. But it is a very weak agonist compared to the exogenousα-galactosylceramide. Therefore, many researchers are trying to search for other immunopotentiators or design chemical modification to glycolipid for treatment of diseases. On this basis, we suppose that modification the structure ofα-GalCer and iGb3 might improve their stimulatory activity. In this study, we compared the chemical structure betweenα-GalCer and iGb3. Furthermore, we assessed the stimulating activity of chemically-modified iGb3 analogues on murine hepatic and spleen NKT cells in order to investigate whether the two iGb3 analogues can be used as novel non-specific immunoadjvants for anti-tumor and anti-infectious therapy.Mothod(1) Screening theα-GalCer and iGb3 analogues by ELISA. Six to eight weeks old WT male C57BL/6 mice weighted 22-26g were grouped according different glycolipid treatment, each group has three mice. The serum levels of IFN-γand IL-4 at various time points after a single intraperitoneal injection of iGb3, S-iGb3, 4,5-2H-iGb3, 4,5-2Br-iGb3, 2′′′-dh-iGb3, 3′′′-dh-iGb3, 4′′′-dh-iGb3, 3′-NAc-GalCer, 3′-Azido-GalCer, 3′-Amino-GalCer 4′-NAc-GalCer or Biotin-GalCer were measured by ELISA.(2) Investgation the NKT cell activation stimulated by iGb3 and its analogues.C57BL/6 mice were treated with iGb3, 4-HO-iGb3 or 4′′′-dh-iGb3 intraperitoneally at the concentration of 4μg/ml. Mice were sacrificed at 4h, 6h, 12h and 48h separately. Hepatic and spleen lymphocytes were separated. The amount of NKT cells was evaluated by flow cytometry via staining with anti-NK1.1 and anti-CD3 mAb(3) Verification whether iGb3 and its analogues influence the productin of cytokines by NKT cells.C57BL/6 mice were treated with iGb3 and its analogues at the concentration of 4μg/ml. Mice were then sacrificed at indicated time points. Hepatic and spleen lymphocytes were separated, and the percentage of IFN-γ+ and IL-4+ positive cells in CD3+NK1.1+ hepatic lymphocytes was detected by flow cytometry.(4) The anti-tumor effect of iGb3 and its analogues.Isolate DC cells derived from mice marrow were loaded with iGb3 analogues for two hours, and were then injected into mice through tail vein. The volume of tumors was detected when treatment with iGb3 and its analogues for one week and four weeks.Results1. 4HO-iGb3 and 4′′′-dh-iGb3 elicit Th1-baised responses in serum.2. 4HO-iGb3 and 4′′′-dh-iGb3 up-regulate CD69 expression on NKT cells and induce more IFN-γ-producing NKT cells.iGb3 and its two analogues did not induce more NKT cells, but CD69 expression was up-regulated on CD3+ NK1.1+ NKT cells when treatment with iGb3, 4-HO-iGb3 and 4′′′-dh-iGb3. 4-HO-iGb3 and 4′′′-dh-iGb3 treatment increased CD69 expression as early as 4 h time point. IFN-γ-producing NKT cells were only slightly increased at various time points with iGb3 stimulation. While the two chemically-modified iGb3 analogues, especially 4′′′-dh-iGb3 induced significantly greater intracellular IFN-γNKT cells in liver and spleen. iGb3 and its two analogues promoted IL-4 production significantly at 4h after treatment. However, there were no differences in percentages of IL-4-producing NKT cells among iGb3 and its two analogues groups at each time points.3. DC loaded with 4′′′-dh-iGb3 significantly inhibits tumor growth. DC cells loaded with 4′′′-dh-iGb3 significantly inhibit B16 tumor growth compared with DC loaded with iGb3 or PBS.Conclusions1. 4HO-iGb3 and 4′′′-dh-iGb3 could elicit Th1-baised responses.2. Chemical modified iGb3 analogues, especially 4′′′-dh-iGb3, stimulate NKT cell activation and induce more IFN-γ-producing NKT cells.3. DC cells loaded with 4′′′-dh-iGb3 significantly inhibit tumor growth.