Transplantation Tolerance through the Modulation of Dendritic Cells

Allogeneic T cell stimulation requires antigen recognition through the T cell receptor and costimulatory signals provided by antigen presenting cells (APCs). For naïve T cells stimulation, interaction between CD28 on T cells and CD80/86 on dendritic cells (DCs) provides an essential costimulatory signal. While, activated effector T cells or memory T cells stimulation is more dependent on costimulation via ICOS on T cells and ICOSL on APCs. Engagement of the T cell receptor without costimulation can lead to anergy and the subsequent induction of regulatory T cells (Tregs). With the aim of generating tolerogenic DCs for the induction of transplantation tolerance specifically to the indirect allorecognition pathway, a negative-vaccine approach composed of two strategies was developed. First: an intra-cellular approach that prevents T cells costimulation composed of a fusion protein – ICOS-KDEL was constructed. ICOS-KDEL is a fusion gene between the human extracellular domain of ICOS and the endoplasmic reticulum (ER) retention signal, KDEL. Mammalian cells transfected with the construct showed knocking down of surface ICOSL expression due to ER retention. Second: an approach to provide the antigen to be recognized by responding T cells in an indirect allorecognition pathway. This was achieved by constructing a fusion protein - invariant chain fusion gene (Ii-A2) - between the immunodominant MHC derived peptide HLA-A2103-120 and the invariant chain of MHC class II. Mammalian cells transfected with the Ii-A2 fusion gene expressed A2 peptide on their surface. The two fusion genes were cloned into one bicistronic vector so that transfection of a single plasmid resulted in the expression of both genes. Functional analysis of the developed bicistronic vector was not possible due to the unavailability of the antigen-specific T cell clones. The last part of this project studied the kinetics of ICOSL expression by human DCs and the role of activated T cells to regulate ICOSL surface expression. The study showed that ICOSL down regulation is a negative feedback loop that regulates the immune response. Activated T cells up-regulate ICOS receptor that binds to ICOSL on APC. This was followed by down-regulation of ICOSL expression causing modulation of the immune response. The kinetic changes of ICOSL expression by DC were related to the maturation state of the DCs. It was shown that ICOS induced ICOSL down regulation could be a novel regulatory mechanism in human DC. The work presented in chapter 7 of this thesis will be presented as an oral presentation and a poster format in the Euroscicon meeting: Understanding dendritic cells and their ability to regulate immune responses, November 201