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International Immunology Advance Access originally published online on March 30, 2006
International Immunology 2006 18(6):837-846; doi:10.1093/intimm/dxl020
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Perforin and Fas induced by IFN{gamma} and TNF{alpha} mediate beta cell death by OT-I CTL

Mark D McKenzie1, Nadine L Dudek1, Lina Mariana1, Mark MW Chong1, Joseph A Trapani2, Thomas WH Kay1 and Helen E Thomas1

1 St Vincent's Institute, Fitzroy, Melbourne, Australia
2 Cancer Immunology Program, Research Division, Peter MacCallum Cancer Centre, East Melbourne, Australia

Correspondence to: T. W. H. Kay; E-mail: tkay{at}svi.edu.au


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Direct interaction between auto-reactive CTL and specific peptide–MHC class I complexes on pancreatic beta cells is critical in mediating beta cell destruction in type I diabetes. We used mice with genetic modifications in three major pathways used by CTL, perforin, Fas and pro-inflammatory cytokines to assess the relative contribution of these mechanisms to beta cell death. In vitro-activated ovalbumin (OVA)-specific CTL, from OT-I TCR-transgenic mice, specifically killed transgenic beta cells expressing OVA (from RIP-mOVA mice) in a 16-h cytotoxicity assay. Perforin-deficient CTL had a reduced ability to kill OVA-expressing islets in vitro (22.1 ± 3.8%) compared with wild-type CTL (71.4 ± 4.6%). Fas-deficient islets were only slightly protected from wild-type CTL but were completely protected from the residual killing observed with perforin-deficient CTL. Residual cytotoxicity in perforin-deficient CTL was also prevented by overexpression of SOCS-1, which blocks multiple cytokine signaling pathways. It was also prevented by pre-incubation with anti-tumor necrosis factor-alpha (anti-TNF{alpha}) antibody or by blocking IFN{gamma} responsiveness through expressing a dominant negative IFN{gamma} receptor. Perforin-deficient CTL produced IFN{gamma} and TNF{alpha} that was shown to directly induce islet Fas expression during the assays. This suggests that Fas-deficiency, SOCS-1 overexpression and blockade of IFN{gamma} and TNF{alpha} all protect beta cells from residual cytotoxicity of perforin-deficient CTL by blocking Fas upregulation. These findings indicate that wild-type CTL destroy antigen-expressing islets via a perforin-dependent mechanism. However, in the absence of perforin, the Fas/FasL pathway provides an alternative mechanism dependent on islet cell Fas upregulation by cytokines IFN{gamma} and TNF{alpha}.

Keywords: apoptosis, autoimmunity, cytotoxic T cells, diabetes, ovalbumin


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Type I diabetes (T1D) results from the autoimmune destruction of insulin-producing islet beta cells of the pancreas by apoptosis (1). Death of beta cells in T1D is due to cell types and molecular mechanisms (2) that have been identified mainly in animal models. In the non-obese diabetic (NOD) mouse, that shares many characteristics with human disease, auto-reactive CD8+ CTL are essential for beta cell destruction. Diabetes is prevented in NOD mice lacking MHC class I on their beta cells (3–5) and pathogenic CD8+ T cells specific for proinsulin (6) or islet-specific glucose 6-phosphatase catalytic subunit-related protein (IGRP) (7) can induce diabetes. In addition to NOD, there are other models of diabetes mediated by CTL that may provide insights into the mechanism of beta cell death in T1D. These involve transgenic expression of antigens under control of the insulin promoter including ovalbumin (OVA) (8–10), influenza virus hemagglutinin (HA) (11,12) and LCMV glycoprotein (13,14) in beta cells.

Previous studies using perforin-deficient mice have indicated that perforin is critical for the effector phase of disease (15–17). NOD mice lacking perforin display a significantly reduced incidence (16% compared with 77%) and delayed onset of diabetes despite developing normal insulitis (16). However, the fact that some perforin-deficient mice still develop T1D suggests that, in the absence of perforin, islet infiltration can lead to beta cell destruction by other mechanisms that appear from these data to be less effective than perforin. Blocking Fas signaling in beta cells of NOD mice, by overexpression of a dominant negative form of Fas itself or the intracellular signaling molecule Fas-associated death domain, partially protects from diabetes in NOD mice (18,19). In contrast, specific deletion of Fas from beta cells has no effect on diabetes mediated by CD4+ T cells specific for influenza HA expressed transgenically in beta cells of C57Bl/6 (B6) mice (20). Pro-inflammatory cytokines may also be important in T1D progression. In vitro several cytokines including IL-1, IFN{gamma} and tumor necrosis factor-alpha (TNF{alpha}) have been shown to have effects on beta cells including upregulation of Fas (21,22), MHC class I (23) and chemokine expression (24) as well as inducing direct cytotoxicity (25–28). Yet, despite the numerous effects of cytokines on beta cells, there has been surprisingly little effect of genetic deficiency of individual cytokines or their receptors, such as IFN{gamma}, on progression to diabetes in NOD mice (29–31). Blocking multiple cytokines by overexpression of suppressor of cytokine signaling-1 (SOCS-1) in beta cells has been shown to partially prevent diabetes in non-transgenic NOD mice (32) and completely prevent diabetes mediated by CD8+ T cells (33).

T cell clones or T cells from TCR-transgenic mice can be used as simplified models for determining the mechanism of beta cell destruction used by CD4+ or CD8+ T cells. Spontaneous diabetes in NOD mice is more complex involving polyclonal T cells of both CD4 and CD8 lineage as well as probable contributions from other cells (34,35). T cell clones also have the advantage of delivering effector molecules to the surface of the target cell in an antigen-driven manner at concentrations likely to be more physiological than purified or recombinant effector molecules. One of the aims of our work has been to identify T cells that kill by defined mechanisms to use as reagents for developing and testing therapeutic strategies for beta cell preservation. We have utilized a well-characterized TCR-transgenic model in which naive OVA-specific CD8+ T cells from the TCR-transgenic mouse line OT-I cause rapid diabetes due to CTL-dependent beta cell death when adoptively transferred into recipients expressing a membrane-bound form of OVA in their beta cells (RIP-mOVA) (8–10). Despite the wealth of information concerning the immunology of this model, the precise effector mechanisms used by activated OVA-specific CTL in mediating beta cell destruction remain unknown. It is known that OT-I CTL kill tumor cell lines in vitro in a perforin-dependent manner (36–38); however, the killing of standard tumor cell targets is unlikely to be identical to primary beta cell targets (17).

We have performed a detailed analysis of the mechanisms used by OT-I CTL to kill pancreatic islets in vitro by using beta cells genetically modified to be protected from common mechanisms of cell death. Our data show that CTL are armed with a number of cytotoxic mechanisms that can destroy target beta cells in an antigen-specific manner.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice, cell lines and reagents
All mice used were between 6 and 16 weeks old and were bred and/or maintained under specific pathogen-free conditions at St Vincent's Institute of Medical Research. B6, B6.MRLlpr/lpr (B6lpr/lpr) and OT-I mice were obtained from the Walter and Eliza Hall Institute animal breeding facility. OT-I TCR-transgenic mice express predominantly H-2Kb restricted CD8+ T cells specific for the OVA peptide, SIINFEKL (OVA257–264). RIP-mOVA mice expressing a membrane-anchored form of OVA under the control of the rat insulin promoter (RIP) in pancreatic beta cells (obtained from the Walter and Eliza Hall Institute) have previously been described (8). OT-I perforin-deficient (OT-I pfp–/–) TCR-transgenic mice were provided by Prof. J Trapani (Peter MacCallum Cancer Institute). The RIP-SOCS-1 transgene was constructed by subcloning the SOCS-1 cDNA into the ClaI restriction site of the RIP7 expression vector (39). B6 mice expressing RIP-SOCS-1 (B6.RIP-SOCS-1) were generated by microinjection of the transgene directly into B6 embryos. RIP-{Delta}{gamma}R mice that express a dominant negative IFN{gamma} receptor ({Delta}{gamma}R) under control of the RIP promoter were generated by backcrossing the NOD-RIP-{Delta}{gamma}R mice (40) onto the B6 strain for 16 generations. Mice deficient in IL-1RI were obtained from M. Labow, Roche and backcrossed onto a B6 background for six generations.

Recombinant murine IFN{gamma} (used at 100 units ml–1) and recombinant murine TNF{alpha} (used at 250 units ml–1) were obtained from Genentech (South San Francisco, CA, USA). TNF{alpha} activity was neutralized with the anti-TNF{alpha} antibody XTP (rat IgG1, Geeta Chaudri, University of Sydney, Australia) added to the targets at 20 µg ml–1 prior to the addition of activated CTL.

The parent H-2Kb EL4 and the stably OVA-transfected EG-7 cell lines were obtained from American Type Culture Collection (Manassas, VA, USA).

Islet isolation
Islets of Langerhans were isolated from mice according to methods of Liu and Shapiro (41). In brief, the common bile duct was cannulated, and the pancreas was distended by intraductal injection of 3 ml of 1.2 U ml–1 of collagenase P (Roche, Basel, Switzerland). Pancreata were dissected and digested at 37°C for 15 min. Islets were separated from the pancreatic digest on a histopaque-1077 density gradient (Sigma-Aldrich, St Louis, MO, USA). Islets were washed and hand picked and cultured overnight at 37°C and 5% CO2 in CMRL medium-1066 (Life technologies, Gaithersburg, MD, USA) supplemented with 100 U ml–1 penicillin, 100 µg ml–1 streptomycin, 2 mM glutamine and 10% FCS.

Generation of OT-I CTL
Splenocytes from wild-type and perforin-deficient OT-I TCR-transgenic mice were stimulated in vitro with irradiated (3000 rads) B6 splenocytes pulsed with 0.1 µM OVA257–264 peptide. Cells were cultured at 37°C in 5% CO2 in RPMI-1640 (Life technologies) supplemented with 100 U ml–1 penicillin, 100 µg ml–1 streptomycin, non-essential amino acids and 10% FCS (RF10). On day 3 of culture 10 U ml–1 of recombinant human IL-2 (rhIL-2; National Cancer Institute, Rockville, MD, USA) was added and activated cells were further expanded in the presence of rhIL-2. Dead or dying cells were removed the day before the assay on a Ficoll-paque density gradient (Amersham Bioscience, Uppsala, Sweden). On day 7 of culture activated OT-I CTL were used in 51Cr cytotoxicity assays.

51Cr release assays
Isolated whole islets or target cell lines were washed in RF10 media and loaded with 150 µCi of sodium chromate [51Cr] (Amersham Pharmacia Biotech, Piscataway, NJ, USA) for 90 min; 45 min into loading targets were pulsed with 0.1 µM of control HSV498–505 (SSIEFARL) peptide (Auspep, Australia) or OVA257–264 (SIINFEKL) peptide (Auspep). Titration of peptide established 0.1 µM as the optimal concentration resulting in maximum killing of target cells. After 90 min targets were washed to remove excess 51Cr and peptide and resuspended in pre-warmed RF10. Targets were then plated out at 104 cells per well (10 islets) in triplicate in a 96-well U-bottom plate in 100 µl. The desired concentration of effectors was added in 100 µl and incubated for 16 h at 37°C and 5% CO2. Media alone and 2% Triton X-100 was added to target cells for determination of spontaneous and maximum cell lysis, respectively. After incubation, plates were centrifuged and 100 µl of supernatant was harvested and counted by a {gamma}-counter. The percentage spontaneous release when compared with maximum was between 4 and 10% when using target cell lines, and between 10 and 25% when using whole islet targets. The percentage-specific lysis was calculated by the following formula:

Formula

Flow cytometry
Isolated islets were either untreated or treated with in vitro-activated perforin-deficient OT-I CTL (20:1 effector to target ratio) or recombinant cytokines for 16 h. To neutralize TNF{alpha} activity islets were pre-treated with anti-TNF{alpha} antibody (XTP). Islets were then washed and T cells removed by handpicking islets several times into fresh media. Islets were dispersed into single cells using trypsin [0.2% (Calbiochem, La Jolla, CA, USA), 10 mmol l–1 EDTA in HBSS]. Dispersed islets were then washed free of trypsin and allowed to recover in culture medium for 1 h before staining. Hamster anti-mouse Fas (Jo2; PharMingen, San Diego, CA, USA) followed by biotinylated anti-hamster Ig (Pharmingen) and PE-conjugated streptavidin (Caltag, Burlingame, CA, USA) was used for analysis of surface Fas expression. Islet cells were also analyzed for expression of class I MHC expression using biotinylated anti-mouse MHC class I/H-2Db (28-14-8; Pharmingen) followed by PE-conjugated streptavidin. Dead cells and leukocytes were excluded by staining with propidium iodide (3.3 µg ml–1) and anti-CD45 (3F11, Pharmingen), respectively. Cells were analyzed on a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA).

Real-time reverse transcription–PCR analyses
Total RNA was isolated using TRIzol reagent (Life technologies). Real-time reverse transcription (RT)–PCR analysis was performed with primer and probe Assay-on-demand sets (Applied Biosystems) for IFN{gamma} and IL-1ß. The primers 5'-TCATGCACCACCATCGGA-3' and 5'-GAGGCAACCTGACCACTCTCC-3' and probe 5'-6-FAM-AATGGGCTTTCCGAATTCACTGGAGC-BH-Q1-3' were used for TNF{alpha}. Beta-2-microglobulin (ß2m) was used as a housekeeping reference gene. Analysis was performed on a Rotor-Gene RG-3000 cycler (Corbett Research, Australia). Results represent mean ± SD of duplicates from three independent samples.

ELISA
To measure TNF{alpha} and IFN{gamma} secretion, 1 x 106 activated perforin-deficient OT-I CTL were incubated with 100 B6 islets pulsed with 0.1 µM of OVA257–264 peptide. Supernatants were harvested after 16 h and assayed by ELISA for TNF{alpha} (MTA00) and IFN{gamma} (MIF00) according to manufacturer's instructions (R&D Systems, Minneapolis, MN, USA).

Statistical analysis
Analyses of data were performed using the program GraphPad Prism (GraphPad Software, San Diego, CA, USA). Cytotoxicity assays were analyzed with paired t tests. Fas and MHC class I expression was analyzed by one-way analysis of variance (ANOVA) with Bonferroni's post-test for comparison of multiple columns.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Killing of OVA-expressing target cell lines and islets in vitro is significantly reduced in the absence of perforin
We first examined cytotoxic activity of in vitro-stimulated OT-I CTL from wild-type and perforin-deficient mice. Splenic CD8+ T cells from wild-type and perforin-deficient OT-I mice were stimulated with irradiated B6 splenocytes pulsed with OVA257–264 peptide. After 7 days' culture, we tested the ability of activated cells to lyse target cell lines. As target cells we used the H-2Kb line EL4 in the presence of either OVA257–264 peptide or an irrelevant H-2Kb-binding peptide (HSV498–505), and EG-7 cells, which have been stably transfected to express OVA. Stimulated OT-I CTL specifically killed the OVA-expressing target cell lines, and not the control cells after 5 h in vitro (Fig. 1A). Cytotoxicity was reduced when perforin-deficient CTL were used, or when the perforin inhibitor concanamycin A (CMA) (42) was added to the CTL, suggesting that perforin plays a major role in killing of target cell lines by OT-I CTL.


Figure 1
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  		Fig. 1 Killing of OVA-expressing target cell lines and islets in vitro is significantly reduced in the absence of perforin. Activated OT-I CTL from wild-type (OT-I) and perforin-deficient (OT-I pfp–/–) mice were tested for cytotoxicity against (A) peptide-pulsed EL4 and OVA-expressing EG-7 target cell lines at an effector to target ratio (E:T) of 20:1 for 5 h. Wild-type OT-I effectors were also pre-incubated with concanamycin A (CMA) at 100 ng ml–1, a reagent that prevents perforin release. Each sample was performed in triplicate and data shown is representative of at least three experiments. (B) Peptide-pulsed B6 islets and RIP-mOVA transgenic islets for 16 h. Each sample was performed in triplicate. Data from two experiments were pooled. The mean ± SEM is shown. *P < 0.05, **P < 0.01, #P < 0.0005, wild-type OT-I versus perforin-deficient OT-I (paired t test).

 
We then extended our analysis of OT-I cytotoxicity to primary islet targets. To generate islet target cells, we either pulsed B6 islets with OVA257–264 or isolated islets from RIP-mOVA transgenic mice, which express a membrane-anchored form of OVA in their beta cells. Killing was not observed after 5 h, the time typically used for target cells (data not shown). CTL-mediated cytotoxicity was therefore measured after a 16-h incubation with whole islets, a time found to yield satisfactory results. It is unclear whether this longer incubation period was necessary to allow CTL to access all cells within the islet, a cluster of ~1000 cells, or because islet cells are intrinsically more resistant to cytotoxic mechanisms than cell lines like EL4. This resistance is not only due to low basal class I MHC levels as pre-incubation with IFN{gamma} did not substantially accelerate cytotoxicity (data not shown). Activated OT-I CTL specifically killed OVA257–264-pulsed B6 and transgenic RIP-mOVA islets in a dose-dependent manner (Fig. 1B). Islets pulsed with control peptide were not killed. This killing was H-2Kb restricted and MHC class I dependent, as it did not occur when using NOD islets (H-2Kd haplotype) or islets lacking MHC class I (ß2mnull) (data not shown).

When perforin-deficient CTL were used, there was significantly reduced killing compared with wild-type OT-I CTL (Fig. 1). There was residual cytotoxicity of up to 25% specific lysis with perforin-deficient CTL.

Perforin-deficient OT-I CTL do not kill Fas-deficient islets in vitro
Because Fas/FasL is an important pathway in cytotoxicity induced by CTL, we hypothesized that the reduced killing by perforin-deficient OT-I CTL may be due to Fas or to pro-inflammatory cytokines. To test this we examined the cytotoxic activity of wild-type and perforin-deficient OT-I effectors against islets isolated from Fas-deficient B6lpr/lpr mice (Fig. 2). Wild-type OT-I cells killed OVA257–264-pulsed B6lpr/lpr islets with only slightly reduced efficiency in comparison to B6 islets. In contrast perforin-deficient OT-I CTL were unable to kill B6lpr/lpr islets, suggesting that the residual islet destruction observed in the absence of perforin is due to Fas.


Figure 2
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  		Fig. 2 Perforin-deficient OT-I CTL cannot kill Fas-deficient islets in vitro. Activated OT-I CTL from wild-type (OT-I) and perforin-deficient OT-I (OT-I pfp–/–) mice were tested for cytotoxicity against peptide-pulsed islets from B6 and Fas-deficient B6lpr/lpr mice for 16 h. Each sample was performed in triplicate. Data from two experiments were pooled. The mean ± SEM is shown. *P < 0.05, **P < 0.005, #P < 0.0001 (paired t test).

 
Overexpression of SOCS-1 in beta cells protects islets from killing in the absence of perforin
SOCS-1 is a negative regulator of cytokines that signal through the Jak-STAT pathway and has been shown to inhibit the action of multiple cytokines (43). We have previously produced transgenic NOD mice that overexpress SOCS-1 in beta cells and shown that IFN{gamma}-induced MHC class I upregulation and IFN{gamma} + TNF{alpha}-induced Fas upregulation on beta cells was blocked (33). To examine the effects of SOCS-1 overexpression in the OT-I model, we generated B6.RIP-SOCS-1 transgenic mice by directly microinjecting the transgene into B6 embryos. Two transgenic lines were generated, both expressing SOCS-1 in beta cells. To test the ability of the SOCS-1 transgene to inhibit cytokine signaling, we treated islets from B6.RIP-SOCS-1 mice and littermate controls with IFN{gamma} and stained cells for MHC class I expression. While MHC class I expression rose on all cells from non-transgenic B6 mice, and on non-beta cells from B6.RIP-SOCS-1 transgenic mice, expression levels on the majority of beta cells of transgenic mice remained at basal levels. When analyzed by flow cytometry, 18% of beta cells remained responsive to IFN{gamma}, suggesting an incomplete pattern of SOCS-1 expression (Fig. 3).


Figure 3
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  		Fig. 3 RIP-SOCS-1 inhibits IFN{gamma}-induced MHC class I upregulation in beta cells. Purified islets from B6.RIP-SOCS-1 mice were treated with 100 U ml–1 IFN{gamma} for 48 h then MHC class I expression was measured by flow cytometry. A representative experiment from one of two lines (B6.RIP-SOCS-1#21) is shown. Beta cells (circled in one panel) were identified based on their high autofluorescence (FL-1 channel) compared with other islet cells (48). Dead cells and leukocytes were excluded by staining with propidium iodide (3.3 µg ml–1) and anti-CD45 (3F11, Pharmingen), respectively.

 
SOCS-1 overexpression in beta cells prevents diabetes in CD8+ TCR-transgenic NOD8.3 mice, although pancreas infiltration is not affected (33). CD8+ T cells from NOD8.3 mice recognize an epitope of IGRP (IGRP206–214) and these mice display an accelerated form of diabetes in comparison to NOD mice (7). Recent unpublished data from our laboratory has suggested that SOCS-1 overexpression prevents both perforin- and Fas-mediated mechanisms induced by NOD8.3 T cells (N. Dudek, T. Kay, unpublished results). We performed in vitro cytotoxicity assays on islets isolated from B6.RIP-SOCS-1 mice to address whether this was also the case for OT-I CTL. Activated wild-type OT-I CTL were able to kill OVA257–264-pulsed B6.RIP-SOCS-1 islets with only slightly reduced efficiency compared with B6 islets (Fig. 4). However, perforin-deficient OT-I CTL were unable to kill OVA257–264-pulsed B6.RIP-SOCS-1 islets. These data suggest that blocking multiple cytokines, including IFN{gamma}, with SOCS-1 can prevent perforin-independent death induced by OT-I CTL, but has little if any effect on perforin-dependent cell death. This is different to islet cell death induced by NOD8.3 T cells for which perforin-dependent cytotoxicity was inhibited by blockade of the effect of cytokines on target beta cells.


Figure 4
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  		Fig. 4 SOCS-1 protects beta cells from perforin-deficient OT-I CTL. Activated OT-I CTL from wild-type (OT-I) and perforin-deficient (OT-I pfp–/–) mice were tested for cytotoxicity against peptide-pulsed B6 or B6.RIP-SOCS-1 islets at an E:T ratio of 10:1 and 20:1 for 16 h. Each sample was performed in triplicate. Data were pooled from two experiments. The mean ± SEM is shown. *P < 0.0005 (paired t test).

 
Blocking individual pro-inflammatory cytokines, IFN{gamma} and TNF{alpha} prevents islet destruction by perforin-deficient OT-I CTL
To test which individual cytokines are likely to be responsible for the effect of SOCS-1, IFN{gamma}, TNF{alpha} and IL-1 were blocked individually. B6.RIP-{Delta}{gamma}R islets express a dominant negative form of the IFN{gamma} receptor on pancreatic beta cells. Beta cells from these islets are unresponsive to physiological doses of IFN{gamma} (40). Figure 5A shows the killing of peptide-pulsed B6.RIP-{Delta}{gamma}R islets in comparison to B6 islets. There was no significant difference in lysis of OVA257–264-pulsed B6 and B6.RIP-{Delta}{gamma}R islets mediated by wild-type OT-I CTL. In contrast, perforin-deficient OT-I CTL were unable to kill B6.RIP-{Delta}{gamma}R islets suggesting that IFN{gamma} facilitates islet destruction in the absence of perforin.


Figure 5
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  		Fig. 5 Blocking IFN{gamma} or TNF{alpha} prevents islet destruction mediated by perforin-deficient OT-I CTL. Activated OT-I CTL from wild-type (OT-I) and perforin-deficient (OT-I pfp–/–) mice were tested for cytotoxicity against (A) peptide-pulsed islets from B6 mice and B6.RIP-{Delta}{gamma}R mice at E:T ratios of 10:1 and 20:1 for 16 h; (B) peptide-pulsed B6 islets in the presence or absence of anti-TNF{alpha} antibody at an E:T ratio of 20:1 for 16 h; (C) peptide-pulsed B6 or B6.IL-1R-deficient islets at various E:T ratios for 16 h. Each sample was performed in triplicate. Data were pooled from two to three similar experiments. The mean ± SEM is shown. *P < 0.05, #P < 0.0001 (paired t test).

 
A similar experiment using peptide-pulsed B6 islets was performed in the presence or absence of antagonist anti-TNF{alpha} antibody (Fig. 5B). Killing of OVA257–264 expressing islets mediated by wild-type OT-I CTL was slightly reduced with anti-TNF{alpha} treatment, suggesting that TNF{alpha} plays an apparent but minor role in this cytotoxicity. However, in the absence of perforin, killing was significantly reduced to background levels with anti-TNF{alpha} antibody treatment, indicating that TNF{alpha} is critical for cytotoxicity in the absence of perforin.

We also tested the role of IL-1 action on islet cells by using islets isolated from B6.IL-1 receptor I-deficient mice. B6.IL-1R-deficient mice were equally susceptible to killing mediated by wild-type OT-I CTL (Fig. 5C). A slight reduction was observed in the killing mediated by perforin-deficient OT-I CTL; however, this does not reduce the killing to basal levels, as observed with inhibition of IFN{gamma} or TNF{alpha}. This suggests that IL-1 is less critical for OT-I cytotoxicity.

IFN{gamma} and TNF{alpha} produced by perforin-deficient OT-I CTL cause upregulation of MHC class I and Fas on islet cells in vitro
These studies suggested that IFN{gamma} and TNF{alpha} are necessary for cytotoxicity induced by OT-I CTL in the absence of perforin. We therefore examined IFN{gamma} and TNF{alpha} mRNA production by antigen-stimulated perforin-deficient OT-I CTL in vitro by RT–PCR prior to and following antigen challenge. Figure 6 shows that in response to OVA257–264 antigen presented on B6 islets, mRNA levels for IFN{gamma} and TNF{alpha} are substantially increased (day 7 + OVA257–264), compared with day 0 or in vitro-activated cells (day 7). This increase in mRNA expression correlated with increased cytokine protein measured by standard sandwich ELISA (IFN{gamma} 185.8 ± 23.7 ng ml–1 and TNF{alpha} 1738.4 ± 123.4 pg ml–1). No detectable IFN{gamma} and TNF{alpha} were found in supernatants from cultures of OT-I CTL alone, OT-I CTL cultured with B6 islets pulsed with control HSV498–505 peptide or from islets alone (data not shown). IL-1ß expression did not increase in these cells. IL-1ß expression is higher in the day 0 samples probably due to macrophages present within the unfractionated spleen cells.


Figure 6
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  		Fig. 6 Perforin-deficient OT-I CTL produce IFN{gamma} and TNF{alpha} and not IL-1ß. RNA was isolated from naive whole splenocytes (day 0) and in vitro-activated perforin-deficient OT-I CTL prior to day 7 and following a 16-h incubation with B6 islets pulsed with OVA257–264 (day 7 + OVA257–264). Real-time RT–PCR analysis was performed with primers and probes for IFN{gamma}, TNF{alpha} and IL-1ß. ß2-microglobulin was used as a housekeeping reference gene. Results represent duplicates from three independent samples.

 
IFN{gamma} and TNF{alpha} can induce expression of key genes involved in diabetes, including MHC class I and Fas. In vitro, a combination of IFN{gamma} + TNF{alpha} is required for functional Fas expression, and either IFN{gamma} or TNF{alpha} alone induces MHC class I expression. Perforin-deficient OT-I CTL were incubated with islets for 16 h, the length of time used for the cytotoxicity assays. We used perforin-deficient OT-I CTL because these are less efficient at killing islets over this time, leaving sufficient islet cell mass for studying gene expression by flow cytometry and also to study the mechanism of killing in the absence of perforin. Cell-surface Fas expression on islets pulsed with OVA257–264 peptide and exposed to perforin-deficient OT-I CTL was upregulated in islets from wild-type B6 mice (Fig. 7A and B). Fas expression on these islets was not as high as islets treated with recombinant cytokines IFN{gamma} and TNF{alpha}; however, it was significantly higher than untreated islets. In contrast Fas expression on B6.RIP-SOCS-1 islets was not upregulated. Fas expression did not increase on B6lpr/lpr islets, and B6.IL-1R-deficient islets had similar levels to wild-type B6 islets. Fas expression induced by OT-I CTL on B6 islets was reduced by anti-TNF{alpha} antibody (Fig. 7C). Together these data suggest that IFN{gamma} and TNF{alpha} produced by perforin-deficient OT-I CTL led to upregulation of Fas on islets.


Figure 7
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  		Fig. 7 Perforin-deficient OT-I CTL directly cause Fas and MHC class I upregulation on islet cells in vitro. OVA257–264 peptide-pulsed islets from B6, B6lpr/lpr (lpr), B6.RIPSOCS-1 (SOCS-1) and B6.IL-1R-deficient (IL-1R) mice were co-cultured with in vitro-activated perforin-deficient OT-I CTL or recombinant cytokines for 16 h and analyzed for islet cell Fas and MHC class I expression by flow cytometry. (A) representative plots showing Fas expression on B6 islets after incubation with or without perforin-deficient OT-I CTL or recombinant cytokines (B) Fas expression on B6, lpr, SOCS-1, and IL-1R islet cells; (C) Fas expression on B6 islets treated with anti-TNF{alpha} antibody; (D) MHC class I expression on B6, lpr, SOCS-1 and IL-1R islet cells; (E) MHC class I expression on B6 islets treated with anti-TNF{alpha} antibody. The mean ± SEM is shown. Results represent data from between one and six independent samples from several experiments. *P < 0.001, #P < 0.01 (one-way ANOVA).

 
We then examined MHC class I expression after incubation of islets pulsed with OVA257–264 peptide with perforin-deficient OT-I CTL. MHC class I was upregulated on islets from B6, B6lpr/lpr and B6.IL-1R mice (Fig. 7D). MHC class I was not upregulated on the majority of beta cells from SOCS-1 overexpressing islets, however, MHC class I upregulation was observed to the same extent as in wild-type islets on the non-beta cells in SOCS-1 islets. This is consistent with the fact that SOCS-1 is expressed only in the majority but not all beta cells from the transgenic mice, and not in other islet cells including alpha cells, which make up the bulk of the non-beta cell population. Upregulation of MHC class I was not prevented with anti-TNF{alpha} antibody treatment suggesting that MHC class I upregulation was due primarily to effects of IFN{gamma} (Fig. 7E). These results suggest upregulation of genes such as MHC class I and Fas on the surface of the islet cells occurs during the incubation period.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The aim of this study was to determine the relative contribution of immune effector mechanisms used by OT-I CTL to destroy OVA-expressing beta cells in vitro in order to develop beta cell protective therapies. We have shown that OT-I CTL kill antigen-expressing islets predominantly via a perforin-dependent mechanism. However, in the absence of perforin, the Fas/FasL pathway provides an alternative mechanism dependent on islet cell Fas upregulation by cytokines IFN{gamma} and TNF{alpha}.

Because of the importance of CTL in the pathogenesis of diabetes and because of the usefulness of transgenic technology in expressing antigens of interest in the beta cell, numerous transgenic models of CTL-mediated diabetes have been developed. In addition, CTL have been extensively studied in the NOD mouse providing the opportunity to compare mechanisms of beta cell destruction in several well-characterized models to determine if the mechanisms used are generally the same or if they vary depending on the antigen targeted and the nature of the CTL. These models have also provided an opportunity to compare CTL mechanisms used to destroy beta cells with those used to kill other cell types.

Our current studies are consistent with the idea that CTL use both perforin and Fas pathways to destroy pancreatic beta cells. Our results show that wild-type OT-I CTL kill predominantly via perforin in cytotoxicity assays because either deficiency of perforin or inhibition of perforin by CMA substantially reduced the cytotoxicity of OT-I CTL. In contrast when Fas alone was eliminated and perforin killing was intact, there was no impact on killing. When both perforin and Fas pathways were eliminated no cytotoxicity was observed in vitro. This supports the idea that perforin is the predominant pathway used by CTL to destroy beta cells, but the Fas pathway can mediate beta cell death in the absence of perforin. This is also consistent with what is seen in vivo where perforin deficiency reduces the frequency of diabetes to 16%, whereas Fas has a much more modest effect. Thus perforin remains an important target of efforts to decrease CTL killing in T1D whether by reducing perforin delivery to beta cells or reducing the effect of perforin and granzymes once they are inside the beta cell.

Anti-TNF{alpha} antibodies or measures taken to reduce the impact of IFN{gamma} on beta cells reduced cytotoxicity by perforin-deficient OT-I CTL. This may be through upregulation of Fas or by other mechanisms such as direct cytotoxicity mediated by induction of inducible NO synthase and other pathways resulting in generation of toxic free radicals. Islets, unlike other target cell types, do not constitutively express Fas; it is upregulated by IFN{gamma} together with TNF{alpha} or IL-1 (21,22). Fas was found on the surface of islet cells by flow cytometry at the end of the CTL assay indicating that the time of the assay was sufficient for Fas upregulation. In addition IFN{gamma} and TNF{alpha} mRNA and protein were increased in the cultures at that time. Islets genetically deficient in Fas (lpr) were protected against perforin-deficient OT-I cytotoxicity with no residual cytotoxicity seen that could be due to mechanisms independent of perforin and Fas. The data therefore are consistent with the role of cytokines being able to upregulate Fas at least under the conditions of this in vitro assay. Many studies have demonstrated that the pro-inflammatory cytokines IFN{gamma}, TNF{alpha} and IL-1 can damage pancreatic islets in vitro, however, this is only detectable over a prolonged incubation period. Cytokines alone do not induce lysis of islets in the 16-h time frame in our experiments (data not shown). It is unlikely that TNF{alpha}-mediated apoptosis via the caspase pathway accounts for residual cytotoxicity of perforin-deficient OT-I cells because TNF does not normally signal cell death pathways in primary beta cells unless protein synthesis is inhibited or the NF{kappa}B pathway is blocked (44,45).

It is also possible that inhibition of cytokines may prevent MHC class I upregulation on beta cells in the CTL assay and therefore block killing because of decreased recognition. SOCS-1 overexpression in beta cells protects from diabetes in NOD and completely protects from diabetes in a NOD8.3 TCR-transgenic model. Perforin-mediated killing is MHC class I dependent and therefore it was surprising that wild-type OT-I CTL were able to kill SOCS-1 islets, which only express basal levels of MHC class I, as effectively as non-transgenic B6 islets. This is different to the protective effect of SOCS-1 overexpression on killing by NOD8.3 T cells and suggests that the dependence on MHC class I expression of killing of islet targets may vary depending on the CTL and antigen being studied. We reasoned that basal levels of MHC class I, the high affinity of OT-I CTL (46) in comparison to NOD8.3 CTL (47), as well as the concentration of cognate peptide in these in vitro assays were sufficient to facilitate killing.

There is an emerging picture that CTL from islet-specific models including OT-I mice, NOD8.3 mice and influenza HA-specific clone 4 TCR-transgenic mice (11,12) destroy beta cells by similar mechanisms, although there are significant differences between them. Both perforin- and Fas-dependent mechanisms are significant in in vitro cytotoxicity assays. When both are blocked cytotoxicity is minimal, although it is possible that additional mechanisms might emerge in vivo over longer periods of observation. It is possible that the kinetics of these cytotoxic pathways, and delivery and concentration of the effector molecules may differ in vitro and in vivo. Nevertheless the low levels of basal MHC class I protein expressed on beta cells and the lack of constitutive Fas expression mean that these pathways are potentially targets for beta cell protection. Blockade of effector mechanisms as well as blockade of recognition of beta cells by CTL remain potential therapeutic approaches against autoimmune beta cell destruction. The multiple pathways of cytotoxicity demonstrated in the current study will need to be taken into account to achieve efficacy. However, the effectiveness of perforin deficiency in reducing islet cell death in several models makes it a prime target and offers hope that blockage of beta cell death may be achievable.


   Acknowledgements
 
We thank Melanie Rowe and Kylie Tolley for expert animal husbandry. This work was supported by grants from the National Health and Medical Research Council of Australia and the Juvenile Diabetes Research Foundation through a Joint Program Grant.


   Abbreviations
 
B6, C57Bl/6
CMA, concanamycin A
HA, hemagglutinin
IGRP, islet-specific glucose 6-phosphatase catalytic subunit-related protein
NOD, non-obese diabetic
OVA, ovalbumin
RIP, rat insulin promoter
rhIL-2, recombinant human IL-2
SOCS-1, suppressor of cytokine signaling-1
TNF{alpha}, tumor necrosis factor-alpha
T1D, type 1 diabetes

   Notes
 
Transmitting editor: A. Cooke

Received 2 September 2005, accepted 2 March 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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