CD8+ cytotoxic T cells play a critical role in initiating insulin-dependent diabetes mellitus. The relative contribution of each of the major cytotoxic pathways, perforin/granzyme and Fas/Fas ligand (FasL), in the induction of autoimmune diabetes remains controversial. To evaluate the role of each lytic pathway in β cell lysis and induction of diabetes, we have used a transgenic mouse model in which β cells expressing the influenza virus hemagglutinin (HA) are destroyed by HA-specific CD8+ T cells from clone-4 TCR-transgenic mice. Upon adoptive transfer of CD8+ T cells from perforin-deficient clone-4 TCR mice, there was a 30-fold increase in the number of T cells required to induce diabetes. In contrast, elimination of the Fas/FasL pathway of cytotoxicity had little consequence. When both pathways of cytolysis were eliminated, mice did not become diabetic. Using a model of spontaneous diabetes, which occurs in double transgenic neonates that express both clone-4 TCR and Ins-HA transgenes, mice deficient in either the perforin or FasL/Fas lytic pathway become diabetic soon after birth. This indicates that, in the neonate, large numbers of autoreactive CD8+ T cells can lead to destruction of islet β cells by either pathway.

Insulin-dependent diabetes mellitus (IDDM)3 is a T cell-mediated autoimmune disease characterized by the destruction of insulin-producing pancreatic β cells located in the pancreatic islets of Langerhans (1, 2, 3). Such destruction requires both CD4+ and CD8+ T cell subsets (4, 5, 6, 7, 8, 9, 10). Several findings have suggested a crucial role for the CD8+ T cell subset in the early induction phase of IDDM (11, 12, 13, 14, 15). It is proposed that, during this phase, β cells are destroyed by cytotoxic CD8+ T cells, leading to the release of pancreatic Ags. These Ags are picked up and presented on professional class II-positive APCs, which can subsequently activate CD4+ T cells. Although CD4+ T cells may not damage the class II-negative β cells directly (16), they can recruit other effector cells, such as activated macrophages, that can damage β cells by secreting IL-1β and NO (17, 18, 19), or possibly other cytokines, such as TNF and IFN-γ (20, 21, 22, 23, 24). In addition, CD4+ T cells can expand or recruit other CD8+ T cells. Thus, cytotoxicity by CD8+ T cells is believed to be critical to the onset of diabetes.

Two major molecular pathways of CD8+ T cell-mediated cytotoxicity have been defined: 1) the exocytosis of granules containing perforin and granzyme molecules, and 2) the ligation of Fas ligand (FasL) on T cells with the apoptosis-inducing Fas molecule on target cells (25, 26, 27, 28, 29). Several laboratories have investigated the contribution of each of these cytotoxic mechanisms in autoimmune diabetes. Recent studies using perforin-deficient mice suggest that perforin-dependent cytotoxicity is a crucial effector mechanism for pancreatic β cell elimination in both a transgenic model of virus-induced autoimmune diabetes (30) and the nonobese diabetic (NOD) model of spontaneous diabetes (31). However, other studies have implicated the Fas/FasL cytotoxic pathway in this disease. Although normal pancreatic β cells do not express Fas (32), it can be up-regulated by exposure to IL-1β (33, 34, 35, 36). Also, protection from spontaneous and CD8+ T cell-transferred diabetes was shown in Fas-negative NOD lpr/lpr mice, suggesting an important role for the Fas/FasL pathway in this particular model of spontaneous autoimmune diabetes (37, 38). More recently, the role of Fas in IDDM has been questioned, since so far all the experiments implicating this lytic pathway were performed in Fas-mutant lpr/lpr recipients, which have an abnormal immune system. Indeed, it was demonstrated that, although such mice do not develop diabetes, pancreata from NOD lpr/lpr mice were destroyed upon transfer into diabetic NOD mice, suggesting that Fas-deficient islets are susceptible to autoimmune destruction (39, 40). In one such study, the Fas-deficient islets were somewhat more resistant to destruction, suggesting a minor role for Fas-induced lysis in diabetes (39).

To reconcile these apparently contradictory findings, we have directly compared both molecular pathways of CD8+ T cell-mediated cytotoxicity in one model of CD8+ T cell-mediated IDDM. In this model, mice that express the influenza virus hemagglutinin (HA) under the control of the rat insulin promoter (Ins-HA mice), develop diabetes soon after the introduction of activated HA-specific CD8+ T cells derived from clone-4 TCR transgenic mice (41, 42, 43). By comparing the degree of islet destruction following transfer of the same number of normal clone-4 TCR CTLs, perforin-deficient clone-4 TCR per−/− CTLs or FasL mutant clone-4 TCR gld/gld, it was observed that elimination of the perforin/granzyme cytotoxic pathway had a more profound impact on the degree of β cell destruction than did the Fas/FasL pathway. However, if given sufficiently high numbers of clone-4 TCR per−/− CTLs, Ins-HA recipient mice became diabetic even if the perforin/granzyme pathway was blocked. Only by blocking both pathways of lysis were Ins-HA mice completely protected from diabetes. In agreement with these results, the elimination of either lytic pathway did not effect the initiation of spontaneous diabetes that occurs in double transgenic neonates, expressing both clone-4 TCR and Ins-HA transgenes.

BALB/c mice were purchased from the breeding colony of The Scripps Research Institute (TSRI). Ins-HA and clone-4 TCR-transgenic mice were generated and characterized as previously described (41, 42) and bred onto the BALB/c background for at least ten generations. Perforin-deficient mice were kindly provided by Drs. W. R. Clarke (UCLA) and J. T. Harty (University of Iowa) and were bred to the clone-4 TCR-transgenic mice (clone-4 TCR per−/−). BALB/c lpr/lpr and BALB/c gld/gld were kindly provided by Dr. Chisari (TSRI) and were bred to the Ins-HA-transgenic mice (Ins-HA lpr/lpr) and clone-4 TCR-transgenic mice, respectively (clone-4 TCR gld/gld).

The perforin, Fas, and FasL genotype were determined by PCR using genomic DNA prepared from tails. Primers for the perforin gene (5′-TGGCCTAGGGTTCACATCCAG- 3′, 5′-CGTGAGAGGTCAGCATCCTTC-3′, and5′-ATATTGGCTGCAGGGTCGCTC-3′) yield a 500-bp fragment for the wild-type and a 350-bp fragment for the mutated perforin allele.

Primers for the Fas gene (5′-GGTTACAAAAGGTCACCGAT-3′, 5′-TTAACTCTAGCCAGGAATAC-3′, and 5′-GGCAGAACT ATTGAGCATAG-3′) yield a 200-bp fragment for the wild-type and a 440-bp fragment for the mutated allele.

Primers for the FasL gene (5′-TCTGATCAATTTTGAGGAATCTAAGGCC-3′ and 5′-CATGAGGTCTTTGTGGCTCATGTA-3′) yield a 178-bp fragment. This PCR fragment was subsequently digested with 5 U of the restriction enzyme StuI (New England Biolabs, Beverly, MA.) and separated on a 3% low melting agar gel (Promega, Madison, WI.). The restriction enzyme StuI recognizes the wild-type allele but not the mutated allele. Wild-type sequence yields a 26-bp and a 152-bp fragment, whereas the mutated allele yields a 178-bp fragment. All mice were bred and maintained under specific pathogen-free conditions in the Scripps Research Institute vivarium. All experimental procedures were conducted according to the guidelines laid out in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

L1210 Fas+ (H-2d) and L1210 Fas (H-2d) were a gift from Drs. R. Dutton and L. Carter (Trudeau Institute, Saranac Lake, NY) and were maintained in DMEM supplemented with 10% (v/v) heat inactivated FCS, 2 mM glutamine, 5 × 10−5 M β-mercaptoethanol, 50 mg/ml gentamicin (Gemini Bio-products, Calabasas, CA), and 200 μg/ml of the neomycin analogue G418 (Life Technologies, Gaithersburg, MD). Cells were cultured in a humidified incubator at 37°C with 10% v/v CO2 and were used as targets in CTL assays as described (44, 45).

Synthetic influenza virus A/PR/8/34 (H1N1) HA peptide (IYSTVASSL, aa 518–526, restricted by H-2Kd) was synthesized by the core facility of TSRI using an Applied Biosystems model 430A synthesizer (Foster City, CA). Purity was greater than 85%, as determined by mass spectrometry and reverse phase HPLC analysis on a Vydac C18 column (Hesperia, CA).

BALB/c splenocytes were irradiated (3000 rad) and pulsed for 1 h with 5 μg/ml of KdHA peptide. Splenocytes were washed three times with RPMI containing 10% (v/v) heat-inactivated FCS, 2 mM glutamine, 5 × 10−5 M β-mercaptoethanol, and 50 mg/ml gentamicin (Gemini Bio-products) and seeded at 6 × 106 cells/well into 24-well tissue culture plates containing 2 × 106 responder clone-4 TCR, clone-4 TCR per−/− or clone-4 TCR gld/gld splenocytes per well. Cells were cultured in a humidified incubator at 37°C with 5% v/v CO2. After 5 days, KdHA-specific CTL activity of effector cells was assessed in a 5-h 51Cr release assay using L1210 Fas+ and L1210 Fas target cells in the absence or presence of 5 μg/ml KdHA peptide. Relative cytotoxic activity (%) was calculated as follows: 100 × (sample release − spontaneous release/maximum release − spontaneous release). Four days after activation in vitro, effector T cells were adoptively transferred i.v. into the tail of sublethally irradiated (750 rad) 8-wk-old Ins-HA and Ins-HA lpr/lpr recipients.

The glucose concentration in blood obtained from the retroorbital plexus of mice was measured using the Accu-ChekIII (Boehringer-Mannheim, La Jolla, CA). Animals were considered diabetic if blood glucose levels were above 250 mg/dl.

Thy 1.2+/+ recipient mice were injected i.v. with 3 × 106 clone-4 TCR Thy1.1+/+ CTLs in 200 μl PBS. Four days later, animals were sacrificed, and single cell suspensions were made from spleen and lymph nodes. The presence of adoptively transferred clone-4 TCR Thy1.1+/+ CTL was detected by double staining with FITC-conjugated anti-CD8 and PE-conjugated anti-Thy1.1+/+ Abs (PharMingen, La Jolla, CA). Cells were analyzed with a FACScan and CELLQuest software (Becton Dickinson, Mountain View, CA.).

Spleen and pancreas were excised and fixed overnight in 10% (v/v) formalin solution (Sigma, St. Louis, MO) and processed for paraffin embedding. Paraffin-embedded tissue was cut using a regular microtome. Paraffin sections were deparaffinized in xylene and rehydrated by washing in graded ethanol in distilled water. Nonspecific binding sites were blocked by incubating with 10% (v/v) goat serum in PBS for 30 min. Sections were incubated for 1 h with guinea pig Abs against mouse insulin (Dako, Carpinteria, CA.) or glucagon (Chemicon International, Temecula, CA). After washing for 10 min in PBS, sections were incubated with secondary biotinylated F(ab′)2 goat anti-guinea pig IgG Abs (Vector Labs, Burlingame, CA) for 1 h and detected using streptavidin-conjugated HRP (Jackson ImmunoResearch, Avondale, PA) and diaminobenzidine as a chromagen (DAB; Sigma).

To confirm that clone-4 TCR CTLs are able to kill through both the perforin/granzyme and the Fas/FasL cytotoxic pathways, activated clone-4 TCR CTLs were tested for cytolytic activity using L1210Fas+ and L1210Fas target cells pulsed with or without cognate peptide (KdHA), in the presence or absence of EGTA. EGTA inhibits the exocytosis of CTL granules containing perforin and granzymes, which is a calcium-dependent process. However, it does not inhibit the Fas/FasL cytotoxic pathway. Clone-4 TCR CTLs are able to kill through both cytotoxic pathways (Fig. 1, A and D); clone-4 TCR per−/− kill only through the Fas/FasL cytotoxic pathway (Fig. 1, B and E); and clone-4 TCR gld/gld only through the perforin/granzyme cytotoxic pathway (Fig. 1, C and F), as expected. When the perforin/granzyme pathway is blocked by adding EGTA (Fig. 1, D–F), there is substantial Fas/FasL killing observed (Fig. 1, D and E). There is no residual cytolysis left when both cytotoxic pathways are blocked (Fig. 1 F), suggesting that the blocking is complete and that the perforin/granzyme and Fas/FasL pathways are the major cytotoxic pathways for lysis by these CTL. No other cytotoxic pathway is observed (e.g., TNF-mediated cytotoxicity), under the conditions tested in this CTL assay.

FIGURE 1.

Clone-4 TCR CTLs can kill through both perforin/granzyme and Fas/FasL cytotoxic pathways in vitro. Splenocytes from transgenic clone-4 TCR, clone-4 TCR per−/−, and clone-4 TCR gld/gld mice were cultured with homologous, irradiated (3000 rad) APCs pulsed with 5 μg/ml of KdHA peptide. On day 4, clone-4 TCR CTLs (A and D), clone-4 TCR per−/− CTLs (B and E) and clone-4 TCR gld/gld CTLs (C and F) were used as effectors for comparative lysis of L1210 Fas target cells pulsed with (□) or without (▪) KdHA peptide and L1210 Fas+ target cells pulsed with (•) or without (○) KdHA peptide in the absence (A, B, and C) or presence (D, E, and F) of 4 mM EGTA and 3 mM MgCl2, as shown.

FIGURE 1.

Clone-4 TCR CTLs can kill through both perforin/granzyme and Fas/FasL cytotoxic pathways in vitro. Splenocytes from transgenic clone-4 TCR, clone-4 TCR per−/−, and clone-4 TCR gld/gld mice were cultured with homologous, irradiated (3000 rad) APCs pulsed with 5 μg/ml of KdHA peptide. On day 4, clone-4 TCR CTLs (A and D), clone-4 TCR per−/− CTLs (B and E) and clone-4 TCR gld/gld CTLs (C and F) were used as effectors for comparative lysis of L1210 Fas target cells pulsed with (□) or without (▪) KdHA peptide and L1210 Fas+ target cells pulsed with (•) or without (○) KdHA peptide in the absence (A, B, and C) or presence (D, E, and F) of 4 mM EGTA and 3 mM MgCl2, as shown.

Close modal

To investigate the relative contribution of perforin/granzyme vs Fas/FasL-mediated cytotoxicity in the induction of diabetes mediated through recognition of HA expressed by the pancreatic islets, we used an experimental protocol that involved adoptive transfer of HA-specific clone-4 TCR cells into Ins-HA mice. To evaluate the role of perforin/granzyme-mediated cytotoxicity, clone-4 TCR cells deficient in perforin were used. To evaluate the role of Fas-mediated cytotoxicity, two different strategies were employed. Either clone-4 TCR gld/gld T cells deficient in FasL were used for adoptive transfer into Ins-HA recipients, or normal clone-4 TCR T cells were transferred into Ins-HA lpr/lpr recipients deficient in Fas. The latter protocol required that recipients undergo sublethal irradiation to eliminate the abnormal CD4CD8B220+ T cells that accumulate in the Fas-deficient Ins-HA lpr/lpr mice, since such cells express high levels of FasL and could affect the viability of transferred cells (46, 47, 48, 49, 50, 51). Varying numbers of clone-4 TCR, clone-4 TCR per−/−, and clone-4 TCR gld/gld CTLs were adoptively transferred i.v. into sublethally irradiated Ins-HA or the Fas-deficient Ins-HA lpr/lpr mice (Fig. 2). When both cytotoxic pathways were intact (clone-4 TCR → Ins-HA), transfer of as few as 0.07 × 106 clone-4 TCR CTLs could cause disease. However, transfer of similar numbers of perforin-deficient clone-4 TCR CTLs resulted in a decrease in the incidence of diabetes (clone-4 TCR per−/− → Ins-HA). However, by increasing the number of perforin-deficient cells 30-fold, diabetes was observed. Moreover, if Fas was eliminated in the recipient pancreas, by using Fas-deficient Ins-HA lpr/lpr mice (clone-4 TCR → Ins-HA lpr/lpr), more than a 100-fold clone-4 TCR cells were required to cause diabetes. When both cytotoxic pathways were blocked (clone-4 TCR per−/− → Ins-HA lpr/lpr), no diabetes was observed. These results suggest that elimination of either cytotoxic pathway profoundly debilitated the ability of the clone-4 TCR cells to cause diabetes. However, when FasL was lacking on the T cell (clone-4 TCR gld/gld → Ins-HA), autoimmune diabetes was quickly induced in 100% of all recipient mice at a cell number of 0.2 × 106 or higher. Thus, depending upon whether the Fas/FasL cytotoxic pathway is blocked at the level of the T cell (FasL-deficient cells), or at the level of the islet (Fas-deficient pancreata), a different conclusion could be made regarding the contribution of this killing pathway in CD8+ T cell-mediated diabetes.

FIGURE 2.

Clone-4 TCR CTLs can destroy islet β cells in vivo through both perforin/granzyme and Fas/FasL cytotoxic pathways. Titration of the number of clone-4 TCR (▪, •), clone-4 TCR per−/− (○, □), and clone-4 TCR gld/gld CTLs (▴) required to cause diabetes following adoptive transfer into irradiated Ins-HA (▪, ○, ▴) and Ins-HA lpr/lpr (•, □) recipients. Animals were considered diabetic if blood glucose levels were above 250 mg/dl. All diabetic animals died within 7–10 days. Every group represents at least nine animals divided over three independent experiments.

FIGURE 2.

Clone-4 TCR CTLs can destroy islet β cells in vivo through both perforin/granzyme and Fas/FasL cytotoxic pathways. Titration of the number of clone-4 TCR (▪, •), clone-4 TCR per−/− (○, □), and clone-4 TCR gld/gld CTLs (▴) required to cause diabetes following adoptive transfer into irradiated Ins-HA (▪, ○, ▴) and Ins-HA lpr/lpr (•, □) recipients. Animals were considered diabetic if blood glucose levels were above 250 mg/dl. All diabetic animals died within 7–10 days. Every group represents at least nine animals divided over three independent experiments.

Close modal

One possible explanation for the contradictory results described above may involve the pleiotropic effects of Fas deficiency in the recipient mice. Although Ins-HA lpr/lpr recipients were sublethally irradiated to prevent lymphadenopathy, it was possible that the Fas deficiency in the recipient may have affected the activity or viability of adoptively transferred clone-4 TCR CTLs. To test this hypothesis, activated clone-4 TCR CTLs, expressing the Thy1.1 allele, were adoptively transferred into Thy1.2-positive, sublethally irradiated BALB/c, BALB/c lpr/lpr, Ins-HA, and Ins-HA lpr/lpr recipient mice. Four days later, the presence of these transferred cells in spleen and peripheral lymph nodes was determined by flow cytometry using anti-Thy1.1 and anti-CD8 Abs (Fig. 3). There was consistently an approximately 2-fold decrease in the numbers of CTLs recovered from BALB/c lpr/lpr mice as compared with BALB/c recipients. There was also a small decrease in the numbers of cells recovered from Ins-HA mice compared with BALB/c recipients, suggesting that deletion was attributed to the presence of HA in the pancreas. Interestingly, there was more than a 6-fold decrease in the number of cells recovered from Ins-HA lpr/lpr mice as compared with Ins-HA, suggesting elimination rather than inactivation of T cells. This deletion appears to be dependent on the presence of cognate Ag, since it occurs at a slower rate in lpr/lpr mice as compared with Ins-HA lpr/lpr mice.

FIGURE 3.

Clone-4 TCR cells are rejected following adoptive transfer into sublethally irradiated Ins-HA lpr/lpr recipient mice. Clone-4 TCR Thy1.1+/+ CTL (3 × 106) were adoptively transferred into sublethally irradiated (750 rad) BALB/c, BALB/c lpr/lpr, Ins-HA, and Ins-HA lpr/lpr mice. Four days later, animals were sacrificed, and splenocytes were stained with anti-CD8 and anti-Thy1.1 Abs and analyzed by flow cytometry. Data are representative of two separate experiments using a total of eight mice per group.

FIGURE 3.

Clone-4 TCR cells are rejected following adoptive transfer into sublethally irradiated Ins-HA lpr/lpr recipient mice. Clone-4 TCR Thy1.1+/+ CTL (3 × 106) were adoptively transferred into sublethally irradiated (750 rad) BALB/c, BALB/c lpr/lpr, Ins-HA, and Ins-HA lpr/lpr mice. Four days later, animals were sacrificed, and splenocytes were stained with anti-CD8 and anti-Thy1.1 Abs and analyzed by flow cytometry. Data are representative of two separate experiments using a total of eight mice per group.

Close modal

To study whether there are morphological differences between the mechanism of islet destruction by the perforin/granzyme or Fas/FasL cytotoxic pathways, 2 × 106 activated clone-4 TCR, clone-4 TCR per−/−, or clone-4 TCR gld/gld CTLs were adoptively transferred into sublethally irradiated Ins-HA or Ins-HA lpr/lpr recipient mice. This number of CTLs was sufficient to cause diabetes in all recipients. To provide information early on during the process of β cell destruction, mice were sacrificed 4 days after adoptive transfer, and sections of pancreata were analyzed by immunohistochemistry using anti-insulin and anti-glucagon Abs (Fig. 4). The extent of β cell destruction and islet infiltration was similar in the Ins-HA mice, which had received either normal clone-4 TCR CTLs (Fig. 4,B) or clone-4 gld/gld CTLs (Fig. 4,E). This suggested that FasL contributed little to the process of β cell destruction. Fig. 4,C (clone-4 per−/− → Ins-HA) and Fig. 4,D (clone-4 → Ins-HA lpr/lpr) show sections of pancreata of Ins-HA and Ins-HA lpr/lpr, respectively. As anticipated, based on the fact that greater numbers of cells were required to cause disease under these two experimental conditions, more insulin-positive β cells are present, and the islet appears more intact with less infiltration, as compared with Fig. 4, B and E. Serial sections were also stained for glucagon (Fig. 4, F-J) to examine whether other pancreatic cells, not expressing the HA Ag, were killed. Glucagon-positive α cells remained intact in recipients in which the Fas/FasL pathway was blocked (Fig. 4, I and J), suggesting that perforin cytotoxicity was highly specific for the β cells. Some bystander killing of α cells may have occurred when the Fas/FasL pathway was operative (Fig. 4, G and H). However, extensive analysis of sections of different regions of the pancreas would be necessary to determine the significance of this apparent difference in numbers of α cells.

FIGURE 4.

Immunohistochemical analysis of β cell destruction by different cytotoxic pathways of CD8+ T cells. Immunohistological analysis of serial sections of pancreata taken from Ins-HA (A–C, E–H, and J) and Ins-HA lpr/lpr (D and I) mice. Pancreata were paraffin embedded, and serial sections were stained for insulin (A–E) or glucagon (F–J) by the immunoperoxidase technique, using DAB as chromagen, and counterstained with hematoxylin. A, A control pancreatic section (×400) of an Ins-HA mouse that did not receive any CTL. No infiltrates are observed, and the islets are intact, as shown by uniform insulin staining. B, A pancreatic section (×200) from an Ins-HA recipient mouse 4 days after transfer of 2 × 106 clone-4 TCR CTLs. Note the massive cellular infiltration and destruction of the islet, leaving only a few insulin-positive β cells. C, A pancreatic section (×200) from an Ins-HA recipient mouse 4 days after transfer of 2 × 106 clone-4 TCR per−/− CTLs. More insulin-positive cells are present as compared with B, and the islet structure appears to be more intact with less cellular infiltration, although β islet destruction is visible. D, A pancreatic section (×400) from an Ins-HA lpr/lpr recipient mouse 4 days after transfer of 2 × 106 clone-4 TCR CTLs. The islet appears more intact as compared with B, and as evidenced by the fact that there is less infiltration and more insulin-positive cells. E, A pancreatic section (×200) from an Ins-HA recipient mouse stained for insulin 4 days after transfer of 2 × 106 clone-4 TCR gld/gld CTLs. Note the massive infiltration and destruction of the islet leaving only a few insulin-positive β cells, as also observed in B. F–J, Control pancreatic sections (F and J, ×400; G, H, and J, ×200) taken from Ins-HA or Ins-HA lpr/lpr recipient mice stained for glucagon. Glucagon positive cells are present in all recipients.

FIGURE 4.

Immunohistochemical analysis of β cell destruction by different cytotoxic pathways of CD8+ T cells. Immunohistological analysis of serial sections of pancreata taken from Ins-HA (A–C, E–H, and J) and Ins-HA lpr/lpr (D and I) mice. Pancreata were paraffin embedded, and serial sections were stained for insulin (A–E) or glucagon (F–J) by the immunoperoxidase technique, using DAB as chromagen, and counterstained with hematoxylin. A, A control pancreatic section (×400) of an Ins-HA mouse that did not receive any CTL. No infiltrates are observed, and the islets are intact, as shown by uniform insulin staining. B, A pancreatic section (×200) from an Ins-HA recipient mouse 4 days after transfer of 2 × 106 clone-4 TCR CTLs. Note the massive cellular infiltration and destruction of the islet, leaving only a few insulin-positive β cells. C, A pancreatic section (×200) from an Ins-HA recipient mouse 4 days after transfer of 2 × 106 clone-4 TCR per−/− CTLs. More insulin-positive cells are present as compared with B, and the islet structure appears to be more intact with less cellular infiltration, although β islet destruction is visible. D, A pancreatic section (×400) from an Ins-HA lpr/lpr recipient mouse 4 days after transfer of 2 × 106 clone-4 TCR CTLs. The islet appears more intact as compared with B, and as evidenced by the fact that there is less infiltration and more insulin-positive cells. E, A pancreatic section (×200) from an Ins-HA recipient mouse stained for insulin 4 days after transfer of 2 × 106 clone-4 TCR gld/gld CTLs. Note the massive infiltration and destruction of the islet leaving only a few insulin-positive β cells, as also observed in B. F–J, Control pancreatic sections (F and J, ×400; G, H, and J, ×200) taken from Ins-HA or Ins-HA lpr/lpr recipient mice stained for glucagon. Glucagon positive cells are present in all recipients.

Close modal

Previous studies from our lab have shown that neonatal double transgenic (clone-4 TCR × Ins-HA)F1 mice develop spontaneous diabetes after birth and die within 7–14 days (42). To investigate which cytotoxic pathway is responsible for this spontaneous diabetes, neonatal double transgenic mice were bred that lacked either the perforin pathway or the Fas/FasL cytotoxic pathway. Regardless of which cytotoxic pathway was blocked, double transgenic neonates still developed spontaneous diabetes and died within 7–14 days. These data suggest that either cytotoxic pathway is able to induce spontaneous neonatal autoimmune diabetes in a model in which large numbers of maturing CD8+ T cells recognize Ag expressed by islet β cells.

The purpose of this study was to evaluate the relative significance of the perforin/granzyme and Fas/FasL cytotoxic pathways on initiation of autoimmune diabetes. Conflicting data exist within the literature concerning the relative contribution of each pathway in the destruction of islet β cells. It is difficult to reconcile these studies since they were performed under a variety of different conditions and in different models. We initiated the current study to directly evaluate both cytolytic pathways in a single model. By using clone-4 TCR deficient in either pathway, and titering the numbers of cells transferred into Ins-HA recipients, it was possible to quantitatively compare the efficiency of each cytolytic pathway in β cell destruction. The incidence of diabetes decreased substantially when the perforin/granzyme pathway was blocked and the number of adoptively transferred HA-specific CTLs was decreased to less than two million. Thirtyfold more CD8+ T cells were required to cause diabetes if the perforin/granzyme pathway was eliminated. This suggests that the Fas/FasL pathway is 30-fold less efficient in β cell destruction than the perforin/granzyme pathway, yet can still lead to diabetes. In agreement with these results, we showed that, when either pathway is blocked in double transgenic (clone-4 TCR × Ins-HA)F1 neonates, diabetes is still induced. The conclusion from both protocols is that, when large numbers of autoreactive T cells are present, either cytotoxic pathway is sufficient to cause diabetes.

A major role for perforin in diabetes was observed by Kägi et al., in both a virus-induced (lymphocytic choriomeningitis virus (LCMV)) and the NOD model of spontaneous disease. In the latter study, a minor role for Fas induced lysis was proposed, since some perforin-deficient NOD mice still developed diabetes, albeit with slower kinetics. However, in the LCMV-induced model, diabetes was not induced following transfer of 5 × 106 perforin-deficient T cells (30). Diabetes could be induced in this model if dendritic cells pulsed with Ag were used to immunize perforin-deficient LCMV-glycoprotein (GP) mice (52). It is possible that the differences between the two models, such as amount of transgene expression in the islets or the affinity of the TCR, may be responsible for the different outcomes.

The role of the Fas-mediated cytolysis in IDDM has been the subject of a number of conflicting reports. Initial studies used NOD lpr/lpr mice to assess the contribution of this lytic pathway to diabetes and concluded that Fas was critical to β cell destruction and diabetes development (37, 38). However, there were significant concerns about the numerous immunological abnormalities manifested by lpr/lpr mice and how this might affect T cell function through mechanisms unrelated to the issue of the mechanism of β cell lysis. Indeed, two different groups have shown that Fas-deficient pancreata from NOD lpr/lpr hosts can be readily destroyed by diabetogenic T cells (39, 40). These conflicting findings could be explained by the fact that rejection of activated T cells occurred in irradiated or nonirradiated lpr/lpr hosts, as originally demonstrated by Allison et al. (39), and confirmed in this study. We have extended these studies by demonstrating that such elimination of clone-4 T cells requires the presence of Ag within the host. Thus, following transfer into lpr/lpr mice, activated clone-4 TCR cells become eliminated only if HA is expressed by the host. This indicates that in vivo activation is required in order for the T cells to become subject to Fas-mediated activation-induced cell death (53, 54, 55, 56, 57).

The mechanism of rejection of T cells by lpr/lpr hosts remains ill defined. One possibility could be that the CD4CD8B220+ T cells that preferentially accumulate in lpr/lpr mice express high levels of FasL and can eliminate adoptively transferred T cells (50, 51). However, the recipients Ins-HA lpr/lpr used in our experiments were 8 wk of age and therefore had few such double negative T cells. Another possibility could be that residual lpr/lpr CD4+ or CD8+ T cells, which are more radioresistant to gamma irradiation than conventional lymphocytes (58), eliminate adoptively transferred T cells (59, 60). In addition, recent data show that nonlymphoid organs such as liver and small intestine are capable of FasL expression and can mediate peripheral deletion of activated T cells (61). This may explain why rejection of adoptively transferred cells occurs in Fas mutant lpr/lpr mice.

In conclusion, the perforin/granzyme cytolytic pathway is 30-fold more effective in causing autoimmune diabetes than the Fas/FasL pathway. When enough anti-HA CTLs are transferred, either pathway is able to destroy HA-expressing pancreatic β cells in this specific model of autoimmune diabetes. In the HA model, T cells were activated in vitro using optimal concentrations of peptide. It has been shown previously that, under conditions of partial T cell activation, the Fas/FasL cytotoxicity pathway may be preferentially triggered (62, 63, 64). It is possible that, in spontaneous models of autoimmune diabetes such as the NOD mouse, the Fas/FasL pathway contributes predominantly in the early stages of autoimmunity when Ag may be limiting. As disease progresses and Ag becomes more plentiful as the result of destruction of β cells, perforin-mediated islet destruction may dominate.

We thank Drs. F. Chisari, J. T. Harty, and W. R. Clarke for providing mice and Drs. R. Dutton and L. Carter for cell lines.

1

This work was supported by National Institutes of Health Grant AI 39664/JDF 995010 (to L.S. and N.S.) and DK/CA 50824 (to L.S.). H.T.C.K. is a recipient of a Fulbright grant (The Netherlands). D.J.M. is a recipient of Juvenile Diabetes Foundation Fellowship 394125.

3

Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; FasL, Fas ligand; NOD, nonobese diabetic; HA, hemagglutinin; LCMV, lymphocytic choriomeningitis virus; Ins-HA, transgenic mouse expressing the HA from influenza virus on pancreatic islet β cells under the control of rat insulin promoter; per, −/− perforin deficient.

1
Steinman, L..
1995
. Escape from “horror autotoxicus”: pathogenesis and treatment of autoimmune disease.
Cell
80
:
7
2
Tisch, R., H. McDevitt.
1996
. Insulin-dependent diabetes mellitus.
Cell
85
:
291
3
Bach, J. F..
1994
. Insulin-dependent diabetes mellitus as an autoimmune disease.
Endocr. Rev.
15
:
516
4
Hanafusa, T., S. Sugihara, H. Fujino-Kurrihara, J. I. Miyagawa, A. Miyazaki, T. Yoshioka, K. Yamada, H. Nakajima, H. Asakawa, N. Kono, H. Fujiwara, T. Hamaoka, S. Tarui.
1988
. Induction of insulitis by adoptive transfer with L3T4+ and Lyt2+ T-lymphocytes in T-lymphocyte depleted NOD mice.
Diabetes
37
:
204
5
Miller, B. J., M. C. Appel, J. J. O’Neil, L. S. Wicker.
1988
. Both the Lyt-2+ and L3T4+ T cell subsets are required for the transfer of diabetes in nonobese diabetic mice.
J. Immunol.
140
:
52
6
Bendelac, A., C. Carnaud, C. Boitard, J. F. Bach.
1987
. Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates: requirement for both L3T4+ and Lyt-2+ T cells.
J. Exp. Med.
166
:
823
7
O’Reilly, L. A., P. R. Hutchings, P. R. Crocker, E. Simpson, T. Lund, D. Kioussis, F. Takei, J. Baird, A. Cooke.
1991
. Characterization of pancreatic islet cell infiltrates in NOD mice: effect of cell transfer and transgene expression.
Eur. J. Immunol.
21
:
1171
8
Reich, E. P., R. S. Sherwin, O. Kanagawa, C. A. Janeway, Jr.
1989
. An explanation for the protective effect of the MHC class II I-E molecule in murine diabetes.
Nature
341
:
326
9
Shimizu, J., O. Kanagawa, E. R. Unanue.
1993
. Presentation of β cell antigens to CD4 and CD8 T cells of nonobese diabetic mice.
J. Immunol.
151
:
1723
10
Nagata, M., P. Santamaria, T. Kawamura, T. Utsugi, J. W. Yoon.
1994
. Evidence for the role of CD8+ cytotoxic T cells in the destruction of pancreatic β cells in nonobese diabetic mice.
J. Immunol.
152
:
2042
11
Wong, S. F., I. Visintin, L. Wen, R. A. Flavell, C. A. Janeway, Jr.
1996
. CD8 T cell clones from young nonobese diabetic (NOD) islets can transfer rapid onset of diabetes in NOD mice in the absence of CD4 cells.
J. Exp. Med.
183
:
67
12
Jarpe, A. J., M. R. Hickman, J. T. Anderson, W. E. Winter, A. B. Peck.
1991
. Flow cytometric enumeration of mononuclear cell populations infiltrating the islets of Langerhans in prediabetic NOD mice: development of a model of autoimmune insulitis for type I diabetes.
Reg. Immunol.
3
:
305
13
Wicker, L. S., E. H. Leiter, J. A. Todd, R. J. Renjilian, E. Peterson, P. A. Fischer, P. L. Podolin, M. Zijlstra, R. Jaenisch, L. B. Peterson.
1994
. β2-microglobulin-deficient NOD mice do not develop insulitis or diabetes.
Diabetes
43
:
500
14
Serreze, D. V., E. H. Leiter, G. J. Christianson, D. Greiner, D. C. Roopenian.
1994
. Major histocompatibility complex class I-deficient NOD-β2mnull mice are diabetes and insulitis resistant.
Diabetes
43
:
505
15
Sumida, T., M. Furukawa, A. Sakamoto, T. Namekawa, T. Maeda, M. Zijlstra, I. Iwamoto, T. Koike, S. Yoshida, H. Tomioka, M. Taniguchi.
1994
. Prevention of insulitis and diabetes in β2-microglobulin-deficient non-obese diabetic mice.
Int. Immunol.
6
:
1445
16
Serreze, D. V., E. H. Leiter.
1994
. Genetic and pathogenic basis of autoimmune diabetes in NOD mice.
Curr. Opin. Immunol.
6
:
900
17
Kolb, H., V. Kolb-Bachofen.
1992
. Nitric oxide: a pathogenetic factor in autoimmunity.
Immunol. Today
13
:
157
18
Reddy, S., S. Kaill, C. A. Poole, J. Ross.
1997
. Inducible nitric oxide synthase in pancreatic islets of the non-obese diabetic mouse: a light and confocal microscopical study of its ontogeny, co-localization and up-regulation following cytokine administration.
J. Histochem.
29
:
53
19
Rabinovitch, A., W. L. Suarez-Pinzon, O. Sorensen, R. C. Bleackley.
1996
. Inducible nitric oxide synthase (iNOS) in pancreatic islets of nonobese diabetic mice: identification of iNOS expressing cells and relationships to cytokines expressed in the islets.
Endocrinology
137
:
2093
20
Yang, X. D., R. Tisch, S. M. Singer, Z. A. Cao, R. S. Liblau, R. D. Schreiber, H. O. McDevitt.
1994
. Effect of tumor necrosis factor α on insulin-dependent diabetes mellitus in NOD mice. I. The early development of autoimmunity and the diabetogenic process.
J. Exp. Med.
180
:
995
21
Ohashi, P., S. Oehen, P. Aichele, H. Pircher, B. Odermatt, P. Herrera, Y. Higuchi, K. Buerki, H. Hengartner, R. M. Zinkernagel.
1993
. Induction of diabetes is influenced by the infectious virus and local expression of MHC class I and tumor necrosis factor α.
J. Immunol.
150
:
5185
22
Campbell, I. L., A. Iscaro, L. C. Harrison.
1988
. IFN-γ and tumor necrosis factor-α: cytotoxicity to murine islets of Langerhans.
J. Immunol.
141
:
2325
23
Sarvetnick, N., J. Shizuru, D. Liggitt, L. Martin, B. McIntyre, A. Gregory, T. Parslow, T. Stewart.
1990
. Loss of pancreatic islet tolerance induced by β-cell expression of interferon-γ.
Nature
346
:
844
24
Von Herrath, M. G., M. B. A. Oldstone.
1997
. Interferon-γ is essential for destruction of β cells and development of insulin-dependent diabetes mellitus.
J. Exp. Med.
185
:
531
25
Lowin, B., M. Hahne, C. Mattmann, J. Tschopp.
1994
. Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways.
Nature
370
:
650
26
Kägi, D., F. Vignaux, B. Ledermann, K. Bürki, V. Depraetere, S. Nagata, H. Hentgartner, P. Golstein.
1994
. Fas and perforin pathways as major mechanisms of T-cell mediated cytotoxicity.
Science
265
:
528
27
Kägi, D., B. Ledermann, K. Bürki, H. Hengartner, R. M. Zinkernagel.
1994
. CD8+ T cell mediated protection against an intracellular bacterium by perforin-dependent cytotoxicity.
Eur. J. Immunol.
24
:
3068
28
Walsh, C. M., M. Matloubian, C. C. Liu, R. Ueda, C. G. Kurahara, J. L. Christensen, M. T. F. Huang, J. D. Young, R. Ahmed, W.R. Clark.
1994
. Immune function in mice lacking the perforin gene.
Proc. Natl. Acad. Sci. USA
91
:
10854
29
Kojima, H., N. Shinohara, S. Hanaoka, Y. Someya-Shirota, Y. Takagaki, H. Ohno, T. Saito, T. Katayama, H. Yagita, K. Okumura, Y. Shinkai, F. W. Alt, S. Yonehara, H. Takayama.
1994
. Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes.
Immunity
1
:
357
30
Kägi, D., B. Odermatt, P. S. Ohashi, R. M. Zinkernagel, H. Hengartner.
1996
. Development of insulitis without diabetes in transgenic mice lacking perforin-dependent cytotoxicity.
J. Exp. Med.
183
:
2143
31
Kägi, D., B. Odermatt, P. Seiler, R. M. Zinkernagel, T. W. Mak, H. Hengartner.
1997
. Reduced incidence and delayed onset of diabetes in perforin-deficient nonobese diabetic mice.
J. Exp. Med.
186
:
989
32
Leithauser, F., J. Dhein, G. Mechtersheimer, K. Koretz, S. Bruderlein, C. Henne, A. Schmidt, K. M. Debatin, P. H. Krammer, P. Moller.
1993
. Constitutive and induced expression of Apo-1, a new member of the nerve growth factor/tumor necrosis factor receptor superfamily, in normal and neoplastic cells.
Lab. Invest.
69
:
415
33
Stassi, G., R. De Maria, G. Trucco, W. Rudert, R. Testi, A. Galluzzo, C. Giordano, M. Trucco.
1997
. Nitric oxide primes pancreatic β cells for Fas-mediated destruction in insulin-dependent diabetes mellitus.
J. Exp. Med.
8
:
1193
34
Corbett, J. A., M. L. McDaniel.
1992
. Does nitric oxide mediate autoimmune destruction of beta-cells?: possible therapeutic interventions in IDDM.
Diabetes
41
:
897
35
Bendtzen, K., T. Mandrup-Poulsen, J. Nerup, J. H. Nielsen, C. A. Dinarello, M. Svenson.
1986
. Cytotoxicity of human pI 7 interleukin-1 for pancreatic islets of Langerhans.
Science
232
:
1545
36
Yamada, K., N. Takane-Gyotoku, X. Yuan, F. Ichikawa, C. Inada, K. Nonaka.
1996
. Mouse islet cell lysis mediated by interleukin-1 induced Fas.
Diabetologia
39
:
1306
37
Chervonsky, A. V., Y. Wang, F. S. Wong, I. Visintin, R. A. Flavell, C. A. Janeway, Jr, L. A. Matis.
1997
. The role of Fas in autoimmune diabetes.
Cell
89
:
17
38
Itoh, N., A. Imagawa, T. Hanafusa, M. Waguri, K. Yamamoto, H. Iwahashi, M. Moriwaki, H. Nakajima, J. Miyagawa, M. Namba, S. Makino, S. Nagata, N. Kono, Y. Matsuzawa.
1997
. Requirement of Fas for the development of autoimmune diabetes in nonobese diabetic mice.
J. Exp. Med.
186
:
613
39
Allison, J., A. Strasser.
1998
. Mechanisms of β cell death in diabetes: a minor role for CD95.
Proc. Natl. Acad. Sci. USA
95
:
13818
40
Kim, Y. H., S. Kim, K. A. Kim, H. Yagita, N. Kayagaki, K. W. Kim, M. S. Lee.
1999
. Apoptosis of pancreatic β cells detected in accelerated diabetes of NOD mice: no role of Fas-Fas ligand interaction in autoimmune diabetes.
Eur. J. Immunol.
29
:
455
41
Lo, D., J. Freedman, S. Hesse, R. D. Palmiter, R. L. Brinster, L. A. Sherman.
1992
. Peripheral tolerance to an islet cell-specific hemagglutinin transgene affects both CD4+ and CD8+ T cells.
Eur. J. Immunol.
22
:
1013
42
Morgan, D. J., R. Liblau, B. Scott, S. Fleck, H. O. McDevitt, D. Lo, L. A. Sherman.
1996
. CD8+ T cell-mediated spontaneous diabetes in neonatal mice.
J. Immunol.
157
:
978
43
Morgan, D. J., H. T. C. Kreuwel, S. Fleck, H. I. Levitsky, D. M. Pardoll, L. A. Sherman.
1998
. Activation of low avidity CTL specific for a self epitope results in tumor rejection but not autoimmunity.
J. Immunol.
160
:
643
44
Walsh, C. M., A. A. Glass, V. Chiu, W. R. Clark.
1994
. The role of the Fas lytic pathway in a perforin-less CTL hybridoma.
J. Immunol.
153
:
2506
45
Rouvier, E., M. F. Luciani, P. Golstein.
1993
. Fas involvement in Ca2+-independent T cell-mediated cytotoxicity.
J. Exp. Med.
177
:
195
46
Murphy, E. D..
1981
. Lymphoproliferation (lpr) and other single-locus models for murine lupus.
Immunol. Defects Lab. Anim.
2
:
143
47
Theofilopoulos, A. N., F. J. Dixon.
1985
. Murine models of systemic lupus erythematosus.
Adv. Immunol.
37
:
269
48
Cohen, P. L., R. A. Eisenberg.
1991
.
lpr and gld: single gene models of systemic autoimmunity and lymphoproliferative disease. Annu. Rev. Immunol.
9
:
243
49
Steinberg, A. D..
1994
. MRL-lpr/lpr disease: theories meet Fas.
Semin. Immunol.
6
:
55
50
Watanabe, D., T. Suda, H. Hashimoto, S. Nagata.
1995
. Constitutive activation of the Fas ligand in mouse lymphoproliferative disorders.
EMBO J.
14
:
12
51
Chu, J. L., P. Ramos, A. Rosendorff, J. Nikolic-Zugic, E. Lacy, A. Matsuzawa, K. B. Elkon.
1995
. Massive up-regulation of the Fas Ligand in lpr and gld mice: implications for Fas regulation and the graft-versus-host disease-like wasting syndrome.
J. Exp. Med.
181
:
393
52
Ludewig, B., B. Odermatt, S. Landmann, H. Hentgartner, R. M. Zinkernagel.
1998
. Dendritic cells induce autoimmune diabetes and maintain disease via de novo formation of local lymphoid tissue.
J. Exp. Med.
188
:
1493
53
Russell, J. H., B. Rush, C. Weaver, R. Wang.
1993
. Mature T cells of autoimmune lpr/lpr mice have a defect in antigen-stimulated suicide.
Proc. Natl. Acad. Sci. USA
90
:
4409
54
Dhein, J., H. Walczak, C. Baumler, K. M. Debatin, P. H. Krammer.
1995
. Autocrine T-cell suicide mediated by APO-1/(Fas/CD95).
Nature
373
:
438
55
Brunner, T., R. J. Mogil, D. LaFace, N. J. Yoo, A. Mahboubi, F. Echeverri, S. J. Martin, W. R. Force, D. H. Lynch, C. F. Ware, D. F. Green.
1995
. Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas.
Nature
373
:
441
56
Ju, S. T., D. J. Panka, H. Cui, R. Ettinger, M. el-Khatib, D. H. Sherr, B. Z. Stanger, A. Marshak-Rothstein.
1995
. Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation.
Nature
373
:
444
57
Alderson, M. R., T. W. Tough, T. Davis-Smith, S. Braddy, B. Falk, K. A. Schooley, R. G. Goodwin, C. A. Smith, F. Ramsdell, D. H. Lynch.
1995
. Fas ligand mediates activation-induced cell death in human T lymphocytes.
J. Exp. Med.
181
:
71
58
Reap, E. A., K. Roof, K. Maynor, M. Borrero, J. Booker, P. L. Cohen.
1997
. Radiation and stress-induced apoptosis: a role for Fas/Fas ligand interactions.
Proc. Natl. Acad. Sci. USA
94
:
5750
59
Bobe, P., D. Bonardelle, M. Reynes, F. Godeau, J. Mahiou, V. Joulin, N. Kiger.
1997
. Fas-mediated liver damage in MRL hemopoietic chimeras undergoing lpr-mediated graft-versus-host disease.
J. Immunol.
159
:
4197
60
Wang, J. K. M., B. Zhu, S. T. Ju, J. Tschopp, A. Marschak-Rothstein.
1997
. CD4+ T cells reactivated with superantigen are both more sensitive to FasL-mediated killing and express a higher level of FasL.
Cell. Immunol.
179
:
153
61
Bonfoco, E., P. M. Stuart, T. Brunner, T. Lin, T. S. Griffith, Y. Gao, H. Nakajima, P. A. Henkart, T. A. Ferguson, D. R. Green.
1998
. Inducible nonlymphoid expression of Fas Ligand is responsible for superantigen-induced peripheral deletion of T cells.
Immunity
9
:
711
62
Cao, W. S., S. Tykodi1, M. T. Esser, V. L. Braciale, T. J. Braciale.
1995
. Partial activation of CD8+ T cells by a self-derived peptide.
Nature
378
:
295
63
Esser, M.T., B. Krishnamurthy, V. L. Braciale.
1996
. Distinct T cell receptor signaling requirements for perforin- or FasL-mediated cytotoxicity.
J. Exp. Med.
183
:
1697
64
Brossart, P., M. J. Bevan.
1996
. Selective activation of Fas/Fas ligand-mediated cytotoxicity by a self peptide.
J. Exp. Med.
183
:
2449