Regulatory Role Of BTLA Signal In The Initiation And Early Phase Of T Cell Activation

BTLA is a new coinhibitory receptor with similarities to CTLA-4and PD-1whichwas discovered at2003and belonged to Ig superfamily. It’s gene of human is located onchromosome3q13.2and shares48%homology with mouse. BTLA mRNA is expressedstrongly in spleen and lymph node tissues but very weakly or undetectably by severalsomatic tissues such as kidney, liver, lung, brain, heart and muscle. BTLA is widelyexpressed on T cells, B cells, dendritic cells, macrophages and NK cells.BTLA contains a signal sequence, extracelluar variable(V)-like immuno-globulin(Ig) domain, transmembrane region and intracellular domain of about100amino acids. Three tyrosine residues in the cytoplasmic domain are contained in BTLA:the first is in a potential Grb2interaction site and the other two are in ITIM sequenceand ITSM sequence. Some research showed that its interaction with HVEM may beinvolved in suppression or termination of T cell response by inhibiting T cellproliferation, cytokine secretion and delaying cell cycle progression. FollowingAg-specific activation of CD4+T cells, BTLA is most highly induced genes selectivelyexpressed in anergic CD4+T cells, suggesting that BTLA signal is important in theinduction and maintainance of T cell anergy. Otherwise, BTLA not only plays animportant role in regulation of peripheral immunological tolerance, but also preventsautoimmune diseases, inhibits immune responses of inflammation and transplantation.BTLA is expressed on naive and activated T cells, so Murphy suggested that BTLAmay regulate all phases of T cell activation, especially the early phase. In this study, wefirstly analyze the influence of BTLA-HVEM cis-complex on the detection of BTLAexpression level on cell surface. Then we study the regulatory role of BTLA signal inthe initiation and early phase of cell activation. Part Ⅰ: BTLA-HVEM cis-complex affect the examination of BTLA on cellsurfaceObjective: BTLA and HVEM are both expressed on U937and T cells. Weinvestigate the influence of BTLA-HVEM cis-complex on the detection of BTLAexpression.Methods: T cells from PBMC were enriched by negative selection using magneticbeads. Then BTLA mAbs8H9and MIH26were used to measure the expression ofBTLA on T cells, U937cells by method of flow cytometry. The transfectant293T/BTLA was examined by8H9mAb.Results: BTLA was expressed on~90%T cells and U937cells when analyzed bymAb MIH26. However, BTLA was expressed on42.2%of T cells and30.8%of U937cells when detected by mAb8H9. Furthermore,0.02μg and0.05μg8H9mAb can detect80%and91.4%expression of BTLA on293T/BTLA transfectant.Conclusion: The results suggest that BTLA could interact with HVEM in cis onU937and T cells. The cis-complex could affect the detection of BTLA expression whenwe use8H9whereas using mAb MIH26could not affect the detection.Part Ⅱ: Regulatory role of BTLA in the initiation and early phase of T cellactivationObjective: To observe the expression of BTLA on T cells during T cell activationand further analyze its inhibitory effects on T cell activation in different phasesespecially the early phase.Methods: Kinetics expression of BTLA, CTLA-4, PD-1on CD3mAb and CD28mAb stimulated T cells were detected by flow cytometry. T cells were stimulated withanti-CD3and anti-CD28in the presence of BTLA mAb8H9, then early activationmarker CD25and CD69were examined by flow cytometry. T cells were stimulatedwith CD3mAb and CD28mAb in the presence of BTLA mAb8H9or HVEM-Fc and Tcell proliferation was tested by CCK8assay in the different culture time. Then wedetected the influence of8H9on IL-2and IFN-γ secretion of T cells stimulated byanti-CD3mAb and CD28mAb by ELISA. BTLA mAb8H9were added at24h,48h,72h after T cells were pre-activated with anti-CD3and anti-CD28, then we investigatethe regulatory role of BTLA signal on T cell activation.Results: BTLA was found to express highly and constituently on T cells and its expression was decreased gently at the beginning and then increased to a high levelquickly. CTLA-4and PD-1are expressed weakly or undetectably on na ve T cells andtheir expression were induced on activated T cells. Anti-BTLA mAb8H9inhibitedexpression of early activation marker CD25and CD69on CD3mAb and CD28mAbstimulated T cells. T cell proliferation was inhibited When T cells were stimulated withanti-CD3and anti-CD28mAb in the presence of BTLA mAb8H9or HVEM-Fc for1d,2d,3d,4d. Moreover,8H9mAb could inhibit IL-2and IFN-γ secretion of T cellsstimulated by anti-CD3mAb and CD28mAb.8H9could still exhibit inhibitory effect onT cell proliferation after24h,48h,72h of preactivation by CD3mAb plus CD28mAbstimulation.Conclusion: BTLA plays importantly negative regulatory role in the initiation andearly phase of T cell activation and enhances the threshold of T cell activation.