Xenotransplantation of microencapsulated fetal pig islet-like cell clusters (FP ICCs) offers a potential cellular therapy for type 1 diabetes. Although microcapsules prevent direct contact of the host immune system with the xenografted tissue, xenograft rejection is still an issue (1). This study aimed to characterise the nature of host immune response when encapsulated FP ICCs were transplanted into an immunocompetent recipient.
Approximately 8000 FP ICCs encapsulated within barium alginate microcapsules were transplanted into the peritoneal cavity of C57BL/6 mice (n=30). Microcapsules were retrieved and the peritoneal fluid collected from recipient mice at days 1, 3, 7, 14 and 21 post-transplantation (n=6 at each time point). The grafts were analysed for pericapsular fibrotic overgrowth (PFO; using a scoring system) and cell viability (live/dead staining). Intra-graft expression of porcine antigens was measured by real-time PCR. Types of murine immune cells and cytokines involved in the immune response were analysed by immunohistochemistry and multiplex bead-based assays respectively. Graft function was assessed ex vivo by insulin secretion studies and presence of porcine C peptide in mouse sera.
PFO was evident from day 7 post-transplantation, with macrophages and fibroblasts being the dominant cells with collagen deposition. This was accompanied by a decrease in cell viability and loss of FP ICC architecture by day 7 post-transplantation. Gene expression analysis on retrieved grafts revealed an increase in the porcine proinflammatory chemokines Macrophage Inflammatory Protein-1 alpha (MIP-1α) and Interleukin-8 (IL-8), as well as the cytokine High Mobility Group Box-1 (HMGB1) and the Heat Shock Protein-90 (HSP90) expression within the first two weeks post-transplantation. The pore size of these alginate microcapsules being ~250 kDa (2) is vulnerable to small molecules such as chemokines/cytokines. Thus, the porcine chemokines/cytokines MIP-1α (~14 kDa), IL-8 (~9 kDa) and HMGB1 (~32 kDa) being less than 250 kDa can easily diffuse through the microcapsule pores and trigger an immune response. A predominant Th2-type immune response was observed, with measurable levels of murine IL-5 and IL 4 peaking at days 1 and 7 respectively. The only murine Th1 type cytokine detected was TNF-α with levels peaking at day 7 post transplantation. Porcine C-peptide was undetectable at all time points with loss of insulin-positive cells evident as early as day 1 post-transplantation. There was no PFO and murine Th1/Th2-type cytokines were undetectable when empty microcapsules were transplanted.
In conclusion, this study demonstrated that the xenogeneic immune response to encapsulated FP ICCs is a result of a chronic inflammatory response probably to the porcine antigens MIP-1α, IL-8, HMGB1 and HSP90 leaking through microcapsule pores thereby mounting a Th2 response leading to PFO.