An immunosufficient murine model for the study of human islets

Abstract

For the sake of therapy of diabetes, it is critical to understand human beta cell function in detail in health and disease. Current studies of human beta cell physiology in vivo are mostly limited to immunodeficient mouse models, which possess significant technical limitations. This study aimed to create a new model for the study of human islets through induction of transplant tolerance in immunosufficient mice. B6 diabetic mice were transplanted with human islets and treated with anti-CD45RB. To assess whether anti-CD45RB-induced transplant tolerance requires B cells, B6 recipients received additional anti-CD20 or B6μMT-/- mice were used. For some anti-CD45RB-treated B6μMT-/- mice, additional anti-CD25 mAb was applied at the early or late stage post-transplant. Immunohistology was performed to show the Foxp3 cells in grafted anti-CD45RB/anti-CD20-treated Foxp3-GFP B6 mice. The results showed that anti-CD45RB alone allowed indefinite graft survival in 26.6% of B6 mice, however 100% of xenografts were accepted in mice treated simultaneously with anti-CD20, and 88.9% of xenografts accepted in anti-CD45RB-treated μMT-/- mice. These μMT-/- mice accepted the islets from another human donor but rejected the islets from baboon. Additional administration of anti-CD25 mAb at the time of transplantation resulted in 100% rejection, whereas 40% of grafts were rejected while the antibody was administrated at days 60 post-transplant. Immunohistologic examination showed Foxp3+ cells accumulated around grafts. We conclude that induction of tolerance to human islets in an immunosufficient mouse model could be generated by targeting murine CD45RB and CD20. This new system will facilitate study of human islets and accelerate the dissection of the critical mechanisms underlying islet health in human disease.

Keywords: Treg; human islet; transplant model; transplant tolerance; xenograft.

Figure 1. Anti-CD45RB promotes long-term survival of xenogenic islet graft, which is enhanced by B cell depletion and is species-specific

(A) Human islets were transplanted under the kidney capsule of B6 mice or B6μMT−/− mice and treated with anti-CD45RB or left untreated. In immune-sufficient B6 recipients, some mice were treated also with anti-CD20 mAb (250 μg, ip, on day 0) alone or in combination with anti-CD45RB. In control mice or anti-CD20 therapy alone, islet xenografts were rejected rapidly (MST = 10.0 ± 2.5 days, MST = 11.8 ± 1.6 days, respectively). 26.6% of grafts survived over 125 days after anti-CD45RB therapy (MST > 77.0 ± 28.8 days) in B6 recipients. In B6μMT−/− recipients, anti-CD45RB treatment resulted in significant prolongation of islet xenograft survival (MST > 109.9 ± 30.3 days) in comparison with control group, p < 0.0001. 100% of grafts showed long-term survival with anti-CD45RB plus anti-CD20 antibody treatment (p < 0.0001 anti-CD45RB or combination vs. control, p< 0.01 anti-CD45RB vs. combination). (B) 120 days after the first human islet transplant into B6μMT−/− recipient tolerized with anti-CD45RB, nephrectomy was performed and hyperglycemia was recovered. Nephrectomized mice were then divided into two groups. One group (n =5) received islets from a second human donor, which were permanently accepted. In a second group (n =3) receiving baboon islets, all islets were rejected.

Figure 2. Histochemistry of grafts from transplanted recipients

Human pancreatic islets were transplanted under the kidney capsule of Foxp3-GFP B6 mice (A–I, magnificantion 100×; J–L, magnificantion 200×) in the presence or absence of treatment with anti-CD45RB (A–I) or anti-CD45RB/anti-CD20(J–L). Graft staining of un-treated recipient at day 7 post transplant (A, D, G), graft staining of antibody-treated recipients at day 14 post transplant (B, E, H) and staining of grafts from tolerant mice more than 100 days post transplant (C, F, I) are shown. In untreated mice, histological analysis of grafts at day 7 post-transplant showed moderate lymphocyte infiltration (A) which was predominately composed of T lymphocytes as indicated by CD3 staining (D). Further immunohistological analysis revealed very few Foxp3+ cells around the grafts (G). In anti-CD45RB treated mice at day 14 post-transplant, hemotoxilin & eosins staining showed minimal cellular infiltration within the grafts (B). Although CD3 staining presented moderate T lymphocyte infiltration, the majority of these T cells were revealed to be Tregs as indicated by the significant Foxp3⁺ positive staining pattern (E, H) and some were collected into larger “nests” in long-term tolerant mice (dotted line in F). CD3 staining is further highlighted in higher magnification insets represented by the boxed area in D and E. In long-term surviving grafts (more than 100 days post-transplant), lymphocytic infiltration continued to accumulate. A feature of these long surviving grafts was the presence of a “nest” of lymphocytes that was not seen in untreated grafts or treated animals at early time points (F). Immunohistochemical analysis showed that these accumulations consisted mainly of Foxp3+ cells (I). Additional analysis of Tregs by immunofluorescence revealed that control mice and anti-CD20 alone treated mice showed negligible Foxp3+ GFP cells (J and K), which was remarkably enhanced by combined anti-CD45RB/anti-CD20 treatment (L). A–C, Hematoxylin & eosin; D–F, CD3 immunohistochemistry (brown dot); G–I, Foxp3 (blue dot) and insulin (brown dot) immunohistochemistry staining; J–L, Foxp3-GFP (green dots indicated by arrow head). In all cases, representative images of all grafted mice analyzed are shown.

Figure 2. Histochemistry of grafts from transplanted recipients

Human pancreatic islets were transplanted under the kidney capsule of Foxp3-GFP B6 mice (A–I, magnificantion 100×; J–L, magnificantion 200×) in the presence or absence of treatment with anti-CD45RB (A–I) or anti-CD45RB/anti-CD20(J–L). Graft staining of un-treated recipient at day 7 post transplant (A, D, G), graft staining of antibody-treated recipients at day 14 post transplant (B, E, H) and staining of grafts from tolerant mice more than 100 days post transplant (C, F, I) are shown. In untreated mice, histological analysis of grafts at day 7 post-transplant showed moderate lymphocyte infiltration (A) which was predominately composed of T lymphocytes as indicated by CD3 staining (D). Further immunohistological analysis revealed very few Foxp3+ cells around the grafts (G). In anti-CD45RB treated mice at day 14 post-transplant, hemotoxilin & eosins staining showed minimal cellular infiltration within the grafts (B). Although CD3 staining presented moderate T lymphocyte infiltration, the majority of these T cells were revealed to be Tregs as indicated by the significant Foxp3⁺ positive staining pattern (E, H) and some were collected into larger “nests” in long-term tolerant mice (dotted line in F). CD3 staining is further highlighted in higher magnification insets represented by the boxed area in D and E. In long-term surviving grafts (more than 100 days post-transplant), lymphocytic infiltration continued to accumulate. A feature of these long surviving grafts was the presence of a “nest” of lymphocytes that was not seen in untreated grafts or treated animals at early time points (F). Immunohistochemical analysis showed that these accumulations consisted mainly of Foxp3+ cells (I). Additional analysis of Tregs by immunofluorescence revealed that control mice and anti-CD20 alone treated mice showed negligible Foxp3+ GFP cells (J and K), which was remarkably enhanced by combined anti-CD45RB/anti-CD20 treatment (L). A–C, Hematoxylin & eosin; D–F, CD3 immunohistochemistry (brown dot); G–I, Foxp3 (blue dot) and insulin (brown dot) immunohistochemistry staining; J–L, Foxp3-GFP (green dots indicated by arrow head). In all cases, representative images of all grafted mice analyzed are shown.

Figure 3. CD4 Tregs promote induction and maintenance of islet xenografts

μMT−/− mice received islet xenografts with anti-CD45RB treatment as in Figure 1 to induce long-term graft survival. Administration of anti-CD25 at day 6 and day 1 before transplantation led to rejection of all xenografts (shown in A). Administration of anti-CD25 at day 63 and 68 post-transplantation resulted in xenograft rejection in 2 of 5(40%) recipients (shown in B).