Study On The Variability Of T Cell Mediated Immune Response Under Different Active States Via B7-H3 Signaling Pathway

Objective In recent years,many studies have shown that T lymphocyte-mediated immune response plays a vital role in the process of anti-inflammatory and anti-tumor etc.It also has already been proved that co-stimulatory signal,the second one of T lymphocyte activation,determines the immune response status of T cells.Positive co-stimulatory signal could maintain the activation status of T cells and enhance T cells immune response;negative co-suppression signal can mediate the formation of T cells immune tolerance and play an important role regarding tumor escape.As the newly discovered co-stimulatory molecule,the role of B7-H3 in T lymphocyte immune response is still controversial.Apart from regulating T cells immune responses,the issues like whether B7-H3 involves in other immune components of the immune microenvironment and plays the biological function are still unknown.In response to the above issues,this paper aims to study the effect of B7-H3 signal on T lymphocyte proliferation in different activation status and related cytokine secretion,to explore the role and mechanism that B7-H3 induced in the T lymphocyte immune response.Methods Isolate peripheral blood mononuclear cells(PBMCs)by Ficoll density gradient centrifugation and separate the pure T cells from them.Provide CD3/CD28 monoclonal antibody co-stimulation culture in vitro,adopt Flow CytoMetry(FCM)to observe the changes of T lymphocyte phenotype CD28 and CTLA-4 under different stimulation time points and analyze T cells activation status.Based on this,add human B7-H3-Fc fusion protein and isotype control Hu-Fc fusion protein on the 0,1st,2nd,3rd and 4th day of CD3 monoclonal antibody pre-stimulation combined with CD28 monoclonal antibody respectively,adopt CCK8 method to detect the T lymphocyte proliferation in each group;Detect the secretion levels of related cytokines(IL-2,IL-10 and IFN-?)by enzyme linked immunosorbent assay(ELISA)and analyze whether T cells under different activation status have different immune responses after undergoing B7-H3 signal stimulation.Results 1.FCM results showed that T cells in PBMCs and pure T cells could be effectively activated under the combined stimulation of CD3 monoclonal antibody and CD28 monoclonal antibody,and the expression of CD28 significantly increased while that of CTLA-4 showed no obvious change.T cells in PBMCs:CD3+CD28+positive rate from(28.30 + 0.034)%to(80.98 + 0.018)%(P<0.05),CD3+CTLA-4+positive rate showed no significant change(P>0.05);Pure T cells:CD3+CD28+ positive rate from(58.84 +0.021)%to(85.14 + 0.041)%(P<0.05),CD3+CTLA-4+ positive rate showed no significant change(P>0.05).2.The CCK8 results show that T cells in PBMCs:the B7-H3 molecule has obvious inhibitory effects on the T cell proliferation(Pre-activation 0d,P<0.05),then the B7-H3molecule has no significant effect on the T cells proliferation i(Pre-activation 2d,P>0.05);Pure T cells:B7-H3 molecules had no significant effect on the proliferation in vitro(Pre-activation 0-2d,P>0.05)3.The ELISA result showed that B7-H3 signal could significantly inhibit the secretion of IL-2 and IL-10 in T lymphocyte resting state.But it had no obvious effect on IFN-? secretion.With the activation of T cells,B7-H3 signal could significantly promote T cells to secrete IL-2 and IFN-y,but did not significantly influence the secretion of IL-10.Conclusion 1.B7-H3 molecule could significantly inhibit the proliferation in vitro of PBMC-derived T lymphocyte,but had no effect on the proliferation of pure T cells,suggesting that B7-H3 molecules can indirectly participate in T lymphocyte immune response by affecting other immunological components in the immune microenvironment;2.By dynamically observing the secretion of related cytokines(IL-2,IL-10 and IFN-?)when B7-H3 molecules act on T cells under different activation status,it is found that the role of B7-H3 changes with the change of T cells activation status,which suggests that the regulation of B7-H3 signal on T cells immunologic function will change under different activation status of T cells.