Pancreatic islet xenograft survival in mice is extended by a combination of alpha-1-antitrypsin and single-dose anti-CD4/CD8 therapy

Abstract

Clinical pancreatic islet transplantation is under evaluation for the treatment of autoimmune diabetes, yet several limitations preclude widespread use. For example, there is a critical shortage of human pancreas donors. Xenotransplantation may solve this problem, yet it evokes a rigorous immune response which can lead to graft rejection. Alpha-1-antitrypsin (AAT), a clinically available and safe circulating anti-inflammatory and tissue protective glycoprotein, facilitates islet alloimmune-tolerance and protects from inflammation in several models. Here, we examine whether human AAT (hAAT), alone or in combination with clinically relevant approaches, achieves long-term islet xenograft survival. Rat-to-mouse islet transplantation was examined in the following groups: untreated (n = 6), hAAT (n = 6, 60-240 mg/kg every 3 days from day -10), low-dose co-stimulation blockade (anti-CD154/LFA-1) and single-dose anti-CD4/CD8 (n = 5-7), either as mono- or combination therapies. Islet grafting was accompanied by blood glucose follow-up. In addition, skin xenografting was performed in order to depict responses that occur in draining lymph nodes. According to our results hAAT monotherapy and hAAT/anti-CD154/LFA-1 combined therapy, did not delay rejection day (11-24 days untreated vs. 10-22 day treated). However, host and donor intragraft inflammatory gene expression was diminished by hAAT therapy in both setups. Single dose T-cell depletion using anti-CD4/CD8 depleting antibodies, which provided 14-15 days of reduced circulating T-cells, significantly delayed rejection day (28-52 days) but did not achieve graft acceptance. In contrast, in combination with hAAT, the group displayed significantly extended rejection days and a high rate of graft acceptance (59, 61, >90, >90, >90). In examination of graft explants, marginal mononuclear-cell infiltration containing regulatory T-cells predominated surviving xenografts. We suggest that temporal T-cell depletion, as in the clinically practiced anti-thymocyte-globulin therapy, combined with hAAT, may promote islet xenograft acceptance. Further studies are required to elucidate the mechanism behind the observed synergy, as well as the applicability of the approach for pig-to-human islet xenotransplantation.

Figure 1. Human AAT monotherapy during pancreatic islet xenotransplantation.

Rat pancreatic islets were grafted into the renal subcapsular space of hyperglycemic mice. Recipients were treated with saline (CT) or human AAT throughout the experiment. (A) Islet graft survival curve (n = 6/group). (B) Graft histology. Representative day-seven explanted grafts from CT and hATT-monotreated mice (n = 3/group). Black arrows, remains of rat pancreatic islets. (C) Mouse gene expression at graft site. Grafts were explanted at indicated times after transplantation. Mean ± SEM from n = 3 grafts/group; *p<0.05, **p<0.01, ***p<0.001. (D) Rat gene expression at graft site. Grafts were explanted at indicated times after transplantation. Mean ± SEM from n = 3 grafts/group; **p<0.01.

Figure 2. DLN response to human AAT monotherapy after skin xenografting.

Mice were either SHAM operated (CT) or recipients of rat skin (Tx) in the absence or presence of human AAT monotherapy. (A) 14-day DLN. FACS analysis. Results expressed as fold change from CT, mean ± SEM from n = 10/group; **p<0.01, ***p<0.001. (B) 72-h DLN. RT-PCR. Results expressed as fold change from CT, mean ± SEM from n = 3/group; **p<0.01, ***p<0.001.

Figure 3. AAT treatment combined with debulking therapy; graft survival.

(A–C) Rat islets were grafted into mice that were treated with anti-CD4/CD8 depleting antibodies, in the absence of AAT therapy (n = 7) or with added AAT therapy (n = 5). (A) CD45⁺CD3⁺ cells from peripheral blood, as monitored by FACS analysis. Results presented as the percent out of initial amount prior to injection. Representative follow-up out of 10 mice. (B) Islet xenograft survival curve. ***p<0.001 between DB and BD/AAT. (C) Glucose follow-up. Representative mouse. Milestones indicated: hAAT treatment stopped, therapy withdrawn followed by glucose follow-up; nephrectomy, graft explantation followed by glucose follow-up; second xenograft, rat islets grafted into the right renal subcapsular space followed by glucose follow-up.

Figure 4. AAT treatment combined with debulking therapy; histology and gene expression.

Rat islets were grafted into mice that were treated with anti-CD4/CD8 depleting antibodies, in the absence of AAT therapy (n = 7) or with added AAT therapy (n = 5), as in Figure 3B. (A) Graft site histology. K, kidney tissue; G, graft site. From left to right, representative syngeneic mouse islet graft (day 35), xenograft (debulking therapy alone, day 25), black arrows indicate immune cell mononuclear infiltration, xenograft (debulking therapy combined with AAT, day 11 after rejection) and xenograft (debulking therapy combined with AAT, day 90). (B) Treg cell content in xenograft sites. Immunofluorescent staining. DB, debulking therapy alone (rejected graft); DB/AAT, combined debulking and AAT therapy (rejected and accepted grafts). Green, foxp3; blue, DAPI nuclear counterstaining. Representative images. (C) Mouse (recipient) gene expression profiles. RT-PCR. CT vs. AAT monotherapy, see Figure 1C, shown over gray background, next to day 90 explants from mice treated by the combination of debulking therapy and AAT (DB/AAT). Results expressed as fold change from CT, mean ± SEM from n = 3/group; *p<0.05, **p<0.01, ***p<0.001. (D) Rat (donor) insulin expression profile. RT-PCR. CT vs. AAT monotherapy, see Figure 1D, are shown over gray background, next to day 90 explants from mice treated by the combination of debulking therapy and AAT (DB/AAT). Results expressed as fold change from CT, mean ± SEM from n = 3/group; **p<0.01.