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ICOS Ligand Costimulation Is Required for T-Cell Encephalitogenicity
Clinical Immunology Vol. 100, No. 3, September, pp. 277–288, 2001 doi:10.1006/clim.2001.5074, available online at http://www.idealibrary.com on
ICOS Ligand Costimulation Is Required for T-Cell Encephalitogenicity Romeo A. Sporici,* Richard L. Beswick,* Carolyn von Allmen,* Catherine A. Rumbley,* Martha Hayden-Ledbetter,† Jeffrey A. Ledbetter,† and Peter J. Perrin* ,1 *Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; and †Pacific Northwest Research Institute, Seattle, Washington 98122
The interaction of ICOS with its ligand on APC provides a costimulatory signal to previously activated T-cells. In these studies, we blocked the ICOS:ICOS ligand interaction with ICOS-Ig during the in vitro activation of MBP-reactive transgenic CD4 ⴙ T-cells. The presence of ICOS-Ig in these cultures inhibited the ability of the transgenic T-cells to transfer EAE, although they entered the brains of the recipient mice. ICOS-Ig increased apoptosis in the transgenic T-cells, especially in the memory population. This enhanced apoptosis was accompanied by an increase in the BAX/ BCL-2 mRNA ratio. ICOS-Ig did not prevent IL2 production, demonstrating that IL-2 production is ICOS ligand independent. IFN-␥ and IL-10 production by the transgenic T-cells, however, was suppressed. Finally, ICOS-Ig injection into mice after the first signs of EAE ameliorated clinical disease. Therefore, ICOSL provides a signal distinct from CD28 costimulation that is required for the activation and viability of encephalitogenic T-cells. © 2001 Academic Press Key Words: EAE/MS; costimulatory molecules; T-lymphocytes; immunotherapy.
INTRODUCTION
Experimental autoimmune encephalomyelitis (EAE)2 is a model of T-cell-mediated demyelination. T-cells that recognize myelin antigens, such as myelin basic protein (MBP), recruit nonspecific T-cells and macrophages into the CNS. These cells effect demyelination, resulting in ascending paralysis. Therefore, EAE is often used as a model for human multiple sclerosis (MS) (1). During T-cell activation, costimulatory and accessory signals determine both whether a T-cell is acti1 To whom correspondence and reprint requests should be addressed at Department of Medicine, Pulmonary, Allergy, and Critical Care Section, 909 BRB II, 421 Curie Boulevard, Philadelphia, PA 19104-6160. E-mail: [email protected]. Fax: 215-8980193. 2 Abbreviations used: Ac, acetylated; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; ICOS, inducible costimulator; ICOSL, ICOS ligand; ip, intraperitoneal; MBP, myelin basic protein; MS, multiple sclerosis.
vated and the outcome of that activation. One major T-cell costimulatory signal is delivered through the interaction of T-cell CD28 with B7 ligands (CD80 and CD86) on APC. Naı¨ve cells have a greater requirement for CD28 costimulation than memory cells do (2, 3). For example, myelin-reactive T-cells are present in both MS patients and controls, demonstrating that autoreactive T-cells can exist in individuals without pathological consequences (2, 4). However, the MBP-reactive T-cells from MS patients are less dependent on CD28-mediated costimulation than those from normal controls. These findings suggest that therapies that prevent CD28 signaling will probably be of limited use in autoimmune diseases like MS and understanding the costimulatory requirements for memory T-cells is desirable. Inducible costimulator (ICOS) is a recently described member of the CD28 family (5–7). Murine ICOS and CD28 share only 19% amino acid identity but there is a strong structural homology between ICOS and the other CD28 family members, CD28 and CD152. Cysteines that are critical for intramolecular and intermolecular disulfide bonds are conserved. Significantly, however, ICOS lacks the MYPPPY motif, a highly conserved hexapeptide, which is critical for CD28 and CD152 binding to CD80 and CD86, suggesting that ICOS interacts with another distinct ligand. ICOSL is a member of the B7 family, which also includes CD80, CD86, and B7H-1 (6, 10). ICOS does not bind CD80 or CD86. Likewise, ICOSL does not bind either T-cell CD28 or CD152. Therefore, although these pathways are related, they do not interact with each other’s receptors and ligands. In contrast to CD28, ICOS is not expressed constitutively but is upregulated following T-cell activation and might be involved primarily in memory T-cell activation (5). For example, ICOS cross-linking during either sensitization or challenge enhanced ear thickness swelling in a contact hypersensitivity response, but this effect was more dramatic during challenge (7). Cytokine production following CD28 and ICOS ligation is significantly different, although the role of ICOS in the production of specific cytokines is still not clear.
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1521-6616/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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ICOS does not influence the production of IL-2 as CD28 does (5, 6). Instead, both IFN-␥ and IL-10 are enhanced by ICOS costimulation (5, 6, 10). For example, crosslinking ICOS with soluble ICOSL (B7RP1-Fc) results in enhanced production of IFN-␥ but not IL-2 (6). However, the inhibition of IL-2 production by ICOS-Ig has been reported (11). In these studies, we assessed the role of ICOSLmediated costimulation in transgenic T-cells bearing a MBP-specific TCR. These transgenic T-cells were activated in the presence or the absence of ICOS-Ig, a soluble receptor that competitively inhibits the ICOS: ICOSL interaction. ICOS-Ig increased the BAX/BCL-2 ratio and increased the apoptosis in these antigenstimulated transgenic T-cells. ICOS-Ig prevented the production of IFN-␥ and IL-10 but not IL-2 and suppressed their ability to adoptively transfer EAE to naı¨ve recipients. Importantly, ICOS-Ig injection after the onset of clinical signs ameliorated EAE. Therefore, ICOSL provides a distinct costimulatory signal that participates in activation and survival of T memory cells. As such, it is a potential therapeutic target for ongoing autoimmune diseases. MATERIALS AND METHODS
Mice Six- to ten-week-old B10.PL mice were purchased from Jackson Laboratory (Bar Harbor, ME). Breeding pairs of MBP-specific TCR transgenic B10.PL mice were provided by Dr. S. Tonegawa (MIT, Cambridge, MA) (12, 13). Mice carrying the transgene were identified using flow cytometry and anti-V8 monoclonal antibody as previously described (MIT, Cambridge, MA) (12, 13). These mice were bred and maintained in our animal colony in compliance with the Animal Care and Use Committee. In conducting this research, the investigators adhered to the Guide for Laboratory Animal Facilities and Care as promulgated by the Committee on the “Guide for Laboratory Animal Resources,” National Academy of Sciences-National Research Council, DHHS, Publication No. (NIH) 86-23.
MBP Ac1-11 (Ac-ASQKRPSQRHG) was purchased from Genemed Synthesis (South San Francisco, CA). Cell Culture and Experimental Autoimmune Encephalomyelitis Spleen cells from transgenic mice were cultured with wild-type spleen cells from B10.PL mice (8 ⫻ 10 6 cells/2 ml in 24-well plates) in complete media with MBP Ac1-11 (25 g/ml) for 48 h. Transgenic and B10.PL spleen cells were cultured in a 1:5 (transgenic:nontransgenic) ratio. Cells and culture supernatants were subsequently harvested for flow cytometry analysis or ELISA assay. In additional experiments, the encephalitogenic potential of these cells was tested as follows. Following 48 h of culture, cells were harvested and naı¨ve syngeneic B10.PL mice received either 20 ⫻ 10 6 or 35 ⫻ 10 6 cells in 0.2 ml ip. These recipient mice were examined daily for signs of clinical disease and graded as previously described (14): 0, no abnormality; 1, flaccid tail; 2, moderate hind limb paralysis; 3, severe hind limb paralysis; 4, complete hind limb paralysis; 5, quadriplegia or premoribund state. Immunohistochemistry Spleen cells from transgenic mice were cultured as above. Following 48 h of culture, cells were harvested and naı¨ve syngeneic B10.PL mice received 20 ⫻ 10 6 cells in 0.2 ml ip. Nine days after injection, the brains were harvested and frozen sections were cut. Sections were blocked with the Avidin/Biotin Blocking Kit (Vector Labs) before the addition of primary antibody (100 l of ICOS-Ig, 8 g/ml). Biotin-labeled mouse antihuman IgG1 Fc (100 l, 8 g/ml) was used for the secondary antibody. For detection, the VectaStain ELITE ABC Kit (Vector Labs) and SigmaFast DAB peroxidase (Sigma, St. Louis) were used according to manufacturer’s protocols. Sections were counterstained with hematoxylin (PolyScientific). Digital images were obtained using a Polaroid DMC ChargedCouple Device (CCD) digital camera. Color images were transferred into Adobe Photoshop 5.5 (Adobe Systems Inc., San Jose, CA).
Reagents
Lymphocyte Proliferation
ICOS-Ig fusion protein was produced from transfected CHO lines and purified over protein A–agarose columns (Gibco-BRL Life Technologies, Inc., Grand Island, NY) as previously described (7). This construct consists of the extracellular domain of human ICOS up to Leu139 and the hinge, CH2, and CH3 domains of human IgG1. Control human IgG1 was purchased from Jackson ImmunoResearch (West Grove, PA). The encephalitogenic peptide
Proliferative responses were assessed by incubating transgenic T-cells and wild-type T-cells (1:5 ratio, 2 ⫻ 10 5 cells/well) with 25 g/ml of MBP Ac1-11 or media alone in the presence or the absence of various concentrations of ICOS-Ig or control human IgG1. The cultures were maintained in 96-well, flat-bottom microtiter plates (Costar, Cambridge, MA) for 72 h at 37°C in humidified 5% CO 2 air. The wells were pulsed with 0.5
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Ci/well of [methyl- 3H]thymidine (New England Nuclear, Cambridge, MA) for the final 4 h of culture. Cells were harvested on glass fibers and incorporation of [methyl- 3H]thymidine was measured with a Betaplate counter (Wallac, Gaithersburg, MD). Results were determined as arithmetic means from quadruplicate cultures. Flow Cytometry Cell surface staining. Cells (10 6) were stained with FITC-conjugated anti-V8.1,8.2 monoclonal antibody MR5-2, anti-CD19 (clone 1D3), anti-macrophage F4/80 (clone CI:A3-1), PE-conjugated anti-CD25 (clone Pc61), PE-conjugated anti-CD69 (clone H1.2F3). Antibodies were purchased from PharMingen (San Diego, CA). Intracellular cytokine staining. Cell suspensions (1 ⫻ 10 7/ml) were incubated in RPMI 1640 plus 10% fetal bovine serum with 10 g/ml Brefeldin A for 3 h at 37°C with 5% CO 2. Following incubation, the cells were harvested, surface stained with FITC-conjugated antiV8, and fixed overnight in 2.5% paraformaldehyde. The following day the cells were permeablized and stained for intracellular cytokines using PE-conjugated anti-cytokine monoclonal antibodies (PharMingen, anti-IL-2 clone 54B6, anti-IL-10 clone JES5-16E3, anti-IFN-␥ clone XMG1.2). Data were acquired and analyzed using a FAC-Scan or FACScalibur instrument equipped with Cell Quest software (version 3.0.1) or FACStar Plus equipped with LYSIS II software (Becton–Dickinson, Mountain View, CA). Determination of Apoptosis by TUNEL Apoptotic cell death was quantified using terminal deoxylnucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) methodology (15). Cells (1 ⫻ 10 6) were stained with PE-conjugated anti-V8 monoclonal antibody and FITC-conjugated anti-CD44 (PharMingen, clone IM7) or FITC conjugated antiCD45Rb (PharMingen, clone 16A). Next, the cells were fixed in 1% formaldehyde, pH 7.4 at 4°C, washed with PBS, and permeabilized with 0.1% saponin and incubated with 50 l of TdT reaction mixture containing 0.1 M sodium cacodylate (pH 7.2), 2 mM CoCl 2, 0.2 mM dithiothreitol, 0.1 mg/ml of BSA, 5 units of TdT (Boehringer-Mannheim, Indianapolis, IN), and 0.3 l of Cy5dUTP (Amersham Pharmacia Biotech, Inc., Pisctaway, NJ) for 2 h at 37°C. The cells were washed twice, suspended in staining buffer, and analyzed by flow cytometry. RNase Protection RNase protection assays were performed using the Riboquant Multi-probe RNase Protection Assay Sys-
FIG. 1. Expression of ICOS ligand on inflammatory cells in the CNS. EAE was induced by adoptive transfer as described under Materials and Methods. After 9 days, brains were harvested from mice with EAE (grade 3) and ICOS-Ig was used to detect ICOS ligand. (A) Negative control, no primary antibody; (B) ICOS-Ig staining of the same region shown in A. (C) ICOS-Ig staining.
tem with the mAPO-2 multiprobe template set (PharMingen). Double-stranded protected RNA products, labeled with 32P, were separated by electrophoresis and signals quantitated with a Phoshpor Imager (Molecular Dynamics, Sunnyvale, CA). Values for specific apoptosis genes were normalized to L32 “housekeeping genes” values.
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FIG. 2. ICOSL expression on mononuclear cells from the brains and spleens of mice with adoptively transferred EAE. Cell suspensions from spleen (righthand panels) and brain (lefthand panels) were prepared as previously described (16). CD3 ⫹ T-cells, CD19 ⫹ B cells, and F4/80 ⫹ macrophages were stained for ICOSL with biotinylated ICOS-Ig antibody and PE-conjugated streptavidin.
ELISA
Statistics
IFN-␥ and IL-10 levels in culture supernatants were assayed with specific Endogen (Cambridge, MA) ELISA kits according to the manufacturer’s instructions. IL-2 levels were assayed with an R & D Systems (Minneapolis, MN) ELISA kit.
Each experiment was performed three to five times except the last experiment (Fig. 10), which was performed twice. Treatment effects on disease severity were assessed with the Mann–Whitney sum of ranks test. Differences in disease incidence
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FIG. 3. Blockade of the ICOSL in vitro decreased encephalitogenicity. (A) MBP-reactive transgenic T-cells were cultured as described under Materials and Methods in the presence of either 25 g/ml ICOS-Ig or 25 g/ml control human IgG1. After 48 h 20 ⫻ 10 6 cells were injected ip into B10.PL syngeneic recipients and the clinical disease severity was assessed daily. Both the incidence (Fisher’s exact test, P ⫽ 0.015) and severity (Mann–Whitney sum of ranks, P ⬍ 0.01) of the disease were lower in animals receiving the cells pretreated with ICOS-Ig than in those of control animals. (B) The cells were cultured as above and 35 ⫻ 10 6 cells were injected ip into B10.PL syngeneic recipient mice. In this experiment, the control mice developed a more severe clinical disease. None of the mice receiving ICOS-Ig-pretreated cells developed EAE. Difference in disease incidence: Fisher’s exact test, P ⫽ 0.008. Difference in disease severity: Mann–Whitney sum of ranks, P ⬍ 0.01.
were analyzed using Fisher’s exact test. Differences in proliferation were analyzed with the Student’s t test. RESULTS
ICOS Ligand Was Expressed on CNS-Infiltrating Leukocytes in EAE We previously studied the role of CD28, CD152, and B7 molecules in the activation of encephalitogenic T-cells. In the present study, we were interested in the ICOS:ICOSL interaction. As shown in Fig. 1, we adoptively transferred MBP-reactive Tcells after in vitro activation with antigen into naı¨ve, syngeneic B10.PL recipients. After these mice developed clinical signs of EAE, we sacrificed them, prepared tissue sections, and used ICOS-Ig to detect ICOSL expression. As shown in Figs. 1B and 1C, we detected significant expression of ICOSL on the leukocytes in the inflammatory cell infiltrate. To identify the ICOSL ⫹ cells, we performed flow cytometry analysis on mononuclear cells from the brains and spleens of mice with grade 3 EAE. The mononuclear cells were isolated from the brains as previously described (16). Different populations of leukocytes were stained for ICOSL with a biotinylated ICOS-Ig and PE-conjugated streptavidin. As shown in Fig. 2, ICOSL was expressed by 12% of B-cells and 63% of macrophages from the brains of these mice. In contrast only 1.25% of lesion T-cells expressed ICOSL. The percentages of ICOSL ⫹ B-cells, macrophages, and T-cells were higher in the spleen.
Culturing T-Cells with ICOS-Ig Decreased Their Encephalitogenic Potential This encouraged us to investigate the role of ICOSLmediated costimulation in EAE. MBP-reactive transgenic T-cells were cultured with 25 g/ml of MBP Ac1-11 in the presence of either 25 g/ml ICOS-Ig or control human IgG1. After 48 h 20 ⫻ 10 6 cells were injected ip into each nontransgenic B10.PL mouse (Fig. 3A). These recipient mice were observed daily for clinical signs of EAE and a mean clinical score was assigned for each group of mice. Control mice (7/9) developed mild disease, with six mice developing a maximum disease grade of 1 and only one mouse developing a grade of 3. Only one of the mice that received ICOS-Ig treated cells developed EAE and this mouse obtained a maximum score of 2. Because of the mild nature of the disease transferred with 20 ⫻ 10 6 cells, we repeated this experiment, injecting 35 ⫻ 10 6 cells per mouse (Fig. 3B). The control mice in this experiment developed a more severe disease than those in the previous experiment. Five of five control mice developed EAE and the mean maximal score was 2.2. In that experiment, none of the mice that received ICOS-Ig-treated cells developed EAE. To determine whether the failure to induce EAE was the result of transferring mostly apoptotic cells that did not migrate to the CNS, we identified V8 ⫹ transgenic T-cells from the brains and spleens of these mice. Transgenic T-cells were activated as described under Materials and Methods and 35 ⫻ 10 6 cells were transferred into each recipient. On day 9, V8 expression by CD3 ⫹ T-cells from brains and spleens of these mice was assessed by flow cytometry. As shown in Fig. 4, we
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FIG. 4. The presence of transgenic T-cells in the CNS (top panels) and spleens (bottom panels) of mice with adoptively transferred EAE in either human control IgG1 or ICOS-Ig-treated groups. Transgenic T-cells were activated and transferred into each recipient as in Fig. 3A. On day 9, V8 expression by CD3 ⫹ T-cells from the brains and spleens of these mice was assessed by flow cytometry.
consistently found V8 ⫹ T-cells in both the brains and spleens of mice that received ICOS-Ig pretreated cells.
ICOS–ICOSL Interaction Provides a Survival Signal to Both Naı¨ve and Memory Cells
ICOS-Ig Suppressed the Proliferative Response of MBP-Reactive T-Cells
Because CD28 costimulation protects T-cells from apoptosis, we reasoned that ICOS:ICOSL interaction might similarly provide an anti-apoptotic signal. Transgenic MBP-reactive T-cells and syngeneic B10.PL cells were mixed in a 1:5 ratio and cultured with antigen and ICOS-Ig or control human IgG1. After 24 h, cultures were harvested and apoptosis of V8 ⫹ T-cells was measured with the TUNEL assay and flow cytometry. In addition, we examined the CD45RB lo memory V8 ⫹ cells and the CD45RB hi naı¨ve V8 ⫹ cells. The percentage of apoptotic transgenic Tcells, including both the CD45RB lo and the CD45RB hi populations, increased following ICOSL blockade, al-
To assess the mechanism by which ICOS regulates T-cell encephalitogenicity, we examined the effect of ICOS-Ig on proliferation, apoptosis, and cytokine production in the MBP-reactive V8 ⫹ transgenic T-cells. Inhibition of ICOS ligation significantly attenuated the proliferative response of MBP-reactive cells in a dosedependent manner (Fig. 5). At 25 g/ml ICOS-Ig, the concentration used in transfer experiments, the proliferation was inhibited by 40%, which is significant yet incomplete.
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tected in cultures of cells that were not stimulated with MBP Ac1-11 (data not shown). The lack of IFN-␥ production was confirmed by measuring the intracellular cytokine content of V8 ⫹ T-cells in these cultures. As shown in Fig. 9, ICOS-Ig preferentially suppressed IFN-␥ and IL-10 but not IL-2 production in this population. ICOS-Ig Injection during Ongoing EAE Ameliorated Clinical Disease
FIG. 5. ICOS-Ig suppressed the proliferative response to antigen. Transgenic T-cells were mixed with B10.PL spleen cells and cultured with Ac1-11 as described under Materials and Methods. Proliferation was determined by [methyl- 3H]thymidine incorporation during the last 4 h of culture. The baseline proliferation of cells cultured in the absence of MBP was 6757 ⫾ 104 cpm and is not subtracted (* proliferation is less than that in control cultures containing human IgG1, P ⬍ 0.05).
though this effect was more pronounced in the memory population (Fig. 6). Therefore, ICOSL provides a protective anti-apoptotic signal to both naı¨ve and memory T-cells. Effects of ICOS-Ig on BCL-2 Family Members The ratio of proapoptotic BAX to anti-apoptotic BCL-2 determines the susceptibility to apoptosis (17– 20). Cells were cultured as described in Fig. 6 and BAX and BCL-2 mRNA levels were detected with RNase protection assays. An increased expression of BAX (120%) and a decreased expression of BCL-2 (13%) were observed in ICOS-Ig-treated cells (Fig. 7A). Consequently, ICOS-Ig increased the BAX/BCL-2 ratio threefold (Fig. 7B). Effects of ICOS-Ig on Cytokine Production We next explored the production of specific cytokines that are involved in the regulation and development of EAE, including IL-2, IFN-␥, and IL-10. Transgenic Tcells were activated in the presence of either ICOS-Ig or human IgG1 control. Supernatants were harvested after 24 and 48 h and IL-2, IFN-␥, and IL-10 content was determined by ELISA (Fig. 8). IL-2 levels were similar in cultures containing either ICOS-Ig or control human IgG1. On the other hand, the production of both IFN-␥ and IL-10 was suppressed by ICOS-Ig. No IFN-␥ or IL-10 was detected in the ICOS-Ig containing cultures. In addition, no IL-2, IFN-␥, or IL-10 was de-
Finally we asked if ICOSL blockade, after the establishment of clinical disease, could attenuate the subsequent disease course. We induced EAE in B10.PL mice by adoptive transfer of 35 ⫻ 10 6 cells as before. After all the mice had signs of EAE, they were divided into two groups and injected with either 100 g of ICOS-Ig or control human IgG1 (5 mice/group). There was no difference in the clinical disease course of these two groups prior to the injections (Mann–Whitney, P ⬎ 0.05). Furthermore, the mean clinical score obtained for each group prior to injections was 2.4. Each animal received injections on days 10, 11, and 12. As shown in Fig. 10, ICOS-Ig injection resulted in an ameliorated disease course (Mann–Whitney, P ⬍ 0.05). DISCUSSION
Previous studies have demonstrated the role of CD28-mediated costimulation in EAE (21–25). The present studies have shown that the activation of encephalitogenic T-cells also requires ICOS:ICOSL costimulation. CD28:B7 and ICOS:ICOSL costimulation provide distinct and important contributions for the activation of encephalitogenic T-cells. For example, T-cell IFN-␥ production in EAE may be ICOS:ICOSL dependent and relatively CD28:B7 independent. In the present experiments, MBP-specific TCR transgenic T-cells were cultured in vitro with antigen and ICOS-Ig. Blockade of ICOSL prevented T-cell production of IFN-␥ and IL-10 but not IL-2 and prevented the transfer of clinical EAE. In contrast, our previous studies have shown that CD28 is required for T-cell IL-2 production in EAE (16, 21, 26, 27). Blockade of the B7 ligands with CTLA4-Ig or of CD28 with anti-CD28 monoclonal antibody suppressed IL-2 production by MBP-primed T-cells and significantly reduced their ability to adoptively transfer EAE (21, 26, 27). In contrast, CTLA4-Ig had a minimal effect on IFN-␥ production by MBP-primed lymph node cells (21). Likewise, T-cells from wild-type and CD28 “knockout” mice that had been immunized with the neuroantigen myelin/olidgodendrocyte glycoprotein produced comparabIe levels of IFN-␥ but only the CD28 ⫹ T-cells produced IL-2 (16). The C57BL/6
FIG. 6. ICOS-Ig increased apoptosis in both CD45RB hi (naı¨ve) and CD45RB lo (memory) populations of V8 ⫹ T-cells. V8 ⫹ MBP-reactive transgenic T-cells were mixed with wild-type spleen cells from B10.PL syngeneic mice in an 1:5 ratio (transgenic:wild type). The cells were incubated with 25 g/ml of MBP Ac1-11 in the presence of either 25 g/ml of ICOS-Ig (six righthand panels) or 25 g/ml of control human IgG1 (six lefthand panels). After 24 h the cultures were harvested and the TUNEL assay was performed as described under Materials and Methods. TdT ⫹ cells were detected by flow cytometry.
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FIG. 7. ICOS-Ig enhanced the BAX/BCL-2 ratio. Transgenic T-cells were cultured as described in the Fig. 6 legend. BAX and BCL-2 mRNA levels were measured with RNase protection assay with the BD PharMingen mAPO-2 multiprobe template set. The signals were quantitated on a Phosphor Imager and values were normalized to those of the “housekeeping gene” L32.
mice developed EAE but the CD28 “knockout” mice developed experimental autoimmune meningitis in response to MOG. This pattern of cytokine production is in general agreement with that of other reports. Most studies indicate that ICOS:ICOSL does not influence the production of IL-2 as CD28 does (5, 6). Instead, both IFN-␥ and IL-10 are enhanced by ICOS costimulation (5, 6, 10). For example, cross-linking ICOS with B7RP-1-Fc (i.e., with soluble ICOSL) resulted in enhanced production of IFN-␥ but not IL-2 (6). However, others have found inhibition of IL-2 production by ICOS-Ig (11). Our data demonstrated that IL-2 production in EAE is ICOS:ICOSL independent.
The concentration of ICOS-Ig used in these studies resulted in an incomplete inhibition of proliferation without a decrease in IL-2 production. The reduced thymidine incorporation was likely due to the increased apoptosis in these cultures. In vivo studies with ICOS-Ig have suggested that ICOS contributes to both Th1 and Th2 CD4 ⫹ T-cell differentiation (28). For example, injection of Nippostrongylus brasiliensis-infected mice with ICOS-Ig suppressed IFN-␥, IL-4, IL-5, and IL-10 production. The effects of ICOS on cytokine production are independent of the effects of CD28. Virus-specific T-cells from choriomeningitis virus-infected CD28 ⫺/⫺ mice produced a significant amount of IFN-␥, which is abrogated by
FIG. 8. ICOS-Ig suppressed IFN-␥ and IL-10 but not IL-2 production. Transgenic T-cells were activated as previously described. Culture supernatants were harvested at 24 and 48 h. Cytokine production was measured by ELISA. ICOS-Ig suppressed IFN-␥ and IL-10 production. Conversely, there was no effect on IL-2 content. N.D., none detected.
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FIG. 9. The effects of ICOS-Ig on intracellular cytokine content. Transgenic T-cells were activated as previously described. Following 24 h of culture, the intracellular cytokine content of the V8 ⫹ T-cells was measured by flow cytometry as described under Materials and Methods. (A) Human IgG. (B) ICOS-IgG.
ICOS-Ig injection (28), indicating that CD28 is not required for ICOS to mediate its effects on the production of this cytokine. In contrast, studies with ICOS knockout mice revealed deficient Th2 cytokine production and T-celldependent B-cell responses (29 –31). On the other hand, IFN-␥ production is either unchanged or increased in ICOS ⫺/⫺ mice (29 –31). While ICOSL costimulates IFN-␥ production in ICOS ⫹/⫹ mice, IFN-␥ induces the expression of ICOSL.
The expression of both ICOSL and the B7 molecules can be upregulated by IFN-␥ (7). However, the mechanisms underlying ICOSL and B7 upregulation by IFN-␥ appear to be very different. While B7 induction by IFN-␥ is NF-B dependent, ICOSL is NF-B independent (7). ICOSL-mediated costimulation appears to provide an anti-apoptotic survival signal to the encephalitogenic T-cells. ICOS-Ig enhanced apoptosis in both memory and naı¨ve cells. However, ICOS-Ig markedly
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that the effect of ICOS-Ig on cytokine production will change. The ability to costimulate either IL-10 or IFN-␥ suggests that ICOS may be involved in the activation of both pathogenic and regulatory T-cells. In these studies, we injected ICOS-Ig when there was a preponderance of proinflammatory cells. In contrast, regulatory cells may dominate later and ICOS-Ig injection at that point might exacerbate disease. Therefore, we are currently performing a series of in vivo studies to determine the role of ICOSL-mediated costimulation on the activation of pathogenic and regulatory T-cells and cytokines during different stages of EAE. ACKNOWLEDGMENTS FIG. 10. ICOS-Ig injection decreased the severity of ongoing EAE. MBP-reactive transgenic spleen cells were cultured with wildtype spleen cells from B10.PL syngeneic mice in a 1:5 ratio (transgenic:wild type) and MBP Ac1-11 (25 g/ml). After for 48 h, cells were harvested and a total of 35 ⫻ 10 6 cells were injected into each naı¨ve syngeneic B10.PL mouse. These mice were examined daily for signs of clinical disease and graded as previously described. Mice with clinical signs of EAE received 100 g/ml of either ICOS-Ig or human IgG1 control on days 10, 11, and 12. Mice that received ICOS-Ig underwent an attenuated disease after the commencement of injections. Differences in groups were compared for the period of observation following the first injections (days 10 –21, Mann–Whitney sum of ranks, P ⬍ 0.05).
increased apoptosis in the memory population. In contrast, a very small percentage of naı¨ve T-cells were susceptible to apoptosis following ICOSL blockade. These findings are consistent with previous results that CD28 protects naı¨ve but not memory T-cells from apoptosis (32) and suggest an important role for ICOS in memory T-cell survival. The cells found within the CNS following adoptive transfer (Fig. 4) may be those that did not undergo apoptosis in the culture, which were predominantly CD45RB hi. Furthermore, these surviving cells may have expanded in the CNS where MBP is present. The lack of encephalitogenicity is likely due to their inability to produce proinflammatory cytokines such as IFN-␥. BCL-2 and BAX antagonize each other and the predominance of one or another factor may render the cells resistant or prone to apoptosis. Therefore, we examined the expression of BCL-2 and BAX mRNA. ICOS-Ig-treated cells increased BAX (120%) and slightly decreased BCL-2 (13%) expression, resulting in a significant increase in the BAX/BCL-2 ratio. Of particular clinical importance, ICOS-Ig injection during ongoing EAE ameliorated disease. In contrast, ICOS ⫺/⫺ mice develop more severe EAE when immunized with the neuroantigen myelin/oligodendrocyte glycoprotein, presumably due to suppression of a regulatory T-cell population (31). These findings are not necessarily opposed to each other. As the immune response evolves, it is possible
This work was supported by grants from NIAID (R29-AI43296) and the National Multiple Sclerosis Society (RG2873A1 and RG3086A2). REFERENCES 1. Swanborg, R. H., Experimental autoimmune encephalomyelitis in rodents as a model for human demyelinating disease. Clin. Immunol. Immunopathol. 77, 4 –13, 1995. 2. Scholz, C., Patton, K. T., Anderson, D. E., Freeman, G. J., and Hafler, D. A., Expansion of autoreactive T cells in multiple sclerosis is independent of exogenous B7 costimulation. J. Immunol. 160, 1532–1538, 1998. 3. Croft, M., Bradley, L. M., and Swain, S. L., Naive versus memory CD4 T cell response to antigen. Memory cells are less dependent on accessory cell costimulation and can respond to many antigenpresenting cell types including resting B cells. J. Immunol. 152, 2675–2685, 1994. 4. Lovett-Racke, A. E., Trotter, J. L., Lauber, J., Perrin, P. J., June, C. H., and Racke, M. K., Decreased dependence of myelin basic protein-reactive T cells on CD28-mediated costimulation in multiple sclerosis patients. A marker of activated/memory T cells [published erratum appears in J. Clin. Invest. (1998) 101(7), 1542]. J. Clin. Invest. 101, 725–730, 1998. 5. Hutloff, A., Dittrich, A. M., Beier, K. C., Eljaschewitsch, B., Kraft, R., Anagnostopoulos, I., and Kroczek, R. A., ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28. Nature 397, 263–266, 1999. 6. Yoshinaga, S. K., Whoriskey, J. S., Khare, S. D., Sarmiento, U., Guo, J., Horan, T., Shih, G., Zhang, M., Coccia, M., Kohno, T., Tafuri-Bladt, A., Brankow, D., Campbell, P., Chang, D., Chiu, L., Dai, T., Duncan, G., Elliot, G. S., Hui, A., McCabe, S. M., Scully, S., Shahinian, A., Shaklee, C. L., Van, G., Mak, T. W., and Senaldi, G., T-cell co-stimulation through B7RP-1 and ICOS. Nature 402, 827– 832, 1999. 7. Aicher, A., Hayden-Ledbetter, M., Brady, W. A., Pezzutto, A., Richter, G., Magaletti, D., Buckwalter, S., Ledbetter, J. A., and Clark, E. A., Characterization of human inducible costimulator ligand expression and function. J. Immunol. 164, 4689 – 4696, 2000. 8. Swallow, M. M., Wallin, J. J., and Sha, W. C., B7h, a novel costimulatory homolog of B7.1 and B7.2, is induced by TNFalpha. Immunity 11, 423– 432, 1999. 9. Abbas, A. K., and Sharpe, A. H., T-cell stimulation: An abundance of B7s. Nat. Med. 5, 1345–1346, 1999. 10. Dong, H., Zhu, G., Tamada, K., and Chen, L., B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nature Med. 5, 1365–1369, 1999.
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Received May 24, 2001; accepted with revision May 30, 2001; published online July 25, 2001
ICOS Ligand Costimulation Is Required for T-Cell Encephalitogenicity Romeo A. Sporici,* Richard L. Beswick,* Carolyn von Allmen,* Catherine A. Rumbley,* Martha Hayden-Ledbetter,† Jeffrey A. Ledbetter,† and Peter J. Perrin* ,1 *Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; and †Pacific Northwest Research Institute, Seattle, Washington 98122
The interaction of ICOS with its ligand on APC provides a costimulatory signal to previously activated T-cells. In these studies, we blocked the ICOS:ICOS ligand interaction with ICOS-Ig during the in vitro activation of MBP-reactive transgenic CD4 ⴙ T-cells. The presence of ICOS-Ig in these cultures inhibited the ability of the transgenic T-cells to transfer EAE, although they entered the brains of the recipient mice. ICOS-Ig increased apoptosis in the transgenic T-cells, especially in the memory population. This enhanced apoptosis was accompanied by an increase in the BAX/ BCL-2 mRNA ratio. ICOS-Ig did not prevent IL2 production, demonstrating that IL-2 production is ICOS ligand independent. IFN-␥ and IL-10 production by the transgenic T-cells, however, was suppressed. Finally, ICOS-Ig injection into mice after the first signs of EAE ameliorated clinical disease. Therefore, ICOSL provides a signal distinct from CD28 costimulation that is required for the activation and viability of encephalitogenic T-cells. © 2001 Academic Press Key Words: EAE/MS; costimulatory molecules; T-lymphocytes; immunotherapy.
INTRODUCTION
Experimental autoimmune encephalomyelitis (EAE)2 is a model of T-cell-mediated demyelination. T-cells that recognize myelin antigens, such as myelin basic protein (MBP), recruit nonspecific T-cells and macrophages into the CNS. These cells effect demyelination, resulting in ascending paralysis. Therefore, EAE is often used as a model for human multiple sclerosis (MS) (1). During T-cell activation, costimulatory and accessory signals determine both whether a T-cell is acti1 To whom correspondence and reprint requests should be addressed at Department of Medicine, Pulmonary, Allergy, and Critical Care Section, 909 BRB II, 421 Curie Boulevard, Philadelphia, PA 19104-6160. E-mail: [email protected]. Fax: 215-8980193. 2 Abbreviations used: Ac, acetylated; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; ICOS, inducible costimulator; ICOSL, ICOS ligand; ip, intraperitoneal; MBP, myelin basic protein; MS, multiple sclerosis.
vated and the outcome of that activation. One major T-cell costimulatory signal is delivered through the interaction of T-cell CD28 with B7 ligands (CD80 and CD86) on APC. Naı¨ve cells have a greater requirement for CD28 costimulation than memory cells do (2, 3). For example, myelin-reactive T-cells are present in both MS patients and controls, demonstrating that autoreactive T-cells can exist in individuals without pathological consequences (2, 4). However, the MBP-reactive T-cells from MS patients are less dependent on CD28-mediated costimulation than those from normal controls. These findings suggest that therapies that prevent CD28 signaling will probably be of limited use in autoimmune diseases like MS and understanding the costimulatory requirements for memory T-cells is desirable. Inducible costimulator (ICOS) is a recently described member of the CD28 family (5–7). Murine ICOS and CD28 share only 19% amino acid identity but there is a strong structural homology between ICOS and the other CD28 family members, CD28 and CD152. Cysteines that are critical for intramolecular and intermolecular disulfide bonds are conserved. Significantly, however, ICOS lacks the MYPPPY motif, a highly conserved hexapeptide, which is critical for CD28 and CD152 binding to CD80 and CD86, suggesting that ICOS interacts with another distinct ligand. ICOSL is a member of the B7 family, which also includes CD80, CD86, and B7H-1 (6, 10). ICOS does not bind CD80 or CD86. Likewise, ICOSL does not bind either T-cell CD28 or CD152. Therefore, although these pathways are related, they do not interact with each other’s receptors and ligands. In contrast to CD28, ICOS is not expressed constitutively but is upregulated following T-cell activation and might be involved primarily in memory T-cell activation (5). For example, ICOS cross-linking during either sensitization or challenge enhanced ear thickness swelling in a contact hypersensitivity response, but this effect was more dramatic during challenge (7). Cytokine production following CD28 and ICOS ligation is significantly different, although the role of ICOS in the production of specific cytokines is still not clear.
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ICOS does not influence the production of IL-2 as CD28 does (5, 6). Instead, both IFN-␥ and IL-10 are enhanced by ICOS costimulation (5, 6, 10). For example, crosslinking ICOS with soluble ICOSL (B7RP1-Fc) results in enhanced production of IFN-␥ but not IL-2 (6). However, the inhibition of IL-2 production by ICOS-Ig has been reported (11). In these studies, we assessed the role of ICOSLmediated costimulation in transgenic T-cells bearing a MBP-specific TCR. These transgenic T-cells were activated in the presence or the absence of ICOS-Ig, a soluble receptor that competitively inhibits the ICOS: ICOSL interaction. ICOS-Ig increased the BAX/BCL-2 ratio and increased the apoptosis in these antigenstimulated transgenic T-cells. ICOS-Ig prevented the production of IFN-␥ and IL-10 but not IL-2 and suppressed their ability to adoptively transfer EAE to naı¨ve recipients. Importantly, ICOS-Ig injection after the onset of clinical signs ameliorated EAE. Therefore, ICOSL provides a distinct costimulatory signal that participates in activation and survival of T memory cells. As such, it is a potential therapeutic target for ongoing autoimmune diseases. MATERIALS AND METHODS
Mice Six- to ten-week-old B10.PL mice were purchased from Jackson Laboratory (Bar Harbor, ME). Breeding pairs of MBP-specific TCR transgenic B10.PL mice were provided by Dr. S. Tonegawa (MIT, Cambridge, MA) (12, 13). Mice carrying the transgene were identified using flow cytometry and anti-V8 monoclonal antibody as previously described (MIT, Cambridge, MA) (12, 13). These mice were bred and maintained in our animal colony in compliance with the Animal Care and Use Committee. In conducting this research, the investigators adhered to the Guide for Laboratory Animal Facilities and Care as promulgated by the Committee on the “Guide for Laboratory Animal Resources,” National Academy of Sciences-National Research Council, DHHS, Publication No. (NIH) 86-23.
MBP Ac1-11 (Ac-ASQKRPSQRHG) was purchased from Genemed Synthesis (South San Francisco, CA). Cell Culture and Experimental Autoimmune Encephalomyelitis Spleen cells from transgenic mice were cultured with wild-type spleen cells from B10.PL mice (8 ⫻ 10 6 cells/2 ml in 24-well plates) in complete media with MBP Ac1-11 (25 g/ml) for 48 h. Transgenic and B10.PL spleen cells were cultured in a 1:5 (transgenic:nontransgenic) ratio. Cells and culture supernatants were subsequently harvested for flow cytometry analysis or ELISA assay. In additional experiments, the encephalitogenic potential of these cells was tested as follows. Following 48 h of culture, cells were harvested and naı¨ve syngeneic B10.PL mice received either 20 ⫻ 10 6 or 35 ⫻ 10 6 cells in 0.2 ml ip. These recipient mice were examined daily for signs of clinical disease and graded as previously described (14): 0, no abnormality; 1, flaccid tail; 2, moderate hind limb paralysis; 3, severe hind limb paralysis; 4, complete hind limb paralysis; 5, quadriplegia or premoribund state. Immunohistochemistry Spleen cells from transgenic mice were cultured as above. Following 48 h of culture, cells were harvested and naı¨ve syngeneic B10.PL mice received 20 ⫻ 10 6 cells in 0.2 ml ip. Nine days after injection, the brains were harvested and frozen sections were cut. Sections were blocked with the Avidin/Biotin Blocking Kit (Vector Labs) before the addition of primary antibody (100 l of ICOS-Ig, 8 g/ml). Biotin-labeled mouse antihuman IgG1 Fc (100 l, 8 g/ml) was used for the secondary antibody. For detection, the VectaStain ELITE ABC Kit (Vector Labs) and SigmaFast DAB peroxidase (Sigma, St. Louis) were used according to manufacturer’s protocols. Sections were counterstained with hematoxylin (PolyScientific). Digital images were obtained using a Polaroid DMC ChargedCouple Device (CCD) digital camera. Color images were transferred into Adobe Photoshop 5.5 (Adobe Systems Inc., San Jose, CA).
Reagents
Lymphocyte Proliferation
ICOS-Ig fusion protein was produced from transfected CHO lines and purified over protein A–agarose columns (Gibco-BRL Life Technologies, Inc., Grand Island, NY) as previously described (7). This construct consists of the extracellular domain of human ICOS up to Leu139 and the hinge, CH2, and CH3 domains of human IgG1. Control human IgG1 was purchased from Jackson ImmunoResearch (West Grove, PA). The encephalitogenic peptide
Proliferative responses were assessed by incubating transgenic T-cells and wild-type T-cells (1:5 ratio, 2 ⫻ 10 5 cells/well) with 25 g/ml of MBP Ac1-11 or media alone in the presence or the absence of various concentrations of ICOS-Ig or control human IgG1. The cultures were maintained in 96-well, flat-bottom microtiter plates (Costar, Cambridge, MA) for 72 h at 37°C in humidified 5% CO 2 air. The wells were pulsed with 0.5
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Ci/well of [methyl- 3H]thymidine (New England Nuclear, Cambridge, MA) for the final 4 h of culture. Cells were harvested on glass fibers and incorporation of [methyl- 3H]thymidine was measured with a Betaplate counter (Wallac, Gaithersburg, MD). Results were determined as arithmetic means from quadruplicate cultures. Flow Cytometry Cell surface staining. Cells (10 6) were stained with FITC-conjugated anti-V8.1,8.2 monoclonal antibody MR5-2, anti-CD19 (clone 1D3), anti-macrophage F4/80 (clone CI:A3-1), PE-conjugated anti-CD25 (clone Pc61), PE-conjugated anti-CD69 (clone H1.2F3). Antibodies were purchased from PharMingen (San Diego, CA). Intracellular cytokine staining. Cell suspensions (1 ⫻ 10 7/ml) were incubated in RPMI 1640 plus 10% fetal bovine serum with 10 g/ml Brefeldin A for 3 h at 37°C with 5% CO 2. Following incubation, the cells were harvested, surface stained with FITC-conjugated antiV8, and fixed overnight in 2.5% paraformaldehyde. The following day the cells were permeablized and stained for intracellular cytokines using PE-conjugated anti-cytokine monoclonal antibodies (PharMingen, anti-IL-2 clone 54B6, anti-IL-10 clone JES5-16E3, anti-IFN-␥ clone XMG1.2). Data were acquired and analyzed using a FAC-Scan or FACScalibur instrument equipped with Cell Quest software (version 3.0.1) or FACStar Plus equipped with LYSIS II software (Becton–Dickinson, Mountain View, CA). Determination of Apoptosis by TUNEL Apoptotic cell death was quantified using terminal deoxylnucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) methodology (15). Cells (1 ⫻ 10 6) were stained with PE-conjugated anti-V8 monoclonal antibody and FITC-conjugated anti-CD44 (PharMingen, clone IM7) or FITC conjugated antiCD45Rb (PharMingen, clone 16A). Next, the cells were fixed in 1% formaldehyde, pH 7.4 at 4°C, washed with PBS, and permeabilized with 0.1% saponin and incubated with 50 l of TdT reaction mixture containing 0.1 M sodium cacodylate (pH 7.2), 2 mM CoCl 2, 0.2 mM dithiothreitol, 0.1 mg/ml of BSA, 5 units of TdT (Boehringer-Mannheim, Indianapolis, IN), and 0.3 l of Cy5dUTP (Amersham Pharmacia Biotech, Inc., Pisctaway, NJ) for 2 h at 37°C. The cells were washed twice, suspended in staining buffer, and analyzed by flow cytometry. RNase Protection RNase protection assays were performed using the Riboquant Multi-probe RNase Protection Assay Sys-
FIG. 1. Expression of ICOS ligand on inflammatory cells in the CNS. EAE was induced by adoptive transfer as described under Materials and Methods. After 9 days, brains were harvested from mice with EAE (grade 3) and ICOS-Ig was used to detect ICOS ligand. (A) Negative control, no primary antibody; (B) ICOS-Ig staining of the same region shown in A. (C) ICOS-Ig staining.
tem with the mAPO-2 multiprobe template set (PharMingen). Double-stranded protected RNA products, labeled with 32P, were separated by electrophoresis and signals quantitated with a Phoshpor Imager (Molecular Dynamics, Sunnyvale, CA). Values for specific apoptosis genes were normalized to L32 “housekeeping genes” values.
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FIG. 2. ICOSL expression on mononuclear cells from the brains and spleens of mice with adoptively transferred EAE. Cell suspensions from spleen (righthand panels) and brain (lefthand panels) were prepared as previously described (16). CD3 ⫹ T-cells, CD19 ⫹ B cells, and F4/80 ⫹ macrophages were stained for ICOSL with biotinylated ICOS-Ig antibody and PE-conjugated streptavidin.
ELISA
Statistics
IFN-␥ and IL-10 levels in culture supernatants were assayed with specific Endogen (Cambridge, MA) ELISA kits according to the manufacturer’s instructions. IL-2 levels were assayed with an R & D Systems (Minneapolis, MN) ELISA kit.
Each experiment was performed three to five times except the last experiment (Fig. 10), which was performed twice. Treatment effects on disease severity were assessed with the Mann–Whitney sum of ranks test. Differences in disease incidence
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FIG. 3. Blockade of the ICOSL in vitro decreased encephalitogenicity. (A) MBP-reactive transgenic T-cells were cultured as described under Materials and Methods in the presence of either 25 g/ml ICOS-Ig or 25 g/ml control human IgG1. After 48 h 20 ⫻ 10 6 cells were injected ip into B10.PL syngeneic recipients and the clinical disease severity was assessed daily. Both the incidence (Fisher’s exact test, P ⫽ 0.015) and severity (Mann–Whitney sum of ranks, P ⬍ 0.01) of the disease were lower in animals receiving the cells pretreated with ICOS-Ig than in those of control animals. (B) The cells were cultured as above and 35 ⫻ 10 6 cells were injected ip into B10.PL syngeneic recipient mice. In this experiment, the control mice developed a more severe clinical disease. None of the mice receiving ICOS-Ig-pretreated cells developed EAE. Difference in disease incidence: Fisher’s exact test, P ⫽ 0.008. Difference in disease severity: Mann–Whitney sum of ranks, P ⬍ 0.01.
were analyzed using Fisher’s exact test. Differences in proliferation were analyzed with the Student’s t test. RESULTS
ICOS Ligand Was Expressed on CNS-Infiltrating Leukocytes in EAE We previously studied the role of CD28, CD152, and B7 molecules in the activation of encephalitogenic T-cells. In the present study, we were interested in the ICOS:ICOSL interaction. As shown in Fig. 1, we adoptively transferred MBP-reactive Tcells after in vitro activation with antigen into naı¨ve, syngeneic B10.PL recipients. After these mice developed clinical signs of EAE, we sacrificed them, prepared tissue sections, and used ICOS-Ig to detect ICOSL expression. As shown in Figs. 1B and 1C, we detected significant expression of ICOSL on the leukocytes in the inflammatory cell infiltrate. To identify the ICOSL ⫹ cells, we performed flow cytometry analysis on mononuclear cells from the brains and spleens of mice with grade 3 EAE. The mononuclear cells were isolated from the brains as previously described (16). Different populations of leukocytes were stained for ICOSL with a biotinylated ICOS-Ig and PE-conjugated streptavidin. As shown in Fig. 2, ICOSL was expressed by 12% of B-cells and 63% of macrophages from the brains of these mice. In contrast only 1.25% of lesion T-cells expressed ICOSL. The percentages of ICOSL ⫹ B-cells, macrophages, and T-cells were higher in the spleen.
Culturing T-Cells with ICOS-Ig Decreased Their Encephalitogenic Potential This encouraged us to investigate the role of ICOSLmediated costimulation in EAE. MBP-reactive transgenic T-cells were cultured with 25 g/ml of MBP Ac1-11 in the presence of either 25 g/ml ICOS-Ig or control human IgG1. After 48 h 20 ⫻ 10 6 cells were injected ip into each nontransgenic B10.PL mouse (Fig. 3A). These recipient mice were observed daily for clinical signs of EAE and a mean clinical score was assigned for each group of mice. Control mice (7/9) developed mild disease, with six mice developing a maximum disease grade of 1 and only one mouse developing a grade of 3. Only one of the mice that received ICOS-Ig treated cells developed EAE and this mouse obtained a maximum score of 2. Because of the mild nature of the disease transferred with 20 ⫻ 10 6 cells, we repeated this experiment, injecting 35 ⫻ 10 6 cells per mouse (Fig. 3B). The control mice in this experiment developed a more severe disease than those in the previous experiment. Five of five control mice developed EAE and the mean maximal score was 2.2. In that experiment, none of the mice that received ICOS-Ig-treated cells developed EAE. To determine whether the failure to induce EAE was the result of transferring mostly apoptotic cells that did not migrate to the CNS, we identified V8 ⫹ transgenic T-cells from the brains and spleens of these mice. Transgenic T-cells were activated as described under Materials and Methods and 35 ⫻ 10 6 cells were transferred into each recipient. On day 9, V8 expression by CD3 ⫹ T-cells from brains and spleens of these mice was assessed by flow cytometry. As shown in Fig. 4, we
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FIG. 4. The presence of transgenic T-cells in the CNS (top panels) and spleens (bottom panels) of mice with adoptively transferred EAE in either human control IgG1 or ICOS-Ig-treated groups. Transgenic T-cells were activated and transferred into each recipient as in Fig. 3A. On day 9, V8 expression by CD3 ⫹ T-cells from the brains and spleens of these mice was assessed by flow cytometry.
consistently found V8 ⫹ T-cells in both the brains and spleens of mice that received ICOS-Ig pretreated cells.
ICOS–ICOSL Interaction Provides a Survival Signal to Both Naı¨ve and Memory Cells
ICOS-Ig Suppressed the Proliferative Response of MBP-Reactive T-Cells
Because CD28 costimulation protects T-cells from apoptosis, we reasoned that ICOS:ICOSL interaction might similarly provide an anti-apoptotic signal. Transgenic MBP-reactive T-cells and syngeneic B10.PL cells were mixed in a 1:5 ratio and cultured with antigen and ICOS-Ig or control human IgG1. After 24 h, cultures were harvested and apoptosis of V8 ⫹ T-cells was measured with the TUNEL assay and flow cytometry. In addition, we examined the CD45RB lo memory V8 ⫹ cells and the CD45RB hi naı¨ve V8 ⫹ cells. The percentage of apoptotic transgenic Tcells, including both the CD45RB lo and the CD45RB hi populations, increased following ICOSL blockade, al-
To assess the mechanism by which ICOS regulates T-cell encephalitogenicity, we examined the effect of ICOS-Ig on proliferation, apoptosis, and cytokine production in the MBP-reactive V8 ⫹ transgenic T-cells. Inhibition of ICOS ligation significantly attenuated the proliferative response of MBP-reactive cells in a dosedependent manner (Fig. 5). At 25 g/ml ICOS-Ig, the concentration used in transfer experiments, the proliferation was inhibited by 40%, which is significant yet incomplete.
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tected in cultures of cells that were not stimulated with MBP Ac1-11 (data not shown). The lack of IFN-␥ production was confirmed by measuring the intracellular cytokine content of V8 ⫹ T-cells in these cultures. As shown in Fig. 9, ICOS-Ig preferentially suppressed IFN-␥ and IL-10 but not IL-2 production in this population. ICOS-Ig Injection during Ongoing EAE Ameliorated Clinical Disease
FIG. 5. ICOS-Ig suppressed the proliferative response to antigen. Transgenic T-cells were mixed with B10.PL spleen cells and cultured with Ac1-11 as described under Materials and Methods. Proliferation was determined by [methyl- 3H]thymidine incorporation during the last 4 h of culture. The baseline proliferation of cells cultured in the absence of MBP was 6757 ⫾ 104 cpm and is not subtracted (* proliferation is less than that in control cultures containing human IgG1, P ⬍ 0.05).
though this effect was more pronounced in the memory population (Fig. 6). Therefore, ICOSL provides a protective anti-apoptotic signal to both naı¨ve and memory T-cells. Effects of ICOS-Ig on BCL-2 Family Members The ratio of proapoptotic BAX to anti-apoptotic BCL-2 determines the susceptibility to apoptosis (17– 20). Cells were cultured as described in Fig. 6 and BAX and BCL-2 mRNA levels were detected with RNase protection assays. An increased expression of BAX (120%) and a decreased expression of BCL-2 (13%) were observed in ICOS-Ig-treated cells (Fig. 7A). Consequently, ICOS-Ig increased the BAX/BCL-2 ratio threefold (Fig. 7B). Effects of ICOS-Ig on Cytokine Production We next explored the production of specific cytokines that are involved in the regulation and development of EAE, including IL-2, IFN-␥, and IL-10. Transgenic Tcells were activated in the presence of either ICOS-Ig or human IgG1 control. Supernatants were harvested after 24 and 48 h and IL-2, IFN-␥, and IL-10 content was determined by ELISA (Fig. 8). IL-2 levels were similar in cultures containing either ICOS-Ig or control human IgG1. On the other hand, the production of both IFN-␥ and IL-10 was suppressed by ICOS-Ig. No IFN-␥ or IL-10 was detected in the ICOS-Ig containing cultures. In addition, no IL-2, IFN-␥, or IL-10 was de-
Finally we asked if ICOSL blockade, after the establishment of clinical disease, could attenuate the subsequent disease course. We induced EAE in B10.PL mice by adoptive transfer of 35 ⫻ 10 6 cells as before. After all the mice had signs of EAE, they were divided into two groups and injected with either 100 g of ICOS-Ig or control human IgG1 (5 mice/group). There was no difference in the clinical disease course of these two groups prior to the injections (Mann–Whitney, P ⬎ 0.05). Furthermore, the mean clinical score obtained for each group prior to injections was 2.4. Each animal received injections on days 10, 11, and 12. As shown in Fig. 10, ICOS-Ig injection resulted in an ameliorated disease course (Mann–Whitney, P ⬍ 0.05). DISCUSSION
Previous studies have demonstrated the role of CD28-mediated costimulation in EAE (21–25). The present studies have shown that the activation of encephalitogenic T-cells also requires ICOS:ICOSL costimulation. CD28:B7 and ICOS:ICOSL costimulation provide distinct and important contributions for the activation of encephalitogenic T-cells. For example, T-cell IFN-␥ production in EAE may be ICOS:ICOSL dependent and relatively CD28:B7 independent. In the present experiments, MBP-specific TCR transgenic T-cells were cultured in vitro with antigen and ICOS-Ig. Blockade of ICOSL prevented T-cell production of IFN-␥ and IL-10 but not IL-2 and prevented the transfer of clinical EAE. In contrast, our previous studies have shown that CD28 is required for T-cell IL-2 production in EAE (16, 21, 26, 27). Blockade of the B7 ligands with CTLA4-Ig or of CD28 with anti-CD28 monoclonal antibody suppressed IL-2 production by MBP-primed T-cells and significantly reduced their ability to adoptively transfer EAE (21, 26, 27). In contrast, CTLA4-Ig had a minimal effect on IFN-␥ production by MBP-primed lymph node cells (21). Likewise, T-cells from wild-type and CD28 “knockout” mice that had been immunized with the neuroantigen myelin/olidgodendrocyte glycoprotein produced comparabIe levels of IFN-␥ but only the CD28 ⫹ T-cells produced IL-2 (16). The C57BL/6
FIG. 6. ICOS-Ig increased apoptosis in both CD45RB hi (naı¨ve) and CD45RB lo (memory) populations of V8 ⫹ T-cells. V8 ⫹ MBP-reactive transgenic T-cells were mixed with wild-type spleen cells from B10.PL syngeneic mice in an 1:5 ratio (transgenic:wild type). The cells were incubated with 25 g/ml of MBP Ac1-11 in the presence of either 25 g/ml of ICOS-Ig (six righthand panels) or 25 g/ml of control human IgG1 (six lefthand panels). After 24 h the cultures were harvested and the TUNEL assay was performed as described under Materials and Methods. TdT ⫹ cells were detected by flow cytometry.
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FIG. 7. ICOS-Ig enhanced the BAX/BCL-2 ratio. Transgenic T-cells were cultured as described in the Fig. 6 legend. BAX and BCL-2 mRNA levels were measured with RNase protection assay with the BD PharMingen mAPO-2 multiprobe template set. The signals were quantitated on a Phosphor Imager and values were normalized to those of the “housekeeping gene” L32.
mice developed EAE but the CD28 “knockout” mice developed experimental autoimmune meningitis in response to MOG. This pattern of cytokine production is in general agreement with that of other reports. Most studies indicate that ICOS:ICOSL does not influence the production of IL-2 as CD28 does (5, 6). Instead, both IFN-␥ and IL-10 are enhanced by ICOS costimulation (5, 6, 10). For example, cross-linking ICOS with B7RP-1-Fc (i.e., with soluble ICOSL) resulted in enhanced production of IFN-␥ but not IL-2 (6). However, others have found inhibition of IL-2 production by ICOS-Ig (11). Our data demonstrated that IL-2 production in EAE is ICOS:ICOSL independent.
The concentration of ICOS-Ig used in these studies resulted in an incomplete inhibition of proliferation without a decrease in IL-2 production. The reduced thymidine incorporation was likely due to the increased apoptosis in these cultures. In vivo studies with ICOS-Ig have suggested that ICOS contributes to both Th1 and Th2 CD4 ⫹ T-cell differentiation (28). For example, injection of Nippostrongylus brasiliensis-infected mice with ICOS-Ig suppressed IFN-␥, IL-4, IL-5, and IL-10 production. The effects of ICOS on cytokine production are independent of the effects of CD28. Virus-specific T-cells from choriomeningitis virus-infected CD28 ⫺/⫺ mice produced a significant amount of IFN-␥, which is abrogated by
FIG. 8. ICOS-Ig suppressed IFN-␥ and IL-10 but not IL-2 production. Transgenic T-cells were activated as previously described. Culture supernatants were harvested at 24 and 48 h. Cytokine production was measured by ELISA. ICOS-Ig suppressed IFN-␥ and IL-10 production. Conversely, there was no effect on IL-2 content. N.D., none detected.
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FIG. 9. The effects of ICOS-Ig on intracellular cytokine content. Transgenic T-cells were activated as previously described. Following 24 h of culture, the intracellular cytokine content of the V8 ⫹ T-cells was measured by flow cytometry as described under Materials and Methods. (A) Human IgG. (B) ICOS-IgG.
ICOS-Ig injection (28), indicating that CD28 is not required for ICOS to mediate its effects on the production of this cytokine. In contrast, studies with ICOS knockout mice revealed deficient Th2 cytokine production and T-celldependent B-cell responses (29 –31). On the other hand, IFN-␥ production is either unchanged or increased in ICOS ⫺/⫺ mice (29 –31). While ICOSL costimulates IFN-␥ production in ICOS ⫹/⫹ mice, IFN-␥ induces the expression of ICOSL.
The expression of both ICOSL and the B7 molecules can be upregulated by IFN-␥ (7). However, the mechanisms underlying ICOSL and B7 upregulation by IFN-␥ appear to be very different. While B7 induction by IFN-␥ is NF-B dependent, ICOSL is NF-B independent (7). ICOSL-mediated costimulation appears to provide an anti-apoptotic survival signal to the encephalitogenic T-cells. ICOS-Ig enhanced apoptosis in both memory and naı¨ve cells. However, ICOS-Ig markedly
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that the effect of ICOS-Ig on cytokine production will change. The ability to costimulate either IL-10 or IFN-␥ suggests that ICOS may be involved in the activation of both pathogenic and regulatory T-cells. In these studies, we injected ICOS-Ig when there was a preponderance of proinflammatory cells. In contrast, regulatory cells may dominate later and ICOS-Ig injection at that point might exacerbate disease. Therefore, we are currently performing a series of in vivo studies to determine the role of ICOSL-mediated costimulation on the activation of pathogenic and regulatory T-cells and cytokines during different stages of EAE. ACKNOWLEDGMENTS FIG. 10. ICOS-Ig injection decreased the severity of ongoing EAE. MBP-reactive transgenic spleen cells were cultured with wildtype spleen cells from B10.PL syngeneic mice in a 1:5 ratio (transgenic:wild type) and MBP Ac1-11 (25 g/ml). After for 48 h, cells were harvested and a total of 35 ⫻ 10 6 cells were injected into each naı¨ve syngeneic B10.PL mouse. These mice were examined daily for signs of clinical disease and graded as previously described. Mice with clinical signs of EAE received 100 g/ml of either ICOS-Ig or human IgG1 control on days 10, 11, and 12. Mice that received ICOS-Ig underwent an attenuated disease after the commencement of injections. Differences in groups were compared for the period of observation following the first injections (days 10 –21, Mann–Whitney sum of ranks, P ⬍ 0.05).
increased apoptosis in the memory population. In contrast, a very small percentage of naı¨ve T-cells were susceptible to apoptosis following ICOSL blockade. These findings are consistent with previous results that CD28 protects naı¨ve but not memory T-cells from apoptosis (32) and suggest an important role for ICOS in memory T-cell survival. The cells found within the CNS following adoptive transfer (Fig. 4) may be those that did not undergo apoptosis in the culture, which were predominantly CD45RB hi. Furthermore, these surviving cells may have expanded in the CNS where MBP is present. The lack of encephalitogenicity is likely due to their inability to produce proinflammatory cytokines such as IFN-␥. BCL-2 and BAX antagonize each other and the predominance of one or another factor may render the cells resistant or prone to apoptosis. Therefore, we examined the expression of BCL-2 and BAX mRNA. ICOS-Ig-treated cells increased BAX (120%) and slightly decreased BCL-2 (13%) expression, resulting in a significant increase in the BAX/BCL-2 ratio. Of particular clinical importance, ICOS-Ig injection during ongoing EAE ameliorated disease. In contrast, ICOS ⫺/⫺ mice develop more severe EAE when immunized with the neuroantigen myelin/oligodendrocyte glycoprotein, presumably due to suppression of a regulatory T-cell population (31). These findings are not necessarily opposed to each other. As the immune response evolves, it is possible
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Received May 24, 2001; accepted with revision May 30, 2001; published online July 25, 2001