After 3 days of incubation, T cell proliferation was assessed by measuring CFSE dilution using flow cytometry, gating on CD4 + T cells, and by checking under a microscope the numbers and sizes of cell clusters formed by the proliferating cells. The CFSE-labeled, anti-CD3 mAb-activated cells were then aliquoted into wells of a 96-well plate at a concentration of 0.4 × 10 6 cells/well, and incubated with different numbers of RPCs (RPCs/T cell ratio: 0, 1:10, 1:20, 1:40, and 1:80) in triplicate. After washing, 2.0 μg/mL of anti-CD3 mAb (BD Biosciences, San Jose, CA) was added to the CFSE-labeled spleen cells to activate T cells. 18 For mouse-activated T cell proliferation assays, naive C57BL/6 mouse spleen cells were first labeled by incubating them with 0.3 μM of CFSE (Invitrogen, Carlsbad, CA) at 37☌ for 8 minutes. This may represent a novel mechanism by which RPCs contribute to preservation of retinal integrity in diseases, including DR.Ī conventional carboxyfluorescein succinimidyl ester (CFSE)–based T cell proliferation assay was used to test the T cell inhibitory activity of RPCs using previously described methods, with minor modifications. These results reveal a new function of RPCs, and its regulation under hyperglycemic conditions. Hyperglycemic conditions impaired the T cell inhibitory activity of RPCs.
RPCs are highly immunosuppressive and they protected RECs from inflammation-mediated apoptosis. Incubation of RPCs with either high glucose or MGO reduced the activity of RPCs to inhibit activated T cell proliferation. There were significantly reduced numbers of inflammation-induced apoptosis-detected RECs in the presence of RPCs. RPCs also produce IL-10, and neutralization of IL-10 reduced their immunosuppressive activity. RPCs express PD-L1, and blocking PD-L1 reduced RPCs' efficacy of T cell inhibition. The T cell inhibitory activity of RPCs was decreased, but was not abolished, in transwell experiments. RPCs profoundly inhibited activated T cell proliferation and inflammatory cytokine production. Finally, to test whether hyperglycemic conditions impair the immunomodulatory activity of RPCs, RPCs pre-incubated in high glucose or methylglyoxal (MGO) were evaluated using the T cell proliferation assays. The efficacy of RPCs in protecting retinal endothelial cells (RECs) from inflammation-induced apoptosis was assessed by apoptosis detection staining. To elucidate the underlying mechanisms, transwell systems, blocking mAbs against PD-L1 and IL-10 were used. Isolated mouse and human RPCs were tested in T cell function assays to evaluate their capability of inhibiting T cell responses. To test the hypothesis that retinal pericytes (RPCs) are immunosuppressive therefore, their loss of function under hyperglycemic conditions favors retinal inflammation and contributes to the pathogenesis of diabetic retinopathy (DR).
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