Characterisation of the Xenogeneic Immune Response to Microencapsulated Fetal Pig Islet-Like Cell Clusters Transplanted into Immunocompetent C57BL/6 Mice

Abstract

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, poor graft survival is still an issue. This study aimed to characterise the nature of the host immune cells present on the engrafted microcapsules and effects on encapsulated FP ICCs that were transplanted into immunocompetent mice. Encapsulated FP ICCs were transplanted into the peritoneal cavity of C57BL/6 mice. Grafts retrieved at days 1, 3, 7, 14 and 21 post-transplantation were analysed for pericapsular fibrotic overgrowth (PFO), cell viability, intragraft porcine gene expression, macrophages, myofibroblasts and intraperitoneal murine cytokines. Graft function was assessed ex vivo by insulin secretion studies. Xenogeneic immune response to encapsulated FP ICCs was associated with enhanced intragraft mRNA expression of porcine antigens MIP-1α, IL-8, HMGB1 and HSP90 seen within the first two weeks post-transplantation. This was associated with the recruitment of host macrophages, infiltration of myofibroblasts and collagen deposition leading to PFO which was evident from day 7 post-transplantation. This was accompanied by a decrease in cell viability and loss of FP ICC architecture. The only pro-inflammatory cytokine detected in the murine peritoneal flushing was TNF-α with levels peaking at day 7 post transplantation. This correlated with the onset of PFO at day 7 implying activated macrophages as its source. The anti-inflammatory cytokines detected were IL-5 and IL-4 with levels peaking at days 1 and 7, respectively. Porcine C-peptide was undetectable at all time points post-transplantation. PFO was absent and murine intraperitoneal cytokines were undetectable when empty microcapsules were transplanted. In conclusion, this study demonstrated that the macrophages are direct effectors of the xenogeneic immune response to encapsulated FP ICCs leading to PFO mediated by a combination of both pro- and anti-inflammatory cytokines.

Immunological Challenges Facing Translation of Alginate Encapsulated Porcine Islet Xenotransplantation to Human Clinical Trials

“…Previous studies had established that an immune response is not elicited ensuing transplantation of empty barium alginate capsules [ 66 , 67 ]. These fi ndings further validate the hypothesis that antigenic DAMP shedding may play a role in mediating graft failure after encapsulated FP ICC transplantation given that the alginate used was pure, capsules were generated as per labspecifi c protocol, and the islets depicted in microscopy and histologic cross-sections were adequately enclosed [ 42 ]. Furthermore, Evaluation of alginate microcapsule pore size by Vaithilingam et al revealed that molecules smaller than 250 kDa were still able to diffuse through the alginate-cation cross-linked membrane [ 66 ].…”

“…Within the graft, an increase in the mRNA expression of porcine antigenic transcripts such as MIP-1 α, IL-8, HMGB1 and HSP90 substantiated claims that release of DAMPs originating from islets small enough to traverse the microcapsule membrane is a likely phenomenon in the post-transplantation setting [ 42 ]. The prevalence of macrophages comprising of the capsular overgrowth, the upregulation of chemotactic molecules specifi c for the recruitment of monocytes such as MIP-1 α, and the detection of peaks in the pro-infl ammatory cytokine TNF-α corresponding with peaks in mRNA expression of HSP90 [ 42 ] all suggest a delayed hypersensitivity response composed of macrophages and NK-cells targeting FP ICCs [ 68 , 69 ]. The ability of HSP proteins to activate and prompt infl ammatory cytokine secretion upon contact with macrophages has been described in the literature [ 70 , 71 ].…”

“…Furthermore, Evaluation of alginate microcapsule pore size by Vaithilingam et al revealed that molecules smaller than 250 kDa were still able to diffuse through the alginate-cation cross-linked membrane [ 66 ]. Within the graft, an increase in the mRNA expression of porcine antigenic transcripts such as MIP-1 α, IL-8, HMGB1 and HSP90 substantiated claims that release of DAMPs originating from islets small enough to traverse the microcapsule membrane is a likely phenomenon in the post-transplantation setting [ 42 ]. The prevalence of macrophages comprising of the capsular overgrowth, the upregulation of chemotactic molecules specifi c for the recruitment of monocytes such as MIP-1 α, and the detection of peaks in the pro-infl ammatory cytokine TNF-α corresponding with peaks in mRNA expression of HSP90 [ 42 ] all suggest a delayed hypersensitivity response composed of macrophages and NK-cells targeting FP ICCs [ 68 , 69 ].…”

“…These fi ndings suggest capsule permeable porcine antigens must act as a key player in compromising the immune-compatibility of the graft. Furthermore, an increase in intragraft expression of porcine islet DAMPs [ 42 ], similarly capable of extrusion into the extra-capsular environment to potentially interact with host sentinel cells, forms the basis for a possible link between PFO, islet necrosis, and secretion of antigenic DAMPs [ 3 ].…”

“…FP ICCs do not differentiate into fully functional insulin secreting β-cells until approximately 3 weeks post transplantation, as detectable by fl uctuations in porcine C-peptide levels in the host serum-a principal disadvantage of FP ICCs [ 42 ]. Levels of porcine C-peptide are directly indicative of the levels of insulin originating from porcine β-cells.…”