Anti-inflammatory effect of YP-P10: upregulation of CD25, FoxP3 and TGFβ/IL-10 co-expression in Behçet’s patients’ blood-derived CD4⁺T cells and downregulation of il-17f and ifng genes by Th17 cells

Abstract

Purpose : The synthetic peptide YP-P10 has previously been demonstrated to exert a protective role in in vivo models of dry eye disease (DED) and was shown to downregulate healthy human peripheral blood (PB)-derived CD4⁺T cells in vitro, decreasing expression levels of IL-17A¹ in the presence of inflammatory stimuli.
The purpose of this study is to investigate the effect of YP-P10 on PB-derived CD4⁺T cells from donors with a systemic inflammatory disease (Behçet’s disease BD; n=14) vs. healthy donors (n=6), and to determine if there is a direct effect on purified human Th17 cells from healthy donors at the level of gene expression.

Methods : Isolated PB mononuclear cells (PBMC; n=14) were obtained from our in-house Biobank, originally donated as part of a clinical study. PBMC were cultured with YP-P10 (500nM) for 1hr prior to adding PMA/ionomycin for 18hr. Immunophenotyping was performed to identify CD4⁺T cells (CD3⁺/CD8^-), and intracellular cytokine staining for functional T-cell subsets (Th17, Treg), acquired for flow cytometry (BD Fortessa) and analysed with FlowJo (BD). Purified healthy human Th17 cells (EasySep^TM, Stemcell, UK) with and without YP-P10 were stimulated for 18hr prior to undergoing single cell RNAseq, applying 10X Genomics platform and analysed with Partek Flow software.

Results : For unstimulated healthy PBMC, YP-P10 had no overall effect on the levels of Treg (CD4⁺CD25⁺FoxP3⁺) but there was a significant increase in % of IL-10-expressing Treg in the presence of YP-P10 post stimulation (n=6; mean % increase from baseline = 21%; P<0.04). There was also a significant increase in TGFb secretion in 3 donors’ PBMC. In 9/14 BD-PBMC there was a significant increase in %IL-10-expressing Treg induced by YP-P10 (P=0.016). By heatmap analysis, 120 genes were differentially expressed, with a decrease in il-17f, ifng and upregulation of ctla-4, tgfb1 and and tgfbr2 in response to YP-P10 in stimulated Th17 cells.

Conclusions : YP-P10 exerts an anti-inflammatory effect on stimulated healthy and patient-derived CD4⁺T cells by downregulating pro-inflammatory cytokines and upregulating Treg-associated cytokines at the protein and RNA levels.


¹Calder et al (2023) ARVO abstract ‘YP-P10 Peptide selectively decreased Th2 and Th17 cells in an in vitro model of human blood-derived effector CD4⁺T-cell subsets’

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.