Regulation Of T Cell Proliferation And Apoptosis By Microglia Via The OX40-OX40L Costimulatory Pathway

Background:Immune privilege of the CNS is thought to be maintained by the tight endothelial junctions of the blood-brain-barrier(BBB),the presence of an immunosuppressive microenvironment.This strict regulation of CNS immune reactivity is overcomed in brain injury or inflammation,in which large numbers of leukocytes are recruited to the CNS,often leading to specific immune response.So microglia play key roles in microbe infection, multiple sclerosis ,autoimmune encephalitis and cerebrovascular diseases.Moreover,the CNS parenchyma is devoid of lymphatic vessels and dendritic cells(DCs) which, in most tissues,process antigens to lymphoid organs to initiate immune responses, so naive T cells are initiated in peripheral lymphoid organs.Once activated or primed T cells readily cross the BBB and are restimulated upon encounter of the target antigen presented by local APCs,specific immune response will occur in CNS.And T cells infiltrating the CNS fail to proliferate,because effector T cells are usually short-lived and undergo rapid apoptosis upon restimulation,so the maintenance of activated T cell response is more important.One of the most debated and controversial issues is how and to what extent,cells residing in the CNS participate in the stimulation and reactivation of CNS-targeted T cells.Extensive study of microglia has revealed that microglia are the most important APCs in CNS.Upregulation of MHCâ…¡, costimulatory/adhesion molecules,such as CD80 (B7-1),CD86 (B7-2), CD40, ICAM-1,microglia have been recognized to behave as efficient APCs in vivo.Thus,microglia most likely participate in the maintenance of activated T cell response in CNS,but the molecular events underlying the interaction of microglia and infiltrating T cells remain poorly defined.Optimal activation of naive T cells requires not only interaction between the TCR and antigen-MHC complexes, but also costimulation provided by costimulatory ligandsexpressed by APCs . In the absence of costimulatory signals, naive T cells that recognize antigen will become anergic and die.The best characterized second signal is the interaction of CD28/CTLA-4-B7-1/B7-2.CD28 provides a critical signal for the regulating of naive T cell activation.In spite of the considerable body of evidence established the importance of CD28/CTLA-4-B7-1/B7-2 and CD40L-CD40 in primary T cell activation,these molecules appear to be far less important in the generation and maintenance of memory and effector T cell functions. OX40-OX40L are newly identified molecules ,they play a significant role in the later response of T cells activation and the function maintenance of the activated T cells (including memory and effector T cells). OX40 is expressed preferentially by activated T cells,and can be induced by antigen stimulation. The OX40 ligand is induciblely expressed by B cells, DCs,macrophage and endothelia. Studies of OX40 show that OX40 costimulation is necessary for the activation and function of effector T cells, maintenance of memeory T cells,and it plays a key role on these antigen-experience T cells proliferation, lymphocytes trafficking, secretion of cytokines and chemokines. OX40-deficient T cells show robust proliferation and cell division 2-3 days after TCR ligation, but reduced proliferation and survival by 5-6 days.Combining with the clues that the molecular mechanisms of maintenance of activated T cell immune response,we speculate that microglia ,the most efficient APC, will play key roles in this process via the OX40-OX40L costimulatory pathway.Objective:To detect the expression changes of costimulatory molecule OX40L between unactivated and activated microglia stimulated by LPS and IFN- Y ;To explore the role of OX40L expressed on microglia in the proliferation and apoptosis of the T cells;To determine the role and mechanism of OX40-OX40L costimulatory pathway in the maintenance of activated T cell response.Methods:1. Observation of the production of TNF- a and NO from N9 murine microglia cell line,supernatants were measured following LPS(500 ng/ml) > m-IFN-y(100 U/ml) stimulation.Expression changes of OX40L mRNA were detected by RT-PCR,the expression of OX40L protein were observed by immunofluorescence and FACS.2. Primary murine microglia were separated from newborn mice cortex.The purity ofmicroglia were identified by immunocytochemistry. Primary microglia were stimulated with LPS(500 ng/ml) and m-IFN-y(100 U/ml).Immunocytochemistry was used to detect the expression of OX40L.3. T cells were purified from spleen of mice by density gradient centrifugation and nylon wool depletion .Efficacy of purification was estimated by FACS,the viable cells were assessed by trypan blue dye exclusion.T cells were activated in the presence of ConA,the expression of OX40 was detected by FACS.4. Activated T cells were co-cultured with irradiated N9 expressing OX40L.Blocking the signal pathway OX40-OX40L with anti-OX40 mAb,proliferation was measured by (3H) -TdR incorporation.T cells were labeled with FITC- Annexin V /PI and assessed by FACS.Results:1. OX40L mRNA was detected low expression and it was significantly up-regulated after U^ m-IFN-y stimulation. FACS showed that OX40L expressed 24h after N9 cells activation.2. OX40L expression was dose-dependent with LPS and IFN stimulation.3. Immunofluorescence showed that OX40L was expressed on the cell membrane ,4. Primary culture of microglia was successful with positive staining with GSA-IB4.After stimulation by LPS and m-IFN-y,OX40L expression was inducible.5. OX40L expressed on N9 could promote T cell proliferation and suppress its apoptosis.Conclusion:We first found that OX40L was expressed on activated N9 cell line and primary murine microglia in vitro, OX40L on microglia could interact with OX40 on activated T cells,resulting in the proliferation of T cells and maintenance of T cell response in CNS.Suppression of T cell apoptosis may be one of the mechanisms.This result will help elucidate the molecular mechanisms of the interacton between resident cells in CNS and T cells,and further understand the expression and function of costimulatory molecule OX40L.