Ex vivo gene transfer of viral interleukin-10 to BB rat islets: no protection after transplantation to diabetic BB rats

Abstract

Allogeneic and autoimmune islet destruction limits the success of islet transplantation in autoimmune diabetic patients. This study was designed to investigate whether ex vivo gene transfer of viral interleukin-10 (vIL-10) protects BioBreeding (BB) rat islets from autoimmune destruction after transplantation into diabetic BB recipients. Islets were transduced with adenoviral constructs (Ad) expressing the enhanced green fluorescent protein (eGFP), alpha-1 antitrypsin (AAT) or vIL-10. Transduction efficiency was demonstrated by eGFP-positive cells and vIL-10 production. Islet function was determined in vitro by measuring insulin content and insulin secretion and in vivo by grafting AdvIL-10-transduced islets into syngeneic streptozotocin (SZ)-diabetic, congenic Lewis (LEW.1 W) rats. Finally, gene-modified BB rat islets were grafted into autoimmune diabetic BB rats. Ad-transduction efficiency of islets increased with virus titre and did not interfere with insulin content and insulin secretion. Ad-transduction did not induce Fas on islet cells. AdvIL-10-transduced LEW.1 W rat islets survived permanently in SZ-diabetic LEW.1 W rats. In diabetic BB rats AdvIL-10-transduced BB rat islets were rapidly destroyed. Prolongation of islet culture prior to transplantation improved the survival of gene-modified islets in BB rats. Several genes including those coding for chemokines and other peptides associated with inflammation were down-regulated in islets after prolonged culture, possibly contributing to improved islet graft function in vivo. Islets transduced ex vivo with vIL-10 are principally able to cure SZ-diabetic rats. Autoimmune islet destruction in diabetic BB rats is not prevented by ex vivo vIL-10 gene transfer to grafted islets. Graft survival in autoimmune diabetic rats may be enhanced by improvements in culture conditions prior to transplantation.

1

Representative flow cytometric analysis of the enhanced green fluorescent protein (eGFP) in islet cells from BB/OK rat islets. Islet cells were prepared 24 hrs after transduction of BB/OK rat islets with different titres of an adenoviral construct expressing the eGFP. The results are shown as one parameter histogram of the green fluorescence intensity. (A) Control; (B) MOI 10; (C) MOI 100 and (D) MOI 1000.

2

Cumulative release of vIL-10 from transduced BB/OK rat islets, 24 hrs after gene transfer with different MOI of AdvIL-10. n = 10; *P < 0.05, ***P < 0.001 versus control (non-transduced islets); ^###P < 0.001 versus MOI 100.

3

Expression of Fas (CD95) on the surface of BB/OK rat islet cells of non-transduced control islets (n= 9), AdAAT- transduced islets (n= 4) or AdvIL-10-transduced islets (n= 4). Fas expression was measured by flow cytometry 24 hrs after gene transfer. ^##P < 0.01, #P < 0.05 versus AdAAT-transduced islets.

4

Blood glucose levels in SZ-diabetic LEW.1 W rats (A and B) or autoimmune diabetic BB/OK rats (C and D) transplanted with 2000 syngeneic non-transduced control or AdAAT- or AdvIL-10-transduced islets. Transplantation of islets was performed 24 hrs after gene transfer. In C and D individual rats redeveloping hyperglycaemia after transplantation are separately indicated with their first hyperglycaemic value (controls —•; AdvIL-10, MOI 10 —δ). (A) LEW.1 W rats receiving non-transduced LEW.1 W control islets (n= 6). (B) LEW.1 W rats receiving AdvIL-10-transduced LEW.1 W islets (n= 10). (C) BB/OK rats receiving non-transduced BB/OK control islets (n= 8) or AdAAT-transduced BB/OK islets (MOI 100, n= 4). (D) BB/OK rats receiving AdvIL-10-transduced BB/OK islets [MOI 10 (n= 3), MOI 100, (n= 5), MOI 1000 (n= 2)]. Grey area = mean blood glucose of LEW.1 W rats with stable normoglycaemia after transplantation of LEW.1 W control rat islets + 2SD, ITx = Islet Transplantation, Nx = Nephrectomy.

5

Blood glucose levels in autoimmune diabetic BB/OK rats transplanted with 2000 syngeneic AdAAT- or AdvIL-10- transduced islets. Transplantation of islets was performed 24 hrs or 96 hrs after gene transfer. Individual rats redeveloping hyperglycaemia after transplantation are separately indicated with their first hyperglycaemic value (—•). (A) BB/OK rats receiving BB/OK islets 24 hrs (n = 4) and 96 hrs (n = 6) after AdAAT gene transfer (MOI 100). (B) BB/OK rats receiving BB/OK islets 24 hrs (n = 5) and 96 hrs (n = 10) after AdvIL-10 gene transfer (MOI 100). Grey area = mean blood glucose of LEW.1 W rats with stable normoglycaemia after transplantation of LEW.1 W control rat islets + 2SD, ITx = Islet Transplantation.

6

(A) Unsupervised two-dimensional cluster analysis. (B) Zoom in section of differentially regulated genes; red = increased expression, green = repressed expression, black = unaltered gene expression, grey = no signal detection. X-axis: different BB/OK rat islet samples; y-axis: gene names. As data input only those genes were used that were detectable in at least 80% of all experiments (representing 742 genes in total). The red rectangles numbered 1 and 2 represent two prominent subclusters of different sets of samples that appear to have similar expression profiles. Cluster 1 represents profiles derived from islet samples harvested 24 hrs after transduction and cluster 2 represents profiles derived from samples harvested 96 hrs after gene transfer.