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Autoantigen-Specific CD4⁺CD28^low T Cell Subset Prevents Autoimmune Exocrinopathy in Murine Sjögren’s Syndrome¹

Kaoru Saegusa^*,, Naozumi Ishimaru^*, Kumiko Yanagi^*, Norio Haneji^*, Mizuho Nishino, Miyuki Azuma, Ichiro Saito^* and Yoshio Hayashi^2,*

Departments of ^* Pathology and Pediatric Dentistry, Tokushima University School of Dentistry, Kuramotocho, Tokushima, Japan; and Department of Immunology, National Children’s Medical Reseach Center, Tokyo, Japan

	Abstract

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Organ-specific autoimmune exocrinopathy resembling Sjögren’s syndrome (SS) that spontaneously develops in NFS/sld mutant mice thymectomized 3 day after birth is dependent on Th1-type CD4⁺ T cells. We previously reported that a cleavage product of 120-kDa -fodrin may be an important autoantigen in the pathogenesis of SS in both an animal model and the patients. We demonstrate that in an animal model of SS with overt exocrinopathy, a unique CD4⁺ T cell subset expressing CD28^low is dramatically increased in spleen cells before the disease onset, but that the CD4⁺ T cells of diseased mice were virtually all CD28^high. We found that the spleen cells in these mice before the disease onset showed a significant increase in autoantigen-specific T cell proliferation. Analysis of in vitro cytokine production by spleen cells indicated, before the disease onset, severely impaired production of IL-2 and IFN- in the animal model, whereas high levels of IL-4 were observed. Expression of cytokine genes, including IL-4, IL-10, and TGF-ß, was detected in FACS-sorted CD4⁺CD28^low T cells by RT-PCR analysis. Transfer of CD4⁺CD28^low T cells into the animal model actually prevented the development of autoimmune lesions including autoantibody production. These results suggest that a CD4⁺CD28^low T cell subset that is continuously activated by an organ-specific autoantigen may play a regulatory role in the development of organ-specific autoimmune disease in an animal model of SS.

	Introduction

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Maintenance of peripheral tolerance is an important defense mechanism of the immune system (1, 2, 3). Although active suppression mediated by T cells has been proposed as a mechanism for maintaining peripheral tolerance (4), failure to isolate regulatory T cells involved in Ag-specific inhibition of immune responses has made it difficult to elucidate the mechanisms underlying active suppression. T cell activation leading to T cell proliferation and cytokine production requires two independent signals (5, 6). The first signal involves the recognition, by a specific TCR/CD3 complex, of Ag presented by MHC molecules on the surface of APCs. One key costimulatory signal is provided by interaction of the T cell surface receptor CD28, with its natural ligands, B7-1/B7-2 on APCs or T cells (7, 8, 9). Recent studies have demonstrated that CD28 costimulation is crucial to autoantigen-specific T cell activation and to the development of collagen-induced arthritis and experimental autoimmune encephalomyelitis (10, 11). On the other hand, antigenic stimulation augments surface expression of CD28 and initiates the synthesis of certain immunomodulatory cytokines, such as IL-2, IL-4, and IFN-. These cytokines are known to be regulated by the CD28 pathway of T cell activation and are essential in promoting a wide range of immune responses including autoimmunity (12, 13).

Sjögren’s syndrome (SS)³ is a chronic autoimmune exocrinopathy chiefly affecting the salivary and lacrimal glands and leading to clinical symptoms of dryness of the mouth and eyes (sicca syndrome) (14, 15). We have established and analyzed an animal model for SS in NFS/sld mutant mice thymectomized 3 days after birth (3d-Tx) (16, 17). However, the role of CD28 costimulation for autoantigen-specific T cell activation in this murine SS model has not yet been analyzed. We previously reported that autoimmune lesions in the murine SS model are mediated by CD4⁺ T cells, and autoreactive T cells have been found to contain mRNAs for Th1-type cytokines, including IL-2 and IFN-, but not for Th2-type cytokines (18, 19). We have identified a 120-kDa -fodrin autoantigen in salivary gland tissue from this model and identified T cell responses specific to this protein, in addition to in vitro production of IL-2 and IFN- (20).

The present study was conducted to elucidate the mechanisms underlying active suppression through the regulatory T cells involved in autoantigen-specific inhibition of immune responses in a murine model of SS.

	Materials and Methods

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Mice

Female NFS/N strain carrying the mutant gene sld (16) were reared in our specific pathogen-free mouse colony, and given food and water ad libitum. Thymectomy was performed on day 3 after birth (3d-Tx) in NFS/sld mice. A total of 176 mice, consisting of 112 3d-Tx and 64 nonthymectomized (non-Tx) NFS/sld mice, were investigated in the present study. They were killed by cervical dislocation sequentially during the time intervals ranging from 1, 2, 3, 4, 8, 12, 16, and 20 wk after the thymectomy. BALB/c mice (n = 24), purchased from Charles River Japan (Atsugi, Japan), were used as controls. 3d-Tx (n = 7) and non-Tx (n = 6) C57BL/6 mice, purchased from Charles River Japan (Atsugi), were analyzed as controls.

Histology

All organs were removed from the mice, fixed with 4% phosphate-buffered formaldehyde (pH 7.2), and prepared for histologic examination. The sections were stained with hematoxylin and eosin. Histological grading of the inflammatory lesions was done according to the method proposed by White and Casarett (21), as follows: score 1 indicates that one to five foci being composed of more than 20 mononuclear cells per focus were seen; score 2 indicates that more than five such foci were seen, but without significant parenchymal destruction; score 3 indicates degeneration of parenchymal tissue; score 4 indicates extensive infiltration of the glands with mononuclear cells and extensive parenchymal destruction. These slides were scored by three independent, well-trained pathologists in a blinded manner.

Flow cytometric analysis

Spleen cells and regional lymph node (LN) cells from 3d-Tx and non-Tx NFS/sld mice were analyzed by flow cytometry. Single cell suspensions were stained with Abs conjugated to PE (anti-CD3, Life Technologies, Grand Island, NY; anti-CD4, Cedarlane Laboratories, Ontario, Canada; B220, PharMingen, San Diego, CA) and FITC (anti-CD8, Cedarlane Laboratories; Thy-1.2, anti-CD44, anti-CD45RB, anti-Mel-14, anti-CD25, anti-CD69, and anti-CD28, PharMingen), and analyzed with EPICS (Coulter, Miami, FL). Viable cells (1 x 10⁵) were gated according to size and scatter to eliminate dead cells and debris from analysis. Analysis of intracellular cytokines by flow cytometry was performed as described (4, 22). Briefly, cells (10⁶ per ml) were activated with immobilized anti-CD3 mAb (Cedarlane Laboratories) for 4 h. Monensin (Wako Pure Chemical, Osaka, Japan) was added at 2 µM, and 2 h later cells were collected, washed, and fixed with 4% paraformaldehyde for 10 min at room temparature and then permeabilized with 0.1% saponin in PBS at 4°C for 10 min. For intracellular cytokine staining, cells were incubated with anti-IL-2 FITC (8 µg/ml; PharMingen), anti-IL-4 PE (5 µg/ml; PharMingen), and anti-IFN- FITC (1 µg/ml; PharMingen), respectively. Samples were analyzed on an EPICS (Coulter). Cell sorting was performed using FACStar^Plus with LYSIS-II software. The purity of sorted cells exceeded 95%.

Proliferation assay

Single cell suspensions of spleen cells from 3d-Tx and non-Tx NFS/sld mice were cultured in 96-well flat-bottom microtiter plates (5 x 10⁵ cells/well) in RPMI 1640 containing 10% FCS, penicillin/streptomycin, and 2-ME. A total of 5 x 10⁵ was added to 96-well flat-bottom microtiter plate (Nunc, Roskilde, Denmark) immobilized with or without anti-CD3 mAb (Cedarlane Laboratories), and recombinant -fodrin Ag (20). For proliferation assay, prepared cells were cultured for 72 h under stimulation of anti-CD3 mAb or recombinant -fodrin protein, and pulsed with 1 µCi/well of [³H]thymidine (NEN Life Science Products, Boston, MA) during final 20 h of the culture. [³H]Thymidine incorporation was evaluated using an automated beta liquid scintillation counter. Data were expressed as the mean ± SD determined in triplicate wells. We further examined the preventive effects of neutralizing Abs to IL-4 (11B11) and IL-10 (provided from Dr. H. Ishida, National Utano Hospital, Kyoto, Japan) for autoantigen-specific proliferative T cell responses.

Measurement of cytokine production

Cytokine production from spleen cells of 3d-Tx and non-Tx NFS/sld mice was tested by two-step sandwich ELISA using a mouse IL-2, IL-4, and IFN- kit (Genzyme, Cambridge, MA). In brief, culture supernatants from spleen cells activated with immobilized anti-CD3 mAb (Cedarlane Laboratories) for 3 days were added to microtiter plates precoated with anti-IL-2, IL-4, and IFN- capture Ab and incubated overnight at 4°C. After addition of biotinylated detecting Ab and incubation at room temparature for 45 min, avidin-peroxidase was added and incubated at room temperature for 30 min. Plates were washed extensively with 1% Tween in PBS between each step. Finally, ABTS (2,2'-azinodi-3-ethylbenzthiazoline sulfonate) substrate containing H₂O₂ was added and the colorimetric reaction was read at an absorbance of 450 nm using an automatic microplate reader (Bio-Rad, Hercules, CA). The concentrations of IL-2 (pg/ml), IL-4 (pg/ml), and IFN- (U/ml) were calculated according to the standard curves produced by various concentrations of recombinant cytokines.

RT-PCR

To characterize the splenic CD4⁺CD28^low T cell subset, FACS-sorted fraction was diced coarsely, rinsed in ice-cold RNase-free saline, and then promptly frozen in liquid nitrogen. Cells were homogenized in guanidine thiocyanate solution, and total RNA was prepared essentially as reported previously (23). Briefly, aliquots of tissues were lysed in 200 µl of ice-cold lysis solution D containing 4 M guanidine thiocyanate, 25 mM sodium citrate (pH 7), 0.1 M 2-ME, and 0.05% (w/v) sarcosyl. The mixture was chilled on ice and centrifuged at 10,000 x g for 20 min at 4°C, and the supernatant was treated with an equal volume of isopropanol at -20°C for 90 min. The mixture was centrifuged, and the pellets were incubated with solution D at 20°C for 60 min. The RNA was washed with 75% ethanol in diethyl pyrocarbonate (Aldrich Chemical, Milwaukee, WI)-treated water, centrifuged, and stored at -80°C until further processing. RNA was reverse transcribed into cDNA. The cDNA reaction mixture was diluted with 90 µl of PCR buffer, and mixed with 50 pM concentrations of the 5' and 3' primers, 1.25 mM dNTP, 20 mM MgCl₂, and 2 U of thermostable Taq polymerase (Perkin-Elmer/Cetus, Norwalk, CT). The primers used were synthesized by the phosphoramide method in a DNA synthesizer (model 391 PCR-MATE; Applied Biosystems, Foster City, CA), and purified on Sephadex G-50 columns (Pharmacia LKB Biotechnology, Piscataway, NJ) and HPLC. The sequences of the primers were specific, as confirmed by a computer-associated search of updated versions of a GenBank, and were chosen to have a balanced nucleotide composition with a GC content of 40–60%. The cDNA was subjected to enzymatic amplification in a DNA thermal cycler (Perkin-Elmer/Cetus) by using specific primers. The specific primers used were as follows: IFN-, CCT CAG ACT CTT TGA ACT CT and CAG CGA CTC CTT TTC CGC TT; IL-2, ATG TAC AGC ATG CAG CTC GCA TCC TGT GTC A and GTT GTC AGA GCC CTT TAG TTT TAC AAC; IL-4, TCT CAA CCC CCA GCT AGT TGT CAT and CCA GGC ATC GAA AAG CCC G; IL-10, ATG CAG GAC TTT AAG GGT TAC TTG GGT T and ATT YCG GAG AGA GGT ACA AAC GAG GTT T; TGF-ß1, GCG GAC TAC TAT GCT AAA GAG G and GAT TTC CGT CTC CTT GGT; TGF-ß2, ATC TGG TCC CGG TGG CGC T and TCT GTA GAA AGT GGG CGG; GAPDH, CCA TCA CCA TCT TCC AGG AGC GAG and CAC AGT CTT CTG GGT GGC AGT GAT; ß-actin, GTG GGC CGC TCT AGG CAC CA and CGG TTG GCC TTA GGG TTC AGG GGG. The amplified DNA reaction mixture was subjected to 1.7% agarose gel electrophoresis, and the amplified product was visualized by UV fluorescence after staining with ethidium bromide.

Effect of cell transfer with CD4⁺CD28^low T cell subset

3d-Tx NFS/sld SS model mice (n = 7, at 4 wk old) were i.p. transferred with FACS-sorted CD4⁺CD28^low splenic T cells (5 x 10⁶) at 2 and/or 3 wk of age, and analyzed at 8 wk old. For control, sorted CD4⁺ and CD8⁺ spleen cells at 6 wk old were injected i.p. into SS model mice (n = 5, 5 x 10⁶ cells each). T cell activation markers (CD44, CD45RB^low, Mel-14^low) were examined in the spleens on CD4 in each group. In addition, autoantibody production against the 120-kDa -fodrin in sera was tested both in treated and nontreated mice.

	Results

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Proliferative T cell response to -fodrin autoantigen

The autoimmune lesions in the salivary and lacrimal glands develop at 4 wk old or later, whereas no inflammatory lesions are observed in non-Tx NFS/sld mice at any age, as previously described in detail (17, 18, 19). To address the role of -fodrin autoantigen-reactive T cells, we examined the proliferative T cell responses against -fodrin autoantigen in the spleen cells of different ages. We found that the spleen cells in 3d-Tx NFS/sld mice showed a significant increase in autoantigen-specific T cell proliferation before the onset of disease (2 or 3 wk old), which increased with age (Fig. 1A), but not in non-Tx control mice. No significant differences were observed in the proliferative response stimulated with Con A between 3d-Tx and non-Tx NFS/sld mice (Fig. 1A). No proliferative T cell response to -fodrin was observed in either 3d-Tx or non-Tx C57BL/6 control mice (Fig. 1B).

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Proliferative T cell response to the recombinant -fodrin autoantigen develops in 3d-Tx NFS/sld mice at 2, 3, 6, and 8 wk of age, but not in non-Tx NFS/sld mice (*, p < 0.001, Student’s t test). A significant increase in proliferative T cell response to the recombinant -fodrin autoantigen was clearly observed in splenic CD4+ T cells before the disease onset (2 and 3 wk old) in 3d-Tx NFS/sld mice, compared with those in non-Tx NFS/sld mice. No difference was observed in Con A-stimulated blastogenesis measured in spleen cells from 3d-Tx and non-Tx NFS/sld mice (A). Five mice of each age group were analyzed. Data are expressed as cpm per culture ± SD in triplicate. No proliferative T cell response to recombinant -fodrin was observed in spleen cells from 3d-Tx and non-Tx C57BL/6 mice (B). Data are expressed as cpm per culture ± SD in triplicate.

To clarify whether self-reactive T cells are spontaneously activated in 3d-Tx NFS/sld mice, we analyzed CD4⁺ T cells bearing activation markers in the spleens and regional LN by flow cytometry. The results showed that the activation markers (CD44^high, CD45RB^low, Mel-14^low) were significantly up-regulated in the spleens and LN cells gated on CD4 before the disease onset in 3d-Tx NFS/sld mice, but not in non-Tx NFS/sld or 3d-Tx C57BL/6 mice (Fig. 2, A and B). This sugested that the effector CD4⁺ T cells appear in the periphery as an early event after the thymectomy. Expression of these markers increased with age. An early activation marker, CD4⁺CD69⁺, was only present at 1 and 2 wk of age after 3d-Tx, but was undetectable from 3 wk of age onward (data not shown).

View larger version (46K): [in this window] [in a new window]   FIGURE 2. Kinetic analysis of flow cytometry of spleen cells from 3d-Tx and non-Tx NFS/sld mice gated on CD4+. Up-regulation of activation markers (CD44high, CD45RBlow, and Mel-14low) was clearly observed in the splenic CD4+ T cells before the disease onset (2 and 3 wk old) in 3d-Tx NFS/sld mice, compared with those of non-Tx NFS/sld mice (A). Five mice of each age group were analyzed. No significant up-regulation of activation markers was observed in the splenic CD4+ T cells from 3d-Tx and non-Tx C57BL/6 mice (B).

We then searched for expression of the CD28 costimulatory signal in the spleens of 3d-Tx NFS/sld mice compared with that in non-Tx mice. A CD4⁺ T cell subset expressing CD28^low was detected in the spleen cells of 3d-Tx NFS/sld mice restricted before the disease onset (1–3 wk old) (Fig. 3A and Table I). This unique subset was not found on the CD8⁺ T cells at any age of 3d-Tx NFS/sld, or on the CD4⁺/CD8⁺ T cells of non-Tx NFS/sld or 3d-Tx C57BL/6 mice. Fig. 3B shows FACS-sorted CD4⁺CD28^low T cell subset of spleen cells from model mice at 3 wk of age. Very few CD4⁺CD28^low T cell subsets appeared after 4 wk old, when organ-specific autoimmune lesions begin to develop. We also confirmed that the CD4⁺CD28^low T cell subset is clearly different from CD4⁺CD25⁺ T cells in the murine SS model (Fig. 3C).

View larger version (41K): [in this window] [in a new window]   FIGURE 3. Identification of a CD4+CD28low T cell subset in spleen cells from 3d-Tx NFS/sld mice by flow cytometry only before the disease onset and not in non-Tx NFS/sld mice (A). Representative profile of the FACS-sorted CD4+CD28low T cell subset in spleen cells from 3d-Tx NFS/sld mice at 3 wk of age (B). The CD4+CD28low T cell subset is clearly different from the CD4+CD25+ T cells in the murine SS model at 2 and 3 wk before the disease onset. Five mice in the same age group were analyzed (C).

Culture supernatants from anti-CD3 mAb-stimulated splenic T cells obtained from the murine SS model before the disease onset contained high levels of IL-4, but low levels of IL-2 and IFN- by ELISA (Fig. 4A). Flow cytometry revealed a high proportion of intracellular cytokine production of IL-4 by splenic CD4⁺ T cells of the murine SS model mice only before the disease onset (Fig. 4B). When we compared with the effect of different doses of CD28 costimulation on the splenic T cell response (8 wk old) to -fodrin autoantigen and anti-CD3, it was found that low dose CD28 costimulation (0.1 and 1 µg/ml) had an inhibitory effect on -fodrin autoantigen-specific proliferation, but not on the anti-CD3 mAb-stimulated response (Fig. 5A), whereas CTLA-4Ig had no effects (Fig. 5B). In addition, -fodrin autoantigen-induced T cell responsiveness (8 wk old), but not anti-CD3 mAb-induced responsiveness, was clearly inhibited by culture supernatants from nonstimulated splenic T cells before the disease onset (3 wk old) (Fig. 6A), and neutralizing Abs to IL-4 and IL-10 blocked these -fodrin autoantigen-specific T cell responses (Fig. 6B). The FACS-sorted CD4⁺CD28^low T cell subset showed increased expression of cytokine genes, including IL-4, IL-10, IFN-, and TGF-ß, whereas CD4⁺CD28^high T cells expressed IFN- and IL-4 by RT-PCR (Fig. 7).

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Detection of the Th2-type cytokine profile in the SS animal model before the disease onset. Culture supernatants from anti-CD3 mAb-stimulated splenic T cells in 3d-Tx NFS/sld mice (3 wk old) contained a high level of IL-4, but low levels of IL-2 and IFN-, as measured by ELISA (*, p < 0.005; **, p < 0.001, Student’s t test) (A). A high proportion of intracellular cytokine production of IL-4 from splenic CD4+ T cells was observed in 3d-Tx NFS/sld mice before the disease onset (3 wk old) (B). Five mice in each age group were analyzed.

View larger version (20K): [in this window] [in a new window]   FIGURE 5. Low dose CD28 costimulation (0.1 and 1 µg/ml) had an inhibitory effect on -fodrin autoantigen-specific T cell proliferation compared with no costimulation (0 µg/ml) from the SS model mice, but not on the anti-CD3 mAb-stimulated T cell response (*, p < 0.05; **, p < 0.01, Student’s t test) (A). CTLA-4Ig (UC10-4F10) had no inhibitory effect on either the autoantigen-specific or the anti-CD3 mAb-stimulated T cell response (B). Spleen cells from 3d-Tx NFS/sld mice (n = 5) at 8 wk old of age were used.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. Autoantigen-specific immunosuppressive effects of culture supernatants before the disease onset. Splenic T cell responsiveness to -fodrin autoantigen (8 wk old) was clearly inhibited by the nonstimulated T cell culture supernatants before the disease onset (3 wk old). Splenic T cell culture supernatants (100 µl, 1 x 107 cells/well) before the disease onset were diluted at different concentrations (1/200, 1/20, and 1/2), and examined. T cell responsiveness was significantly inhibited by the culture supernatants diluted at 1/20 and 1/2, compared with those diluted at 1/200. Data are expressed as cpm per culture ± SD in triplicate (*, p < 0.01; **, p < 0.001, Student’s t test). Anti-CD3 mAb-stimulated T cell responses were not inhibited in the same manner (A). Neutralizing Abs to IL-4 and IL-10 blocked splenic T cell responsiveness to -fodrin autoantigen by the culture supernatants from splenic T cells before the disease onset (3 wk old). Spleen cells from 8-wk-old 3d-Tx NFS/sld mice (n = 5) were analyzed. Data are expressed as cpm per culture ± SD in triplicate (*, p < 0.01; **, p < 0.001, Student’s t test) (B).

View larger version (32K): [in this window] [in a new window]   FIGURE 7. Cytokine gene expressions in the FACS-sorted CD4+CD28low T cell subset from spleen cells at 2- and/or 3-wk-old 3d-Tx NFS/sld mice, as assessed by RT-PCR analysis. The FACS-sorted CD4+CD28low T cell subset expressed for the cytokine genes including IL-4, IL-10, and TGF-ß, whereas CD4+CD28high T cells expressed IL-2, IFN-, and IL-4. Con-A-stimulated splenic T cells expressed IL-2, IFN-, IL-4, IL-10, and TGF-ß mRNAs.

3d-Tx NFS/sld SS model mice were injected with FACS-sorted CD4⁺CD28^low T cells (5 x 10⁶) at 4 wk old (n = 7) and examined histopathologically at 8 wk old. They were compared with control mice injected with CD4⁺ and CD8⁺ splenic T cells at 6 wk old (5 x 10⁶; n = 5 each). Transfer of CD4⁺CD28^low T cells was effective in preventing the development of autoimmune lesions in the parotid (p < 0.05), submandibular (p < 0.01), and lacrimal (p < 0.01) glands of the SS model mice, but not in the control group (Table II). The activation markers (CD44^high, CD45RB^low, Mel-14^low) were clearly down-regulated in spleen cells gated on CD4 from transferred SS model mice, and production of serum autoantibody against 120-kDa -fodrin was inhibited (Fig. 8, A and B).

View larger version (29K): [in this window] [in a new window]   FIGURE 8. Preventive effect of transfer of FACS-sorted CD4+CD28low T cells into the SS animal model. Down-regulation of activation markers (CD44high, CD45RBlow, Mel-14low) was observed in spleens cells gated on CD4 from recipient SS model mice. The differences bewteen groups were statistically significant at p < 0.01 (Student’s t test) (A). Inhibition of serum autoantibody production to 120-kDa -fodrin was observed in two different recipient mice (1, 2), as shown by immunoblotting (B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we identified a unique CD4⁺ T cell subset expressing CD28^low in the spleens before the disease onset of murine SS in 3d-Tx NFS/sld mice. The CD4⁺CD28^low subset was completely absent after 4 wk old, when organ-specific autoimmune lesions begin to develop. It can be speculated that the CD4⁺ T cell subset expressing CD28^low is generated when autoreactive T cells to the organ-specific autoantigen are selectively activated in the periphery in vivo. Previous studies have demonstrated that CD28 signaling can prevent anergy in Ag-specific T cell responses (31). Blocking CD28 ligation with Fab fragments of anti-CD28 mAb has been described as a strategy to suppress an inappropriate immune response, and treatment in vitro led to inhibition of cytokine production and induction of a state of T cell hyporesponsiveness (32). Culture supernatants from anti-CD3 mAb-stimulated splenic T cells in the SS model contained a high level of IL-4, but low levels of IL-2, and IFN- only before the disease onset. Moreover, spleen cells from the murine SS model before the disease onset showed a significant increase in -fodrin autoantigen-specific T cell proliferation, and neutralizing Abs to IL-4 and IL-10 clearly blocked these responses. These findings strongly suggest that in the murine SS model before the disease onset, a peripheral CD4⁺ T cell subset expressing CD28^low elicited a predominant Th2 response with IL-4 and IL-10 production. Moreover, the FACS-sorted CD4⁺CD28^low T cell subset expresses cytokine genes, including IL-4, IL-10, IFN-, and TGF-ß, according to the results of RT-PCR. It is well known that IL-10 and TGF-ß are regulatory molecules in various immune responses (33, 34, 35, 36). Cell to cell interaction between CD4⁺ T cells and APC has been demonstrated to result in the secretion of IL-10, which is capable of shifting the Th1/Th2 balance toward Th2 (37). It is possible that the CD4⁺ regulatory T cells expressing CD28^low that we have described in this study protect against SS autoimmune lesions by secretion of immunomodulatory cytokines, such as IL-4, IL-10, and/or TGF-ß. Indeed, we demonstrated evidence that transfer of CD4⁺CD28^low T cells is effective in preventing the development of autoimmune lesions in a murine SS model including autoantibody production. Further studies are required to determine how they mediate immunosuppressive activities through unknown signaling events.

Much attention has been focused on the role of CD28 as a costimulatory receptor during various T cell activation (38, 39, 40), and blockage of CD28/B7 interactions has been demonstrated to lead to prolonged survival of both allo- and xenografts in graft rejection in vivo (41, 42). Studies in CD28-deficient mice have confirmed the requirement for costimulation through CD28 for both autoantigen-specific T cell activation and T cell-dependent B cell responses (43, 44). These investigations have suggested that CD28 is required not only for initiation of a successful immune response, but for maintenance of a response driven by Ag receptor engagement (44, 45). It has been proposed that B7-CD28 interaction delivers a positive signal for T cell responses, whereas B7-CTLA-4 interaction delivers a negative signal (46, 47). Since we found that CD4⁺ T cells stimulated with low dose CD28 costimulation (0.1 and 1 µg/ml), but not with CTLA-4Ig, had an inhibitory effect on the autoantigen-specific proliferative response in vitro, it is unlikely that B7-CTLA-4 interaction delivered a negative signal for autoantigen-specific T cell responses in vivo.

Taken together, these studies suggest that a unique CD4⁺ T cell subset expressing CD28^low may play a regulatory role in the maintenance of peripheral tolerance against organ-specific autoantigen, in which autoantigen-specific T cell responses may be a critical event early in the development of the autoimmune lesions.

	Footnotes

 
¹ This work was supported in part by a Grant-in-Aid for Scientific Research (08407057, 12307040) from the Ministry of Education, Science, and Culture of Japan.

² Address correspondence and reprint requests to Dr. Yoshio Hayashi, Department of Pathology, Tokushima University School of Dentistry, 3 Kuramotocho, Tokushima 770, Japan.

³ Abbreviations used in this paper: SS, Sjögren’s syndrome; 3d-Tx, mice thymectomized 3 days after birth; LN, lymph node; MBP, myelin basic protein; non-Tx, nonthymectomized.

Received for publication December 27, 1999. Accepted for publication May 23, 2000.

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