June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
YP-P10 Peptide selectively decreased Th2 and Th17 cells in an in vitro model of human blood-derived effector CD4+T-cell subsets.
Author Affiliations & Notes
  • Virginia L Calder
    Institute of Ophthalmology, University College London, London, London, United Kingdom
  • Malihe Eskandarpour
    Institute of Ophthalmology, University College London, London, London, United Kingdom
  • Larry Park
    Naason Science Inc, Osong-eup, Korea (the Republic of)
  • Laxmikant Gharat
    Yuyu Pharma Inc, Jung-gu, Seoul, Korea (the Republic of)
  • Footnotes
    Commercial Relationships   Virginia Calder Yuyu Pharma, Code F (Financial Support); Malihe Eskandarpour None; Larry Park Yuyu Pharma, Code C (Consultant/Contractor), Naason Science Inc, Code E (Employment); Laxmikant Gharat Yuyu Pharma, Code E (Employment)
  • Footnotes
    Support  UCL/Yuyu Pharma Research Collaborative Grant
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3919. doi:
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      Virginia L Calder, Malihe Eskandarpour, Larry Park, Laxmikant Gharat; YP-P10 Peptide selectively decreased Th2 and Th17 cells in an in vitro model of human blood-derived effector CD4+T-cell subsets.. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3919.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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