Generation of tolerogenic human DC through Rapamycin conditioning and genetic modification with HLA-G.

Abstract

Dendritic cells (DC) are potent antigen presenting cells involved in the initiation of the alloimmune response and organ transplant rejection. This thesis, has investigated pharmacological and genetic approaches to manipulate DC in order to generate tolerogenic DC which elicit poor allostimulatory activity as potential cell therapy agents to treat allograft rejection. In the first aspect of this study, human monocyte-derived DC were used to study the influence of Rapamycin (RAPA) on DC phenotype and function. This study showed that RAPA when added to monocytes prior to DC differentiation or after DC maturation generated tolerogenic DC as evidenced by the ability of these cells to induce T cell hyporesponsiveness. However, T cell hyporesponsiveness was associated with downregulation of costimulatory molecules only when added prior to differentiation and surprisingly was not influenced by the induction of CD4 ⁺FoxP3 ⁺ T cells. To assess the effects of RAPA on DC function in the transplant setting an in vivo chimeric model of ovine vascularised skin allograft transplantation was established in immunocompromised NOD/SCID mice as a host. This model was established as a preliminary model to acquire in vivo data prior to testing the effect of pharmacologically modified DC in the preclinical ovine model of renal allograft transplantation, also established in the host laboratory. Firstly, comparison of ovine DC obtained from cannulation of the prefemoral lymphatic vessels in sheep demonstrated that RAPA-modified ovine DC acted as poor stimulators of allogeneic ovine T cells similar to human DC treated with RAPA. Secondly, in NOD/SCID mice engrafted with ovine skin, the infusion of allogeneic ovine T cells together with RAPA-modified ovine DC reduced histological rejection in comparison to control DC. In the second aspect of this study, the effects of genetic manipulation of DC were investigated. In order to investigate the effects of genetic modification of DC, two isoforms of the human HLA-G molecule, HLA-G1 (membrane bound) and HLA-G5 (soluble isoform) were used to generate adenoviral vectors. Unexpectedly, both HLA-G isoforms expressed by human DC transfectants were unable to induce allogeneic T cell hyporesponsiveness in the mixed lymphocyte reaction (MLR). Surprisingly, in the MLR the allogeneic T cells acquired HLA-G1, but not HLA-G5, indicating that direct cell contact and membrane transfer from DC to T cells occurred (Trogocytosis). In addition to HLA-G1, costimulatory molecules (CD40, CD80, CD86 and MHC Class II) were also cotransferred from DC to allogeneic T cells. Accordingly, in secondary proliferation assays T cells immunoselected after co-culture with allogeneic untransfected DC (TUT) demonstrated potent antigen presenting activity when used as stimulators of autologous T cells (analogous to the indirect pathway of antigen presentation). In contrast to TUT, immunoselected T cells that acquired HLA-G1 (THLA-G1) upon co-culture with DCtransfectants showed poor stimulatory capacity. Thus the data reported in this thesis supports the proposed novel concept that HLA-G acquired by T cells through genetically modified DC, functions to autoregulate T cells via T-T cell interaction through the HLA-G receptor ILT2 (negative signalling receptor) expressed on T cells. In conclusion, this thesis has firstly provided supportive evidence that the pharmacological modification of human and ovine DC with RAPA has potential therapeutic effects on allograft rejection. Secondly, the genetic modification of DC to induce expression of HLA-G has specifically allowed the transfer of this molecule to T cells by trogocytosis and the inhibition of alloreactive T cell expansion.