Ex-vivo generation of regulatory T-cells expressing transcription factor foxp3 and their application in cornea transplantation

Abstract

This project investigated the therapeutic potential of allo-antigen specific regulatory T-cells in the context of penetrating keratoplasty. Two methods of ex-vivo generation of T regulatory cells have been explored in this study. Using recombinant retroviruses gene transfer of the transcription factor foxp3 was achieved into allo-antigen specific primary rat T-cells. However, expansion of foxp3 gene modified cells necessary for in-vitro and in-vivo studies, was found to be too cumbersome. The second approach exploited a non-depleting anti-CD4 antibody. Allo-primed mixed lymphocyte cultures were treated with low-dose anti-CD4 antibody resulting in an outgrowth of foxp3 positive T-cells. CFSE labelled responder T-cells targeted with anti-CD4 treatment indeed confirmed preferential growth of regulatory T-lymphocytes. Tregs generated by this procedure were studied by flowcytometry and found to express high levels of CD25, Ox-40 and ICOS, confirming their suppressor T-cell phenotype. To study Treg effects on the outcome of allo-graft survival, a full allogeneic cornea transplant model was established. Two different strain combinations were tested. First, a high responder LEW-DA model was set-up but was found to be too fragile and unreliable to serve as a preclinical model. A second novel strain combination was tested and discovered to be of low-responder characteristics and remarkably robust and highly suitable for in-vivo applications. The rejection process of the BN-PVG model was comprehensively studied using flowcytometric analysis of draining lymph nodes. Moreover, a gentle digestion protocol was established to isolate viable graft infiltration lymphocytes. Applying multi-parameter FACS six distinct cell populations were observed among which were CD8+ cytotoxic T-cells, CD4+ T-cells, CD3- CD8+/- CD161high NK, CD3+ CD8+ CD161dull NK-T-cells, CD161dull large granular lymphocytes and MHC-2 positive cells. Additionally a serum analysis found evidence for IgM, IgG1 and IgG2a allo-antibodies in rejection animals. Finally ex-vivo generated Tregs were tested towards their ability to prevent allo-graft rejection. CD3 sorted regulatory T-cell preparations with direct allo-antigen specificity did not prevent or delay rejection of allogeneic corneal grafts. The implications of this finding for the clinical application of adoptive Treg therapies have been discussed in detail.