Cytotoxic T-Cells From T-Cell Receptor Transgenic NOD8.3 Mice Destroy -Cells via the Perforin and Fas Pathways Nadine L. Dudek, 1 Helen E. Thomas, 1 Lina Mariana, 1 Robyn M. Sutherland, 2 Janette Allison, 1 Eugene Estella, 1 Eveline Angstetra, 1 Joseph A. Trapani, 3 Pere Santamaria, 4 Andrew M. Lew, 2 and Thomas W.H. Kay 1,5 Cytotoxic T-cells are the major mediators of -cell destruc- tion in type 1 diabetes, but the molecular mechanisms are not definitively established. We have examined the contri- bution of perforin and Fas ligand to -cell destruction using islet-specific CD8  T-cells from T-cell receptor trans- genic NOD8.3 mice. NOD8.3 T-cells killed Fas-deficient islets in vitro and in vivo. Perforin-deficient NOD8.3 T-cells were able to destroy wild-type but not Fas-deficient islets in vitro. These results imply that NOD8.3 T-cells use both pathways and that Fas is required for -cell killing only when perforin is missing. Consistent with this theory, transgenic NOD8.3 mice with -cells that do not respond to Fas ligation were not protected from diabetes. We next investigated the mechanism of protection provided by over- expression of suppressor of cytokine signaling-1 (SOCS-1) in -cells of NOD8.3 mice. SOCS-1 islets remained intact when grafted into NOD8.3 mice and were less efficiently killed in vitro. However, addition of exogenous peptide rendered SOCS-1 islets susceptible to 8.3 T-cell–mediated lysis. Therefore, NOD8.3 T-cells use both perforin and Fas pathways to kill -cells and the surprising blockade of NOD8.3 T-cell–mediated -cell death by SOCS-1 overex- pression may be due in part to reduced target cell recogni- tion. Diabetes 55:2412–2418, 2006 T ype 1 diabetes results from the selective destruc- tion of insulin-secreting -cells within the pan- creas. Autoreactive T-cells play a major role in -cell death both in humans and in the NOD mouse model of type 1 diabetes. Although efficient disease progression requires both CD4 + and CD8 + T-cells (1,2), the relative contribution of effector mechanisms used by these activated T-cells is unclear. Evidence from the NOD mouse implicates perforin, a major component of the lytic granules of cytotoxic T-cells (CTLs) and natural killer cells, as an important mediator of -cell death. Although genetic deficiency of perforin significantly reduces the incidence of diabetes in NOD mice, a small percentage of knockout animals still develop disease (3). Proinflamma- tory cytokines such as interferon (IFN)-, tumor necrosis factor-, and interleukin-1 are important candidates in non-perforin– dependent killing. Combinations of these cytokines are toxic to -cells in vitro (4) and may enhance recognition of -cells by upregulation of major histocom- patibility complex (MHC) class I molecules and cell death receptors including Fas. We have studied the effector mechanisms utilized by a population of CD8 + T-cells recognizing an epitope within the islet-specific glucose 6-phosphatase catalytic subunit– related protein (IGRP). In wild-type NOD mice, up to 40% of CD8 + T-cells infiltrating the islets recognize IGRP before the onset of disease (5– 8). Quantitation of IGRP- specific T-cells in the peripheral blood using MHC class I tetramers can be used to predict which NOD mice will develop diabetes (9), suggesting that this is an important T-cell specificity in the NOD model. The expression of an IGRP-specific T-cell receptor (TCR) in NOD mice (NOD8.3) accelerates the onset and increases the inci- dence of spontaneous disease (10). Such TCR transgenic mice provide simplified models of diabetes in which the contribution of a single population of CD4 + or CD8 + T-cells can be studied in isolation. These mice develop diabetes with high disease penetrance and at an acceler- ated rate but provide a basis for understanding the mech- anisms involved in nontransgenic mice and possibly in humans. We have previously reported that overexpression of suppressor of cytokine signaling-1 (SOCS-1) in -cells of NOD8.3 mice completely prevents diabetes, although in- sulitis and T-cell activation appear to proceed normally (11). We have now analyzed the effector mechanisms used by 8.3 T-cells to understand which of these can be blocked by SOCS-1 in vivo. The surprising result is that 8.3 T-cells clearly use both the perforin-granzyme and Fas pathways to kill -cells, indicating that both of these pathways are blocked by overexpression of SOCS-1. RESEARCH DESIGN AND METHODS NOD mice expressing a SOCS-1 transgene under the control of the rat insulin promoter (RIP-SOCS-1) (11) and NOD8.3 mice expressing the TCR rear- rangements of the H-2K d –restricted, -cell–reactive, CD8 + T-cell clone NY8.3 have been described (12). Perforin knockout NOD mice were obtained from The Jackson Laboratories type 1 diabetes repository and crossed to NOD8.3 transgenic mice (NOD8.3.PO o/o ). NODlpr (13), RIP-B7-NOD.scid (14), RIP- From 1 St. Vincent’s Institute, Fitzroy, Victoria, Australia; the 2 Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia; the 3 Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; the 4 Julia McFarlane Diabetes Research Center and Department of Microbiology and Infectious Disease, University of Calgary Faculty of Medicine, Calgary, Alberta, Canada; and 5 The University of Melbourne Department of Medicine, St. Vincent’s Hospital, Fitzroy, Victoria, Australia. Address correspondence and reprint requests to Professor Thomas W.H. Kay, St. Vincent’s Institute, 41 Victoria Parade, Fitzroy, Victoria 3065, Austra- lia. E-mail: tkay@svi.edu.au. Received for publication 24 January 2006 and accepted in revised form 8 June 2006. CTL, cytotoxic T-cell; dn, dominant-negative; FasL, Fas ligand; FADD, Fas-associated death domain; IGRP, islet-specific glucose 6-phosphatase catalytic subunit–related protein; IFN, interferon; MHC, major histocompati- bility complex; PLN, pancreatic lymph node; RIP, rat insulin promotor; SOCS-1, suppressor of cytokine signaling-1; TCR, T-cell receptor. DOI: 10.2337/db06-0109 © 2006 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. 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