Transgenic anti-CD4 monoclonal antibody secretion by mouse segmental pancreas allografts promotes long term survival
Introduction
Although Type 1 diabetes can be successfully treated with insulin therapy, only pancreas or islet transplantation can permanently cure patients and prevent the end-stage organ failure often associated with long term diabetes. The results for pancreas transplantation have improved dramatically in the last 10 years, with the 1 year survival rate for pancreas plus kidney transplants now equivalent to that of kidney transplants alone [1]. However the results for pancreas only transplants, which could prevent renal failure and vascular disease, still lag behind, with 15% graft loss due to rejection occurring at 1 year compared with 2% for simultaneous kidney plus pancreas transplants [1]. Current research to improve pancreas grafts centres on the supply of islet tissue [2] and immunosuppression that is less toxic to islet tissue [3]. The problems of developing immunosuppression to combat both T cell-mediated autoimmune responses and allograft rejection apply to whole and segmental pancreas transplantation and to islet cell replacement [4]. CD4 T cells have been shown to play a central role in the rejection of both pancreas allografts and xenografts placed under the kidney capsule in mice [5], [6]. Clinical results suggest that anti-CD4 antibody may be more effective and less toxic than anti-CD3 as an immunosuppressive agent for pancreas transplantation [1]. Our previous studies in STZ-induced diabetic mice have shown prolonged survival of vascularised pancreas allografts in mice treated with either anti-lymphocyte serum or anti-CD4 antibody [7]. Thus, we have used a segmental pancreas allograft model in the mouse to compare the effects of transgenic delivery of anti-CD4 antibody with systemic injection of monoclonal antibody at the time of transplant only. This transplant model provides immediate results in that recipient blood glucose levels return to normal within hours when transplants are successful [7]. The recently established line of transgenic mice expressing a rat anti-CD4 antibody (GK2c) have sustained secretion of anti-CD4 antibody from the pancreatic islets and acinar tissue [8]. Transgenic delivery of anti-CD4 antibody provides a secreted immunosuppressive product within the graft, a delivery system that has proved effective in other studies for CTLA4Ig [9], [10] and a method that may be applicable in future to genetically modified islet xenotransplantation.
Section snippets
Objectives
Treatment with the immunosuppressive antibody GK1.5 specific for CD4 T cells is known to be effective in prolonging islet and pancreas allograft survival in the mouse [5], [7]. Since low dose, locally delivered immunosuppression has been reported to be more effective than systemically delivered treatment [11], our aim was to investigate the immunosuppressive effects of transgenic anti-CD4 secreted in situ by the pancreas allograft and to compare this with anti-CD4 delivered by i.p. injection.
Mice
CBA (H-2k) were used as pancreas recipients. BALB/c (H-2d), bm1 (H-2b/bm1) C57Bl/6 (H-2b) and GK5 (H-2b/bm1) mice were used as donors. Mice transgenic for a rat anti-mouse CD4 antibody have been described previously [8]. The entire κ light chain of GK1.5 was used. The V-region and first half of the CH1 region was fused to the latter half of the CH1, hinge, CH2 and CH3 regions of mouse IgG2c. These donor mice had no peripheral CD4 T cells [8]. Recipient mice were treated with a single i.v.
Statistics
Mean survival times (MST) were compared between different groups of pancreas grafts by multiple analysis of variance without replication (ANOVA, StatView 4.5, Abacus Concepts, Inc., Berkeley, CA, USA). If the ANOVA was significant, individual comparisons were made by the Student's t-test and the level of significance was corrected by the Bonferroni method [16].
Long-term pancreas allograft survival in anti-CD4 antibody-treated diabetic mice
Pancreas survival data are summarized in Table 1. BALB/c pancreas grafts to untreated CBA mice survived for a mean of 27 days. Survival of bm1 allografts in untreated CBA mice was similar to that of BALB/c allografts (MST 30 days compared with 27 days, P=0.8). This was to be expected since both allografts were fully MHC mismatched. Wild-type C57BL/6 (H-2b) to CBA grafts showed inconsistent improvement in graft survival (MST=44±35 days, P>0.05 compared with untreated controls) while BALB/c
Discussion
Although T cell-mediated acute rejection has been studied in detail in allografts, where the host immune system was modified by anti-CD4 antibody treatment of the recipients to allow graft survival [17], [18], [19], transgenic technology offers the exciting possibility of modifying the donor tissue to provide its own immunosuppression. The findings presented here show that transgenic expression of a chimeric anti-CD4 antibody (GK2c) within a transplanted organ can protect the allograft from
Acknowledgements
PLM, W-RH and LM-S were supported by the National Heart Foundation of Australia and the National Health and Medical Research Council of Australia (NHMRC). YZ was funded by the JDFI-NIH Angelo and Susan Alberti program grant and grants from Diabetes Australia. AML and JLB were funded by NHMRC.
We thank Steven Mihajlovic and Ellen Tsui for assistance with histology and Fleur Rodda and Michelle Latimer for assistance with mouse maintenance. We also thank Ms Julie Maguire (Monash Medical School,
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