Abstract
Purpose: :
Evidences from literature shows that GITR–GITRL interaction plays important role in delivering co–stimulatory signals controlling regulatory T cells. Human GITR ligand has recently been shown to be constitutively expressed in retinal tissues. By expressing eYFP tagged human GITRL in human retinal pigment epithelial (RPE) cells, we investigated the biological significance of expression of GITRL on human ocular tissue.
Methods: :
Full length GITRL was amplified from a human ovarian cDNA library using GITRL specific primers and subcloned in a pEYFPC1 vector for expressing GITRL as an eYFP tagged protein in RPE cells. Using an RPE/T cell co–culture system we studied the invitro proliferative responses of purified CD3+ T cells or CD3+CD25–T cells co–cultured with lethally irradiated RPE cells in the presence or absence of anti–CD3 and anti CD28 antibody. For cytokine measurement, culture supernatants were collected from parallel RPE/T cell co–culture experiments.
Results: :
Both immunofluorescence microscopy and flow cytometry confirmed that GITRL was predominantly expressed on surface of RPE cells. Expression of GITRL on the surface of RPE cells abrogated RPE mediated immunosuppression of CD3+ T cells, a well–established function of ocular tissues implicated as a possible mechanism for ocular immune privilege. This abrogation of immunosuppression by RPE was solely dependent on GITR–GITRL interaction and could not be mimicked by anti–CD28 antibody. There was no difference in this abrogation of immunosuppression by GITRL bearing RPE on either CD3+CD25+ T cells or CD3+CD25– T cells. Analysis of cytokine profiles revealed high level of TGF–beta during the immunosuppression of RPE cells while GITR–GITRL interaction abrogated the RPE cell induced TGF–beta expression. In addition to enhanced proliferation of CD3+ T cells, the abrogation of immunosuppression of RPE cells by GITRL was marked by up–regulation of an array of pro–inflammatory cytokines/chemokines.
Conclusions: :
These results suggest that GITR–GITRL interaction provides a unique pro–inflammatory co–stimulation that abrogates RPE mediated immune suppression and may signal through a different pathway than that of CD28–B7 co–stimulation. This study suggested GITRL could be a potential candidate for regulation of the ocular immune privilege and might have implications to further understanding the nature of the balance between ocular immune privilege and ocular inflammation.
Keywords: immune tolerance/privilege • autoimmune disease • retinal pigment epithelium