Xenografted islet cell clusters from INSLEA29Y transgenic pigs rescue diabetes and prevent immune rejection in humanized mice

Abstract

Islet transplantation is a potential treatment for type 1 diabetes, but the shortage of donor organs limits its routine application. As potential donor animals, we generated transgenic pigs expressing LEA29Y, a high-affinity variant of the T-cell costimulation inhibitor CTLA-4Ig, under the control of the porcine insulin gene promoter. Neonatal islet cell clusters (ICCs) from INSLEA29Y transgenic (LEA-tg) pigs and wild-type controls were transplanted into streptozotocin-induced hyperglycemic NOD-scid IL2Rγ(null) mice. Cloned LEA-tg pigs are healthy and exhibit a strong β-cell-specific transgene expression. LEA-tg ICCs displayed the same potential to normalize glucose homeostasis as wild-type ICCs after transplantation. After adoptive transfer of human peripheral blood mononuclear cells, transplanted LEA-tg ICCs were completely protected from rejection, whereas reoccurrence of hyperglycemia was observed in 80% of mice transplanted with wild-type ICCs. In the current study, we provide the first proof-of-principle report on transgenic pigs with β-cell-specific expression of LEA29Y and their successful application as donors in a xenotransplantation model. This approach may represent a major step toward the development of a novel strategy for pig-to-human islet transplantation without side effects of systemic immunosuppression.

FIG. 1.

Generation of INSLEA29Y transgenic (LEA-tg) pigs. A: The vector consisted of a 1.3-kb regulatory sequence from the porcine INS gene, the LEA29Y coding sequence, and the poly-adenylation box from the bovine GH gene. Regulatory sequences are depicted as lines, whereas exonic structures are boxed. Untranslated regions are shaded. The selection cassette provides resistance to neomycin. Binding sites for primers are indicated as arrows, and the probe for Southern blot hybridization is shown as a bold line. B: Southern blotting of seven founders was performed on XbaI-digested genomic DNA with a probe binding to the neomycin resistance cassette. C: Immunohistochemical staining for LEA29Y on tissue sections from a neonatal transgenic pig (aged 2 days, pancreas, C2), an adult founder animal (age 3 months; pancreas, liver, lung, kidney, and spleen, C4, 5–8), and from age-matched wild-type control pigs (pancreas, C1, 3). Scale bar: 100 μm. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Grafted LEA-tg ICCs display physiological β-cell function. Course of blood glucose levels after transplantation (A), IPGTT (performed 10 days after the development of normoglycemia (B), and immunohistochemistry of grafted ICCs (7–9 days after transplantation [C] and 4.0–4.5 months after transplantation [normoglycemic animals] [D]) in mice transplanted with wild-type (Tx, wt) and in mice transplanted with LEA-tg (Tx, LEA-tg) ICCs. Mice of both transplantation groups developed stable normoglycemia (A) and restored glucose tolerance (B, bottom), by porcine insulin secretion (B, top). The area under the curve (AUC) for glucose and insulin (B) during IPGTT was comparable in both transplantation groups. C: Immunohistochemical staining of serial sections from the transplantation site against insulin and IgG revealed insulin/LEA29Y expression in a minor proportion of ICCs a few days after transplantation. D: In contrast, after the development of normoglycemia, the transplanted cells have differentiated into a widespread insulin-positive stained tissue in both transplantation groups with LEA29Y transgene expression restricted to the grafted ICCs from transgenic pigs. Scale bar: 100 μm. n = 5 for each transplantation group. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

LEA29Y expression prevents reoccurrence of hyperglycemia after transfer of human PBMCs. A: Engraftment of human PBMCs (as indicated by FACS staining for human CD45⁺ cells in both spleen and bone marrow cells) did not significantly differ between mice transplanted with wild-type (Tx, wt) and in mice transplanted with LEA-tg (Tx, LEA-tg) ICCs. B: Four of five Tx, wt mice, but no Tx, LEA-tg mice, developed hyperglycemia within 28 days after human PBMC transfer. After removal of the graft-bearing kidney (uninephrectomy, Unx) of normoglycemic animals, all mice returned to severe hyperglycemia, indicating the absence of endogenous β-cell regeneration. C: Life-table analysis revealed a significantly (P = 0.016) higher proportion of hyperglycemia reoccurrence in Tx, wt as compared with Tx, LEA-tg mice. D: Furthermore, the area under the curve (AUC) of glucose and insulin during the IPGTT was unchanged before and 27 days after the transfer of human PBMCs in Tx, LEA-tg mice. n = 4–5 animals for each transplantation group. †One animal died at day 26 as a result of graft-versus-host disease.

FIG. 4.

LEA29Y expressing ICCs are almost completely preserved from mononuclear cell infiltration.Characteristic insulin (red) and CD3⁺, CD45⁺, CD4⁺, and CD8⁺ cell (brown) staining pattern of serial sections from the transplantation sites of a mouse transplanted with wild-type ICCs (Tx, wt; rejection at day 12 after PBMC transfer) vs. an animal with LEA29Y transgenic ICCs (Tx, LEA-tg, day 29 post PBMC transfer). In Tx, wt only few ICCs were detectable with vast T-cell (CD3⁺, CD4⁺, and CD8⁺) and CD45⁺ cell infiltration in the graft region. In contrast, Tx LEA-tg ICCs appeared completely preserved with T-cell and leukocyte accumulation restricted to the subcapsular area (day 29 after Tx). The localization of tissue sections shown in the insets is marked by an asterisk. Scale bar: 100 μm, insets: scale bar 20 μm. (A high-quality digital representation of this figure is available in the online issue.)