Modification of dendritic cells for the induction of tolerance

Allogeneic T cell stimulation requires not only antigen-specific signals but also costimulatory signals, most importantly between CD80/86 on the antigen presenting cell and CD28 or CTLA4 on the T cell. Engagement of the T cell receptor without costimulation can lead to anergy and the subsequent induction of regulatory T cells (Tregs). T cell activation is also controlled by dendritic cell (DC) expression of the tryptophan-catabolising, immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Depletion of this essential amino acid, and/or the production of tryptophan metabolites (kynurenines) inhibits T cell proliferation. With the aim of generating tolerogenic DCs for the induction of donor-specific transplantation tolerance, lentiviral vectors were used in this study for gene transfer into murine DCs. Firstly, an intracellular approach that prevents costimulation was developed: A fusion protein- CTLA4-KDEL- consisting of the extracellular domain of CTLA4 and the KDEL peptide [an endoplasmic reticulum (ER) retention signal] was expressed in DCs and prevented surface CD80/86 expression due to ER retention. A second approach used in the study involved the lentiviral gene transfer of IDO into DCs. The induction of donor-specific tolerance (mediated by Tregs with direct and indirect pathway allospecificity) was demonstrated, both in vitro and in vivo, using CTLA4-KDEL-expressing DCs. Linked suppression was observed and mediated by donor-specific Tregs. Although IDO-expressing DCs did not induce and/or sustain allogeneic T cell proliferation, this anergy lacked donor specificity. It was demonstrated that IDO+CD80/86+ DCs fail to induce and/or expand FoxP3+CTLA4+ Treg populations. Furthermore, IDO+CD80/86- DCs failed to sustain pre-induced populations of FoxP3+CTLA4+ Tregs (associated with a high rate of cell death). The capacity of CTLA4-KDEL- and IDO-expressing DCs to induce tolerance to allografts was investigated using a murine corneal allograft model, and administration of CTLA4-KDEL-expressing DCs prevented allograft rejection. The study demonstrates the clinical potential of CTLA4-KDEL-expressing DCs to induce Treg-mediated, donor-specific transplantation tolerance.EThOS - Electronic Theses Online ServiceRoche Organ Transplantation Research Foundation (ROTRF)GBUnited Kingdo