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Original Article On the Edge of Autoimmunity T-Cell Stimulation by Steady-State Dendritic Cells Prevents Autoimmune Diabetes Dunja Bruder,1 Astrid M. Westendorf,1 Wiebke Hansen,1 Silvia Prettin,1 Achim D. Gruber,2 Yingjie Qian,3 Harald von Boehmer,4 Karsten Mahnke,3 and Jan Buer1,5 Targeting of antigens to immature dendritic cells has been this process, i.e., pancreata from diabetic mice are infil- shown to result in antigen-specific T-cell tolerance in vivo. trated with T-cells (2), and crossing of diabetes-prone In the INS-HA/TCR-HA transgenic mouse model for type 1 NOD mice on the SCID (3) background interferes with diabetes, we tested the potential of the dendritic cell– development of diabetes. Moreover, the disease can be specific monoclonal antibody DEC-205 conjugated to the induced in animals by transferal of autoreactive T-cells hemagglutinin (HA) antigen (DEC-HA) to prevent disease (4), and neonatal thymectomy prevents the disease (5). onset. Whereas untreated INS-HA/TCR-HA mice all de- velop insulitis, and ϳ40% of these mice become diabetic, In the INS-HA/TCR-HA transgenic mouse model, which repeated injection of newborn mice with DEC-HA pro- is based on the concomitant expression of hemagglutinin tected almost all mice from disease development. Histolog- (HA) in the ␤-cells of the pancreas and an HA-specific ical examination of the pancreata revealed significant major histocompatibility complex class II–restricted trans- reduction of peri-islet infiltrations in DEC-HA–treated genic T-cell receptor (TCR) specific for this antigen, one mice, and the islet structure remained intact. Moreover, -؉ observes 2–3 weeks after birth a rather strong accumula HA-specific CD4 T-cells from anti–DEC-HA–treated INS- tion of lymphocytes around the pancreatic islets. This HA/TCR-HA mice exhibited increased expression of Foxp3, infiltrate can persist for long periods of time, but the cytotoxic T-lymphocyte–associated antigen-4, and the im- ␤ munosuppressive cytokines interleukin-10 and transform- -cells undergo destruction in only one-third of the mice, ing growth factor-␤. The findings indicate that targeting of and diabetes develops (6). In this particular model, the the HA antigen to immature dendritic cells in vivo leads to diabetes incidence decreases in INS-HA/TCR-HA mice on Ϫ Ϫ a relative increase of antigen-specific Foxp3؉ regulatory the RAG / genetic background. Furthermore, HA-specific T-cells that suppress the development of type 1 diabetes. CD4ϩ T-cells isolated from double-transgenic mice exhibit Our results provide a basis for the development of novel a reduced proliferative capacity (7). These observations strategies focusing on prevention rather than treatment of suggest the possibility that immunoregulation by CD4ϩ autoimmune diseases. Diabetes 54:3395–3401, 2005 T-cells contributes to the control of ␤-cell destruction. This assumption is consistent with results showing that the development of diabetes can be provoked by maneu- ype 1, or insulin-dependent, diabetes is regarded vers that bypass immunoregulation, such as treatment as an immune-mediated disease in which ␤-cells with cyclophosphamide or transfer of HA-specific T-cells of the pancreatic islets of Langerhans are de- into lymphopenic mice expressing HA in the pancreas (7). stroyed as a consequence of inflammatory stim- Likewise, in the NOD mouse model for type 1 diabetes, an T imbalance between diabetogenic effectors and immuno- uli (1). Several observations demonstrated a pathological role of T-cells specific for ␤-cell–associated antigens in regulatory T-cell subsets has been described (8,9). The induction of antigen-specific tolerance is a tempting approach to prevent type 1 diabetes, particularly because From the 1Department of Mucosal Immunity, German Research Centre for some autoantigens triggering the disease are currently Biotechnology, Braunschweig, Germany; the 2Department of Veterinary Pa- being defined (10,11). In this context, the in vivo induction thology, Free University Berlin, Berlin, Germany; the 3Department of Derma- tology, University of Heidelberg, Heidelberg, Germany; the 4Dana Faber of antigen-specific regulatory T-cells that may prevent Cancer Institute, Harvard Medical School, Boston, Massachusetts; and the autoimmunity is of particular interest. Recently, it has 5Institute of Medical Microbiology, Hannover Medical School, Hannover, been shown that it is possible to artificially generate or Germany. Address correspondence and reprint requests to Dunja Bruder, Department expand suppressor T-cells inside (12,13) and outside (14) of Mucosal Immunity, German Research Centre for Biotechnology, Mas- the thymus, resulting in tolerance in the absence of cheroder Weg 1, D-38124 Braunschweig, Germany. E-mail: [email protected]. immunity. Another approach uses the immunomodulatory Received for publication 23 May 2005 and accepted in revised form 6 September 2005. properties of immature dendritic cells to induce T-cell D.B. and A.M.W. contributed equally to this work. tolerance rather than immunity. This attempt is based on CTLA, cytotoxic T-lymphocyte–associated antigen; DEC-HA, DEC-205 con- the use of a monoclonal antibody directed against the jugated to the hemagglutinin antigen; HA, hemagglutinin; IL, interleukin; TCR, endocytosis receptor DEC-205 that targets antigens in vivo T-cell receptor; TGF, transforming growth factor; TRP2, tyrosinase-related protein-2. nearly exclusively to dendritic cells (15). In contrast to © 2005 by the American Diabetes Association. other methods using in vitro–generated, i.e., mature 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 and/or activated, dendritic cells, targeting of antigens to with 18 U.S.C. Section 1734 solely to indicate this fact. DEC-205 allows efficient loading and antigen presentation DIABETES, VOL. 54, DECEMBER 2005 3395 PREVENTION OF AUTOIMMUNE DIABETES by immature, so-called steady-state dendritic cells. Results Histology. Pancreata were fixed with neutral buffered formalin, embedded in obtained in adoptive transfer models suggested that pre- paraffin, sectioned, and stained with hematoxylin and eosin. Staining for intracellular insulin was performed with a guinea pig anti-human insulin sentation of antigens by steady-state dendritic cells leads antibody from Dako and revealed with a horseradish peroxidase–conjugated to short-term proliferation of antigen-specific T-cells fol- goat anti–guinea pig conjugate. Histological details were quantified morpho- lowed by deletion (15,16). Such targeting of antigen can metrically using a Colorview II digital camera on a BX 41 Olympus light also lead to an increase in regulatory T-cells either by microscope and SIS image analysis software (Munster, Germany). Immuno- expansion of already existing cells or by conversion of histochemical stains for insulin were used to measure the size (area) of islets nonregulatory into regulatory T-cells (14,17). containing insulin-producing ␤-cells (controls) or, where appropriate, the area of the largest fragments of disrupted islands (INS-HA/TCR-HA). Areas were In this report, we show that repeated treatment of given (in ␮m2) as the means Ϯ SD, n ϭ 5. INS-HA/TCR-HA mice with anti–DEC-205 conjugated to Antibodies and flow cytometry. The monoclonal antibodies 6.5 (anti–TCR- the HA antigen (DEC-HA) conjugates protected mice from HA) and CD62L (Mel-14) were purified from hybridoma supernatants by developing spontaneous diabetes. In untreated or DEC- protein G affinity chromatography. All other antibodies were obtained from 205–treated controls, accumulation of lymphocytes BD Biosciences (San Jose, CA). Two- and three-color flow cytometry was performed on a FACSCalibur (BD Biosciences). For quantitative real-time around the pancreatic islets resulted in progression to ϩ ϩ diabetes in 40% of mice, whereas in DEC-HA–treated mice, RT-PCR, CD4 6.5 T-cells were sorted with a MoFlow cell sorter (Cytoma- ϩ tion, Fort Collins, CO), and purity was Ͼ97%. there was an increase of Foxp3 T-cells and of the Real-time RT-PCR. Total RNA was prepared from 2 ϫ 105 sorted T-cells immunosuppressive cytokines interleukin (IL)-10 and using an RNeasy kit (Qiagen, Hilden, Germany). cDNA synthesis was per- transforming growth factor (TGF)-␤, which efficiently pre- formed using Superscript II reverse transcriptase, oligo dT, and random vented the onset of diabetes. Thus, our results emphasize hexamer primers (Invitrogen) according to the manufacturer’s recommenda- the potential of targeting to tolerogenic dendritic cells as a tions. Quantitative real-time RT-PCR was performed in an ABI PRISM cycler (Applied Biosystems), using a SYBR Green PCR kit from Stratagene and tool to prevent autoimmunity in predisposed individuals. specific primers optimized to amplify 90- to 250-bp fragments from the different genes analyzed. A threshold was set in the linear part of the amplification curve, and the number of cycles needed to reach it was RESEARCH DESIGN AND METHODS calculated for every gene. Relative mRNA level were determined, using BALB/c mice were obtained from Harlan (Borchen, Germany). TCR-HA included standard curves for each individual gene and further normalization transgenic mice expressing a TCR-␣␤ specific for peptide 110–120 from to ribosomal protein S9. Melting curves established the purity of the amplified influenza HA presented by I-Ed have been described previously (18). TCR-HA band. Primer sequences are: Foxp3 (5Ј-CTG GCG AAG GGC TCG GTA GTC mice were crossed to INS-HA mice expressing the HA protein from the same CT-3Ј,5Ј-CTC CCA GAG CCC ATG GCA GAA GT-3Ј), cytotoxic T-lymphocyte– virus in pancreatic ␤-cells under the control of the rat insulin promoter (19) to associated antigen (CTLA-4; 5Ј-GGG CTG GGT CTT TAC ACT CAT TTT-3Ј, generate INS-HA/TCR-HA mice. The incidence of spontaneous diabetes in 5Ј-CTT CCT GTG GCA TTA ACT TTG TGT-3Ј), IL-10 (5Ј-CTG GAC AAC ATA double-transgenic mice was 40%.

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