CREATION OF TOLEROGENIC ANTIGEN PRESENTING CELLS VIA INTRACELLULAR CTLA4: A NOVEL STRATEGY WITH POTENTIAL CLINICAL UTILITY IN TRANSPLANTATION.

Abstract

P900 Activation of T lymphocytes requires the recognition of peptide-MHC complexes and co-stimulatory signals provided by antigen-presenting cells (APCs). It has been shown that T cell activation without co-stimulation can lead to T cell anergy, a state of T cell hyporesponsiveness. In this study, we developed a novel strategy to inhibit expression of B7 molecules (CD80/86) by transfecting APCs (artificial APCs, B cells and dendritic cells) with a gene construct encoding a modified CTLA4 molecule (CTLA4-KDEL) that is targeted to the endoplasmic reticulum (ER). Our approach is to express within the cells CTLA4-KDEL, since we reason that the CTLA4-KDEL will bind to the newly synthesised CD80/86 molecules in the ER and prevent them from reaching the cell surface. Our in-vitro and in-vivo data using CTLA4-KDEL in various APCs show that this strategy does indeed block B7 expression by APC and that antigen-reactive (both allospecific and peptide-specific) T cells not only are hyporesponsive to antigen presented by these cells but also are rendered anergic. Following the exposure to CTLA4-KDEL APCs, the cytokine profiles skew toward Th2 profiles (with CTLA4-KDEL-transfected B cells, the Th2 profiles and a high IL-10 level were seen). In addition, we also showed that these anergic T cells generated by CTLA4-KDEL-transfected dendritic cells can regulate in allogeneic antigen-specific manner via a contact-dependent mechanism but not IL-10 or TGF-β dependent mechanism. A recent report has indicated that cross-linking of CD80/86 by CTLA4-Ig results in upregulation of the indoleamine 2, 3-dioxygenase (IDO) enzyme leading to increased tryptophan metabolism. We are interested to determine if this is the mechanism by which our CTLA4-KDEL-transfected dendritic cells are tolerogenic. Our data suggest that cell expressing CTLA4-KDEL do not upregulate the IDO enzyme, unlike the cells treated with soluble CTLA4-Ig. This gene-based strategy to inhibit expression of key surface receptors in order to generate “B7-deficient” APCs has significant advantages over the currently described approaches of using immature dendritic cells for treatment of transplant rejection.