Studies of immune biology of the common marmoset: a novel non-human primate transplant model.

Abstract

Donor-specific immune tolerance is a highly desirable goal in clinical transplantation. Dendritic cells (DC) are potent immune system regulators, and promoting both anti-donor immunity and immune tolerance. DC are therefore an important target for potential tolerance-inducing therapies, which must be validated in non-human primate models before clinical trials. The common marmoset is a small, New World primate which our group is developing as a novel transplant model. The scope of this thesis involves the development of methodology and characterisation of critical aspects of marmoset immune biology pertinent to transplantation and DC immunotherapy. Chapter 1 discusses the context of this thesis and contains a comprehensive literature review. Chapter 2 outlines methodology and materials utilised in this thesis. Chapter 3 describes a new technique for genotyping marmoset major histocompatibility complex (MHC) Class II DRB genes, to facilitate choosing mismatched donor and recipient animals for transplant studies. Genotype-based matching was predictive of in-vitro immune reactivity, and therefore validated as a method for selecting immunologically disparate animal pairs. Two new alleles were also identified. This work has given rise to two publications, and the methodology subsequently extended to marmoset MHC Class I and other Class II genes by others in our group. Chapter 4 describes the first-ever studies of propagation of marmoset DC in-vitro from peripheral blood DC precursors (monocytes and stem cells) mobilised by the growth factor G-CSF. These methods enabled large-scale DC production from small volumes of peripheral blood. Marmoset DC were characterised extensively by morphology, phenotype and function, with many similarities to human and NHP DC. As with all animal models, specific differences were also identified. In particular, marmoset monocyte-derived DC were maturation-resistant, whereas stem-cell derived DC were semi-mature. This work establishes that marmoset DC exist within the paradigm of human and NHP DC systems, and is therefore a feasible model for DC-based tolerance studies. Chapter 5 describes for the first time the in-vivo propagation of marmoset DC following treatment with the growth factor FLT3-Ligand. A three-colour flow cytometry strategy for identifying and sorting marmoset putative peripheral blood myeloid DC was validated. The rare myeloid DC population was expanded massively by FLT3-Ligand, and could be isolated in numbers sufficient for therapeutic use. These DC had typical myeloid DC morphology and were capable of immune stimulation in-vitro. In addition, new markers for plasmacytoid DC were evaluated. This work forms the basis for ongoing studies of in-vivo marmoset DC. Chapter 6 describes the culmination of the studies outlined in earlier chapters, with the first-ever studies of DC immunotherapy in marmoset monkeys. Three donor and recipient pairs were chosen on the basis of MHC genotype mismatch. Donor animals were treated with G-CSF and immature monocyte-derived DC propagated in-vitro. Recipient animals were treated with intravenous infusion of unmodified immature donor DC, and immune responses monitored. Two animals exhibited reduction in anti-donor (and third party) immune responses, whereas one animal was initially sensitized to donor cells. These preliminary studies establish the feasibility of DC-based immunotherapy in this model, and demonstrate that DC-induced immune modification can occur and be successfully monitored. Thus, the work presented in this thesis creates a platform from which future studies of DC-based immune tolerance strategies can be developed in this novel transplant model.