Preliminary Study Of The Role Of Soluble DC-SIGN In Regulation Of T Lymphocytes

20 years ago, natural immune was regarded as a degenerate immune mechanism, and had been got little concern of immunologists, on the contrary, adaptive immune attained a great attention owing to its high specific antigen identification system and complex molecular and cellular interactions. Over the last decade, natural immune aquired more and more favour. Natural immune system is a complex network structure composed by a variety of recognition and effector moleculars which constitute the first line of defense against infection by mutual cooperation; and detect dangerous signals to guide the property and intensity of adaptive immune response. As the first-line defense against potential pathogens, natural immune is more complex, more important and more extensive specificity, consequently a wider recognition spectrum, than we thought previously. More and more evidence opened out that natural immune played a important leader role to adaptive immune response.Important players in this host defense system are'collectins', a class of soluble innate immune proteins.DC-SIGN belongs to c-type lectin family and is a typeⅡmenbrance protein which contains 404 amino acids and is of 44KD in molecular weight.DC-SIGN consists of extracelluar domain,transmenbrane region and cytoplasmic region.The extracellular domain contains carbohydrate recognition domain (CRD),which can combined with pathogen sugar moieties specificly, including manann,fucose and so on, and neck domain or hinge domain. DC-SIGN is exclusively expressed by the dendritic cells (dendritic cell, DC) and macrophages and activated B cells In addition to this, as well as in a variety of organizations such as synovial fluid, placenta, lymphatic tissue, dermis, cerical intraepithelial. As a membrance receptor, DC-SIGN involves in uptake of antigens, adhesion to endothelial cells, migration and maturity of DCs, moreover, DC-SIGN plays an important role in interaction with naive T cells and triggering T cell response. In addition, DC-SIGN participates in various of pathogens such as HIV, HCV and mycobacterium tuberculosis infecting and spreading among cells. In recent years, Our research group has been studying immunoregulation of MBL(mannan-binding lectin) which is a soluble pattern recognition receptors existed in the serum. MBL belongs to C-type lectin family as well as DC -SIGN that brings some new thoughts,such as whether a form of soluble DC-SIGN exists and whether it can regulate adaptive immune response directly?In the first place we prokaryotically expressed recombinant sDC-SIGN and following we established the double-monoantibodies sandwich ELISA testing systeme.The last step,we apply the detection system to test plasma samples. We have confirmed preliminaryly that there is soluble DC-SIGN existed in human plasma.Further, we studied the effect of sDC-SIGN on inmmunoregulation of T cells from three levels including cell proliferation, cytokines secretion and cell activation.PartⅠestablishment and application of sandwich ELISA testing systemIn preliminary studies we have prepared 2 monoclonal antibodies which is against sDC-SIGN designated as 1C6 and 4H3. Further we compared the sensitivity and specificity of two mAbs with commercial antibody by ELSIA and WesternBlot.sDC-SIGN protein which we have expressed prokaryotically is composed of carbohydrate recognition domain and neck domain.In the sugar-binding experiments,mAb 1C6 showed a higher affinity to sC-SIGN compared with mAb 4H3 that suggest the site which 4H3 recognited is very close to the CRD domain.For a high sensitivity of sandwich ELISA testing system, we labled 2 mAbs with Biotin and optimized experiment conditions.At final mAb 1C6 was determined as capture antibody and biotin-labeled 4H3 as detection antibody. Next, the sandwich ELISA testing system was applied to detect plasma samples which we collected including 218 cases of the general population and 106 cases of disordered endocrine population. Results were exciting that soluble DC-SIGN do exist in human plasma the general population 219 cases plasma samples. Following up the victory,we studied the role of soluble DC-SIGN in regulation of T lymphocytes.PartⅡstudy of the role of soluble DC-SIGN in regulation of T lymphocytesStudy showed that SP-A and SP-D which belong to as well as DC-SIGN can function as a immunoregulator to natural immunity and adaptive immunity,such as SP-A and SP-D restrain T cell proliferation through IL-2-dependent or IL-2-independent way. In the earlier experiment we have detected sDC-SIGN in human plasma.Being is profound!! At present there is little reports on the role of immunoregulation of DC-SIGN,let alone direct action on T lymphocytes. According to the homology of structure and function of type-C lectin family members, we hypothesized that sDC-SIGN may also have an impact on adaptive Immune.Based on previous work, we expressed sDC-SIGN prokaryotically first; and then we used density gradient centrifugation to separate peripheral blood cells mononuclear cells (PBMC) from healthy volunteers followed by purifying CD3+ T lymphocytes with negative choose method of MACS. T lymphocytes were treated with specific stimulants anti-CD3/CD28 monoclonal antibody and non-specific stimulant ConA respectively, and supernatant was collected 72h later for further cytokines detection including IL-2, IFN-γ, IL-10, IL-6 and IL-17a.On the side,we analysed the effect of sDC-SIGN on the proliferation of T cells by CFSE simultaneously test the expression of CD69 which is the early activation sign of T cells by FCM. The result revealed, sitimulating CD3+ T cells with Con A or anti-CD3/CD28 antibody, sDC-SIGN inhibited the proliferation of T cells,especially CD8+ T cells,moreover, sDC-SIGN downregulated the production of IL-2, IFN-γ, IL-10 and IL-17A, simultaneously promoted the secretion of IL-6. Data suggested sDC-SIGN probably influences differentiation of T cell through altering the dynamic balance among cytokines.Beyond that we also found sDC-SIGN downregulated the expression of CD69.All of this provide evidence for further illustrating the role of immunoregulation of sDC-SIGN and fertilizing the guidance of natural immune to adaptive immune.PartⅢstudy of the mechanism of the effect of soluble DC-SIGN on regulation of T lymphocytesWe have confirmed that sDC-SIGN existed in human plasma and could influence the proliferation, secretion of cytokines and activation of T cells. Further, we found out probable structure domain that involved in the combination of sDC-SIGN with T cells. sDC-SIGN protein which we have expressed prokaryotically is composed of carbohydrate recognition domain and neck domain.The CRD of membranoustype DC-SIGN can bind to sugar moieties specificly. Neck area mainly involved in oligosaccharifying the sugar recognited and modified the specificity of sugar to make for the combination between DC-SIGN and sugar ligands.The result of Cell ELISA revealed the affinity of sDC-SIGN to T cells was higher with Ca2+ compared to that without Ca2+. Analysis of cytokines showed the participation of Ca2+ was able to reverse the effect of sDC-SIGN on cytokines secretion,including IL-2, IFN-γ, IL-10, IL-17A and IL-6 of T cells. that stay relevant with the result of Cell ELISA. Above all suggested CRD was not the functional domain,otherwise,the combination between CRD and its ligand was Ca2+ -independent. In addition, we found rhIL-2 was able to reverse the effect of sDC-SIGN on cytokines secretion of T cells, thereafter, we hypothesize the inhibiting of sDC-SIGN to the proliferation of T cells did not relay on IL-2.In conclusion,we found a soluble form of DC-SIGN in human plasma which played a part in the proliferation, activation and the secretion of cytokines of T cells via a direct combination between non-CRD and T cells.