Infections That Induce Autoimmune Diabetes in BBDR Rats Modulate CD4  CD25  T Cell Populations 1,2 Danny Zipris, ‡ Jan-Luuk Hillebrands, ¶ Raymond M. Welsh,* § Jan Rozing, ¶ Jenny X. Xie, ‡ John P. Mordes, ‡ Dale L. Greiner,* ‡ and Aldo A. Rossini 3 * †‡ Viruses are believed to contribute to the pathogenesis of autoimmune type 1A diabetes in humans. This pathogenic process can be modeled in the BBDR rat, which develops pancreatic insulitis and type 1A-like diabetes after infection with Kilham’s rat virus (RV). The mechanism is unknown, but does not involve infection of the pancreatic islets. We first documented that RV infection of BBDR rats induces diabetes, whereas infection with its close homologue H-1 does not. Both viruses induced similar humoral and cellular immune responses in the host, but only RV also caused a decrease in splenic CD4  CD25  T cells in both BBDR rats and normal WF rats. Surprisingly, RV infection increased CD4  CD25  T cells in pancreatic lymph nodes of BBDR but not WF rats. This increase appeared to be due to the accumulation of nonproliferating CD4  CD25  T cells. The results imply that the reduction in splenic CD4  CD25  cells observed in RV-infected animals is virus specific, whereas the increase in pancreatic lymph node CD4  CD25  cells is both virus and rat strain specific. The data suggest that RV but not H-1 infection alters T cell regulation in BBDR rats and permits the expression of autoimmune diabetes. More generally, the results suggest a mechanism that could link an underlying genetic predisposition to environmental perturbation and transform a “regulated predisposition” into autoimmune diabetes, namely, failure to maintain regulatory CD4  CD25  T cell function. The Journal of Immunology, 2003, 170: 3592–3602. I t is generally agreed that environmental perturbants, specif- ically viral infection, play a role in the expression of type 1 diabetes mellitus in genetically susceptible individuals (1). Unfortunately, although data from epidemiological studies and the analysis of discordant monozygotic twins strongly support the con- cept, little is known with certainty regarding underlying mecha- nisms. Human studies of the interaction of environment and ge- netic predisposition are exceedingly difficult. The most widely used animal model of autoimmune diabetes, the nonobese diabetic (NOD) 4 mouse, models the process poorly because the over- whelming majority of perturbants reduce the frequency of dia- betes and often prevent it entirely (2). Among murine viruses, encephalomyocarditis virus (3), mouse hepatitis virus (4), lym- phocytic choriomeningitis virus (5), and others reduce the fre- quency of disease (6). Only pancreatotropic Coxsackie virus has been documented to accelerate disease in these mice (7), but this murine infection is associated with a significant degree of inflammation and exocrine pancreatic necrosis (8) not charac- teristic of the prediabetic state in humans who develop type 1A diabetes (9, 10). Recent attempts to address the problem have relied on trans- genic mouse systems. For example, using a tetracycline-inducible promoter for TNF- and CD80 expression, Green et al. (11) were able to synchronize environmental perturbation with the expres- sion of diabetes. Using this system, they defined periods of im- munoregulation and anti-islet aggression and suggested that failure to maintain regulatory CD4 + CD25 + T cell function plays a critical role in disease progression and expression. To study the problem of environmental factors in diabetes in- duction and the possible role of regulatory CD4 + CD25 + T cells in a less artificial system, we have used the BBDR rat. Spontaneous type 1 diabetes does not occur in viral Ab-free BBDR rats (12), but the disorder can be induced in about one-third of these animals by infection with the UMass strain of rat virus (RV-UMass, formerly designated Kilham’s rat virus) (13, 14). Induction of diabetes is not associated with infection of the islets of Langerhans (15) and the combination of RV-UMass infection plus injection of the immune activator poly(I:C) leads to insulitis and diabetes in nearly all treated animals (16). RV is a parvovirus, a ssDNA virus that infects several animal species including rodents (17) and humans (18). RV, H-1, and the recently described rat parvovirus-1 (RPV-1, formerly designated orphan parvovirus) are the three autonomous parvoviruses that in- fect rats (17). RV encodes three overlapping structural proteins, VP1, VP2, and VP3, and two overlapping nonstructural proteins, NS1 and NS2 (17). RV is most homologous with H-1; RV and H-1 VP proteins are 80% homologous and their NS proteins are 100% homologous. The mechanism by which RV-UMass infection leads to the ex- pression of autoimmune diabetes in the BBDR rat is not yet fully understood. The virus infects lymphoid organs and endothelial Programs in *Immunology and Virology and † Molecular Medicine and Departments of ‡ Medicine and § Pathology, University of Massachusetts Medical School, Worces- ter, MA 01655; and ¶ Division of Cell Biology, Section of Immunology, Faculty of Medical Sciences, University of Groningen, Groningen, The Netherlands Received for publication April 24, 2002. Accepted for publication January 30, 2003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by Grants DK49106, DK36024, and DK25306 and Center Grant DK32520 from the National Institutes of Health. J.-L.H. was supported by the Ter Meulen Fund, Royal Netherlands Academy of Arts and Sciences (Project 6419) and the Dutch Diabetes Foundation (Project 2001.05.003). 2 The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health. 3 Address correspondence and reprint requests to Dr. Aldo A. Rossini, Diabetes Di- vision, University of Massachusetts Medical School, 373 Plantation Street, Suite 218, Worcester, MA 01605. E-mail address: aldo.rossini@umassmed.edu 4 Abbreviations used in this paper: NOD, nonobese diabetic; RV-UMass, UMass strain of rat virus; RPV-1 rat parovirus 1; BrdU, 5-bromo-2'-deoxyuridine. The Journal of Immunology Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00