The Effects Of BAP31 On The Development And Function Of T Cells

B cell receptor associated protein 31(BAP31/BCAP31)is an ubiquitously expressed polytopic integral protein of the endoplasmic reticulum(ER).The study showed that expression of the BAP31 gene was analyzed from various mouse tissues and cell lines,such as lymphocytes,thymic stromal cells,cerebellar Purkinje neuron bodies,dendrites and thyroid follicular epithelial cells.BAP31 functions as a participant in the transportation of a variety of molecules from the ER to Golgi apparatus,such as newly synthesized IgD,cellubrevin,class I MHC,CD11b/CD18 and protein tyrosine phosphatases like B(PTPLB).BAP31 is involved in the apoptosis and Endoplasmic reticulum associated degradation(ERAD)pathways.The thymus is an important organ of T cell differentiation and development.Lymphocytes development in thymus must go through the negative and positive selection process.However,the pro-T cells without the expression of both the CD4 and CD8 are termed DN(double-negative).Positive selection for thymocytes bearing receptors capable of binding self-MHC molecules,results in MHC restriction.The cells that fail positive selection are eliminated within the thymus by apoptosis and survive thymic selection develop into immature single-positive CD4+ thymocytes or single-positive CD8+ thymocytes.These single-positive cells undergo additional negative selection and migrate from the cortex to the medulla,where they pass from the thymus into the peripheral immune system.A couple of publications indicate that BAP31 participates in class I MHC molecules transportation from the ER to Golgi apparatus.Since class I MHC molecules play an important role in T cell development process,whether BAP31 may be involved in T cell development?In order to clarify if BAP31 affects T cell development and function we created a BAP31 conditional knockout mouse and crossed with a Lck-Cre mouse to knockdown the expression of BAP31 specifically in the thymus organs.The results are as follows after the corresponding analyses:BAP31 is involved in T cell activation through TCR signal pathways:In order to clarify whether the BAP31 is involved in T cell activation through TCR signal pathways,we analysed the animal model and found that BAP31-/-mice had a significant reduction in the thymic weight,size and total numbers.Analysis by flow cytometry showed that the proportion of CD4+ and CD8+ conventional T cells was not affected by lack of BAP31 in thymus.Similarly there was no significant effect of BAP31 deletion on the DN1-4 thymic subsets but increased thymocytes susceptibility to apoptosis in BAP31-/-mice.In addition,we found that effector/memory T cells(CD44hiCD62Llow)in CD4+ and CD8+ T cells increased in the BAP31 deficieny mice,and the naive T cell population(CD44lowCD62Lhi)correspondingly reduced.Analysis by flow cytometry indicated that BAP31 deficiency resulted in significant lack of mature T lymphocytes in the spleen and LN.The above results show that BAP31 is probably involved in T lymphocyte activation and proliferation.FACS and RT-qPCR revealed a profound reduction of the BAP31-/-T cells in producing two major cytokines IL-2 and IFN-y.Our subsequent analysis by WB evidence revealed a decrease in activation of some key signaling events,including phosphorylation of ZAP70,Lck and LAT upon the stimulation of TCR by anti-CD3 plus anti-CD28 Abs.Furthermore,stimulation of T cells with anti-CD3/CD28 Abs results in a decreased phosphorylation of several downstream signaling molecules,including IKKo?/? AKT,GSK-3? and MAPK kinases in total T cells.Consistently,the deficiency of BAP31 inhibited the activation of the major downstream transcription of NF-KB-p65 and C-Jun,the factors involved in T cell activation.To clarify how BAP31 regulated the expressions of TCR signaling,we examined the membrane proteins on the cell surface of mature T cells,including CD3,TCR?,TCR?,CD4,CD8,CD28,CD27,4-IBB and CTLA-4.The results show that these membrane proteins in BAP31-/-mice were expressed in relative lower levels as detected by FACS except for CTLA-4 that was expressed in relative higher levels.Meanwhile,our results indicate that some T cell activation markers CD25/CD69/CD44/CD62L expressed lower levels in BAP31-/-mice.To establish whether BAP31 directly associates with the TCR complex,an 'immunoprecipitation of the TCR was performed.Results demonstrate that BAP31 and TCR?,TCR?,ZAP70 did not form a detectable complex in both resting and activated T cells.The above results indicate that BAP31 plays an essential role in T lymphocytes activation by regulating TCR signaling.In order to further elucidate BAP31 effect of T cells function,the above mice were used to establish the Lewis lung cancer model.Normal control group and tumor control group were established simultaneously.Body weight,tumor size and tumor weight were detected.NK cell function was detected by NK cells mediated cytotoxicity assay.The amount of CD4+/CD8+T lymphocytes and B lymphocyte were analyzed by applying FACS.The proliferating functions of T and B lymphocytes were analyzed with lymphocyte proliferation assay.The results shown that compared with control group,proliferation function and amount of T cell and B cell,and killing activity of NK cell of tumor group decreased in different degree.Compared with tumor group,lack of BAP31 could significantly inhibit the killing activity of NK cells,inhibit proliferation function of T cell,and significantly reduced the amount of CD4+T cells.