1-Antitrypsin Monotherapy Induces Immune Tolerance During Islet Allograft Transplantation in Mice

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

Eli C. Lewis*†, Mark Mizrahi†, Michel Toledano†, Nathaniel DeFelice†, Joanne L. Wright‡, Andrew Churg‡, Leland Shapiro*, and Charles A. Dinarello*§

*Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262; †Department of Clinical Biochemistry, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel; and ‡Department of Pathology, University of British Columbia, Vancouver, BC, Canada V6T 2B5

Contributed by Charles A. Dinarello, August 7, 2008 (sent for review June 2, 2008) Human pancreatic islet transplantation offers diabetic patients 13). We reported that AAT prevented the demise of islet beta tight glucose control but has low graft survival rates. The immu- cells from normal mice, enabling insulin secretion in the pres- nosuppressive drugs that are administered to graft recipients lack ence of IL-1␤ and IFN-␥ and reducing cytokine and chemokine the antiinflammatory benefits of corticosteroids because of their secretion (13). Administered to animals, AAT reduced the diabetogenic effects. The serum protease inhibitor ␣1-antitrypsin susceptibility of islets to inflammation and prolonged islet (AAT) possesses antiinflammatory properties and reduces allograft survival (13). Importantly, as reported elsewhere (14) cytokine-mediated islet damage. In the present study, diabetic and verified in the present study, AAT treatment allows for IL-2 mice were grafted with allogeneic islets and treated with AAT activity while suppressing inflammation. After 14 days of treatment, mice remained In the present study, the impact of extended human AAT .(24 ؍ monotherapy (n normoglycemic and islet allografts were functional for up to 120 (hAAT) monotherapy on the process of allograft rejection was treatment-free days. After graft removal and retransplantation, examined in the setting of a normal immune system. Unexpect- mice accepted same-strain islets but rejected third-strain islets, edly, hAAT therapy withdrawal revealed the remarkable induc- thus confirming that specific immune tolerance had been induced. tion of treatment-induced strain-specific immune tolerance. Explanted grafts exhibited a population of T regulatory cells in transplant sites. According to RT-PCR, grafts contained high levels Results of mRNA for foxp3, cytotoxic T lymphocyte antigen-4, TGF-␤, IL-10, Prolonged Administration of hAAT to Diabetic Islet Allograft Recipient and IL-1 receptor antagonist; expression of proinflammatory me- Mice Results in Strain-Specific Immune Tolerance. To examine the diators was low or absent. After implantation of skin allografts, outcome of islet allograft transplantation during extended AAT-treated mice had greater numbers of foxp3-positive cells in monotherapy with hAAT, mice heterozygous for tissue-specific draining lymph nodes (DLNs) compared with control treatment hAAT [hAAT transgene (hAAT-Tg)], which exhibit levels of mice. Moreover, dendritic cells in DLNs exhibited an immature circulating hAAT that are below detection, were used as graft phenotype with decreased CD86 activation marker. Although the recipients. hAAT-Tg mice (H-2b) were rendered diabetic, trans- number of CD3 transcripts decreased in the DLNs, AAT did not planted with allogeneic islets (H-2d), and treated with serial affect IL-2 activity in vitro. Thus, AAT monotherapy provides doses of hAAT (n ϭ 24) or albumin (n ϭ 6). As shown in Fig. allografts with antiinflammatory conditions that favor develop- 1A, control albumin-treated mice rejected allografts by day 12. ment of antigen-specific T regulatory cells. Because AAT treatment In contrast, all hAAT-treated mice that were treated for the in humans is safe, its use during human islet transplantation may various durations indicated exhibited extended normoglycemia. be considered. The shortest course of hAAT therapy, 14 days, resulted in delayed loss of function in 50% of the transplants and in graft dendritic cells ͉ diabetes ͉ interleukin-1 ͉ interleukin-10 ͉ T-regulatory cells acceptance in the remainder. A 21-day course resulted in a single delayed graft failure event of the six transplanted islet grafts. All slet loss of function in most islet transplant patients steadily 12 mice that received hAAT treatment for 30 days or more Iprogresses after grafting and results in a low 5-year graft achieved graft acceptance (treatment duration, 30 days, n ϭ 8; survival rate (1, 2). The immunosuppressive agents used for islet 41 days, n ϭ 1; 52 days, n ϭ 2; 60 days, n ϭ 1). transplantation exclude diabetogenic corticosteroids, and the Removal of grafts by nephrectomy restored hyperglycemia. grafted cells lack the benefit of antiinflammatory therapy (3). Shown as an example in Fig. 1C Left and Right, by day 12 after Because islets are particularly prone to injury during inflamma- transplantation the albumin-treated mouse (broken line) had tory conditions (4), the functioning islet mass rapidly decreases mounted an acute allograft rejection response and developed after transplantation before antigenic recognition (5). As dam- overt hyperglycemia, whereas the hAAT-treated mice main- age intensifies, necrotic islet cells become a source of damage- tained normoglycemia for the duration of therapy. After with- associated molecular patterns, which induce the production of injurious cytokines and chemokines that facilitate alloantigen presentation to the recipient (6); the host immune system is thus Author contributions: E.C.L., M.M., M.T., N.D., L.S., and C.A.D. designed research; E.C.L., M.M., M.T., N.D., and C.A.D. performed research; E.C.L., J.L.W., A.C., L.S., and C.A.D. alerted to the alloantigen-rich site (7). contributed new reagents/analytical tools; E.C.L., L.S., and C.A.D. analyzed data; and E.C.L., The favorable state of immune tolerance can be elaborated by M.M., M.T., N.D., L.S., and C.A.D. wrote the paper. a shift in balance between effector T cells and protective The authors declare no conflict of interest. regulatory T (Treg) cells (8), a process that requires, contrary to Freely available online through the PNAS open access option. immunosuppression, the uninterrupted activity of IL-2 (9, 10). §To whom correspondence should be addressed at: Division of Infectious Diseases, B168, By reducing the intensity of inflammation while allowing IL-2 University of Colorado Health Sciences Center, Denver, CO 80262. E-mail: cdinarello@ activity, one may provide the conditions for prolonged allograft mac.com. survival and tolerance induction (8). This article contains supporting information online at www.pnas.org/cgi/content/short/ In addition to its ability to inhibit serine proteases, ␣1- 0807627105/DCSupplemental. antitrypsin (AAT) possesses antiinflammatory properties (11– © 2008 by The National Academy of Sciences of the USA

16236–16241 ͉ PNAS ͉ October 21, 2008 ͉ vol. 105 ͉ no. 42 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807627105 Downloaded by guest on October 2, 2021 A B 30-day therapy (n = 12) 100

80 21-day therapy (n = 6)

40 14-day therapy (n = 6)

20 Control (n = 6) 0 0 1 2 3 4 5 6 7 8 9 101112 Weeks from transplantation C Nephrectomy 600 600 Nephrectomy ALB Treatment Second Allograft Treatment ALB 400 stopped (same strain) 400 stopped hAAT Second Allograft 200 200 hAAT (3rd strain)

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 Days from allograft transplantation Days from allograft transplantation

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Fig. 1. Extended AAT monotherapy induces strain-speciﬁc immune tolerance toward islet allografts in mice. Islet allograft transplantation was performed and IMMUNOLOGY blood glucose was followed in mice that received albumin (ALB) (n ϭ 6) or hAAT monotherapy (n ϭ 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 ϭ 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).

drawal of hAAT, continued graft-derived insulin production was shown), and a predominance of CD4- or CD8-positive cells, observed (Fig. 1 B, second column, and C, days 52–72), raising interspersed with CD25-positive cells (data not shown). the possibility that allospecific immune tolerance was achieved. To examine this possibility, grafts were removed and a second Inflammatory and Antiinflammatory Gene Expression in Islet Allo- grafting procedure was undertaken in the subcapsular space of grafts. We next examined the steady-state levels of inflamma- the remaining kidney without further treatment, using the same tion-related genes in explanted islet grafts. Fig. 2 depicts a strain of islets that had been originally transplanted (n ϭ 4; H-2d) comparison between mRNA patterns present early after trans- (Fig. 1B and illustrated in an example in Fig. 1C Left). In each plantation in albumin-treated mice (days 1, 3, 5, and 7) and those case, after reengraftment, recipient mice remained normogly- present in long-lasting hAAT-treated islet grafts (representative cemic for Ͼ50 days. To ascertain that antigen-specific immune day 72). As shown, transcripts of genes coding for islet-injurious tolerance had been induced, islets from a third strain (H-2k) were ligands were low in grafts from hAAT-treated mice. These used as the source of the second graft in three hyperglycemic include IL-1␤, in addition to CD14, a marker for invading mice without further hAAT treatment (Fig. 1 B and C Right). As macrophages, IL-2 from invading T cells, and intercellular shown, all three mice exhibited acute rejection of third-strain adhesion molecule-1 (ICAM-1). In addition, mRNA transcripts allografts. that encode for the proneutrophilic CXC chemokines, KC and According to histology, a ‘‘cuff’’ of mononuclear cells sur- macrophage inflammatory protein-2 (MIP-2), were undetect- rounded the entire islet mass in all explanted grafts (Fig. 1D). able in long-lasting islet allografts. Islet allograft explants from The mononuclear cells were located at the intersection between hAAT-treated mice also exhibited elevated expression of IL-1 the renal parenchyma and capsule, flanking an intact islet graft receptor antagonist (IL-1Ra) and isoforms of IL-18-binding mass. By staining for several cell-specific markers, we found the protein (IL-18BP), both reported to protect islet allografts (15, near absence of activated macrophages (CD11b) (data not 16). In contrast, explants of albumin-treated mice exhibited

Lewis et al. PNAS ͉ October 21, 2008 ͉ vol. 105 ͉ no. 42 ͉ 16237 Downloaded by guest on October 2, 2021 Treatment: ALB AAT A Treatment: ALB AAT Days from transplant: 1 53 7 72 Days from transplant: 1 53 7 72 IL-1 foxp3 CD14 TGF 1 IL-2 CTLA-4 ICAM-1 KC B Day 14 Day 30 MIP-2 foxp3 IL-1Ra CTLA-4 IL-18BP IL-10 GAPDH K G K G K G K G K G Insulin Fig. 4. Identification of hAAT-induced IL-10-expressing Treg cells in nonre- Fig. 2. Effect of hAAT monotherapy on gene expression profile in islet jecting islet allografts. (A) RT-PCR of explanted islet allografts in albumin allografts. RT-PCR of explanted islet allografts from albumin (ALB)-treated (ALB)-treated graft recipients during acute allorejection (left four columns; control and hAAT-treated mice. The left four columns show the initial days days 1–7) and hAAT-treated graft recipient 20 days after withdrawal of hAAT after islet transplantation into control mice. The right column shows day 72 treatment (right column; day 72; see Fig. 2). Data are representative of n ϭ 6 after islet transplantation into hAAT-treated mice (see Fig. 1). Data are (ALB) and n ϭ 3 (hAAT; representative time point between days 30 and 72 representative of n ϭ 6 (ALB) and n ϭ 3 (hAAT; time points between days 30 after transplantation). (B) Intragraft gene expression profile throughout and 72 after transplantation). 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. either low or undetectable expression of IL-1Ra and IL-18BP (Fig. 2, days 1, 3, 5, and 7). contain the grafted islets (Fig. 4B, K), foxp3-positive cells were Cell-Specific Effects of AAT. The expression of antiinflammatory also observed. CTLA-4 expression was present only inside the molecules observed here belongs to grafts that were explanted graft (G). Of particular importance, IL-10 transcript levels were several weeks after withdrawal of hAAT treatment (see Fig. 1). closely associated with foxp3 expression, suggesting that the It is therefore more likely that the intragraft antiinflammatory identified Treg cells are also inducers of IL-10. gene expression profile reflects the cellular components that accumulated in the antigen-rich site. To examine the effects of Time-Dependent Distribution of Treg Cells Between Draining Lymph hAAT on major cell subpopulations, we performed in vitro Nodes (DLNs) and Allografts. To examine the effect of hAAT on assays for lymphocytic and nonlymphocytic responses. As shown Treg cell development during transplantation, foxp3-GFP in Fig. 3, IL-2-stimulated human peripheral blood mononuclear knock-in mice were used as graft recipients (C57BL/6 back- cells (PBMCs) were able to produce IFN-␥ and proliferate, as ground, H-2b). A vigorous allorecognition response was evoked expected, in the presence of hAAT. Similarly, mouse splenocytes by implanting wild-type skin grafts (H-2d) under the surface of responded to Con A with secretion of IFN-␥, as well as increased both left and right thighs. Animals were treated with hAAT (n ϭ cell proliferation and cell clumping, each response unaffected by 13) or albumin (n ϭ 13) using the same dosing schedule used in hAAT [supporting information (SI) Fig. S1a]. In contrast to the islet transplantation protocol (see Fig. 1). Inguinal DLNs lymphocytic responses, peritoneal macrophages responded to were removed on various days after grafting and CD4ϩ-sorted ␥ hAAT by secreting significantly less IFN- -induced nitric oxide cells were examined by FACS and by RT-PCR for foxp3-positive in a concentration-dependent manner (Fig. S1b). cells. As shown in Fig. 5A, between transplantation and 3 days after engraftment of islets, the number of foxp3-positive cells in Treg-Related Gene Expression in hAAT-Treated Islet Allografts. In the the DLNs unvaryingly decreases in both the albumin control and unique set of genes expressed within grafts of hAAT-treated hAAT-treated graft recipient mice. However, between days 4 recipient mice, we also observed the expression of genes indic- and 9, DLNs from hAAT-treated mice had more foxp3-positive ative of Treg cells (Fig. 4). As shown, grafts from hAAT-treated cells. In the days that followed, the gap in the size of the Treg mice (Fig. 4A, representative day 72) exhibit a significantly population was restored. Gene expression analysis corroborated ␤ elevated expression of foxp3, TGF- , and cytotoxic T lympho- FACS findings (Fig. 5A Inset). cyte antigen-4 (CTLA-4), representing the expected phenotype By using foxp3-GFP knock-in mice as Matrigel skin graft of Treg cells (17–20). In contrast, the expression of these genes recipients, we were able to observe Treg cells infiltrating allo- was either below detection or terminated early in grafts from grafts by day 10 after transplantation. This model offers a albumin-treated mice (days 1, 3, 5, and 7). As depicted in Fig. 4B, particular advantage because invading fluorescent cells can be the presence of foxp3-positive cells was observed as early as day directly identified in freshly obtained, unstained specimens by 14 of hAAT therapy in sections that contained the graft site (Fig. using fluorescent microscopy. As shown in Fig. 5B, invading 4B, G). Notably, in renal tissue from kidney portions that did not foxp3-positive cells localized to grafts in hAAT-treated animals (Fig. 5B Lower). Fur is autofluorescent and can be observed. Total intensity of infiltrating cells can be appreciated by DAPI 100 0.15 AAT counterstaining. Similarly, as shown in Fig. 5C, islet allografts 75 0.10 CT that had been transplanted into foxp3-GFP knock-in recipient 50 0.05 mice also contained foxp3-positive cells in the cuff site. 25 0 0.00 Early Local and Systemic Effects of hAAT. We next studied the events 0 2 4 6 8 10 0 2 4 6 8 10 that might precede the changes observed in the DLNs. Islets IL-2 ng/ml IL-2 ng/ml embedded into Matrigel allow for examination of islet-driven Fig. 3. Cell-specific effects of hAAT. Inducible IFN-␥ levels (Left) and cell cellular invasion during the first 48 h after implantation. Allo- proliferation (Right) assessed in Con A-primed PBMCs that were stimulated geneic islets were introduced into hAAT-containing Matrigel with increasing concentrations of IL-2 in the presence of 0.5 mg/ml hAAT or plugs (12.5–100 ␮g of hAAT per graft) and implanted s.c. into albumin (CT). Data are mean Ϯ SEM of three individual donors. mice. Grafts were retrieved 48 h after transplantation and

16238 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807627105 Lewis et al. Downloaded by guest on October 2, 2021 IL-10 CD86 CD3 ** 0.3 0.4 0.8 * 0.3 * 0.6 0.2 0.2 0.4 0.1 0.1 0.2 0.0 0.0 0.0 -CTAAT -CTAAT -CTAAT

72 hrs 72 hrs 72 hrs

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 Ϯ SEM from three experiments. *, P Ͻ 0.05; **, P Ͻ 0.01 between CT and AAT.

origin because VEGF mRNA copies coincided with near absence of host genomic DNA (Fig. 6). In addition to local events that reflect a dampened inflammatory response and suboptimal antigen presentation environment, we searched for other indications that may support the generation of Tregs. Mice were subjected to 10 days of hAAT treatment to reproduce the circulating cytokine environment of a treated islet graft recipient. Control mice received albumin, and serum levels of cytokines were measured. As shown in Fig. S2, serum levels of Th17-related cytokines, IL-17 and IL-23, were 3-fold lower compared with levels in control mice. Serum Fig. 5. Time-dependent hAAT-induced distribution of Treg cells between DLNs and allograft. foxp3-GFP knock-in mice (H-2b) were grafted with wild- IL-6 and monocyte chemoattractant protein-1 were also de- type BALB/c tissue (H-2d). Mice received a 10-day hAAT treatment or albumin creased. However, serum IL-10 levels increased 2-fold and the protocol (see Fig. 1). (A) Inguinal DLN. FACS analysis of CD4ϩ-sorted foxp3- levels of I-309, a chemoattractant for Treg cells, increased 2-fold. GFP-positive DLN cells. (Inset) RT-PCR for foxp3 mRNA transcripts in DLNs. To examine the circulating cytokines evoked during a vigorous Shown are representative time points. (B) Matrigel-skin graft. Treg cells in inflammatory response, mice were treated for 10 days with Matrigel grafts on day 10 identiﬁed by ﬂuorescent microscopy of unstained hAAT or albumin and then challenged with LPS. Serum cyto- material (Left) plus DAPI-counterstained material (Right). (C) Islet graft. Day kines were assessed after 2 h (see Fig. S2). Once again, I-309 and

14 Treg cells identified in the cuff site (see Fig. 1D). Anti-GFP antibody MIP-2 levels exhibited the same changes observed in hAAT- IMMUNOLOGY immunostaining and DAPI counterstaining is shown. Representative image of treated but nonchallenged mice. Most strikingly, circulating three hAAT-treated grafts is shown. Grafts from albumin-treated mice contained no cuff (data not shown). IL-1Ra levels increased 3-fold. The effect of hAAT treatment on serum IL-6 and IL-10 levels were also measured after a sterile inflammatory response (Fig. S2). In this procedure, the inflam- intragraft mRNA levels were assessed. As shown in Fig. 6, a matory response results in increased levels of IL-1␤-dependent dose-dependent decrease in CD14 mRNA levels had occurred, IL-6 (21). However, mice treated with hAAT exhibited a 30% reflecting hAAT-dependent decrease in macrophage popula- decrease in serum IL-6 protein levels and a 27% increase in tion. The recipient mice carry the hAAT genomic insert, and serum IL-10 protein levels. invading host cells can thus be identified. Indeed, the amount of invading cells decreased in the presence of hAAT, as corrobo- Effect of AAT on Dendritic Cell Migration, Maturation, and Function. rated by histological examination of the explanted Matrigel at To investigate the implications of a dampened antigen presen- 48 h (data not shown). Copies of insulin mRNA transcripts tation process during transplantation, we studied dendritic cell correlated with the amount of added hAAT, representing im- activation in vitro and in vivo. By using transgenic GFP-positive donor skin grafts and subsequent PCR amplification of DNA proved hAAT-mediated beta cell viability. hAAT treatment also isolated from DLNs, we evaluated the migration of graft-derived resulted in a dose-dependent increase in VEGF mRNA levels. cells toward DLNs in vivo (Fig. S3a). Mice received hAAT 1 day VEGF mRNA in the Matrigel-islet graft is likely to be of islet cell before grafting, as during islet allograft transplantation. Graft- derived DNA was present in DLNs after transplantation in both control and hAAT-treated mice. We next examined the effect of hAAT on the transcript levels µ AAT g/ml 0 12.5 25 50 100 of CD86, CD3, and IL-10 in the DLNs of kidneys in mice receiving skin grafts into the renal subcapsular space (Fig. 7). CD14 (RT-PCR) hAAT (genomic) For background gene expression, DLNs from nontransplanted mice were examined. Seventy-two hours after transplantation, Insulin (RT-PCR) CD86 mRNA transcript levels were reduced 2-fold by hAAT VEGF (RT-PCR) treatment. At the same time point, DLNs contained 2-fold less total CD3 mRNA transcripts. Notably, a 2.5-fold rise in IL-10 Fig. 6. Early local and systemic effects of hAAT. Wild-type islet-Matrigel gene expression was observed in DLNs from hAAT-treated grafts containing increasing concentrations of hAAT (indicated, amount per Matrigel) were explanted 48 h after transplantation into hAAT-Tg recipients. grafted mice. (Upper) Identification of CD14-positive cells (RT-PCR) and identification of To examine the direct effect of hAAT on dendritic cell host cells inside the graft (genomic). (Lower) RT-PCR depiction of insulin and activation and maturation, dendritic cells were cultured in vitro VEGF intragraft transcripts. with LPS in the absence or presence of hAAT (Fig. S3b). By

Lewis et al. PNAS ͉ October 21, 2008 ͉ vol. 105 ͉ no. 42 ͉ 16239 Downloaded by guest on October 2, 2021 FACS analysis, LPS stimulation in the presence of hAAT foreign cells, uninterrupted CD3-positive cell infiltration oc- resulted in a marked decrease in the levels of inducible surface curred, suggesting that the isolated event of lymphocyte activa- MHC class II and CD86. tion is, indeed, intact in the presence of AAT. To address the possibility that hAAT treatment had specifi- IL-1Ra transcripts were elevated in the explanted long-lasting cally induced IL-10 production in Treg cells in vivo,hAATor islet allografts. IL-1␤ has been shown to break tolerance (30) and albumin was administered 3 days before LPS challenge to facilitate islet antigen presentation (6). In other studies, IL-1Ra foxp3-GFP knock-in mice. Sixteen hours later, spleens were inhibited mouse islet allograft rejection (15), increased islet beta harvested and splenocytes were isolated to examine IL-10 re- cell function during transplantation (31), and improved disease lease in a cytometric secretion assay. LPS administration alone parameters in patients with type 2 diabetes (32). AAT was shown resulted in foxp3-positive cells that released IL-10 (6.1 Ϯ 0.1%, to induce IL-1Ra in human PBMCs (33). The explanted hAAT- compared with 0.2 Ϯ 0.1% without LPS). The number of treated grafts also exhibited elevated CTLA-4 mRNA levels. The IL-10-secreting Treg cells increased in hAAT-treated mice to engagement of CTLA-4 participates in the induction of periph- 10.6 Ϯ 1.2% (mean Ϯ SEM; P ϭ 0.0167). eral tolerance (34) and induces the production of TGF-␤ (35). In addition, AAT treatment reduced the production of IL-6, as Discussion shown here and reported by others (36). In the presence of In the present study, we demonstrate that monotherapy with TGF-␤, high levels of IL-6 interfere with Treg differentiation in hAAT during islet allograft transplantation results in the induc- favor of Th17 cells, as reported by Bettelli et al. (37). Because tion of antigen-specific immune tolerance. Therapy comprised a IL-1␤ is required for Th17 survival (38), it is possible that AAT 1-day preconditioning dose with hAAT followed by serial main- provides a cytokine milieu that actively supports Treg predom- tenance injections every 3 days extending for a minimum of 14 inance. Indeed, one of the most significant changes observed in days. A population of mononuclear cells that included a sub- sera from hAAT-treated mice in a protein-blotting array of 36 population of Treg cells surrounded the islet mass in the form of cytokines was reduced levels of circulating IL-17 and IL-23. ␤ a cellular cuff, which had high expression levels of foxp3, TGF- , Although AAT inhibits caspase-3 in a rat beta cell line (39), CTLA-4, and IL-10, but decreased levels of inflammatory genes. an antiapoptotic function of AAT per se is probably incapable of In early days after transplantation, islets embedded into Matrigel inducing tolerance, because global inhibition of apoptosis during plugs that contained hAAT exhibited a dose-dependent im- an inflammatory and/or immune response would promote an provement in islet viability and a marked decrease in monocyte/ expanded reactive T cell population. We therefore propose that macrophage infiltration. These data suggest that immunogenic- the cytoprotection observed in the presence of AAT is by virtue ity of implanted islets is considerably reduced by hAAT. By using of its antiinflammatory properties. skin allografts to evoke a vigorous immune reaction, we ob- In support of the inclusion of AAT therapy for transplant served that the number of Tregs rises in the draining lymph patients are the long-term safety studies, extending over several nodes of hAAT-treated graft recipients. years of weekly AAT infusions to patients with various degrees Several studies suggest that Tregs participate in controlling of AAT deficiency (40–42); no evidence of compromised host allograft stability (reviewed in ref. 22). To generate Tregs, it is defense can be demonstrated. We therefore suggest that AAT essential that IL-2 activity be maintained. Indeed, as demon- can be considered for testing in human islet transplantation. strated in vitro and as supported by other studies (23), IL-2 activity is unaffected by the presence of hAAT. Also, pivotal to Methods the differentiation of naive T cells into Treg cells in the DLNs, Mice. hAAT-Tg mice, background strain C57BL/6, were engineered as de- we confirm that the process of cell migration from the site of scribed in ref. 43 and studied as detailed in SI Methods. Circulating levels of transplantation into the DLNs occurs without interruption dur- hAAT in heterozygous hAAT-Tg mice were determined by a specific ELISA for ing hAAT therapy. human AAT, as described in ref. 13. Serum levels were below the limit of It has been shown that dendritic cell-expanded T regulatory detection (10 ng/ml). Foxp3-GFP knock-in mice were kindly provided by T. B. cells restore normoglycemia in diabetic NOD mice (24). In a Strom (Harvard Institutes of Medicine, Boston, MA) (37). Wild-type BALB/c, noninflammatory environment, dendritic cells remain immature CBA/Ca, and DBA/2 mice were purchased from The Jackson Laboratory. Ex- and promote antigen-specific immune tolerance (25); dendritic periments were approved by the University of Colorado Institutional Animal cells that migrate to the DLNs express cell surface molecules Care and Use Committee. such as CD86 (also known as B7) (26). Here, we show that soon after grafting of islets into hAAT-treated animals, CD86 and Islet Allograft Transplantation. Renal subcapsular islet transplantation was MHC class II are diminished in the DLNs. Our findings coin- performed as described in ref. 13. Briefly, hAAT-Tg heterozygote mice weigh- cided with a decrease in total CD3 transcript levels in the ing 25–30 g were rendered diabetic by a single i.p. injection of streptozotocin (225 mg/kg; Sigma–Aldrich). Donor islets were isolated and collected on associated DLNs and a marked increase in IL-10 expression. We 100-␮m cell strainer (Falcon; BD Biosciences Discovery Labware), as described previously reported that hAAT reduces isolation-induced ϩ in ref. 13. A total of 450 hand-picked isolated islets from DBA/2, BALB/c, CD45 -islet-cell surface MHC class II levels (13). We further C57BL/6, or CBA/Ca donor mice were grafted under the renal subcapsular show in the present study that hAAT inhibits dendritic cell space. hAAT treatment was initiated 1 day before transplantation and every maturation in vitro. As we described in ref. 13, the administration third day (2 mg per mouse; Aralast; Baxter). Control hAAT-Tg mice received of hAAT before day 3 is essential for the prolongation of islet the same amount of human serum albumin (Abbott). In the experiments in allograft survival. Here, cellular infiltration into islet grafts which monotherapy exceeded 14 days, the amount of hAAT was increased by within the initial 3 days of engraftment was blocked by hAAT in 0.5 mg every third day until a 6-mg maintenance dose was reached. Islet a dose-dependent manner. AAT has been described as blocking allograft rejection was defined as the day blood glucose exceeded 300 mg/dl phagocytic cell invasion (13) and both production (27) and after a period of at least 3 days of normoglycemia. activity (28) of IL-8 in human cells. Importantly, IL-8 is pro- As donor tissue, 1 mm3 of freshly prepared skin derived from duced in large amounts by human islets during and after islet Skin Allografts. shaved avascular portion of the abdominal midline was used. A graft was isolation and thus contributes to early graft destruction (29). inserted into the s.c. space of each thigh in foxp3-GFP knock-in mice through Administration of AAT before inoculation of foreign cells into a 1-mm-long incision. Incision site was sealed with a 3-0 suture. the peritoneal cavity reduced the number of infiltrating CD3- positive cells by 50% (13). Conversely, in the same model, by Immunocyte Responses in Vitro. PBMCs were isolated from healthy individuals, allowing neutrophil and macrophage invasion to reach comple- as described in ref. 44. Studies of human blood were approved by the Colorado tion and administering AAT 3 days after the inoculation of Multiple Institutional Review Board. Splenocytes and resident peritoneal

16240 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807627105 Lewis et al. Downloaded by guest on October 2, 2021 macrophages were obtained from C57BL/6 mice, as described in ref. 13. For ACKNOWLEDGMENTS. We thank Owen Bowers, Tania Azam, Nathan Farb, response assays, see SI Methods. Galit Shahaf, and Chad Ficek for their excellent technical contributions and For Matrigel-islet grafts and Matrigel-skin grafts, DLN analysis and assess- support. We thank Terry Strom for providing foxp3-GFP knock-in mice. This ment of explanted renal allografts, histology, RT-PCR, FACS analysis, and work was funded by National Institutes of Health Grants AI-15614 and cytokine measurements, see SI Methods. CA-04 6934 (to C.A.D.), Canadian Institutes of Health Research Grant MOP42539 (to A.C.), the Juvenile Diabetes Research Foundation Grant Statistical Analysis. Comparisons between groups were performed by two- 2-2007-103 (to E.C.L.), and the Marc Rich Foundation for Education, Cul- sided t test. ture, and Welfare (E.C.L.).

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