Abstract
Purpose :
A synthetic peptide YP-P10 has previously been demonstrated to exert a protective role in in vivo models of dry eye disease (DED) and is in a Phase 2 study for DED. The purpose of this study is to investigate YP-P10 for its anti-inflammatory effect(s) on adaptive immune cell responses in vitro and elucidate its mechanism of action.
Methods :
Fresh, heparinized healthy bloods (n=4) were obtained (Cambridge Bioscience, UK) and mononuclear cells (PBMC) isolated by Ficoll centrifugation. Viability of lymphocytes, assessed based on exclusion of Zombie dye (Biolegend) for 18hr, remained high in the presence of YP-P10 (less than 500nM). For immune cell responses, PBMC were cultured in the presence of stimuli (anti-CD3/CD28 Ab or PMA/ionomycin or PBS control), adding YP-P10 (0, 10 or 500nM) 1 hr beforehand, and incubated overnight.
Immunophenotyping was then performed to identify B (CD19+) and T cells (CD3+/CD8+/-), and intracellular cytokine staining for functional T-cell subsets. For Th17 cell enrichment, PBMC were cultured with YP-P10 (500nM) for 1hr prior to culture in anti-CD3/28 for 5 days in the presence of IL-6 [30 ng/ml], IL-1β [10ng/ml], IL-23 [20ng/ml] and TGFß [2.5ng/ml). Cells were restimulated with PMA/ionomycin for 4 hr prior to immunophenotyping and acquired for flow cytometry (BD Fortessa) and analysed with FlowJo (BD Version 10.8).
Results :
Gating on live, single-cells, we detected T-cells (70±15%; mean ±SD) and B-cells (30% ±10) in all PBMC in the presence or absence of YP-P10, with levels of CD4+T cells (49.8% ± 2.6) and CD8+T cells (22.7% ± 0.7) similar in all PBMC. YP-P10 significantly decreased Th2 (CD3+CD8-IL-4+) cells (n=4, 9.1% ± 5.4; P<0.046), independent of the mode of stimulation. YP-P10 treatment also showed a moderate reduction in Th17 levels (2.42% ±1.3), despite low baseline levels of Th1 and Th17 subsets after anti-CD3/28 stimulation. However, following Th17 cell enrichment (n=2), YP-P10 significantly decreased levels of Th17 (CD3+CD8-IL-17+) cells (26.38% ± 20.4).
Conclusions :
YP-P10 selectively downregulated both Th2 and Th17 cells and we are currently investigating the relevant pathways involved.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.