Donor- and third party-derived, but not recipient-derived mesenchymal stromal cells, promote corneal allograft survival in rats

Abstract

Purpose: Mesenchymal stromal cells (MSCs) have potent immunomodulatory potential and are being investigated clinically to promote allograft survival. Here, we investigated the ability of MSCs to promote allograft survival in a rat corneal transplant model.

Methods: Recipient-derived (syngeneic-syn), donor-derived (allogeneic-allo) or 3rd party MSCs were isolated from bone marrow of Lewis (LEW), Dark Agouti (DA) or Wistar-Furth rats, respectively. A fully allogeneic rat cornea transplant model (DA to LEW) was used for in vivo studies. Recipient (LEW) rats received one dose of 1x10⁶ MSCs (syn, allo or 3rd party) intravenously on day -7 and a second dose on the day of transplantation (day 0). Graft survival and neovascularization was monitored. Flow cytometry was used to analyze graft-infiltrating cells and cellular distribution in the draining lymph nodes and spleens of MSC-treated transplanted rats and also to analyze allo-antigen or 3rd party antigen-primed T cells following re-stimulation with relevant antigen in vitro. Corneal stromal edema was quantified using histological analysis.

Results: Untreated allografts were uniformly rejected (MST 16.6±1.5d, n=9) and, while syn-MSC treatment showed some evidence of attenuation of rejection (MST 18.3±4d, n=9), no clear pattern emerged following this treatment. In contrast, corneal allograft survival was significantly prolonged in approximately 90% of allo-MSC treated and 80% of 3rd party MSC treated allograft recipients. Flow cytometric analysis showed lower percentages of infiltrating NKT cells in corneas of both allo- and 3rd party MSC treated animals, coupled with a significantly higher frequency of splenic CD4+Foxp3+ regulatory T cells (Tregs), compared to controls. Furthermore, in an in vitro recall response assay following re-stimulation of lymphocytes from allo-MSC treated allograft recipients with allo-antigen, a significantly higher percentage of CD4+Foxp3+ Tregs were present compared to allo-antigen re-stimulated lymphocytes from syn-MSC treated recipients.

Conclusions: Systemic administration of both allo- and 3rd party MSCs prolongs corneal allograft survival with between 80 and 90% of allografts surviving for ≥ 30 days. This can be in part explained by the presence of fewer intragraft NKT cells and a higher frequency of splenic Tregs, coupled with antigen-specific modulation in the case of allo-MSC treatment.

Keywords: 479 cornea: clinical science • 555 immunomodulation/immunoregulation • 721 stem cells