alpha1-Antitrypsin monotherapy induces immune tolerance during islet allograft transplantation in mice

Abstract

Human pancreatic islet transplantation offers diabetic patients tight glucose control but has low graft survival rates. The immunosuppressive drugs that are administered to graft recipients lack the antiinflammatory benefits of corticosteroids because of their diabetogenic effects. The serum protease inhibitor alpha1-antitrypsin (AAT) possesses antiinflammatory properties and reduces cytokine-mediated islet damage. In the present study, diabetic mice were grafted with allogeneic islets and treated with AAT monotherapy (n = 24). After 14 days of treatment, mice remained normoglycemic and islet allografts were functional for up to 120 treatment-free days. After graft removal and retransplantation, mice accepted same-strain islets but rejected third-strain islets, thus confirming that specific immune tolerance had been induced. Explanted grafts exhibited a population of T regulatory cells in transplant sites. According to RT-PCR, grafts contained high levels of mRNA for foxp3, cytotoxic T lymphocyte antigen-4, TGF-beta, IL-10, and IL-1 receptor antagonist; expression of proinflammatory mediators was low or absent. After implantation of skin allografts, AAT-treated mice had greater numbers of foxp3-positive cells in draining lymph nodes (DLNs) compared with control treatment mice. Moreover, dendritic cells in DLNs exhibited an immature phenotype with decreased CD86 activation marker. Although the number of CD3 transcripts decreased in the DLNs, AAT did not affect IL-2 activity in vitro. Thus, AAT monotherapy provides allografts with antiinflammatory conditions that favor development of antigen-specific T regulatory cells. Because AAT treatment in humans is safe, its use during human islet transplantation may be considered.

Fig. 1.

Extended AAT monotherapy induces strain-specific immune tolerance toward islet allografts in mice. Islet allograft transplantation was performed and blood glucose was followed in mice that received albumin (ALB) (n = 6) or hAAT monotherapy (n = 24) for various periods of time. (A) Islet graft survival curve. (B) Summary of uninterrupted normoglycemic intervals achieved during and after hAAT monotherapy (“First graft”) and during a second grafting procedure that was carried out in explanted animals in the absence of therapy (“Second graft”) (n = 7). Double-underlined headings indicate number of hAAT monotherapy and therapy-free days. The outcome of the second grafting procedure is indicated for individual mice. (C) Representative blood glucose follow-up. Albumin (ALB)-treated animals are represented by a dashed line. Day of hAAT treatment withdrawal is indicated. Treatment-free glucose levels were determined during the ensuing days. Graft removal by nephrectomy, resulting in hyperglycemia, is indicated. A second grafting without further hAAT treatment was performed with same strain islet allograft (Left) or third strain islet allograft (Right). Transplantation outcome of the second grafting is monitored for 50 days. (D) Histology. Representative day 72 explanted graft from hAAT-treated mice 20 days after withdrawal of hAAT treatment. H&E stain, image of entire islet graft site, is shown. Islet mass appears flanked by a dense mononuclear cell population (thick arrows).

Fig. 2.

Effect of hAAT monotherapy on gene expression profile in islet allografts. RT-PCR of explanted islet allografts from albumin (ALB)-treated control and hAAT-treated mice. The left four columns show the initial days after islet transplantation into control mice. The right column shows day 72 after islet transplantation into hAAT-treated mice (see Fig. 1). Data are representative of n = 6 (ALB) and n = 3 (hAAT; time points between days 30 and 72 after transplantation).

Fig. 3.

Cell-specific effects of hAAT. Inducible IFN-γ levels (Left) and cell proliferation (Right) assessed in Con A-primed PBMCs that were stimulated with increasing concentrations of IL-2 in the presence of 0.5 mg/ml hAAT or albumin (CT). Data are mean ± SEM of three individual donors.

Fig. 4.

Identification of hAAT-induced IL-10-expressing Treg cells in nonrejecting islet allografts. (A) RT-PCR of explanted islet allografts in albumin (ALB)-treated graft recipients during acute allorejection (left four columns; days 1–7) and hAAT-treated graft recipient 20 days after withdrawal of hAAT treatment (right column; day 72; see Fig. 2). Data are representative of n = 6 (ALB) and n = 3 (hAAT; representative time point between days 30 and 72 after transplantation). (B) Intragraft gene expression profile throughout hAAT therapy. RT-PCR of explanted islet allografts in hAAT-treated graft recipients during hAAT treatment. K, tissue from pole opposite to the grafting site; G, intragraft gene profile.

Fig. 5.

Time-dependent hAAT-induced distribution of Treg cells between DLNs and allograft. foxp3-GFP knock-in mice (H-2^b) were grafted with wild-type BALB/c tissue (H-2^d). Mice received a 10-day hAAT treatment or albumin protocol (see Fig. 1). (A) Inguinal DLN. FACS analysis of CD4⁺-sorted foxp3-GFP-positive DLN cells. (Inset) RT-PCR for foxp3 mRNA transcripts in DLNs. Shown are representative time points. (B) Matrigel-skin graft. Treg cells in Matrigel grafts on day 10 identified by fluorescent microscopy of unstained material (Left) plus DAPI-counterstained material (Right). (C) Islet graft. Day 14 Treg cells identified in the cuff site (see Fig. 1D). Anti-GFP antibody immunostaining and DAPI counterstaining is shown. Representative image of three hAAT-treated grafts is shown. Grafts from albumin-treated mice contained no cuff (data not shown).

Fig. 6.

Early local and systemic effects of hAAT. Wild-type islet-Matrigel grafts containing increasing concentrations of hAAT (indicated, amount per Matrigel) were explanted 48 h after transplantation into hAAT-Tg recipients. (Upper) Identification of CD14-positive cells (RT-PCR) and identification of host cells inside the graft (genomic). (Lower) RT-PCR depiction of insulin and VEGF intragraft transcripts.

Fig. 7.

Effect of AAT on dendritic cell migration and maturation. CD86, MHC class II, and IL-10 expression in renal DLNs. Seventy-two hours after allogeneic skin grafting under the renal capsule, DLNs were harvested and examined by RT-PCR. DLN from nongrafted mice (first bar on left) is compared with 72-h DLN gene expression from untreated (CT) and hAAT-treated (AAT) mice. Shown are mean ± SEM from three experiments. *, P < 0.05; **, P < 0.01 between CT and AAT.