Invitro alteration of rat pancreatic islet immunogenicity in an allogeneic transplant model
Creators: name: Skarsgard, Erik David
Publisher: name: University of British Columbia
Year: 2010

description: Allograft rejection remains the fundamental stumbling block to tissue transplantation. Traditional assumption has been that transplanted tissue alone provides an antigen source (alloantigen), which directly stimulates a host response resulting in graft rejection; accordingly, traditional attempts at circumventing the allograft response have focussed on techniques of recipient immunosuppression. Recently, increasing attention has been given to a subset of non-parenchymal, bone marrow derived lymphoid cells (characterized by their surface expression of class II MHC antigen) which are carried passively with the allograft into an immune competent recipient. A current hypothesis is that these cells, called antigen presenting cells (APCs), participate in the sensitization of the immunologically naive but responsive host to the transplanted tissue, leading ultimately to graft rejection. Therefore, it has been suggested that depletion of APCs from donor tissue prior to transplantation may permit allogeneic transplantation to occur, without host immunosuppression. In contrast to solid organs, pancreatic islets are well suited to this type of immunomodulation prior to transplantation, since they can be maintained in a functional ex vivo state by cell culture. The purpose of this thesis was to evaluate donor islet APC depletion by pre-transplant cell culture and APC-ablative photodynamic therapy (PDT), and to see whether either in vitro technique could prevent rejection in a rat, allogeneic transplant model. Briefly, a donor (Sprague Dawley, RTlu) -recipient (Wistar Furth, RTla) pair with a major histo-incompatible barrier was selected. After collagenase digestion of donor pancreata, islets were isolated from A the digested tissue by centrifugation through a discontinuous dextran gradient followed by hand picking using a dissecting microscope. Once isolated, the islets were either used fresh, placed in tissue culture (Ham's F-12 media, 11 mM glucose, 5% C02/room air at 37 C) for variable periods, or subjected to APC-ablative PDT. Islet APC depletion was assessed by fluorescent immunocytochemistry. Fresh, cultured and PDT treated islets were frozen in liquid N2 then cryostat sectioned and stained for class II MHC + cells (APCs), using an anti-class II mouse monoclonal antibody (OX-6), followed by a fluorescent (fluorescein indothiocyanate) labelled anti-mouse monoclonal. Using this technique, APCs could be identified by fluorescent microscopy on the basis of their enhanced surface staining. While fresh islets demonstrated between 1 and 5 APCs per cryostat section, a culture period of at least 10 days resulted in complete islet APC elimination. Islet allograft studies with fresh and cultured islets were then performed to determine: 1) if pre-transplant islet culture could sufficiently reduce donor tissue immunogenicity to allow successful allografting in immune-competent recipients, and if so, 2) what duration of culture was necessary to permit consistently successful allografting. Allografts of fresh and cultured (4, 7, 10, 14, and 21 day) islets were placed under the renal capsule of immune-competent, recipient rats and after 12 days the grafts were removed and studied histologically for evidence of rejection. While all grafts which were cultured for 10 days or less prior to transplantation were rejected, 4/10-14 day cultured islets, and 4/5-21 day cultured islets demonstrated engraftment. In vivo function of 21 day cultured islet allografts was demonstrated by transplantation of islets via the portal vein, into recipients which had been rendered hyperglycemic by IV streptozotocin. This resulted in an immediate and sustained reversion to euglycemia (as assessed by daily plasma glucose determinations using a glucose analyser) over a 30 day period of study. In contrast, streptozotocin "diabetic" recipients of fresh and 14 day cultured islet allografts demonstrated a brief (7-10 day) period of graft function (euglycemia) prior to a return of hyperglycemia, consistent with graft rejection. Photodynamic therapy (PDT) achieves selective cell ablation by the stimulated emission of singlet oxygen from a light-activated compound (benzoporphyrin) which has been delivered to the cell target. In these experiments, APC elimination was attempted by in vitro islet treatment with OX-6, followed by a specific, secondary antibody (RAMIg) to which BPD had been conjugated. After UV lightactivation the treated islets were frozen, cryostat sectioned and immunostained for Class II MHC + cells. In contrast to control islets which underwent a secondary incubation with either BPD alone or BPD conjugated to an irrelevant secondary antibody, islets which underwent PDT using the specific RAMIg-BPD conjugate demonstrated elimination of APCs as assessed by immunocytochemistry. When syngeneic and allogeneic transplants were performed using islets which had undergone APC "photoablation", the histologic appearance of the grafts was compatible with either inflammation in response to non-viable tissue, or allograft rejection. The temporal disparity between the duration of tissue culture necessary to deplete islet APCs and that required to allow successful islet allografting can be variably explained. One possibility is that failure to stain APCs after a 7-10 day period of culture is not proof that these cells have been destroyed. It is conceivable that culture alters the surface of the APC such that it is no longer identified by anti-Class II MHC immunostains, but nevertheless retains its ability to present alloantigen. Alternatively, one can hypothesize that in vitro culture causes some donor tissue alteration other than APC depletion which renders it less immunogenic. The failure of PDT to permit successful syngeneic or allogeneic transplantation despite its apparent ability to eliminate islet APCs suggests that the treatment itself may cause irreversible islet injury, and that the inflammatory reaction observed is merely in response to non-viable transplanted tissue.