Wensheng Zhang,1,2 Vijay S. Gorantla,1,2 Phil G. Campbell,2,3 Yang Li,1 Yang Yang,1

Chiaki Komatsu,1,2 Lee E. Weiss,2,4 Xin Xiao Zheng,1,5 and Mario G. Solari1,2

Biopatterned CTLA4/Fc MatricesFacilitate Local Immunomodulation,Engraftment, and Glucose HomeostasisAfter Pancreatic Islet TransplantationDiabetes 2016;65:3660–3666 | DOI: 10.2337/db16-0320

Pancreatic islet transplantation (PIT) represents apotential therapy to circumvent the need for exoge-nous insulin in type 1 diabetes. However, PIT remainslimited by lack of donor islets and the need for long-term multidrug immunosuppression to prevent alloim-mune islet rejection. Our goal was to evaluate a localimmunoregulatory strategy that sustains islet allograftsurvival and restores glucose homeostasis in the ab-sence of systemic immunosuppression. Nanogramquantities of murine CTLA4/Fc fusion protein werecontrollably delivered within human acellular dermalmatrix scaffolds using an inkjet-based biopatterningtechnology and cotransplanted with allogeneic isletsunder the renal capsule to create an immunoregula-tory microenvironment around the islet allograft. Weachieved long-term engraftment of small loads of allo-geneic islet cells with 40% of MHC-mismatched mouserecipients maintaining sustained normoglycemia fol-lowing pancreatic b-cell ablation by streptozotocin.Biopatterned CTLA4/Fc local therapy was associatedwith expansion of Foxp3+ regulatory T cells and shiftsin cytokine production and gene expression from proin-flammatory to regulatory profiles, thus substantiallybenefiting islet allografts survival and function. Thisstudy is a new paradigm for targeted therapies in PITthat demonstrates the favorable effects of immune al-terations in the transplant milieu and suggests a uniquestrategy for minimizing systemic immunosuppressionand promoting islet allograft survival.

There is an immediate need for pancreatic islet trans-plantation (PIT) strategies that increase functional effi-cacy of small cell loads by optimizing engraftment andeliminating the need for chronic immunosuppression forgraft survival. Costimulation blockade with cytotoxic Tlymphocyte antigen 4 fusion protein (CTLA4-Ig) has beeneffective in downregulating allo- and autoimmune re-sponses, leading to enhancement of islet allograft survivaland inhibition of type 1 diabetes development (1–4).However, systemic administration of large doses ofCTLA4-Ig not only has long-term side effects but alsohas failed to completely inhibit alloimmune destructionof islet b-cells and to induce tolerance (5). Site-specificimmunomodulation may be a promising strategy to pre-vent acute rejection and minimize indiscriminate damageto essential cells and organs by systemic immunosuppres-sion (6–8).

Our team has developed a cutting-edge biopatterningtechnology that can spatially control the distribution ofphysiologically relevant, pico-to-nanogram level doses ofsignaling molecules immobilized as “solid-phase” patternsto extracellular matrix (ECM) constructs via native bind-ing affinities to direct cell behavior in vitro and tissueformation in vivo (9–11). Our a priori hypothesis is basedon successful prior work where biopatterning of signalingmolecules in solid phase significantly improved outcomesacross multiple applications (12,13). Here, we fabricatedimmunoregulatory microenvironments using murine

1Department of Plastic Surgery, University of Pittsburgh School of Medicine,Pittsburgh, PA2McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pitts-burgh, PA3Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh,PA4Robotics Institute, Carnegie Mellon University, Pittsburgh, PA5Transplantation Medical Center, Zhongnan Hospital, Wuhan University, Wuhan,China

Corresponding authors: Mario G. Solari, solarimg@upmc.edu, and Xin XiaoZheng, xzheng6688@163.com.

Received 9 March 2016 and accepted 7 September 2016.

W.Z., V.S.G., and P.G.C. contributed equally to the study.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

3660 Diabetes Volume 65, December 2016

IMMUNOLOGY

AND

TRANSPLANTATIO

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CTLA4/Fcg2a heavy-chain chimeric fusion protein (CTLA4/Fc)constructs biopatterned within immunoneutral human-derived acellular dermal matrix (ADM). We investigatedthe effects of this CTLA4/Fc microenvironment oninhibiting alloimmune responses after PIT and promot-ing islet allograft survival without additional immuno-suppression. Our work is the first to demonstrate thattargeted, spatially controlled delivery of extremelysmall doses of solid-phase proregulatory agents, suchas biopatterned CTLA4/Fc microenvironments, can pro-mote engraftment, function, and long-term survival ofislet allografts.

RESEARCH DESIGN AND METHODS

Creation of Biopatterned Murine CTLA4/FcMicroenvironment in PITUniform patterns of murine CTLA4/Fc bioink (200 mg/mL)were produced in our laboratory and printed onto 200-mmthick, 53 5 mm pieces of ADM (Synthes, West Chester, PA)using our inkjet-based biopatterning system (9,10,14). Theprinted bioink absorbs into the open-pore structure of thedermal surface and binds within the matrix to produce immo-bilized 3-D patterns that persist for sufficient periods of timeneeded to elicit the desired biological control (9). Bindingretention of CTLA4/Fc was estimated by printing125I-CTLA4/Fc onto ADM using the method similar to otherhormone bioinks (14,15), which demonstrated that biopat-terned CTLA4/Fc persist stabilized release with 50% of boundremaining for 21 days under simulated in vitro conditions.

PITPIT was performed from donor DBA/2(H-2d) mice tostreptozotocin-induced diabetic C57BL/6(H-2b) recipientmice as previously described (16). After prerinsing, ADMsquares were cut into 1 3 5 mm strips and insertedbelow the renal capsule. Approximately 300 islet allo-grafts were injected between strips, so that the biopat-terned CTLA4/Fc could be in direct contact withislet allografts (Fig. 1). Other groups included single doseCTLA4/Fc (20 mg/kg) intraperitoneally (i.p.), CTLA4/FcADM implanted under the contralateral renal capsule, non-printed ADM, and untreated recipients. Allograft functionwas assessed by monitoring blood glucose levels (BGL), andrejection was defined as BGL of.300 mg/dL on two consec-utive measurements (17).

Quantification of Cytokines and GenesThe production of IL-4, IFN-g, IL-6, and IL-17 in serumsamples obtained at 2 and 8 weeks posttransplantationwere assessed by enzyme-linked immunosorbent assay(ELISA). Total RNA was extracted from draining lymph nodesor islet allografts. Levels of specific mRNA IL-4, IFN-g, Gzmb,IL-17, and Foxp3 were quantified by real-time PCR.

Assessment of T-Cell SubsetsCells were harvested from lymph nodes, spleens, orperipheral blood of long-term survival recipients at day150 after transplantation. The expression of cell surface

and intracellular markers (CD3, CD4, CD25, and Foxp3)were analyzed by flow cytometric analysis. To test regulatoryT-cell (Treg) suppression, flow-sorted CD4+CD252 respondercells from naive C57BL/6 mice were cultured with CD4+CD25hi

T cells from long-term islet allograft survival recipients or age-matched naive mice in 96-well plates, using anti-CD3 and anti-CD28 monoclonal antibody (mAb) or irradiated splenocytesfrom donor (DBA/2) or the third party (C3H) as stimulators.Cultures were pulsed for the last 8 h of the incubation with1 mCi/well 3H-thymidine, and 3H-thymidine incorporationwas measured using standard scintillation procedures andexpressed as percent inhibition of proliferation.

Histological AnalysisThe kidney bearing the islet graft was removed fromrecipients and processed for hematoxylin-eosin (H&E) stain-ing and immune-peroxidase histochemistry with anti-mouseFoxp3 mAb or anti-insulin mAb. Samples were evaluated ina blinded fashion at two different levels of sectioning.

Statistical AnalysisData are expressed as mean 6 SD. Comparisons betweentwo samples were performed using the Student t test. Multiplegroups were compared by one-way ANOVA with Tukey multi-ple comparisons test. Allograft survival was analyzed using alog-rank test. Differences are considered significant if P, 0.05.

RESULTS

Localized Delivery of a Biopatterned CTLA4/FcImmunoregulatory Microenvironment Promotes IsletAllograft Survival Across a Full MHC Barrier in MiceIn the MHC-mismatched DBA/2 to C57BL/6 mice PITmodel, treatment with local biopatterned CTLA4/Fc ADMsignificantly prolonged islet allograft survival with mediansurvival time (MST) of 71 days (n = 10; P , 0.001 vs.untreated group; P , 0.05 vs. CTLA4/Fc i.p. group). Allthose recipients maintained normoglycemia for.28 days,40% of which demonstrated long-term (.150 days)euglycemia. Contralaterally implanted CTLA4/Fc ADMdid not induce engraftment of islet allografts, indicatingthat the biopatterned CTLA4/Fc functioned at the trans-plant site but not remotely (Fig. 2A). Furthermore, biopat-terned CTLA4/Fc maintains persistence of detectable lowserum levels longer than systemically delivered CTLA4/Fcas detected by ELISA over time after PIT (Fig. 2B).

Biopatterned CTLA4/Fc Microenvironment AltersT-Cell–Derived Cytokine Production, Favors TregExpansion, and Maintains Treg Suppression After PITThe levels of IL-4 increased and IFN-g and IL-6 decreasedin the local biopatterned CTLA4/Fc treatment group com-pared with the nonprinted ADM group at 2 weeks post-transplantation. After 8 weeks, the alteration of IL-4 andIL-6 remained in biopatterned CTLA4/Fc–treated recipi-ents, when a significant decrease of IL-17 was detected(Fig. 3A). Additionally, there was marked upregulation ofIL-4 and Foxp3 mRNA and downregulation of IFN-g andGzmb mRNA expression in both islet allografts and

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draining lymph nodes of biopatterned CTLA4/Fc–treatedrecipients at 2 weeks posttransplantation (Fig. 3B). T cellsfrom biopatterned CTLA4/Fc–treated, long-term survivingrecipients demonstrated an increase of CD4+Foxp3+ Tregin peripheral blood (6.0 6 0.3%), spleen (8.8 6 0.5%), and

lymph nodes (9.5 6 0.4%) compared with the naive mice(Fig. 3C). Furthermore, CD4+CD25hi Treg isolated fromthese long-term surviving recipients significantly inhibitedthe proliferation of CD4+CD252 T cells activated bythe same donor antigen in a mixed lymphocyte reaction

Figure 1—Creation of biopatterned CTLA4/Fc microenvironment in the islet transplant site. Bioinks consisting of murine CTLA4/Fc wereprinted in a uniform pattern onto the 200-mm thick, 5 3 5 mm ADM. The printed dose of CTLA4/Fc was controlled by a fixed number of50 overprints per each square. Printed side (dermal side) is facing up when the notch and semicircle are oriented at the 6:00 and 4:00position. After prerinsing, ADM biopatterned with CTLA4/Fc was cut into 5 pieces, 1 mm 3 5 mm3 0.2 mm, and inserted into the pouch ofthe renal capsule, and islet allografts were injected between strips. The photo (lower right corner) and immunohistochemical insulin staining(upper right corner) show the positional relationship of the transplanted islets with respect to the ADM and kidney. Scale bar = 100 mm.

Figure 2—Pancreatic islets from DBA/2 mice were transplanted under the renal capsule of diabetic C57BL/6 recipients, which were treatedwith either localized delivery of biopatterned CTLA4/Fc ADM (n = 10), a single i.p. injection of CTLA4/Fc (n = 4), biopatterned CTLA4/FcADM implanted under the contralateral renal capsule (n = 4), nonprinted ADM (n = 6), or no treatment (n = 6). A: Survival of islet allografts.Untreated: MST 15.5 days; nonprinted ADM: MST 15.0 days; CTLA4/Fc i.p.: MST 36.5 days; contralateral CTLA4/Fc ADM: MST 23 days;local biopatterned CTLA4/Fc ADM: MST 71 days. *P < 0.05, **P < 0.01, ***P < 0.001. B: Mean CTLA4/Fc serum concentrations inislet allograft recipient mice given either a single i.p. injection of CTLA4/Fc (20 mg/kg) or implantation of biopatterned CTLA4/Fc ADM(3 mice in each group) immediately after transplantation. Serum samples were collected from recipients at a serial time points. Concen-trations of CTLA4/Fc were determined by a sandwich ELISA using anti-mouse CTLA4 mAb as the capture antibody and horseradishperoxidase–conjugated rat anti-mouse IgG2a mAb as the detection antibody. Serum CTLA4/Fc demonstrates a gradual elevation to1.5 mg/mL and maintained low detectable levels for up to 8 weeks until it was completely undetectable at 12 weeks.

3662 Biopatterned CTLA4/Fc in Islet Transplantation Diabetes Volume 65, December 2016

(71.2 6 2.7% vs. 31.6 6 2.1% inhibition; P , 0.01) butcould not suppress third-party antigen-induced alloreactivity.These Treg also exhibited more potent suppressive abilitythan Treg from naive mice (67.9 6 3.1% vs. 24.7 6 4.0%inhibition; P, 0.01) under nonspecific stimulation (Fig. 3D).

Biopatterned CTLA4/Fc Microenvironment ImprovesAllograft Engraftment and Survival by Local Regulationand ImmunoprotectionA decreased inflammatory cell infiltration and minimaldamage of islet allografts were observed by H&E at day14 posttransplantation in the biopatterned CTLA4/Fc–treatedrecipients compared with the nonprinted ADM–treated recip-ients (Fig. 4A). The low degree of inflammatory infiltra-tion and significant preservation of islet allografts werefurther characterized in the biopatterned CTLA4/Fc–treated

long-term surviving recipients at 150 days posttransplantationby H&E and immunohistochemical analysis. The stronginsulin staining demonstrated the potent function of allo-geneic islets with insulin-producing cells. An increase ofFoxp3-expressing cells suggests that local immune regula-tion is dominant in the protection of islet allografts fromalloimmune destruction (Fig. 4B).

DISCUSSION

Local or spatial control of the transplant microenviron-ment can modulate inflammation, immune cell infiltra-tion, and activation associated alloimmune responses(18). Prior work has shown that T-cell–mediated cytotox-icity may be mitigated through local delivery of CTLA4-Ig(19). In this study, we sought to improve the efficiency of

Figure 3—Cytokine production, gene expression, and Treg assessment in islet allograft recipients. A: Serum samples were obtained fromrecipients at 2 and 8 weeks posttransplantation. Serum levels of IL-4, IFN-g, IL-6, and IL-17 were determined by ELISA. B: Draining lymphnodes and islet allografts were harvested from recipients at 2 weeks posttransplantation. The mRNA expression of IL-4, IFN-g, Gzmb,IL-17, and Foxp3 (relative to that of GAPDH) in draining lymph nodes and islet allografts was determined by quantitative real-time PCR. C:Peripheral blood, spleen, and draining lymph nodes (DLn) were harvested from biopatterned CTLA4/Fc–treated long-term survival recip-ients at 150 days after islet transplantation or from age-matched naive C57BL/6 mice. CD3+CD4+Foxp3+ cells were analyzed by flowcytometry. The percentages are presented by the scatter plot. Bar represents mean value of four samples each group. D: Flow-sortedCD4+CD25hi Treg (>98% purity) from naive or biopatterned CTLA4/Fc–treated long-term survival recipients were cultured with naiveCD4+CD252 T cells in the presence of anti-CD3/CD28 mAb (each 2 mg/mL) or irradiated (3,000 rad) splenocytes from DBA/2 or C3Hmice. T-cell proliferation was measured by 3H-thymidine incorporation after 72 h or 6 days of culture. Data are expressed as the meanpercent inhibition of T-cell proliferation and are representative of three independent experiments, with triplicates in each experiment. *P <0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

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local immune regulation by targeting delivery of CTLA4/Fcto the transplant microenvironment of engrafted b-cells.Our goal was to eliminate systemic nonspecific immuno-suppression to minimize islet toxicity, thereby reducing theislet cell load needed for PIT and supporting long-termengraftment, survival, and endocrine function.

We printed bioinks of CTLA4/Fc on a biodegradableADM with defined surface concentrations and character-ized spatial organization to create a tissue-engineeredmicroenvironment using our established inkjet biopattern-ing technology, which is scalable and readily adaptable toother signaling molecules and/or ECMs (12,14). This tech-nique allows for the solid-phase delivery of therapeutic

proteins in extremely low dosages, with increased bio-activity relative to liquid-phase delivery. Our resultsshow that a biopatterned CTLA4/Fc microenvironmentimplanted along with a low number of islet cells underthe renal capsule achieved significantly prolonged survival,whereas a single i.p. injection of CTLA4/Fc or contralater-ally implanted CTLA4/Fc ADM was less effective, suggest-ing that the immunoregulatory effect is exerted locally byCTLA4/Fc at the site of islet transplantation. Furthermore,prolonged low levels of serum CTLA4/Fc were observedfollowing implantation of biopatterned CTLA4/Fc, indicat-ing that high levels of CTLA4/Fc in the systemic circulationwere not necessary. This would suggest that a persistent

Figure 4—Histological analysis of islet allografts from recipients with biopatterned CTLA4/Fc treatment. A: H&E staining of islet allograftsections at day 14 posttransplantation showed significant reduction of inflammatory cell infiltrates and minimal damage of islet allografts inthe biopatterned CTLA4/Fc–treated recipients. B: A low degree of inflammatory infiltration and significant preservation of islet allograftswere observed in biopatterned CTLA4/Fc–treated long-term survival recipients at 150 days posttransplantation (H&E). Immunohistochem-ical insulin staining demonstrated intact islets positive for insulin granules (brown stain) and confirmed the allogeneic islets are functional.Significant Foxp3+ expression was observed in the long-term surviving islet allograft by immunohistochemical Foxp3 staining, suggestingthe dominant immune regulation. Scale bars = 100 mm.

3664 Biopatterned CTLA4/Fc in Islet Transplantation Diabetes Volume 65, December 2016

presence of solid-phase CTLA4/Fc might be optimal forimmunoregulation.

ADM, as a native biological scaffold maintaining theoriginal dermal ECM architecture and low immunogenicity,was determined to be a suitable surrogate material tosequester biologically active components (9). It has alsobeen shown to support cell growth in vitro and to supportneovascularization following implantation in vivo (20). There-fore, biopatterning technology combined with the features ofADM enables prolonged retention and an extended bio-availability of biopatterned CTLA4/Fc, thus allowing itto be sequestered at nanogram levels. This establishes amicroenvironment that supports transplanted islet b-cellsand exerts local immunomodulation by CTLA4/Fc to pro-mote islet allograft engraftment and long-term function.

Type 1 T-helper cells (Th1) and type 2 T-helper cells(Th2) are the principal regulators of alloimmunity intransplantation. The induction of Th2 cytokines caninhibit Th1-mediated rejection responses, thereby pro-moting allograft acceptance (21). The cytokine profilesfrom biopatterned CTLA4/Fc–treated recipients revealedincreased Th2 cytokine (IL-4) and decreased Th1 cyto-kines (IFN-g) production in serum, with changes beingmore significant in the late posttransplantation period(8 weeks). In addition, the corresponding upregulationof IL-4 gene and downregulation of IFN-g gene expressionwere detected in both islet allografts and draining lymphnodes. This finding suggests that biopatterned CTLA4/Fcdelivered locally acts efficiently to modulate Th1/Th2 cy-tokines and to promote an immune deviation toward Th2responses. Furthermore, histological examination showeddecreased lymphocyte infiltration and long-term pre-served islet allograft with increased Foxp3 staining in thebiopatterned CTLA4/Fc–treated recipients, indicating theprotective effect of biopatterned CTLA4/Fc in situ againstalloreactive effector T-cell responses.

Active immune suppression by Treg is a prominentperiphery tolerance mechanism. Nevertheless, inflamma-tion of local tissue during transplantation not only limitsTreg suppression but also promotes proinflammatoryTh17 responses (22,23). Our data demonstrated that lo-calized biopatterned CTLA4/Fc treatment reduced serumlevels of IL-6 and IL-17 and decreased Gzmb gene andenhanced Foxp3 gene expression in both islet allograftsand draining lymph nodes, accompanied by increasedTreg cells. These findings further support the multifacetedimmunomodulation conferred by localized biopatternedCTLA4/Fc. They may provide microenvironments thatfavor Treg generation in the hostile environment ofan alloimmune response through suppressing cytotoxicT lymphocytes and inhibiting IL-6–mediated Th17 responses.In this study, the expansion of Treg and maintenance of theirsuppressive potency on effector T-cell proliferation againstdonor antigen in a mixed lymphocyte reaction may at leastin part underlie the long-term allograft survival induced bythe local biopatterned CTLA4/Fc therapy. A continuing pres-ence of CTLA4/Fc in the islet allograft could especially benefit

the recruitment of Treg to the allograft and ensure Tregeffective control of antidonor reactivity. These factors en-courage the further investigation of local immunoregulationfor the induction of transplantation tolerance.

In conclusion, for the first time, we demonstrated thatbiopatterning of native matrices, such as ADM, withimmunoregulatory agents, such as CTLA4/Fc, facilitateshighly localized delivery of these agents without diminu-tion of efficacy. Our study confirms that this technologypromotes long-term engraftment of minimal loads of allogeneicislet cells and leads to sustained glucose homeostasis. TheCTLA4/Fc microenvironment promoted expansion of Treg andshifted cytokine production and gene expression from proin-flammatory to regulatory profiles. Our findings suggest that thetargeted delivery of biopatterned CTLA4/Fc microenvironmentscould become a safe and effective strategy for minimizingsystemic immunosuppression and promoting islet allograftsurvival in PIT.

Acknowledgments. The authors thank Kia M. Washington and MaxineMiller (Department of Plastic Surgery, University of Pittsburgh School of Medicine)for providing input and advice to our work.Funding. This study was supported by a JDRF Innovative Grant (5-2012-308to M.G.S. [principal investigator], X.X.Z., V.S.G., and P.G.C. [co-principal investi-gator]) and in part by the Plastic Surgery Foundation and Musculoskeletal Trans-plant Foundation Dermal Tissue Research Grant (349234 to W.Z. [principalinvestigator]).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. W.Z., V.S.G., P.G.C., L.E.W., and X.X.Z. generatedthe original study conception. W.Z., V.S.G., P.G.C., L.E.W., X.X.Z., and M.G.S.designed experiments, analyzed data, and interpreted results. W.Z., V.S.G., andP.G.C. wrote the manuscript and prepared the figures. W.Z., V.S.G., P.G.C.,L.E.W., X.X.Z., and M.G.S. made critical scientific input and revisions forintellectual content. W.Z., Y.L., Y.Y., and C.K. performed animal experiments,immunological assays, and data acquisition. W.Z. and X.X.Z. constructed theCTLA4/Fc fusion protein. P.G.C. and L.E.W. made the biopatterned constructs.M.G.S. coordinated and directed the project. M.G.S. is the guarantor of thiswork and, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.

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