The Role Of PD-1/PD-L1 Pathway On Immune Modulation During The Process Of Atherosclerosis

The idea that inflammation is a driving force in every phase of atherosclerosis has been well established. Immunohistochemical analyses have shown that atherosclerotic plaques contain significant numbers of T cells, including CD4+ and CD8+ subsets, at all ages of lesion development, in both humans and mouse models. However, there is no sufficient knowledge to justify the mechanism of immunomodulation in the progression of atherosclerosis.Pathways in the B7/CD28 family critically regulate the balance between the stimulatory and inhibitory signals needed for defense against microbes and for self-tolerance. These pathways provide second signals that can regulate the activation, inhibition and fine-tuning of T-cell responses. One of the surprises of the past few years has been the abundance of inhibitory pathways within the B7/CD28 family that can attenuate T-cell responses and promote T-cell tolerance. Programmed death-1 (PD-1, CD279) is an immune inhibitory receptor belonging to the B7/CD28 family of costimulatory molecules. PD-1 has two known ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). The interaction between PD-1 on T cells and PD-L on antigen-presenting cells (APCs) play an essencial role in mediating immune suppression. Interferonsα,β, andγare potent upregulators of PD-L1 expression on APCs, endothelial cells and epithelial cells. With the integral role for PD-1 in receiving signals from DCs, the ligation of PD-1 by PD-Ls may control the T-cell tolerance versus activation. Taken together, accumulating evidence suggests that the PD-1/PD-L pathway plays a critical role in attenuating T cell responses and promoting T cell tolerance. Therefore, the PD-1/PD-L pathway is currently under intense investigation because manipulating it has the potential to modulate immune responses in a positive or negative manner.DCs represent the most potent APCs and, thus, are very important in the initiation and maintenance of T cell immunity. As a subpopulation of DCs, myeloid DCs (mDCs) play a pivotal role in adaptive immunity by activating naive T cells. The relative role of PD-L1 versus PD-L2 on the DC is of interest. Using mouse model, Gotsman and colleagues have recently shown that PD-1/PD-L1/2 expression may correlate with the extent of systemic CD4+ T cell response to hypercholesterolemia and the associated development of arterial disease. However, in human body, it remains unclear whether PD-1/PD-L pathway influences immune response to plaque antigens and thereby impacts on the process of atherosclerosis. Here we studied the expression profiles of PD-1 on CD4+ T cells and PD-L1 on mDCs from CAD patients and evaluated the roles of PD-1/PD-L1 in the pathogenesis of this disease.In order to clarify whether this pathway regulates proatherogenic responses in human, we studied the expression profiles of PD-1 and PD-L1 on Peripheral blood mononuclear cells (PBMCs) in 76 patients with coronary artery disease (CAD) and evaluated the roles of PD-1/PD-L1 in the pathogenesis of atherosclerosis. Our study was as follows:1 Sample preparation and analysis of PD-1/PD-L1 expression on PBMCA total of 76 CAD patients who had coronary atherosclerosis, defined as a >50% diameter stenosis in≥1 coronary artery, were enrolled at our Department. Blood sample from each patient was collected in heparinized tubes. Serum cholesterol, concentrations of C-reactive protein (CRP) and white blood cell count were measured according to routine protocols. PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation from fresh heparinized blood samples. The plasma supernatants were collected for the detection of inflammatory cytokines including IFN-γ,IL-2,IL-10 and IL-12, using ELISA kits. PD-1 and PD-L1 expression on CD4+ or CD8+ T cells and PD-L1 expression on mDCs were analyzed by flow cytometry on a FACS Calibur.2 The influence of the PD-1/PD-L1 pathway on the T cell immune function in the process of atherosclerosisCD4+ T cells, CD8+ T cells and mDCs isolated with positive or negative isolation kit were used in this part. Matured mDCs were mixed at different ratios with freshly isolated CD4+ or CD8+ T cells from a third healthy individual, and allogeneic mixed leukocyte reactions (MLR) were performed. Before the measurement of incorporated [3H] thymidine, part of the culture supernatants was collected for cytokine detection with EILSA kit.3 The effects of PD-L1 up-regulation by Interferonαon the mDCs function and T cell responseInterferonαis an upregulator of PD-L1 expression on mDCs in short term and can be used as a positive modulation factor of PD-L1. For up-regulating the expression of PD-L1 on mDCs, IFN-αwas added in the culture medium during the process of mDCs maturation. Then the allogeneic MLR were performed to test the stimulatory ability of mDCs on the proliferation of T cells. In the meantime, the concentration of inflammatory cytokine in the culture supernatants was also detected by ELISA.Results:1 Compared with healthy individuals, the expression of PD-1 was significantly down-regulated on CD4+ or CD8+ T cells. PD-L1 on CD4+ T cells and myeloid dendritic cells (mDCs) of CAD patients were also down-regulated significantly. 2 Plasma levels of IFN-γand IL-2, type 1 effector cytokines secreted by T cells, were significant higher in CAD patients than that of controls. In comparison with healthy subjects, increased IL-12 and decreased IL-10 secretion were also detected in plasma of patients group. The difference between SA group and ACS group maybe the result of enhanced immune responses during the progression of acute coronary syndrome.3 Based on allogeneic MLR, mDCs of CAD patients had a significantly increased capacity to stimulate allogeneic CD4+ and CD8+ T cells at all ratios tested compared to healthy controls. Meanwhile, mDCs of CAD patients showed an increased capacity to produce IL-12 and decreased capacity to produce IL-10. The production of IFN-γand IL-2 by allogeneic CD4+ and CD8+ T cells were also increased compared to healthy controls.4 In vitro stimulation of PD-L1 expression by IFN-αcould not only attenuate the stimulatory ability of mDCs on allogeneic T cell proliferation and its cytokine production, including IFN-γand IL-2, but also influence the production of IL-10 and IL-12 by mDCs.Conclusions:1 The down-regulation of PD-1/PD-L1 on PBMC is closely related with the increased T cell immune responses of CAD patients. This pathway can influence the interactions between T lymphocytes and mDCs, manifested with enhanced T cell proliferation, increased secretion of proinflammatory cytokines and decreased secretion of anti-inflammatory ones.2 The PD-1/PD-L1 pathway may have a key role in the regulation of proatherogenic T cell immunity by intervening APC-dependent T cell activation and associated proinflammatory or antiinflammatory cytokine production. Manipulating this pathway may represent a strategy of immunotherapy for atherosclerosis.