Developing a non-human primate model of dendritic cell based immunotherapy in transplantation: studies in the common marmoset monkey (Callithrix jacchus).

Abstract

Kidney transplantation represents the best treatment for end-stage kidney disease, and in comparison to dialysis treatment has been shown to improve survival, quality of life, and reduce health-care costs over time. However, in order to prevent transplant failure from allograft rejection, immunosuppressive drug therapy is required. Immunosuppression is associated with significant systemic toxicities, and continues to impair optimal patient and graft outcomes. The avoidance or minimisation of immunosuppression via the promotion of tolerance of the allograft, or the use of targeted therapeutic strategies, in clinical transplantation is therefore an important goal that could have many benefits for patients. Dendritic cells (DC) are potent antigen-presenting cells that play a pivotal role in the initiation and maintenance of immune responses, and therapies utilising or targeting DC offer the potential to manipulate immune responses towards tolerance. This thesis seeks to develop the potential of DC based immunotherapies in a small and clinically relevant non-human primate (NHP) transplant model, the common marmoset monkey, and thereby facilitate translation of these therapies towards human clinical trials. Chapter 1 establishes the context for this thesis by outlining the background and providing a comprehensive review of relevant literature. Chapter 2 describes the materials and methods utilised in this thesis. Additional details of methods are contained in relevant chapters. Chapter 3 presents a comprehensive study of renal pathology in a colony of laboratory marmosets, including histology, immunofluorescence and electron microscopy, and correlates this for the first time with serum and urine biochemistry. This work demonstrates that the spontaneously observed glomerular pathology in marmosets represents a benign occurrence that would not impact on the assessment of renal function or histology in a marmoset kidney transplant model. Chapter 4 examines the trafficking behaviour in vivo of intravenously and subcutaneously administered allogeneic marmoset DC propagated in vitro from genetically disparate marmoset donors. The findings indicate that allogeneic marmoset DC do not necessarily exhibit normal trafficking behaviour in vivo, as they are not found in secondary lymphoid tissues at 48 hours, in contrast to similarly administered autologous DC. This finding lends weight to other recent studies of donor DC cellular therapy that indicate that the tolerogenic effects of this therapy are not mediated through cell to cell interactions with recipient T-cells, but rather through providing a source of donor antigen for acquisition and processing by recipient DC. Chapter 5 describes studies to develop a monoclonal antibody to marmoset DC-specific ICAM 3-grabbing non-integrin (DC-SIGN), which is a DC-specific marker. Ultimately, a marmoset cross-reactive commercially available anti-human DC-SIGN antibody (DCN46) was identified, and found to be suitable to utilise in the development of DC-SIGN targeted cell-specific therapy. Using this antibody, marmoset DC-SIGN positive cells were identified in the Lineage⁻ CD11c⁺ Class II⁺ fraction of marmoset spleen; in contrast in vitro propagated marmoset monocyte-derived DC have been confirmed to lack DC-SIGN expression. Chapter 6 describes the successful development of a novel nanocarrier targeted to DC: PLGA nanoparticles that target DC using the human and marmoset DC-SIGN cross-reactive antibody identified in Chapter 5. A series of preliminary studies have demonstrated that DC-SIGN targeted PLGA nanoparticles are taken up by Class II⁺ CD11c⁺ marmoset spleen cells, and that loading of the nanoparticles with the immunomodulatory drug curcumin shows evidence of in vitro immunosuppressive capacity, as shown in mixed leucocyte reaction; however the specificity for DC of immunosuppressive targeted PLGA nanoparticles remains to be demonstrated. Chapter 7 summarises the overall findings from this thesis, and proposes a series of necessary studies to exploit the identified potentials from this work further. Overall, the work in this thesis significantly advances the marmoset NHP model as a means to translate the potential of DC based immunotherapies towards clinical transplantation. The feasibility of DC-targeted therapy using nanoparticles has been established, and represents an opportunity to specifically target DC with immunosuppressive drugs in vivo, and thereby manipulate the immune response towards tolerance, while reducing the burden of non-targeted immunosuppression.