September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Licensing of recipient-derived mesenchymal stem cells enhances their immunosuppressive properties and prolongs corneal allograft survival in rats
Author Affiliations & Notes
  • Oliver Treacy
    National University of Ireland, Galway, Galway, Ireland
  • Nick Murphy
    National University of Ireland, Galway, Galway, Ireland
  • Aideen E Ryan
    National University of Ireland, Galway, Galway, Ireland
  • Paul Lohan
    National University of Ireland, Galway, Galway, Ireland
  • Kevin Lynch
    National University of Ireland, Galway, Galway, Ireland
  • Maurice Morcos
    National University of Ireland, Galway, Galway, Ireland
  • Gerry Fahy
    National University of Ireland, Galway, Galway, Ireland
  • Matthew Griffin
    National University of Ireland, Galway, Galway, Ireland
  • Thomas Ritter
    National University of Ireland, Galway, Galway, Ireland
  • Footnotes
    Commercial Relationships   Oliver Treacy, None; Nick Murphy, None; Aideen Ryan, None; Paul Lohan, None; Kevin Lynch, None; Maurice Morcos, None; Gerry Fahy, None; Matthew Griffin, None; Thomas Ritter, None
  • Footnotes
    Support  Science Foundation Ireland (SFI) Grant 12/IA/1624
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 1435. doi:
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      Oliver Treacy, Nick Murphy, Aideen E Ryan, Paul Lohan, Kevin Lynch, Maurice Morcos, Gerry Fahy, Matthew Griffin, Thomas Ritter; Licensing of recipient-derived mesenchymal stem cells enhances their immunosuppressive properties and prolongs corneal allograft survival in rats. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1435.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : To investigate if activation of recipient-derived mesenchymal stem cells (MSC) with a cocktail of pro-inflammatory cytokines (a procedure known as licensing) enhances their immunosuppressive effects and prolongs corneal allograft survival.

Methods : Recipient (Lewis) rat MSC were isolated from bone marrow and cultured with the recombinant pro-inflammatory cytokines IFN-γ, TNF-α, and/or IL-1β for 72 hours prior to downstream use. Following activation, MSC production of inducible nitric oxide synthase (iNOS), which suppresses T cell responsiveness, was measured by Griess assay and the key immunomodulatory molecules IL-10, programmed death-ligand 1 (PD-L1) and IL-6 were measured by RT-PCR analysis. Furthermore, licensed MSC were co-cultured with lymphocytes to assess their ability to inhibit T cell proliferation. In vivo, licensed MSC were intravenously injected into Lewis rats receiving a fully allogeneic corneal transplant from Dark Agouti (DA) rats. Graft survival was recorded and immunomodulatory effects following MSC injection were analysed in corneas, draining lymph nodes and spleens of graft recipients.

Results : Production of nitric oxide was increased in licensed MSC pre-treated with either TNF-α/IL-1β or IFN-γ/TNF-α/IL-1β (6.61 and 11.33 µM, respectively), compared to untreated MSC (0.2 µM). These combinations also significantly inhibited T cell proliferation (CD4+ T cells-12.9%; CD8+ T cells-13.08% and CD4+ T cells-13.1%; CD8+ T cells-12.9%, respectively) compared to untreated MSC (CD4+ T cells-91.3%; CD8+ T cells-82.8%, p<0.001). Moreover, addition of the iNOS inhibitor S-methylisothiourea (SMT) to the co-cultures abrogated these inhibitory effects. RT-PCR analysis showed that mRNA expression levels of IL-10, PD-L1 and IL-6 were up-regulated in cytokine pre-treated MSC. In vivo, corneal allograft survival was significantly prolonged up to day 30 post-transplantation in 70% of recipients (MST 26.4±6d, n=10) receiving 2 post-transplant injections of TNF-α/IL-1β pre-treated MSC (1 million cells per injection), compared to untreated controls (MST 19±3d, n=10; p<0.0001).

Conclusions : Licensing of MSC induces a strong immunosuppressive phenotype by increasing production of immunosuppressive factors, thereby creating a more potent immunotherapy compared to their untreated counterparts.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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