İnsan Serebral Mikrovasküler Endotel Hücrelerinin T Hücre Ko-İnhibisyonuna Etkisi

Abstract

With continuous stimulation, naive T helper cells (Th) convert into effector and exhausted Th cells with low cytokine production and proliferation capacity. In addition, during this differentiation process, there are changes in the costimulatory and co-inhibitory molecules on T lymphocytes. Cerebral endothelial cells show structural changes when they encounter T lymphocytes. However, it is not clear how T cells with different structural and functional properties change the endothelial structure of the blood brain barrier. In this thesis study, we planned to investigate the interaction of cerebral microvascular endothelial cells and T cells in different activation states and their genotypic and phenotypic changes in the case of ongoing inflammation. Human cerebral microvascular endothelial cell (hCMEC/D3) and acute myeloid leukemia (THP-1) cell lines and CD4+ T lymphocytes purified from peripheral blood polymorphonuclear cells of healthy individuals were used in the study. After hCMEC/D3 cells are cultured with inflammatory cytokines (TNF-α, IFN-γ), various surface molecules (CD40, CD80, CD86, HLA-DR, CD14, PD-L1, PD-L2, CTLA- 4, LAG3, TIM3) by flow cytometry; different transcript variants were analyzed by RTPCR. CD4+ T lymphocytes were co-cultured with THP-1 cells to achieve different phenotypes and functional properties (0, 24, 72 and 120 hours). Afterwards, CD4+ T cells purified again by FACS method were co-cultured with h-CMEC/D3s and expression of selected surface markers, proliferation of cells and levels of various cytokines in supernatants were evaluated by flow cytometry method. Inflammatory cytokines, including IFN-γ, increase the expression percentages of CD40 and CD86 costimulatory molecules of h-CMEC/D3 cells. IFN-γ increased HLA-DR expression of h-CMEC/D3 cells. The percentage of HLA-DR expression in h-CMEC/D3s evaluated after co-cultures of naïve and active CD4+ T cells was increased compared to control. The expression of the co-stimulatory CD40 molecule was most markedly increased by the naive T cells, followed by the effector T cells at the 24th hour, while the tired/memory T cells at the 72nd hour did not affect the CD40 expression. PD-L1 and PD-L2, which are co-inhibitory molecules, are also found at high rates in h- CMEC/D3 in the control condition. PD-L1 expression intensity increased most significantly at 0 and 24 hours.120. Fatigue T cells at 1 hour did not affect PD-L1 and PD-L2 MFY. When cerebral microvascular endothelial cells encounter Th cells, their antigen-presenting capacity increases. Naive and activated Th cells stimulate both costimulation and co-inhibition of h-CMEC/D3 more than exhausted Th cells.