Original Article Phenotype and Functional Characteristics of Islet-Infiltrating B-Cells Suggest the Existence of Immune Regulatory Mechanisms in Islet Milieu Maria Carmen Puertas, 1 Jorge Carrillo, 1 Xavier Pastor, 1 Rosa Maria Ampudia, 1 Aurora Alba, 1 Raquel Planas, 1 Ricardo Pujol-Borrell, 1 Marta Vives-Pi, 1 and Joan Verdaguer 1,2 B-cells participate in the autoimmune response that pre- cedes the onset of type 1 diabetes, but how these cells contribute to disease progression is unclear. In this study, we analyzed the phenotype and functional characteristics of islet-infiltrating B-cells in the diabetes-prone NOD mouse and in the insulitis-prone but diabetes-resistant (NODNOR)F1 mouse. The results indicate that B-cells accumulate in the islets of both mice influenced by sex traits. Phenotypically and functionally, these B-cells are highly affected by the islet inflammatory milieu, which may keep them in a silenced status. Moreover, although islet- infiltrating B-cells seem to be antigen experienced, they can only induce islet-infiltrating T-cell proliferation when they act as accessory cells. Thus, these results strongly suggest that islet-infiltrating B-cells do not activate islet- infiltrating T-cells in situ, although they may affect the progression of the disease otherwise. Diabetes 56:940 –949, 2007 T ype 1 diabetes develops following the selective loss of pancreatic -cells due to a self-destruc- tive mechanism mediated by the patient’s own immune system (1). The NOD (nonobese dia- betic) mouse develops a form of autoimmune diabetes that resembles human type 1 diabetes (2). Studies with this animal model have established that islet-infiltrating -cell– reactive T-cells are the major effectors of -cell damage. However, other immune system cells are also crucial in the disease development. Among these cells, B-cells are essential in the onset and progression of type 1 diabetes (rev. in 3), and although it is not fully understood when and how these cells participate in type 1 diabetes, it is known that they produce autoantibodies against many -cell autoantigens (rev. in 4) and act as antigen-present- ing cells (APCs) (3). On the one hand, the production of specific autoantibodies directly correlates with the pro- gression of type 1 diabetes in both humans and NOD mice (5,6). In addition, in NOD mice, the transmission of maternal islet -cell–reactive autoantibodies during preg- nancy contributes to the disease progression in offspring (7). On the other hand, as APCs, B-cells may be involved in regulating the activation of islet-reactive T-cells. This activity may be more relevant in the genesis and/or devel- opment of type 1 diabetes, since an impaired B-cell– mediated antigen presentation by major histocompatibility complex class II results in a resistance to spontaneous type 1 diabetes development in NOD mice (8 –10). B-cells may also have a tolerogenic function, as the transfer of activated B-cells prevents type 1 diabetes, probably by triggering the apoptosis of -cell–reactive cytotoxic T-cells (CTLs) and/or inhibiting APC activity in NOD mice (11). In fact, the antigenic repertoire and functional status of B-cells may be different depending on the anatomical region and stage of the disease, and the behavior and role during the progression of type 1 diabetes may also be different. B-cells migrate to the pancreatic islets and infiltrate them in the same way as do T-cells, but the reasons remain unknown; however, since most -cell damage occurs by an in situ CTL response, these islet-infiltrating B-cells might also be relevant in the development of the disease. The aim of this study was to discover the phenotypical and func- tional traits of islet-infiltrating B-cells in NOD and (NODNOR)F1 mice (12). The results indicate that islet- infiltrating B-cells accumulate in the islets influenced by sex traits, and their phenotype is compatible with a downregulated population. Moreover, although islet-infil- trating B-cells seem to be antigen experienced, they can only induce islet-infiltrating T-cell proliferation when they act as accessory cells. In conclusion, islet-infiltrating B- cells may not activate islet-infiltrating T-cells in situ but may contribute to the onset of type 1 diabetes otherwise. RESEARCH DESIGN AND METHODS Breeding pairs of NOD, NOR (13), and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained by brother-sister mating under specific pathogen-free conditions, following the European Laws and Directives concerning the protection of animals for experimentation. In our facility, 85% female and 37% male NOD mice developed spontaneous autoimmune diabetes at 32 weeks of age. (NODNOR)F1 mice were gener- ated by crossing NOD with NOR mice. Most (NODNOR)F1 mice developed a nondestructive but severe form of insulitis, although only 19% of (NOD From the 1 Laboratory of Immunobiology for Research and Diagnosis (LIRAD) and Center for Transfusion and Tissue Bank (BST), Institut d’Investigacio Germans Trias i Pujol, Carretera de Can Ruti, Badalona, Barcelona, Spain; and the 2 Immunology Unit, Department of Ciencies Mediques Basiques, Faculty of Medicine, University of Lleida, Lleida, Catalonia, Spain. Address correspondence and reprint requests to Dr. Joan Verdaguer, Unit of Immunology, Departament de Ciencies Mediques Basiques, Facultat de Medi- cina, Universitat de Lleida, Carrer Montserrat Roig 2, 25008 Lleida, Catalonia, Spain. E-mail: joan.verdaguer@cmb.udl.es. Received for publication 30 March 2006 and accepted in revised form 21 December 2006. APC, antigen-presenting cell; CFSE, carboxy-fluorescein diacetate succin- imidyl ester; CTL, cytotoxic T-cell; FITC, fluorescein isothiocyanate; IFN-, -interferon; IL, interleukin; LPS, lipopolysaccharide; mAb, monoclonal anti- body; MHC, major histocompatibility complex. DOI: 10.2337/db06-0428 © 2007 by the American Diabetes Association. 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. 940 DIABETES, VOL. 56, APRIL 2007