Pig embryonic pancreatic tissue as a source for transplantation in diabetes: transient treatment with anti-LFA1, anti-CD48, and FTY720 enables long-term graft maintenance in mice with only mild ongoing immunosuppression

Abstract

Objective: Defining an optimal costimulatory blockade-based immune suppression protocol enabling engraftment and functional development of E42 pig embryonic pancreatic tissue in mice.

Research design and methods: Considering that anti-CD40L was found to be thrombotic in humans, we sought to test alternative costimulatory blockade agents already in clinical use, including CTLA4-Ig, anti-LFA1, and anti-CD48. These agents were tested in conjunction with T-cell debulking by anti-CD4 and anti-CD8 antibodies or with conventional immunosuppressive drugs. Engraftment and functional development of E42 pig pancreatic tissue was monitored by immunohistology and by measuring pig insulin blood levels.

Results: Fetal pig pancreatic tissue harvested at E42, or even as early as at E28, was fiercely rejected in C57BL/6 mice and in Lewis rats. A novel immune suppression comprising anti-LFA1, anti-CD48, and FTY720 afforded optimal growth and functional development. Cessation of treatment with anti-LFA1 and anti-CD48 at 3 months posttransplant did not lead to graft rejection, and graft maintenance could be achieved for >8 months with twice-weekly low-dose FTY720 treatment. These grafts exhibited normal morphology and were functional, as revealed by the high pig insulin blood levels in the transplanted mice and by the ability of the recipients to resist alloxan induced diabetes.

Conclusions: This novel protocol, comprising agents that simulate those approved for clinical use, offer an attractive approach for embryonic xenogeneic transplantation. Further studies in nonhuman primates are warranted.

FIG. 1.

Comparison between E42 and E28 pancreas in growth potential, function, and rejection patterns. A: The difference in size and insulin secretion of the E42 and E28 pancreas transplanted under the kidney capsule of NOD-SCID mice (black [E42] and white [E28] bars, respectively). The graft size was determined 6 weeks following transplantation. B: Histological (hematoxylin and eosin) evaluation of E42 and E28 pancreatic grafts 1 month after transplantation under the kidney capsule of NOD-SCID or C57BL mice. C: Histological (hematoxylin and eosin) evaluation of E28 pancreatic grafts 10 days after transplantation in the omentum of Nude or Lewis rats. (A high-quality representation of this figure is available in the online issue.)

FIG. 2.

Immunostaining of E28 pig pancreatic tissue implanted in the omentum of Nude and Lewis rats. Pig pancreatic epithelial cells, 15 days after transplantation in nude (A) or Lewis (B) rats, visualized using cytokeratin 20 (reddish color)-specific staining. Nuclei are stained with yellow Hoechst. Pig endocrine cells, 4 months after transplantation in Nude (C) or Lewis (D) rats, visualized using insulin (blue) and glucagon (green)-specific staining. Infiltration of the omentum in transplanted Lewis rats 15 days after transplantation is visualized using CD3 for detection T-cells (E) and F4/80 for detection of macrophages (F). (A high-quality representation of this figure is available in the online issue.)

FIG. 3.

Histological examination of E42 pig pancreatic tissue 2 weeks after transplantation under the renal capsule of C57BL with (A) and without (B) immune-suppression with costimulatory blockade (anti-LFA1, anti-CD48, and CTLA4-Ig), FTY720, and T-cell debulking. Slides were stained for pig insulin, cytokeratin (MNF116), CD3⁺ lymphocytes, and macrophages (F4/80), as indicated. (A high-quality representation of this figure is available in the online issue.)

FIG. 4.

Histological examination of E42 pig pancreatic tissue 2–10 months after transplantation under the renal capsule of C57BL mice treated with the costimulatory blockade protocol (anti-LFA1, anti-CD48, and FTY720). Slides were stained for CD3+ lymphocytes (A), macrophages (F4/80) (B), insulin (blue) and glucagon (green) (C), mouse blood vessels (CD31, red), and pig blood vessels (CD31, green) (D), and cytokeratin (broad spectrum, red) and IgM and IgG deposits (green) (E). The inset in D demonstrates positive staining for pig endothelial cells (green) in E42 pancreas. The inset in E demonstrates positive normal staining for IgM and IgG deposits in the glomeruli of the kidney. The data are representative of the experiment shown in Table 1 (n = 11). (A high-quality representation of this figure is available in the online issue.)

FIG. 5.

Porcine insulin blood levels 10 weeks after transplantation of E42 pancreas into immunosuppressed C57BL/6 mice. Recipients were treated with different combinations of costimulatory blockade agents (anti-LFA1, anti-CD48, and CTLA4-Ig) and FTY720, with or without T-cell debulking. Data are presented as means ± SE. *P ≤ 0.05.

FIG. 6.

A: Porcine insulin levels in the serum of C57BL mice transplanted with E42 pancreas and treated with costimulatory blockade agents (anti-LFA1, anti-CD48, and ±CTLA4-Ig), FTY720, with (◇) or without (○) debulking, at different time points after transplantation. Treatment with costimulatory antibodies was stopped at 3 months posttransplant, and graft maintenance was continued twice weekly only with FTY720. Insulin levels in the serum of NOD-SCID mice transplanted with E42 pancreas served as a positive control (♦). The inset demonstrates average pig insulin levels in transplanted mice over a course of 6 months. No statistical difference could be found between the tested groups. B: Porcine insulin levels in the serum of C57BL mice transplanted with E42 pancreas and treated with costimulatory blockade agents with or without debulking at different time points after FTY720 withdrawal. Data are presented as means ± SE.

FIG. 7.

Long-term engrafted (♦) as opposed to nonengrafted (◇) mice resist the alloxan challenge. At the indicated time points, the implanted mice were subjected to nephrectomy of the kidney harboring the implant, leading to prompt induction of hyperglycemia.