A Microsphere-Based Vaccine Prevents and ReversesNew-Onset Autoimmune DiabetesBrett Phillips,1Karen Nylander,1Jo Harnaha,1Jennifer Machen,1Robert Lakomy,1Alexis Styche,1Kimberly Gillis,2Larry Brown,2Debra Lafreniere,2Michael Gallo,2Janet Knox,2Kenneth Hogeland,2Massimo Trucco,1and Nick Giannoukakis1,3OBJECTIVE—This study was aimed at ascertaining the efficacyof antisense oligonucleotide-formulated microspheres to preventtype 1 diabetes and to reverse new-onset disease.RESEARCH DESIGN AND METHODS—Microspheres carry-ing antisense oligonucleotides to CD40, CD80, and CD86 weredelivered into NOD mice. Glycemia was monitored to determinedisease prevention and reversal. In recipients that remainedand/or became diabetes free, spleen and lymph node T-cells wereenriched to determine the prevalence of Foxp3⫹putative regu-latory T-cells (Treg cells). Splenocytes from diabetes-free micro-sphere-treated recipients were adoptively cotransferred withsplenocytes from diabetic NOD mice into NOD-scid recipients.Live-animal in vivo imaging measured the microsphere accumu-lation pattern. To rule out nonspecific systemic immunosuppres-sion, splenocytes from successfully treated recipients werepulsed with ␤-cell antigen or ovalbumin or cocultured withallogeneic splenocytes.RESULTS—The microspheres prevented type 1 diabetes and,most importantly, exhibited a capacity to reverse clinical hy-perglycemia, suggesting reversal of new-onset disease. Themicrospheres augmented Foxp3⫹Treg cells and induced hypo-responsiveness to NOD-derived pancreatic ␤-cell antigen, with-out compromising global immune responses to alloantigens andnominal antigens. T-cells from successfully treated mice sup-pressed adoptive transfer of disease by diabetogenic splenocytesinto secondary immunodeficient recipients. Finally, micro-spheres accumulated within the pancreas and the spleen aftereither intraperitoneal or subcutaneous injection. Dendritic cellsfrom spleen of the microsphere-treated mice exhibit decreasedcell surface CD40, CD80, and CD86.CONCLUSIONS—This novel microsphere formulation repre-sents the first diabetes-suppressive and reversing nucleic acidvaccine that confers an immunoregulatory phenotype to endog-enous dendritic cells. Diabetes 57:1544–1555, 2008Type 1 diabetes is a disorder of glucose homeosta-sis caused by a chronic autoimmune inflamma-tion of the pancreatic islets of Langerhans (1).The ultimate outcome is the loss of insulin-producing cells to numbers below a threshold that iscritically required to maintain physiological glucoregula-tion. Before this threshold, however, escalating inflamma-tion around (peri-insulitis) and in the islets of Langerhans(insulitis) first renders the insulin-producing ␤-cells insen-sitive to glucose and incapable of appropriate insulinproduction mainly due to the actions of cytokines likeinterferon-␥ (IFN-␥), tumor necrosis factor-␣ (TNF-␣), andinterleukin (IL)-1␤ (2,3).On clinical confirmation, a large number of type 1diabetic patients still exhibit evidence of residual ␤-cellmass that, for a limited time, is functionally responsive toglucose and produces insulin (the so-called “honeymoonperiod”) (4). In fact, patients with a residual ␤-cell massmanifest better glycemic control and improved prognosisfor diabetic complications including retinopathy and ne-phropathy. These observations have compelled investiga-tion into agents that can be used at the time of clinicaldiagnosis to preserve residual ␤-cell mass primarily byintervening with the ongoing autoimmunity. The use ofpharmacological systemic immunosuppressive drugs metwith initial success in controlling autoimmunity, however,on withdrawal, the autoimmunity recurred, indicating thatsystemic agents would need to be administered long-termwith their associated adverse effects (5,6). More recently,clinical reversal of hyperglycemia has been achieved byanti-CD3 antibody administration, although some ques-tions linger regarding mechanism of action in the transientimmunodepletion and associated cytokine-related side ef-fects (7,8). Finally, despite the initial observations inadults, administration of a peptide derived from HSP60into new-onset diabetic children failed to exhibit anybenefit compared with control subjects (9,10). A needtherefore remains for a diabetes-suppressive immunother-apeutic agent that does not engender nonspecific systemicimmunosuppression.It is generally accepted that the initial wave of infiltrat-ing immune cells in type 1 diabetes immunopathogenesisconsists mainly of antigen-presenting cells homing into theislets in response to an as-yet-unidentified microenviron-mental anomaly (11). Although not completely resolvedmechanistically and temporally, this anomaly, in a chronicprocess, compels migratory antigen-presenting cells, andmost prominently dendritic cells, to acquire ␤-cell–resi-dent antigens derived from apoptotic and/or necrotic␤-cells. The migratory dendritic cells then undergo anintrinsic “maturation” program that renders them capableFrom the1Diabetes Institute, Division of Immunogenetics, Department ofPediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsyl-vania;2Epic Therapeutics, a wholly owned subsidiary of Baxter HealthcareCorporation, Norwood, Massachusetts; and the3Department of Pathology,University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.Corresponding author: Nick Giannoukakis, PhD, Department of Pathology,University of Pittsburgh School of Medicine, Diabetes Institute, Rangos ResearchCenter, 3460 Fifth Ave., Pittsburgh, PA 15213. E-mail: [email protected] for publication 11 April 2007 and accepted in revised form 22February 2008.Published ahead of print at http://diabetes.diabetesjournals.org on 26 Feb-ruary 2008. DOI: 10.2337/db07-0507.Additional information for this article can be found in an online appendix athttp://dx.doi.org/10.2337/db07-0507.APC, allophycocyanin; AS-MSP, antisense microsphere; FACS, fluores-cence-activated cell sorter; HPLC, high-performance liquid chromatography;IFN-␥, interferon-␥; IL, interleukin; PE, phycoerythrin; PEG, polyethyleneglycol; PLGA, poly-(lactic-co-glycolic acid); PVP, polyvinyl pyrrolidone; SCR-MSP, scrambled control sequences microsphere; TNF-␣, tumor necrosisfactor-␣; Treg cell, regulatory T-cell.© 2008 by the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of pagecharges. This article