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The Journal of Immunology, 2000, 165: 202-210.
Copyright © 2000 by The American Association of Immunologists

Systemic Administration of Agonist Peptide Blocks the Progression of Spontaneous CD8-Mediated Autoimmune Diabetes in Transgenic Mice Without Bystander Damage1

Nadège Bercovici2,*, Agnès Heurtier*, Csaba Vizler*, Nathalie Pardigon{dagger}, Christophe Cambouris*,2, Pierre Desreumaux{ddagger} and Roland Liblau3,*

* Laboratoire d’Immunologie Cellulaire, Institut National de la Santé et de la Recherche Médicale CJF 9711, Paris, France; {dagger} Unité de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Médicale Unité 277, Institut Pasteur, Paris, France; and {ddagger} Département de Gastroenterologie, Centre Hospitalier Universitaire, Lille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-dependent diabetes is an autoimmune disease targeting pancreatic ß-islet cells. Recent data suggest that autoreactive CD8+ T cells are involved in both the early events leading to insulitis and the late destructive phase resulting in diabetes. Although therapeutic injection of protein and synthetic peptides corresponding to CD4+ T cell epitopes has been shown to prevent or block autoimmune disease in several models, down-regulation of an ongoing CD8+ T cell-mediated autoimmune response using this approach has not yet been reported. Using CL4-TCR single transgenic mice, in which most CD8+ T cells express a TCR specific for the influenza virus hemagglutinin HA512–520 peptide:Kd complex, we first show that i.v. injection of soluble HA512–520 peptide induces transient activation followed by apoptosis of Tc1-like CD8+ T cells. We next tested a similar tolerance induction strategy in (CL4-TCR x Ins-HA)F1 double transgenic mice that also express HA in the ß-islet cells and, as a result, spontaneously develop a juvenile onset and lethal diabetes. Soluble HA512–520 peptide treatment, at a time when pathogenic CD8+ T cells have already infiltrated the pancreas, very significantly prolongs survival of the double transgenic pups. In addition, we found that Ag administration eliminates CD8+ T cell infiltrates from the pancreas without histological evidence of bystander damage. Our data indicate that agonist peptide can down-regulate an autoimmune reaction mediated by CD8+ T cells in vivo and block disease progression. Thus, in addition to autoreactive CD4+ T cells, CD8+ T cells may constitute targets for Ag-specific therapy in autoimmune diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Organ-specific autoimmune diseases, such as insulin-dependent diabetes mellitus (IDDM)4 and experimental autoimmune encephalomyelitis, result from the T and/or B cell-mediated destruction of autoantigen-expressing cells. Induction of Ag-specific immunological tolerance has been proposed as a therapy to block disease progression. Systemic or mucosal administration of Ag in a soluble form has been shown to induce tolerance of mature T cells in vivo, through 1) apoptosis, 2) anergy, and 3) immune deviation (1, 2). Administration of glutamic acid decarboxylase (GAD) autoantigen, for example, protects nonobese diabetic (NOD) mice from diabetes (3, 4, 5). Systemic administration of immunodominant self-peptides is sufficient to block disease progression in experimental autoimmune diseases (6, 7, 8, 9, 10, 11). In these experiments, autoreactive CD4+ T cells were specifically targeted by the administration of MHC class II-binding peptides.

Although a major role was initially attributed to the CD4+ T cell subset in the initiation and progression of autoimmune diabetes, recent data using NOD mice and genetically manipulated murine models of IDDM have revealed an important contribution of CD8+ T cells in autoantigen recognition and ß cell destruction (12, 13, 14, 15). Indeed, CD8 recognition of MHC class I self-epitopes on ß cells is crucial for the development of insulitis (16, 17, 18, 19), and cytotoxicity mediated by CD8+ T cells contributes to the progression from insulitis to diabetes (19, 20, 21). These data suggest that down-regulation of autoreactive CD8+ T cells should be considered as a means of blocking the disease process. Administration of MHC class I-restricted agonist or blocking peptides have been shown to prevent virus-induced autoimmune diabetes (22, 23). However, to be of clinical relevance, tolerance has to be induced in activated autoreactive CD8+ T cells during an ongoing diabetogenic response. Although autoreactive CD4+ T cells can be tolerized by administration of soluble peptide after disease onset (6, 7, 8, 9, 10, 11), this has yet to be shown for CD8+ T cells. Recently, Aichele et al. have reported that administration of soluble MHC class I-binding glycoprotein peptide from lymphocytic choriomeningitis virus (LCMV) to C57BL/6 mice previously infected with LCMV activates virus-specific memory CD8+ T cells that mediate cytotoxicity and immunopathology in the spleen (24). This result underscored the fact that following tolerogenic stimulation cytotoxic activity of memory CD8+ T cells can be triggered and be responsible for severe side effects. Whether this phenomenon would occur during the treatment of an ongoing organ-specific autoimmune disease in which the effector CD8+ T cells are localized in the target organ remains to be determined.

We investigated the mechanisms of tolerance induction for CD8+ T cells using transgenic mice expressing a TCR specific for the hemagglutinin HA512–520:Kd complex (CL4-TCR mice). We found that following i.v. injection of soluble HA512–520 peptide in CL4-TCR single transgenic mice, targeted CD8+ T cells were deleted through apoptosis in the lymphoid organs and tolerance was associated with reduced proliferation and IFN-{gamma} secretion in response to HA peptide. The spontaneous autoimmune diabetes that is mediated by these CD8+ T cells in (CL4-TCR x Ins-HA)F1 double transgenic mice that also express HA in the ß-islet cells (25) provides an ideal opportunity to test whether down-regulation of an ongoing CD8+ T cell response can block disease progression. In (CL4-TCR x Ins-HA)F1 mice exhibiting infiltration of the pancreas, i.v. injections of the Kd-binding HA peptide significantly prolonged the survival of mice that would otherwise have died from fulminant diabetes. This effect was achieved by elimination of CD8+ T cells from the pancreas and was not associated with bystander damage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

CL4-TCR mice are transgenic for an influenza virus HA512–520 peptide-specific, H-2Kd-restricted TCR composed of V{alpha}10 and Vß8.2 chains (25). The mice were backcrossed at least eight times onto the BALB/c genetic background. CL4-TCR transgenic mice were included in experiments at 4–8 wk of age except when stated otherwise. Ins-HA transgenic mice express the HA protein specifically in pancreatic ß cells (26). The mice were backcrossed for three generations onto the BALB/c genetic background. Hemizygous CL4-TCR and Ins-HA mice were mated to generate (CL4-TCR x Ins-HA)F1 double transgenic mice. In these crosses, mice were genotyped by PCR amplification on tail DNA before day 3 of birth.

Peptide treatment

HA512–520 peptide (IYSTVASSL) was synthesized by Neosystem Laboratory (Strasbourg, France), and Cw3, a control Kd-binding peptide (RYLKNGKETL), was synthesized by Chiron Technologies (Melbourne, Australia). Both peptides were HPLC purified (>95% pure). For tolerance induction, peptides were dissolved in PBS and injected i.v. Adult CL4-TCR transgenic mice received one i.v. injection of PBS or HA peptide (250 µg). (CL4-TCR x Ins-HA)F1 double transgenic mice received i.v. injections of 30 µg of Cw3 peptide or HA512–520 peptide daily from day 3 to day 5 after birth. In some experiments, CL4-TCR pups were treated similarly as double transgenic mice. In adoptive transfer experiments, adult Ins-HA transgenic mice received an i.v. injection of 2 x 106 in vitro-preactivated CD8+ T cells from CL4-TCR mice as previously described (15). Recipients were treated with three i.v. injections of HA512–520 peptide or Cw3 control peptide (250 µg) at 24, 48, and 72 h posttransfer. Diabetes was assessed by measurement of blood glucose level using a Bayer Glucometer 4 (Bayer, Elkhart, IN). Mice were considered diabetic if blood glucose levels were >3 g/L.

Flow cytometry

Single-cell suspensions from thymus, spleen, and lymph nodes were prepared as previously described (27). Triple staining was performed using PE-conjugated anti-CD4 mAb, FITC-conjugated anti-CD8{alpha} mAb (Caltag, Burlingame, CA), and biotinylated mAbs (PharMingen, San Diego, CA) specific for mouse Vß8.1,8.2 (KJ16), CD90.2 (53-2.1), CD69 (H1.2F3), CD25 (7D4), CD62-L (Mel-14), CD44 (Pgp-1), or CD45RB (23G2) followed by staining with streptavidin-TC (Caltag). Viable cells (3 x 104 to 2 x 105) were collected on a FACScan cytometer and analyzed using the CellQuest software (Becton Dickinson, Mountain View, CA). Apoptotic cells were detected using staining with biotinylated annexin V (Bioproducts Boehringer Ingelheim, Germany), which binds to phosphatidylserine residues present on the outer leaflet of the cell membrane upon initiation of apoptosis (28). Briefly, cells (4 x 105/ml) were stained with annexin V for 20 min at room temperature. After two washes, cells were incubated with strepavidin-PE and FITC-conjugated anti-CD8{alpha} mAb for 30 min in the dark. Cell collection was performed following washes and addition of propidium iodide (0.5 µg/ml).

T cell proliferation assays

Splenocytes from PBS- or HA-injected CL4-TCR mice were restimulated in vitro with increasing concentrations of HA512–520 peptide in RPMI 1640 supplemented with 5% FCS, 2 mM glutamine, 200 U/ml penicillin, 200 µg/ml streptomycin, and 20 mM HEPES (Life Technologies, Grand Island, NY) in 96-well flat-bottom microtiter plates (7x105 cells/well). Proliferation was measured by [3H]thymidine (Amersham, Arlington Heights, IL) incorporation after 24 h. SEMs were <15% of the mean. To measure spontaneous proliferation, splenocytes were incubated under the same conditions but without the addition of HA peptide in vitro. To determine the in vivo half-life of HA512–520 peptide, BALB/c mice were sacrificed at different time points following an i.v. injection of 250 µg HA peptide and their splenocytes used as APCs in T cell proliferation assays. Purified CD8+ T cells from unmanipulated CL4-TCR mice were cultured at an APC-to-T cell ratio of 10:1 without additional HA peptide.

Cytokine measurements

Splenocytes (2x106 cells/ml) from PBS- or HA-injected CL4-TCR mice were cultured with or without HA512–520 peptide (0.4 µg/ml) in 24-well plates. Supernatants were collected at 24, 48, and 72 h of culture and stored at -80°C until used. The presence of IFN-{gamma} and IL-10 in culture supernatants was determined by sandwich ELISAs using Cytoset-matched Ab pairs and recombinant cytokine standards from Biosource International (Camarillo, CA). The following Abs were used: polyclonal rabbit anti-mouse IFN-{gamma} capture Ab and biotinylated anti-IFN-{gamma} mAb (DB-1), anti-IL-10 (JES5-SXC1) capture mAb and biotinylated anti-IL10 (JES5-2A5), anti-IL-4 (BVD4-1D11) capture mAb and biotinylated anti-IL-4 (BVD4-24G2) mAb. The cytokine ELISAs were performed, according to the instructions of the supplier, using Costar (Cambridge, MA) enzyme immunoassay plates, Biosource streptavidin-HRP conjugate, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate (Sigma, St. Louis, MO).

Ex vivo cytotoxic assay

Splenic and lymph node CD8+ T cells from Cw3- or HA-injected mice were negatively purified using rat anti-CD4 mAb, anti-Mac1 mAb (Caltag), and anti-B220 mAb (Cedarlane Laboratories, Hornby, Ontario, Canada) followed by a 30-min incubation with anti-rat IgG-coated Dynabeads (Dynal, Oslo, Norway) and magnetic separation. Cytotoxicity was assessed ex vivo on 51Cr-labeled P815 target cells pulsed or not with HA512–520 peptide as previously described (29).

Immunohistochemistry

Pancreata were fixed in 10% v/v formaldehyde solution and processed for paraffin embedding. Sections (4 µm) were stained with hematoxylin/eosin for general morphology. To detect ß-islet cells, sections were stained for insulin using a three-step protocol. Nonspecific binding was prevented by incubation in 10% normal goat serum (Jackson ImmunoResearch, West Grove, PA) for 20 min; endogenous peroxidase was blocked in 0.3% H2O2/methanol solution for 15 min. Slides were incubated with guinea pig anti-human insulin serum (Linco Research, St. Charles, MO), followed by staining with biotinylated F(ab')2 goat anti-guinea pig IgG (Jackson ImmunoResearch) and revealed with streptavidin-conjugated HRP (Jackson ImmunoResearch) and diaminobenzidine as a chromogen (Dako, Carpinteria, CA). For immunohistochemistry on frozen sections, pancreata from neonates were placed in a solution of PBS containing 1% paraformaldehyde (Sigma) and 5% sucrose at 4°C for 30 min. The organs were then transferred to a 10% sucrose solution for 30 min at 4°C. Rapid freezing was achieved using melting isopenthane in liquid nitrogen. Five-micrometer-thick sections were fixed immediately in cold acetone before storage at -20°C. Frozen sections were rehydrated by washing in PBS for 5 min, and endogenous peroxidase was blocked in PBS containing 0.3% H2O2 for 15 min. To assess for the CD8+ T cell infiltration in the pancreas, frozen sections were treated by sequential incubation in PBS-3% BSA for 20 min followed by treatment with an avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA). Sections were stained with a rat mAb specific for mouse CD8{alpha} (PharMingen). After three washes in PBS, sections were incubated with biotinylated F(ab')2 mouse anti-rat IgG (Jackson ImmunoResearch) and then detected with streptavidin-conjugated HRP and diaminobenzidine. All slides were counterstained with hematoxylin (Sigma).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation-induced apoptosis of anti-HA CD8+ T cells in adult CL4-TCR single transgenic mice following i.v. injection of HA peptide

We and others have shown that i.v. injection of soluble Ag favors induction of tolerance (7, 30, 31, 32). Induction of tolerance in mature T cells is characteristically preceded by an activation phase. Different cellular mechanisms have been implicated in T cell tolerance but the most frequently reported is activation-induced apoptosis of the targeted T cells. We tested whether i.v. injection of HA512–520 peptide could induce tolerance of anti-HA CD8+ T cells in CL4-TCR transgenic mice. First, the duration of efficient presentation of the injected HA peptide by APCs to T cells was determined. BALB/c mice were sacrificed at different time points after an i.v. injection of 250 µg HA peptide. Their splenocytes were used to induce proliferation of purified CD8+ T cells from untreated CL4-TCR mice. As shown in Fig. 1GoA, the agonist peptide can be detected by T cells on recipients’ splenocytes with an in vivo half-life of 27 h.



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  		FIGURE 1. Early activation of CD8+ T cells in the spleen following i.v. injection of HA peptide in CL4-TCR mice. A, In vivo half-life of HA512–520 peptide on APCs. Splenocytes from BALB/c mice injected with 250 µg HA512–520 peptide were harvested at different times to induce proliferation of purified CD8+ T cells from unmanipulated CL4-TCR mice. Maximal [3H]thymidine uptake (39,740 ± 4,776 cpm) was defined as the stimulation induced by APCs from BALB/c mice injected 0.5 h previously with the HA peptide. Results are presented as the mean (±SEM) percentage of the maximal value from three experiments. B and C, CL4-TCR transgenic mice were injected with PBS (gray graphs) or 250 µg HA512–520 peptide (open graphs) and sacrificed at different time points after injection. Splenocytes were analyzed by FACS for (B) forward scatter and the expression of CD25 and (C) Vß8.2 and CD44 after gating on CD8+ T cells. These are representative histograms of two to five independent experiments.

 
CL4-TCR mice were then injected i.v. with 250 µg HA peptide, and the phenotype of anti-HA CD8+ T cells in the lymphoid organs was determined at different time points after injection. Peripheral CD8+ T cells were rapidly activated, marked by elevated levels of IL-2R {alpha}-chain expression (Fig. 1GoB). Down-modulation of the selectin CD62-L was detectable as early as 1 h on 80% (n = 3) of lymph node CD8+ T cells and as early as 3 h in splenic CD8+ T cells after HA peptide injection. CD69 was expressed on 94% (n = 3) of CD8+ T cells in both lymphoid organs at 12 h. Activation of HA-specific CD8+ T cells resulted in transient proliferation and cytotoxicity ex vivo (Fig. 2Go, A and B) and in cytokine secretion upon restimulation with HA peptide in vitro (see below).



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  		FIGURE 2. Early activation and late hyporesponsiveness of CD8+ T cells from HA-treated CL4-TCR mice. A, Proliferation of CD8+ T cells following in vivo activation with the HA peptide was measured by incubating splenocytes from PBS-injected ({square}) or HA-injected ({blacksquare}) CL4-TCR mice for 24 h in vitro without addition of HA peptide. Histograms represent the mean [3H]thymidine uptake (cpm ± SEM) obtained in three to four independent experiments. B, The cytotoxic activity of negatively purified CD8+ T cells from Cw3- or HA-injected CL4-TCR injected mice was assessed ex vivo on P815 cells pulsed (empty and filled bars) or not (hatched bars) with 1 µg/ml HA peptide at an E:T ratio of 30:1. Data are from one mouse representative of three tested per time point and per group. C, At different time points after injection of PBS or HA peptide, splenocytes were restimulated in vitro with HA peptide, and proliferation was measured by thymidine incorporation. The curves represent the mean stimulation index (SI) per CD8+ T cell (±SEM) at different concentrations of HA peptide. The range of cpm/CD8 for medium-only cultures were 11.4 and 26.4 in PBS- and HA-injected mice, respectively. The number of CD8+ T cells in each well was calculated on the basis of the percentage of CD90+CD8+ cells, as determined by FACS analyses. Data are from three to four independent experiments, except for the mouse analyzed 60 days after injection.

 
Whereas the number of CD8- T cells was stable, the number of CD8+ T cells in the spleen of HA peptide-injected mice was considerably reduced at day 1 after injection compared with PBS-injected mice but was >2-fold higher at day 3 (Fig. 3GoA). Similar results were obtained when we assessed the absolute number of CD8+ Vß8+ splenocytes (Fig. 3GoB). Because i.v. injection of peptide induces massive apoptosis of HA-reactive thymocytes (data not shown), the increase in CD8+ T cells at day 3 likely reflects the proliferation of peripheral CD8+ T cells remaining at day 1, which express CD25 (Fig. 1GoB). The ex vivo proliferation of splenocytes from HA-treated CL4-TCR mice due to the in vivo activation of CD8+ T cells (Fig. 2GoA) provides further evidence that these cells expand in vivo during this period.



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  		FIGURE 3. Evolution of CD8+ T cell numbers in the spleen of CL4-TCR mice after HA peptide injection. A, At different time points after injection, the number of CD8+ or CD8- T cells in the spleen of PBS- or HA-injected CL4-TCR mice was determined by staining with anti-CD8{alpha} and anti-CD90 mAbs and FACS analysis. B, The absolute number of CD8+Vß8+ T cells in the spleen of HA-injected CL4-TCR mice is plotted for different time points following injection. Each value represents the mean (±SEM) of two to six HA-injected mice. Seven PBS-injected CL4-TCR mice serve as a control group.

 
Depletion in CD8+ Vß8+ T cells was also evident 7 days after injection (Fig. 3GoB). On day 7, CD8+ T cells expressed a high level of CD44 as well as low levels of CD45RB and CD62-L (Fig. 1GoC and data not shown) indicating that they had encountered the agonist peptide. The number of CD8+ T cells in HA-peptide injected CL4-TCR mice progressively returned to baseline values within 30 to 60 days (Fig. 3Go and data not shown).

Because variations in CD8+ T cell numbers in HA-injected CL4-TCR mice were paralleled in the spleen and lymph nodes, we tested whether the reduction in CD8+ T cells resulted from apoptosis in secondary lymphoid organs. CL4-TCR transgenic mice were injected with HA peptide, sacrificed 12 h or 3 days later, and their CD8+ splenic T cells stained with annexin V and analyzed by flow cytometry. Ex vivo analysis at 12 h (Fig. 4Go, a and b) shows a 2.4-fold increase in the percentage of apoptotic CD8+ T cells in HA-injected mice compared with PBS-injected control mice. As apoptotic cells are rapidly eliminated by phagocytosis in vivo (33), in parallel experiments, CD8+ T cells were first incubated for 4 h in vitro before FACs analysis. As demonstrated in Figs. 4Goc and 4d, a high proportion of CD8+ splenic T cells from HA-injected mice undergo apoptosis. In lymph nodes, an increase in the proportion of annexin V+CD8+ T cells was also observed at 12 h in HA- vs PBS-injected CL4-TCR mice (6.4 ± 2% vs 2.5 ± 0.2% ex vivo and 24.2 ± 5.7% vs 3 ± 1% after 4 h in vitro). Similarly, an increase in CD8+ splenocytes undergoing apoptosis could be detected ex vivo, or after a 4-h in vitro incubation period, 3 days after HA peptide injection (Fig. 4Go, e–h).



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  		FIGURE 4. Apoptosis is induced in activated CD8+ T cells following HA peptide injection. CL4-TCR mice were sacrificed 12 h (a–d) or 3 days (e–h) after injection of PBS (a, c, e, and g) or HA peptide (b, d, f, and h). Splenocytes were stained with anti-CD8{alpha}, annexin V, and propidium iodide ex vivo or after a 4-h in vitro culture period at 37°C and analyzed by FACS. We have set arbitrary gates so that only 2.5% of the control CD8+ T cell population coming from PBS-injected animals would be positive. Data are representative of two independent experiments.

 
These results show that the peripheral CD8+ T cell depletion observed 1 and 3 days after HA peptide injection is associated with an increase in apoptotic CD8+ T cells in the secondary lymphoid organs. This suggests that a high proportion of HA-reactive CD8+ T cells undergo activation-induced cell death (AICD) in vivo following a single i.v. injection of agonist peptide.

Characterization of the remaining HA-specific CD8+ T cells

Although we have shown that a high proportion of HA-reactive CD8+ T cells is deleted following i.v. injection of peptide, some escape both the early (during the first 24 h) and the late (between days 3 and 7) deletion phases. Thus, we tested whether the CD8+ T cells that persist in the spleen of HA-injected CL4-TCR mice were fully responsive to HA peptide restimulation or whether they had been rendered tolerant. As shown in Fig. 2GoC, the proliferative responses to HA peptide restimulation in vitro were strongly reduced on a per cell basis for >30 days following HA peptide treatment. At early time points only, this reduced proliferative capacity was associated with a consistent decrease in Vß8.2 and CD3 surface expression (Fig. 1GoC and data not shown). However, TCR levels returned to baseline values after 7 days (Fig. 1GoC), whereas hyporesponsiveness persisted (Fig. 2GoC), indicating that other functional alterations are involved in the hyporeactivity of the residual CD44high HA-specific CD8+ T cells.

Because type 1 cytokine-secreting cells are usually associated with pathogenicity in organ-specific autoimmune diseases (34), it was of particular interest to analyze the cytokines secreted by splenocytes of CL4-TCR mice after the HA peptide treatment. In vivo activation of CD8+ T cells in 12-h peptide-injected mice is associated with a high level of IFN-{gamma} secretion upon HA peptide restimulation in vitro compared with PBS-injected mice (Fig. 5Go). However, no IFN-{gamma} secretion was detected in cultures from HA- or PBS-injected CL4-TCR mice in the absence of added HA peptide. Concomitant with reduced proliferation, we found a profound and sustained reduction in IFN-{gamma} secretion in CL4-TCR mice from day 7 to day 30 following HA peptide injection (Fig. 5Go). We also found that spleen cells from two of three CL4-TCR mice injected 30–60 days previously with HA peptide produced >1 ng/ml IL-10, whereas this cytokine was not detected in cultures prepared from PBS-injected mice or HA-injected mice analyzed before 30 days postinjection (data not shown). However, no consistent increase in IL-4 secretion was detected in these cultures (data not shown).



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  		FIGURE 5. Induction of tolerance is associated with reduced IFN-{gamma} secretion in HA peptide-treated CL4-TCR mice. At different time points after HA injection, splenocytes from PBS- or HA-treated mice were restimulated in vitro with HA peptide, and the concentration of IFN-{gamma} in the supernatant was determined by ELISA. The data (mean ± SEM of duplicates) are from two to three independent experiments except for day 60 data originating from a single mouse per group. In the absence of HA peptide, the concentration of IFN-{gamma} in the culture supernatants was always <0.016 ng/ml.

 
HA peptide treatment blocks ongoing CD8-mediated diabetes in (CL4-TCR x Ins-HA)F1 double transgenic pups.

Because injection of HA peptide in CL4-TCR transgenic mice can induce tolerance in naive CD8+ T cells, we tested whether this strategy is also effective to tolerize autoreactive CD8+ T cells during an ongoing autoimmune disease. We took advantage of the fact that anti-HA CD8+ T cells from CL4-TCR mice mediate a spontaneous autoimmune diabetes in (CL4-TCR x Ins-HA)F1 double transgenic mice. Indeed, in contrast to most, but not all (35, 36), similar transgenic models of pancreatic CD8+ T cell autoreactivity, the anti-HA CD8+ T cells are neither tolerant nor indifferent to the tissue-specific neoautoantigen. HA-specific CD8+ T cells destroy the HA-expressing ß cells leading to diabetes and death in 100% of the animals within 12 days of life (15, 25). At day 3 after birth, pancreatic infiltration is clearly present, although the percentage of CD8+ T cells in the secondary lymphoid organs is low (15). We injected the HA or Cw3 peptide i.v. and recorded the survival of treated (CL4-TCR x Ins-HA)F1 mice. As shown in Fig. 6Go, A and B, 30 µg HA peptide injected i.v. daily from day 3 to day 5 delays diabetes and very significantly prolonged the survival of the (CL4-TCR x Ins-HA)F1 mice. Whereas 100% of Cw3-treated animals succumbed from diabetes by day 11, 40% of HA-treated mice survived for >30 days. Eight of 23 (35%) HA-treated mice died from diabetes between days 40 and 79 (data not shown). Similarly, i.v. injection of HA peptide blocks diabetes in adult Ins-HA mice adoptively transferred with activated CD8+ T cells from CL4-TCR mice (100% diabetes in Cw3-treated vs 0% (n = 8) in HA peptide-treated Ins-HA recipients; p = 2 x 10-4, Fisher’s exact test).



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  		FIGURE 6. HA peptide treatment delays diabetes in (CL4-TCR x Ins-HA)F1 mice. (CL4-TCR x Ins-HA)F1 mice were either untreated (•) or received i.v. injections of 30 µg Cw3 ({circ}) or HA peptide ({square}) at days 3, 4, and 5 after birth. A, Groups of mice were sacrificed at day 6 of life, and their blood glucose was determined. Blood glucose levels were significantly lower in HA-treated animals (p < 10-4, Student t test). B, In another series of experiments, the survival of untreated, Cw3-treated, and HA-treated double transgenic mice was assessed. The survival of untreated or Cw3-treated mice was significantly shorter than that of HA-treated mice (p < 10-4, Logrank test).

 
HA peptide treatment in (CL4-TCR x Ins-HA)F1 double transgenic pups does not induce bystander damage

Because HA peptide was injected at a time when activated HA-specific CD8+ T cells were inducing disease, we searched for bystander tissue damage that might result from destruction of Kd-expressing neighboring cells presenting the exogeneous HA peptide to the cytotoxic CD8+ T cells. (CL4-TCR x Ins-HA)F1 mice were treated daily with HA peptide from day 3 to day 5 after birth and sacrificed 1 day after the last injection. Pancreata from these animals were analyzed by immunohistochemistry. CD8+ T cell infiltration in the pancreas of Cw3-treated mice was extensive, and few insulin-positive cells were detected in the islets at this time point (Fig. 7Go, A–C, and Table IGo). In contrast, pancreata from HA-treated mice harbor very few infiltrating CD8+ T cells (Fig. 7GoE and Table IGo); importantly, the architecture of the islets is normal with numerous insulin-positive cells (Fig. 7Go, D and F). In addition, the exocrine pancreas was intact. Thus, autoreactive CD8+ T cells were eliminated from the pancreas without evidence of tissue damage in HA-treated (CL4-TCR x Ins-HA)F1 mice.



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  		FIGURE 7. Histology of pancreas from peptide-treated (CL4-TCR x Ins-HA)F1 mice. Sections from the pancreas of 6-day-old (A–C) Cw3-treated and (D–F) HA-treated (CL4-TCR x Ins-HA)F1 double transgenic mice are presented. A and D, Paraffin-embedded pancreatic tissue stained with hematoxylin and eosin (x320); B and E, cryostat sections stained for CD8 expression and counterstained with hematoxylin (x320); C and F, paraffin-embedded pancreatic tissue stained for insulin and counterstained with hematoxylin (x320).

 

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  		Table I. Number of infiltrating CD8+ T cells per islet in treated (CL4-TCR x Ins-HA)F1 pups1

 
We also assessed the effect of HA peptide treatment on CD8+ T cells in secondary lymphoid organs. Specifically, CD8+ T cells found in the spleen were analyzed for expression of CD62-L and transgenic Vß8.2 as markers of recent antigenic stimulation. Before treatment, >90% of CD8+ splenocytes from (CL4-TCR x Ins-HA)F1 mice were CD62-Lhigh and TCRhigh (Table IIGo). HA treatment induced TCR down-modulation on CD8+ T cells in both (CL4-TCR x Ins-HA)F1 and CL4-TCR transgenic mice (Table IIGo), although an intermediate decrease in the proportion of CD62-LhighVß8high CD8+ splenocytes was present at day 6 in Cw3-treated (or untreated) (CL4-TCR x Ins-HA)F1 mice. In addition, CD8+ T cell numbers were moderately decreased in (CL4-TCR x Ins-HA)F1 mice treated with HA vs Cw3 peptide (Table IIGo), suggesting that some of these cells had undergone AICD. Histological analyses of the spleen at day 6 show no signs of bystander destruction; notably, the structure of the follicles is similar in Cw3- and HA-treated (CL4-TCR x Ins-HA)F1 mice as well as in single Ins-HA mice (data not shown). Taken together, these data show that HA peptide treatment induced activation of CD8+ T cells without triggering cytotoxicity against neighboring cells in the pancreas or in the spleen.


View this table:
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  		Table II. Triggering of splenic CD8+ T cells in HA-treated CL4-TCR and (CL4-TCR x Ins-HA)F1 transgenic pups1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The novel findings reported in this study are that peptide-induced tolerance can be used to target activated pathogenic CD8+ T cells during an ongoing autoimmune disease. Furthermore, no bystander damage resulting from CD8+ T cell recognition of MHC class I+ cells loaded in vivo with the injected peptide is observed.

In CL4-TCR mice, injection of soluble HA512–520 peptide induced a rapid and synchronized activation of peripheral CD8+ T cells as early as 1 h after injection. An increase in the number of CD8+ T cells in the secondary lymphoid organs at day 3 preceded the deletion of Ag-specific T cells, as previously reported (32, 37, 38). We also observed an early depletion of CD8+ T cells at day 1 postinjection. Competition between T cell apoptosis and proliferation may occur at this time (39). Indeed, although a high proportion of apoptotic peripheral CD8+ T cells was detected 12 h postinjection, APCs from HA-injected mice are still able to induce proliferation of HA-reactive CD8+ T cells at this early time point. This heterogeneity in the behavior of CD8+ T cells may reflect different levels of transgenic TCR due to incomplete allelic exclusion at the TCR{alpha} locus. Alternatively, it may result from heterogeneity in Ag accessibility and recognition resulting in different levels of T cell activation.

More than 20% of CD8+ T cells were apoptotic 12 h postinjection, consistent with the rapid decrease in CD8+ T cell numbers observed between 12 and 24 h. A second deletion phase resulted in elimination of most of the CD8+ T cells from the spleen and the lymph nodes between days 3 and 7. Apoptosis was probably less synchronized during this period, consistent with fewer apoptotic CD8+ T cells being detected, therefore leading to a slower decline in the number of CD8+ T cells. Apoptotic CD8+ and CD4+ cells have also been detected in secondary lymphoid organs following peptide treatment in other models (30, 31, 32, 37, 39). However, we cannot exclude the possibility that, following HA peptide injection, activated CD8+ T cells also migrate to the liver to die (40).

By day 7, tolerance is established in HA-injected CL4-TCR mice. CD8+ T cells display reduced proliferation and IFN-{gamma} secretion capacity in response to HA peptide. In contrast to anergy, in which cells can retain the capacity to secrete IFN-{gamma} (41, 42, 43), reduced IFN-{gamma} secretion in our model probably results from the deletion of Tc1-like CD8+ cells. Peptide treatment could have induced deletion of high-avidity HA-specific CD8+ T cells selecting for CD8+ T cells that express a lower level of transgenic TCR. However, additional mechanisms could be responsible for the altered response to Ag. Using another TCR transgenic model, Dubois et al. have recently shown that the hyporesponsiveness of tolerized CD8+ T cells is linked to a defect in TCR signal transduction and to low levels of TCR surface expression (44). Although tolerance is relatively long-lasting in our model, the lack of Ag persistence in vivo and thymic output of new HA-specific T cells progressively reverses the tolerance state. Continuous or repetitive administration of peptide would most likely prolong this tolerance state (27).

To demonstrate that this Ag-specific strategy can be used to target activated CD8+ T cells during an autoimmune disease and protect from autoimmune damage, we applied the treatment to prediabetic (CL4-TCR x Ins-HA)F1 mice that exhibited CD8+ T cell infiltration in the pancreas. We have shown that HA peptide treatment significantly enhances the survival of these mice in contrast to mice treated with the Cw3 control peptide, which die from diabetes as rapidly as the untreated littermates. Histological analyses of the pancreata from HA-treated mice showed that most of the ß-islet cells were preserved and that infiltrating CD8+ T cells had been eliminated. These results are consistent with the notion that, in NOD and transgenic models of IDDM, the cytotoxic autoreactive CD8+ T cells are directly involved in ß-islet cell destruction (17, 21, 45, 46). Diabetes was significantly delayed by this short treatment. Newly generated autoreactive CD8+ T cells emerging from the thymus after peptide treatment would contribute to the progressive reversion of the protective effect.

In HA-treated double transgenic mice, the decrease in the number of CD8+ Vß8high T cells observed in the spleen, together with thymic deletion, might be sufficient to prevent recruitment of new infiltrating CD8+ cells to the pancreas. In other systems, autoreactive T cells have been shown to undergo apoptosis in the tissue targeted by the autoimmune process following peptide treatment (47, 48). Whether this also applies to the present model could not be assessed, as apoptotic cells were already numerous in the infiltrated pancreas of untreated animals (our unpublished results), precluding the evaluation of an increase in apoptotic cell numbers. Moreover, apoptosis of T cells would be difficult to distinguish from ß cell apoptosis, which is detectable in such accelerated models of autoimmune diabetes (49, 50).

Alternatively, peptide-induced tolerance in double transgenic mice may result from additional mechanisms. First, although newborn mice are able to mount strong CTL responses (51, 52, 53), differentiation of type 2 cytokine-secreting T cells is preferentially induced by systemic Ag during the neonatal period (54, 55). Several factors could contribute to this phenomenon: 1) differences in the function of APCs (56, 57, 58, 59, 60); 2) lower frequency of T cells (61); 3) greater requirements for accessory cell factors (62); and 4) the high relative Ag dose (54). Second, whereas in CL4-TCR transgenic mice the peptide injection targets naive CD8+ T cells, in double transgenic mice >50% of pancreatic CD8+ T cells (data not shown) and a part of the splenic CD8+ T cells are preactivated. Previous in vivo encounter with Ag changes reactivity of T cells to injected Ag (63) and can favor induction of regulatory subsets secreting IL-4 and/or IL-10 (4, 5, 11). Consistent with this notion, significantly increased levels of IL-10 (but not IL-4) mRNA were detected by RT-PCR in spleens of HA peptide-treated, as compared with untreated, (CL4 x Ins-HA)F1 double transgenic animals (data not shown). Similarly, IL-10 transcripts were detected in the pancreas of three of five HA peptide-treated double transgenic mice.

It has been shown that memory CD8+ T cells are more resistant to tolerance induction than naive T cells (64) and that these cells can mediate cytotoxicity resulting in tissue damage following peptide treatment (24). Therefore, HA peptide administration could enhance cytotoxicity by established CD8+ effector T cells. An important finding in the present report is that peptide treatment did not elicit bystander killing. Both endocrine and exocrine pancreatic tissue remained intact, nor was tissue damage detected in the spleen, where HA peptide is rapidly distributed after i.v. injection.

Altogether, our data provide the first evidence that administration of soluble MHC class I-binding agonist peptide to specifically target autoreactive CD8+ T cells can provide protection from spontaneous autoimmunity. In contrast to other models in which immunopathology is associated with tolerance induction in memory CD8+ T cells, HA peptide treatment eliminates activated CD8+ T cells during ongoing autoimmune diabetes without bystander damage. However, this approach has to be validated in a nontransgenic model of organ-specific autoimmune disease. In that respect, this approach might prove to be effective in NOD mice, in which autoreactive CD8+ T cells participate in disease progression and in which a homogeneous autoreactive CD8+ T cell population has been identified (19, 65, 66, 67). For human autoimmune diseases, where peptide therapy is at an early stage of clinical development, a combination of class I- and class II-binding self-peptides should be considered, to target both autoreactive CD8+ and CD4+ T cells.


   Acknowledgments
 
We thank F. Marotte and A. Lesot for technical assistance. We also thank Drs. A. Freitas and R. Tisch for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale, the French agency for AIDS research (Agence Nationale de Recherches sur le SIDA), and the Fondation de France. N.B. is a recipient of fellowships from the Ministère de la Recherche and the Fondation pour la Recherche Médicale. Back

2 Current address: Immuno-Designed Molecules, 172 rue de Charonne, 75011 Paris, France. Back

3 Address correspondence and reprint requests to Dr. Roland Liblau, Institut National de la Santé et de la Recherche Médicale CJF 9711, 105 boulevard de l’Hôpital, 75013 Paris, France. Back

4 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; HA, hemagglutinin; GAD, glutamic acid decarboxylase; NOD, nonobese diabetic; AICD, activation-induced cell death; LCMV, lymphocytic choriomeningitis virus. Back

Received for publication September 30, 1999. Accepted for publication April 12, 2000.


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