LTβR-Ig Promotes The Maturation Of Immature Dentritic Cells By Combination With LIGHT On DCs

Members of the TNF superfamily of cytokines and their receptors play important roles in the development of the immune system and in immune regulation,inflammation, and autoimmunity.Acting through specific cellular receptors,these cytokines initiate signaling cascades that regulate cell death,survival,and differentiation.LIGHT(lymphotoxin-like,exhibits inducible expression,and competes with HSV glycoprotein D(gD) for HVEM,a receptor expressed by Tlymphocytes;TNFSF14) is a member of the TNF family expressed on immature dendritic cells(DCs) and activated T cells.LIGHT binds three distinct members of the TNF receptor family,including the herpes virus entry mediator(HVEM),the lymphotoxinβreceptor(LTβR),and the soluble decoy receptor 3(DcR3).The functions of the signaling pathways associated with these receptors are often cell-context specific due to their expression by a variety of cell types.Biological activities of LIGHT-LTβR signaling include the in vitro induction of apoptosis,the production of various cytokines and now include developmental roles such as mesenteric lymph node(LN) organogenesis,and the restoration of secondary lymphoid structure and function.In most cases,HVEM signals confer anti-apoptotic or growth-inducing properties to cells.Effects of LIGHT-HVEM signaling define LIGHT as a co-stimulatory molecule for T cell activation,and a modulator of T cell responses. Overexpression of LIGHT on T cells results in extensive T cell proliferative disorders characterized by massive polyclonal expansion of CD4⁺ and CD8⁺ T cells.In contrast, blockade or disruption of LIGHT interaction with its receptors prevents or ameliorates graft-vs-host disease,allograft rejection,and autoimmune disorders including colitis and arthritis.Thus,LIGHT regulates multiple immune functions of innate and adaptive immunity through interactions with different receptors.Although the role of costimulatory molecules interactions in promoting initial T cell activation is well established,recent studies indicate that another means by which co-stimulatory molecules may influence immune responses is through reverse signaling into APC.As to CD28:B7 costimulatory signals,CTLA-4 not only regulates T-cell receptor(TCR) and CD28 signals in T cells but also delivers signals via B7s into DCs to induce indoleamine 2,3-dioxygenase(IDO),an enzyme that degrades tryptophan to byproducts that inhibit T cell proliferation and induction of T cell tolerance;CD28 delivers signals via B7s into DCs to induce IL-6 and IFN-γto enhance T cell-mediated immunity to tumor and microbial infection.These studies suggest that reverse signaling into APC cells is a novel aspect of costimulatory molecules function and may cause a variety of biological effects.As a new member of TNF super family,the studies of LIGHT-HVEM/LTβR/DcR3 now are focus on forward signaling to the receptor-expressing cells.Whether LIGHT-has reverse signaling to APC cells has still not been reported.In this study,we used LTβR-Ig to do in vitro and in vivo functional study on LIGHT-co-stimulatory pathway.We may acquire some original results from this study.It will enrich the knowledge of LIGHT-signal pathway immunoregulation.Partâ… Verification of the specificity of LTβR-Ig for LIGHT on immature DCs Specific binding between LTβR-Ig and LIGHT is the base of our research.To characterize the expression of LIGHT in mouse immature DCs,we stain DCs with PE-labeled rat anti-mouse LIGHT mAb.To determine whether LTβR-Ig could binding LIGHT on DCs,we incubated DCs by LTβR-Ig firstly,and then stain DCs using PE-labeled anti-huam IgG.We found that the percentage of LTβR-Ig⁺ cells is corresponding to LIGHT⁺ cells and the LIGHT mAb could block the engagement of LTβR-Ig to DCs.These results indicated that LTβR-Ig could binding with DCs specificallyPartâ…¡The effect of LTβR-Ig to dendritic cells by LTβR-LIGHT reverse signalingDC-LTβR-Ig showed higher CD40 and CD86 expression than DCs and DC-IgG. These results suggested that LTβR-Ig promots the maturation of DCs.We then analyzed the phagocytic and antigen presenting capacities of three kinds of DCs.To examine DCs phagocytic ability,purified DCs,DC-IgG or DC-LTβR-Ig were incubated with FITC-conjugated OVA and analyzed by FACS.DC-LTβR-Ig displayed lower phagocytic ability than DCs,DC-IgG.To assess DCs antigen presentation capacity,DCs were co-cultured with OVA_323-339-specific T cells in the presence of OVA_323-339 peptide and the proliferation of T cell were quantified to present the antigen presentation capacity.The proliferation of syngeneic T cell was significantly increased in DC-LTβR-Ig compared to that elicited by DCs,DC-IgG,suggesting that antigen-presenting capacity of DC-LTβR-Ig is augmented.Partâ…¢The effect of LTβR-Ig treated DCs to Ag-specific CD4⁺ T cellsTo further examination the effect of LTβR-Ig treated DCs to OVA_323-339-specific T cells,we detected the IL-12,IL-6,IFN-γof DCs by ELISA and real-time PCR.We found IL-6 secreted by DC-LTβR-Ig was increased significantly and this increasing was not the result of LPS pollution.In additional,we found that DC-LTβR-Ig could promote Ag-specific T cells to secrete Th2 type cytokine especially for IL-4 and this effect was diminished when IL-6 was neutralized by anti-IL-6 mAb.These results suggested that LTβR-Ig could promote the maturation of DCs production of IL-4 by Ag-specific T cells through increasing the expression of IL-6 from DCs.Partâ…£The examination of the function of LTβR-Ig treated DCs in vitroTo examination the function of DC-LTβR-Ig in vitro,we injected DC-LTβR-Ig/ DC-IgG and OVA-specific T cells in the present of OVA_323-339 peptide return to C57BL/6 via vena caudalis.Three days later,collected the lymphocytes of spleen and LN analyzed by FACS.The results is consistent with those in vivo.