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The Journal of Immunology, 2005, 175: 1558-1565.
Copyright © 2005 by The American Association of Immunologists

CD226 Is Specifically Expressed on the Surface of Th1 Cells and Regulates Their Expansion and Effector Functions1

Valerie Dardalhon2,*, Anna S. Schubart2,*, Jayagopala Reddy*, Jennifer Hartt Meyers*, Laurent Monney*, Catherine A. Sabatos*, Rakesh Ahuja{ddagger}, Khuong Nguyen3,{dagger}, Gordon J. Freeman{dagger}, Edward A. Greenfield*,§, Raymond A. Sobel and Vijay K. Kuchroo4,*

* Department of Neurology, Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, {dagger} Department of Medical Oncology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, MA 02115; {ddagger} Department of Molecular and Cellular Biology, University of California, Berkeley, CA 94720; § Millennium Pharmaceuticals, Cambridge, MA 02139; and Veterans Administration Health Care System, Palo Alto, and Department of Pathology, Stanford University School of Medicine, Stanford, CA 95305


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Surface molecules that are differentially expressed on Th1 and Th2 cells may be useful in regulating specific immune responses in vivo. Using a panel of mAbs, we have identified murine CD226 as specifically expressed on the surface of differentiated Th1 cells but not Th2 or Th0 cells. Although CD226 is constitutively expressed on CD8 cells, it is up-regulated on CD4 cells upon activation. Th1 differentiation results in enhanced CD226 expression, whereas expression is down-regulated upon Th2 polarization. We demonstrate that CD226 is involved in the regulation of T cell activation; in vivo treatment with anti-CD226 results in significant reduction of Th1 cell expansion and in the induction of APCs that inhibit T cell activation. Furthermore, anti-CD226 treatment delays the onset and reduces the severity of a Th1-mediated autoimmune disease, experimental autoimmune encephalomyelitis. Our data suggest that CD226 is a costimulatory molecule that plays an important role in activation and effector functions of Th1 cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Subpopulations of CD4+ Th cells exert distinct effector functions and are characterized by distinct patterns of cytokine secretion (1). Thl cells produce IL-2 and IFN-{gamma}, which elicits a delayed-type hypersensitivity response and activates macrophages. In contrast, Th2 cells produce IL-4, IL-5, IL-10, and IL-13 and are especially important for IgE production and eosinophilic inflammation. Th1- and Th2-derived cytokines show reciprocal inhibition, e.g., IFN-{gamma} and IL-4 inhibit differentiation of Th cells producing the opposite cytokine, and IL-10 suppresses the synthesis of Th1-derived cytokines by interfering with Ag presentation by macrophages (2). The important roles of the Th subsets in various immunopathological conditions including leprosy, leishmaniasis, schistosomiasis, and organ-specific autoimmune diseases have been demonstrated (3, 4). Differentiation of CD4+ T cells into Th1 and Th2 populations is mainly driven by cytokines. Numerous experimental models have shown that the production of IL-12 by activated macrophages and IFN-{gamma} by NK cells promotes the differentiation of naive T cells into Th1 cells and inhibits their differentiation into Th2 cells (5). Conversely, IL-4 promotes Th2 cell differentiation and inhibits the development of Th1 cells.

In recent years, significant progress has been made in identifying the transcription factors that promote Th1 (STAT4 and T-bet) and Th2 (STAT6, GATA-3, and c-Maf) differentiation (6, 7, 8, 9, 10, 11, 12). However, there are very few cell surface markers that can reliably distinguish between the two cell types. There is evidence that some chemokine receptors (13, 14) and costimulatory molecules (15) are differentially expressed between the two subtypes, but the differences are only quantitative and these receptors can also be detected on the surface of naive T cells. Chandra has been identified as a molecule selectively expressed on Th1 cells, but its function and ligand are still unknown (16). Similarly, T1/ST2 was identified as a unique surface marker for Th2 cells (8). Using a panel of mAb, we recently identified T cell Ig domain, mucin domain 3 and CD94 as cell surface molecules specific for Th1 cells (17, 18). Although CD94 is expressed on NK cells and NK T cells, T cell Ig domain, mucin domain 3 is a novel cell surface molecule selectively expressed on Th1 cells whose function is just beginning to be elucidated.

Th1-specific cell surface molecules likely play an important role in Th1 activation, differentiation, and/or effector functions. Identification of these molecules would be useful not only for detecting and isolating Th1 cells during an immune response but also for regulating their effector functions in vivo. To define molecules that are specifically expressed on the surface of Th1 cells, we used an expression cloning approach to identify the targets of a panel of mAbs that specifically stained Th1 but not Th2 cells. We identified CD226 as a molecule selectively expressed on differentiated Th1 cells but not on Th2 cells.

We show in this study that mouse CD226 is constitutively expressed on CD8+ T cells as well as on subsets of naive CD11b+ macrophages and NK cells. Expression of CD226 is also detected on activated CD4+ T cells. Polarization toward a Th1 phenotype results in enhanced expression of CD226, whereas its expression is down-regulated upon Th2 polarization. We demonstrate that CD226 is involved in regulating T cell activation, and that anti-CD226 mAb treatment delays the onset and reduces the severity of a Th1-mediated autoimmune disease, experimental autoimmune encephalomyelitis (EAE).5


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

The experiments involved the use of Lewis and Lou/M rats and SJL/J, BALB/c, and DO11.10 BALB/c transgenic for the TCR specific for OVA 323–339 mice. The rats were procured from Harlan Sprague Dawley and the mice from The Jackson Laboratory. They were maintained at the animal facilities of Harvard Institutes of Medicine according to the animal protocol guidelines of Harvard University (Boston, MA).

Generation of Th1-specific mAbs

Lewis and Lou/M female rats were immunized three times by a combination of s.c./i.p. injections with 1–5 x 107 Th1-polarized T cell clones (AE7) and/or lines emulsified in CFA. Test bleeds were titered by absorbing out Th2 cell reactive Abs, and the shifts seen by flow cytometry on Th1 vs Th2 polarized cells were compared. Rats showing a differential binding toward Th1>Th2 were selected for fusion. The rats were boosted and 4 days later spleen cells were fused with myeloma cells (clone CRL8006; American Type Culture Collection) using polyethylene glycol 1450 and selected in HAT (hypoxanthine/aminopterin/thymidine) medium. The supernatants from the fusion plate wells were screened for surface expression of molecules on Th1 and Th2 cells by flow cytometry. All hybridoma wells that gave a positive shift on Th1 but not Th2 cells were expanded and subcloned twice by limiting dilution.

T cell clones

The AE7, D10G4, and 7A5 clones and DO11.10 T cells have been previously described (17, 18). The 2D2 Th1 clone is specific for myelin oligodendrocyte glycoprotein 35–55 peptide/I-Ab (19) and LR1F1, Q1.4A11, and Q1.3C11 are Th2 clones specific for an altered peptide ligand of proteolipid protein (PLP) 139–151 (20, 21, 22, 23, 24).

Expression cloning

A eukaryotic expression library was constructed using mRNA from the AE7 Th1 clone and the pAXEF vector (25). We conducted the library screening by expression cloning according to the method previously developed (26). Immunoselected individual plasmids were transfected into COS cells followed by indirect immunofluorescence staining with the anti-CD226 Ab (10E5 mAb). Anti-rat IgG conjugated to FITC was used as a second Ab (Caltag Laboratories). Positive clones were sequenced.

In vitro T cell differentiation

Purified CD4+ DO11.10 T cells were stimulated in vitro with irradiated BALB/c spleen cells and OVA 323–339 peptide (10 µg/ml) for 7 days in the presence of murine (m)IL-12 (5 ng/ml; BD Pharmingen) and anti-mIL-4 (10 µg/ml, clone 11B11) for Th1 differentiation and mIL-4 (10 ng/ml; R&D Systems) and anti-mIL-12 (10 µg/ml; BD Pharmingen) for Th2 differentiation. The cultures were supplemented with rmIL-2 (10 ng/ml) from day 3 as required. After 7–10 days, cells were restimulated in the presence of freshly isolated, irradiated splenocytes and OVA 323–339 under polarizing conditions as described, and this procedure was repeated for a total of four rounds of stimulation/polarization.

Proliferation assays

Female SJL/J mice (8–12 wk old) were injected s.c., with 50 µg of PLP 139–151 peptide emulsified in CFA. Mice were injected i.p. every other day with either 100 µg of anti-CD226 or 100 µg of control rat IgG or PBS. Mice were sacrificed on day 10 and spleen and lymph nodes were removed. Cells were plated at 5 x 105 cells per well in round-bottom 96-well plates with PLP 139–151 peptide added at 0–100 µg/ml for 48 h, after which plates were pulsed with 1 µCi of [3H]thymidine per well for 15–18 h. Proliferative responses were measured as cpm based on [3H]thymidine incorporation using a Wallac scintillation counter (PerkinElmer).

For cell separation experiments, CD11b+ cells were purified by positive selection using magnetic beads (Miltenyi Biotec), and CD4+ T cells were purified by negative selection columns (R&D Systems). CD4+ T cells were plated at a density of 1 x 105 cells/well and CD11b+ cells were plated at a density of 2 x 105 cells/well.

I-As tetramer staining

SJL/J mice were immunized with PLP 139–151 in CFA and treated five times with either PBS, 10E5 mAb or rat IgG on alternate days, starting on the day of immunization. After 10 days, single cell suspensions were prepared from spleens and lymph nodes and stimulated with PLP 139–151 (50 µg/ml) at a density of 5 x 106 cells per ml in DMEM containing 10% FCS, 2 mM L-glutamine, 10 mM HEPES, 2 mM 2-ME, 1000 U/ml penicillin, and 1 mg/ml streptomycin (BioWhittaker). Viable lymphoblasts were harvested by Ficoll-Hypaque density gradient centrifugation on days 4 and 6. Cells were incubated with I-As tetramers (PLP 139–151 or Theiler’s murine encephalomyelitis virus (TMEV) 70–86) conjugated with PE as previously described (27). After staining with anti-allophycocyanin-conjugated anti-CD4 (clone RM4.5) and 7-aminoactinomycin D (BD Pharmingen), cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences). The data were analyzed using FlowJo software (TreeStar) and the percentage of I-As tetramer+ cells were determined in the live CD4+ T cell population by eliminating the dead cell fraction (7-aminoactinomycin D-positive). Because the tetramer positive CD4+ T cells did not differ between the mice treated with PBS alone and the IgG isotype control, these data were pooled.

Cytokine ELISA

Cytokine production was measured for IL-2, IL-4, IL-10, and IFN-{gamma} by quantitative capture ELISA. Purified rat mAb to mouse IL-2 (clone Jes-1A12), IL-4 (clone BVD4-1D11), IL-10 (clone Jes5-2A5), and IFN-{gamma} (clone R4-6A2) were used to coat the ELISA plates (Immulon 4; Dynatech Laboratories). Recombinant mouse cytokines were used to construct standard curves. Biotinylated rat mAb to mouse IL-2 (clone Jes6-5H4), IL-4 (clone BVD6-24G2), IL-10 (clone SXC-1), IFN-{gamma} (clone XMG1.2), and TNF-{alpha} (clone MP6-XT3) were used as the secondary Abs. All the cytokines and their Ab were obtained from BD Pharmingen. Plates were developed using microwell peroxidase substrate (Kirkegaard & Perry Laboratories) and absorbance was measured at 450 nm using a Benchmark microplate reader (Bio-Rad).

Intracellular cytokine staining

Polarized T cells were restimulated with PMA (20 ng/ml) plus ionomycin (500 ng/ml) and 2 mM monensin (GolgiStop; BD Pharmingen) for 4–6 h at 37°C. Cells were washed and stained with anti-CD4 (clone RM4.5-APC) by incubating on ice for 20 min. After two washes in 1x PBS containing 2% FCS and 0.1% sodium azide, the cells were fixed with 4% paraformaldehyde and permeabilized with buffer containing saponin according to the manufacturer’s recommendations (BD Pharmingen). Following permeabilization, PE-conjugated cytokine Abs were added and 20 min after incubation on ice, cells were washed and analyzed by using a FACSCalibur flow cytometer (BD Biosciences). Cytokine Abs and their corresponding isotype controls were obtained from BD Pharmingen. Their respective clones were as follows: IL-2, JES6- and A-95-1; IL-4, 11B11 and R3-34; IL-10, JES5-16E3 and A95-1; IFN-{gamma}, XMG1.2 and R3-34.

Adoptive transfer EAE

Female SJL/J mice (8–12 wk old) were injected s.c. in each flank with 100 µg of PLP 139–151 peptide emulsified in CFA. Ten days later, draining lymph nodes were removed and single cell suspensions were cultured for 3 days in the presence of 10 µg/ml PLP 139–151 and 20 ng/ml IL-12 at a concentration of 107 cells/ml in 6-cm dishes. After 3 days, T cell blasts were purified and 5 x 106 cells in a volume of 100 µl of PBS were injected into the tail vein, followed by i.v. administration of 100 ng of pertussis toxin. Mice were injected i.p. every other day with either 100 µg of 10E5 mAb, 100 µg of control rat IgG or PBS and scored for clinical signs and weight loss as described (28, 29).

Histopathology

Mice were killed at days 15 and 43. Brain and spinal cords from the mice were harvested for histological examination and the mice that died during the observation period were excluded. The tissues were fixed in 10% phosphate-buffered formalin. Histological disease was evaluated by counting the inflammatory foci in the meninges and parenchyma as previously described (30).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Monoclonal Ab 10E5 specifically recognizes CD226 on the surface of Th1 cells

To generate Abs that specifically bind to the surface of Th1 cells, Lewis and Lou/M rats were immunized with the established murine Th1 cell clone AE7 and Th1 cell lines derived from 5B6 and DO11.10 TCR transgenic mice. A panel of 20,000 mAb were produced and screened on polarized Th1 and Th2 T cell clones and cell lines by flow cytometry. One of these mAb, named 10E5, selectively recognized a molecule common to all Th1 clones tested but did not bind to any Th2/Th0 cell clones (Fig. 1). Expression cloning using a cDNA library from Th1 clone AE7 identified CD226 (PTA-1, DNAM-1, TliSA1) as the molecule recognized by the 10E5 mAb. We confirmed the specificity of this Ab to CD226 by transiently transfecting 293 cells with the CD226 cDNA and demonstrating that the 10E5 mAb stained the CD226 transfectants but not untransfected cells (Fig. 2).



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  		FIGURE 1. CD226 is expressed on Th1 but not Th2 or Th0 T cell clones. T cell clones (Th0, Th1, or Th2 specific) were stained with 10E5 mAb hybridoma supernatants or rat IgG2b isotype control Ab. Anti-rat IgG-FITC was used as the secondary Ab. Surface expression of CD226 was then analyzed by flow cytometry.

 


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  		FIGURE 2. 10E5 mAb recognizes CD226. 293 cells were transfected with either pcDNA3.1 encoding CD226 cDNA (293-CD226) or with the vector alone (293-mock). Transiently transfected cells were stained with 10E5 mAb or rat IgG2b isotype control and the surface expression of CD226 was then analyzed by flow cytometry using anti-rat IgG FITC as secondary Ab.

 
CD226 is expressed on naive and activated T cells

We determined the surface expression of CD226 on various cell types by flow cytometry. For this analysis, several cell lines (Th1, Th2, dendritic cells (D2SC/1), macrophages (Raw 264), and B cells (LS102.9)) as well as primary cells derived from naive C57BL/6 mice were costained with 10E5 mAb (anti-CD226) and various other cell-specific surface markers (T cell, NK cell, macrophage, dendritic and B cells) (Fig. 3 and data not shown). CD226 was expressed on essentially all naive CD8+ T cells and on a lower percentage (~40%) of unactivated CD4+ T cells (Fig. 3a). Upon in vitro T cell activation using either plate-bound anti-CD3 with or without anti-CD28 or PMA plus ionomycin, expression of CD226 was up-regulated on CD4+ T cells and maintained at a high level on all CD8+ T cells (Fig. 3b).



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  		FIGURE 3. CD226 is predominantly expressed on T cells. Single cell suspensions were obtained from lymph nodes and spleens from naive C57BL/6 mice and they were costained with 10E5 mAb and PE-conjugated Abs to various cell surface markers (CD4, CD8, DX5, CD11b, CD11c, and B220). Surface expression of these markers was determined by flow cytometry. a, CD226 expression on naive CD4+ and CD8+ T cells. b, CD226 expression on anti-CD3 + anti-CD28 activated CD4+ and CD8+ T cells. c, CD226 expression on naive non-T cells: B cells (B220), macrophages (CD11b), dendritic cells (CD11c), and NK cells (NK1.1 and DX5).

 
Although it was primarily expressed on T cells, CD226 was also expressed on some subsets of primary CD11b+, CD11c+, and NK cells (Fig. 3c). Altogether, these data suggest that the molecule recognized by 10E5 mAb is expressed mainly on T cells.

CD226 is selectively up-regulated on Th1 cells and down-regulated on Th2 cells upon T cell differentiation

Although our initial screening revealed CD226 expression on differentiated Th1 cells but not Th2 cells, it was not clear how the selective expression on Th1 cells was achieved because we found that CD226 was up-regulated on all primary CD4+ T cells upon activation (Fig. 3b). To determine the expression pattern of CD226 during Th cell differentiation, we isolated CD4+ T cells from DO11.10-TCR transgenic mice and activated them in vitro under polarizing conditions to induce their differentiation into either the Th1 or Th2 subset. As early as the second round of Ag-specific restimulation and polarization, we observed a strong reduction in CD226 expression on Th2 cells. After three rounds of polarization, CD226 was significantly up-regulated on Th1 cells, whereas expression on Th2 cells was down-regulated to barely detectable levels (Fig. 4a). This was also true for Th0 cells (Fig. 4a), which produced significant amounts of both IFN-{gamma} and IL-4 (Fig. 4b). Similar trends were observed when Th1 and Th2 cells derived from SJL/J mice were used (data not shown). Thus, T cell differentiation is associated with an up-regulation of CD226 on Th1 cells, whereas CD226 expression is down-regulated on differentiated Th2 and Th0 cells.



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  		FIGURE 4. CD226 expression is up-regulated on Th1 cells and down-regulated on Th2 cells. CD4+ T cells were stimulated in vitro under Th1-, Th2-, or Th0-polarizing conditions. Cells were stained 7–10 days after each round of restimulation with irradiated BALB/c spleen cells and OVA 323–339 peptide (10 µg/ml) in the presence of mIL-12 and anti-mIL-4 for Th1 differentiation or mIL-4 and anti-mIL-12 for Th2 differentiation. Th1, Th2, and Th0 cells were restimulated with PMA plus ionomycin to determine the phenotype of cytokine-producing cells by intracellular staining and by surface staining with CD226 and CD4. a, CD226 expression on CD4+ T cells after each round of restimulation. b, Percentages of IFN-{gamma}- and IL-4-secreting cells after rounds 2 and 4 of restimulation.

 
10E5 mAb inhibits Ag-specific T cell expansion

To determine the role of CD226 in the development of T cell responses, SJL/J mice were immunized with PLP 139–151 peptide emulsified in CFA and treated with either PBS, rat IgG, or 10E5 mAb on alternate days starting on the day of immunization. Immunization of SJL/J mice with PLP 139–151 peptide induces predominantly Th1 responses (21, 24, 27). Ten days postimmunization, lymph nodes and spleen were removed and Ag-specific expansion was quantified based on [3H]thymidine incorporation and I-As/PLP 139–151 tetramer staining.

As shown in Fig. 5a, lymphocytes derived from control mice (treated with either PBS or rat IgG) showed an Ag-specific proliferative response in a typical dose response fashion. In contrast, the spleen cells from mice treated with anti-CD226 showed a marked reduction in response to PLP 139–151 stimulation. We verified this reduced response using I-As/PLP 139–151 tetramers that allowed us to enumerate the frequency of PLP-specific cells by flow cytometry. TMEV 70–86 tetramers were used as negative controls because this peptide also binds I-As molecules. Lymph node cells/splenocytes were isolated 10 days post immunization with PLP 139–151 from the rat IgG/PBS and 10E5 mAb-treated mice and restimulated with PLP 139–151 (10 µg/ml) as in the proliferation assay. Viable lymphoblasts were collected and stained with I-As tetramers. There was a significant reduction in the expansion of PLP 139–151 tetramer positive cells in the mice treated in vivo with 10E5 mAb as compared with control groups (Fig. 5b). Strikingly, IFN-{gamma} secretion was significantly reduced in the cultures derived from mice treated with 10E5 mAb (p = 0.02), whereas secretion of IL-2 (p = 0.03), IL-4 (p = 0.007), and IL-10 (p = 0.03) was increased (Fig. 5c). Taken together, the data suggest that anti-CD226 mAb reduced the expansion of PLP 139–151-specific Th1 cells producing IFN-{gamma} while sparing Th2 cells.



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  		FIGURE 5. Administration of 10E5 mAb in vivo inhibits the expansion of Ag-specific T cells and IFN-{gamma} production. a, Proliferation. SJL/J mice immunized with PLP 139–151 were treated with PBS, rat IgG or 10E5 mAb on alternate days up to day 10. The mice were then sacrificed and splenocytes were stimulated with PLP 139–151. Proliferation was measured after 48 h as CPM based on [3H]thymidine incorporation. {Delta}CPM is presented on the y-axis after subtracting CPM in the medium control alone. b, I-As tetramer staining. SJL/J mice were treated with either isotype control or 10E5 mAb after immunization with PLP 139–151. Ten days later, pooled splenocytes and lymph node cells were stimulated with PLP 139–151 and the frequency of PLP 139–151-specific CD4+ T cells was determined at day 6 by tetramer staining. TMEV 70–86 tetramer was used as a negative control. c, Cytokine ELISA. Supernatants derived from the above cultures were tested on day 3 for cytokine secretion. Each bar represents the mean ± SEM value for a group of mice (n = 3–12 mice per group). IFN-{gamma}, *, p ≤ 0.02; IL-2, *, p ≤ 0.03; IL-4, *, p ≤ 0.007, and IL-10, *, p ≤ 0.03.

 
These findings did not explain whether the reduction in numbers of Ag-specific T cells occurred due to a deletion by the in vivo Ab treatment or to an inhibition of T cell expansion. We therefore purified specific cell populations CD4+ T cells and macrophages (CD11b+) from spleens and lymph nodes of mice treated with either 10E5 mAb or control rat IgG. In vitro experiments were performed, in which equivalent numbers of T cells and APC from 10E5 mAb or control Ab-treated mice were recombined in vitro in various combinations. CD4+ T cells from the control groups showed significant Ag-specific proliferation in the presence of CD11b+ cells derived from control Ab-treated mice (Fig. 6). Interestingly, CD4+ T cells derived from 10E5 mAb-treated mice proliferated ~2-fold less than CD4+ T cells from control Ab-treated animals when activated by Ag in the presence of CD11b+ cells from control Ab-treated mice. This result suggests that the 10E5 mAb in vivo treatment affected the proliferative response of the T cells. Furthermore, CD11b+ cells isolated from the treatment with 10E5 mAb-treated mice were significantly less potent in driving the expansion of CD4+ T cells isolated from either treatment group (Fig. 6), indicating that the treatment with the 10E5 mAb (anti-CD226) interfered with the stimulatory/costimulatory functions of APC. Maximal inhibition, however, was observed when both T cells and macrophages were obtained from the 10E5 mAb-treated animals. We have also confirmed these data with immunized 2D2, myelin oligodendrocyte glycoprotein 35–55-specific TCR transgenic T cells in which 10E5 mAb-treated CD11b+ cells did not activate the transgenic T cells as well as control Ab-treated CD11b+ cells (data not shown).



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  		FIGURE 6. Treatment with 10E5 mAb inhibits T cell proliferation by affecting the function of both T cells and CD11b+ APC. SJL/J mice were immunized with PLP 139–151 peptide in CFA and were treated every other day from day 0–8 with mAb 10E5 or rat IgG/PBS. CD4+ T cells isolated from these groups were restimulated in the presence of CD11b+ cells derived from control Ab- or 10E5 mAb-treated mice. Proliferative responses were measured based on [3H]thymidine incorporation as CPM. Representative data from three independent experiments are shown.

 
Together, these results imply that in vivo administration of 10E5 mAb inhibits Ag-specific T cell proliferation in a developing immune response by interfering with both the stimulatory/costimulatory functions of macrophages and with the functional expansion of T cells.

Preliminary data show that this interference requires an active immune response because CD11b+ cells from unimmunized 10E5-treated mice did not display such an inhibitory effect (data not shown).

Administration of 10E5 mAb suppresses EAE

To determine the role of CD226 on activated Th1 T cells in vivo, we used an adoptive transfer model of the Th1-mediated autoimmune disease EAE, an animal model for multiple sclerosis. Because CD226 is expressed on multiple cell types, a purely T cell-mediated adoptive transfer model was used rather than active immunization, as effects on other cell types would complicate the interpretation of the results. For this purpose, we transferred activated, PLP 139–151-specific Th1 cells into RAG2-deficient SJL/J mice, which are devoid of endogenous B and T cells (both CD8+ and CD4+). Mice were treated five times with PBS, rat IgG, or 10E5 mAb on alternate days starting on the day of transfer (Fig. 7). In two independent experiments, treatment with anti-CD226 significantly delayed disease onset and reduced the severity of the initial phase of EAE (p < 0.01), supporting the in vitro data indicating that the 10E5 mAb interferes with Ag-specific T cell activation/expansion. However, after cessation of treatment, 10E5 mAb-treated RAG2-deficient mice developed a similar disease course to that of the control groups, suggesting that there was no long-term effect of Ab treatment. Histological examination revealed that on day 15 posttransfer the numbers of inflammatory foci in the CNS were reduced in 10E5 mAb-treated mice compared with controls, indicating that the inflammatory response correlated with the clinical disease (Table I). Similar trends were observed on day 43 posttransfer (data not shown). Taken together, our results show that the CD226 pathway is involved in the effector phase of T cell responses, as 10E5 mAb treatment in vivo suppressed both Ag-specific T cell expansion and an autoimmune disease mediated by effector Th1 cells.



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  		FIGURE 7. Treatment with 10E5 mAb inhibits adoptively transferred EAE. Female RAG2-deficient SJL/J mice were injected with 5 x 106 PLP 139–151-specific T cell blasts, followed by injection of 100 ng of pertussis toxin i.v. The mice were treated five times on alternative days with either PBS, rat IgG or 10E5 Ab i.p., and the disease was scored for 15 (a) or 43 (b) days. Disease pattern in two independent experiments is shown (n = 5–7 per group).

 

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  		Table I. Anti-CD226 inhibits clinical and histologic EAE

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Upon activation, naive CD4+ T cells can differentiate into Th1 or Th2 effector cells. Although much is known about the effector functions of these two T cell subpopulations, there is still little knowledge about the identity and function of their cell surface-specific molecules. We have identified CD226 as a molecule expressed selectively on differentiated Th1, but not Th2 or Th0 T cells and demonstrate that CD226 plays an important role in APC and CD4+ Th effector functions.

In humans, CD226 is expressed on T cells (CD4+ > CD8+ T cells), NK cells, monocytes, and subsets of B cells (31) and platelets (32), and it is implicated in CD8+ and NK-mediated cytotoxicity (31) and platelet activation (32). We demonstrate that the expression pattern of CD226 in mice is different, in that it is predominantly expressed on T cells (CD8+>CD4+), and only minor levels of expression are detected on some subsets of NK cells and macrophages, but not on B cells. More importantly, we describe a differential expression of CD226 on Th subtypes. Murine CD226 is specifically up-regulated on CD4+ Th1 cells and down-regulated on Th2 cells. Although CD226 is up-regulated on all activated undifferentiated CD4+ T cells, it is down-regulated upon differentiation to Th2 cells. Th0 cells express significant amounts of IFN-{gamma} but not CD226, suggesting that expression of CD226 is not regulated by IFN-{gamma}. Using mice deficient for IFN-{gamma}, IL-4, IL-10, and TNF-{alpha}, we observed a normal expression level of CD226 on T cells during T cell activation (data not shown), suggesting that these cytokines do not directly regulate CD226 expression. However, the addition of IL-12 and anti-IL-4 to Th0 polarized cells after the second round of restimulation during in vitro differentiation resulted in the induction of CD226 expression (data not shown). In contrast, Th2 cytokines failed to suppress CD226 expression on polarized Th1 cells (data not shown). These observations indicate that Th1-polarizing conditions can induce CD226 expression on unpolarized cells, but CD226 expression cannot be reversed on Th1 cells even after addition of Th2 cytokines. In addition, neither ectopic expression of CD226 in Th2 cells by retroviral infection nor the addition of the CD226-specific mAb 10E5 to in vitro cultures modified Th differentiation, suggesting that stimulation through CD226 does not regulate Th2 differentiation (data not shown). In contrast, Shibuya et al. (33) recently demonstrated that overexpression of a dominant negative mutation in the signaling domain of CD226 in naive human CD4+ T cells strongly suppressed Th1 differentiation, suggesting that functional signaling through CD226 is a prerequisite for Th1 differentiation. To account for these data, we propose that CD226 may play a role in Th1 effector cell generation and function, but its presence does not interfere with the differentiation of Th2 cells.

To study the role of CD226 during the initiation and effector phases of a pathogenic immune response, we examined its role on Th1 cells in vivo following immunization of SJL/J mice with PLP 139–151. Administration of anti-CD226 mAb 10E5 during the first 8 days after immunization with PLP 139–151 inhibited the expansion of PLP-specific CD4+ T cells as determined by the frequency of PLP 139–151-specific cells monitored with a PLP 139–151 loaded tetramer. To explain this inhibition of T cell proliferation, we purified CD11b+ and CD4+ T cells from either 10E5 mAb or control Ab-treated immunized mice and performed reconstitution experiments. These experiments not only revealed a suppressive effect of 10E5 mAb treatment on the proliferation of CD4+ T cells, but also a suppressive function on macrophages, resulting in reduced Ag-specific proliferation of T cells in the presence of CD11b+ cells derived from 10E5 mAb-treated mice. It is interesting to note that even though CD226 seems to be expressed on only a low percentage of CD11b+ cells, the anti-CD226 mAb seemed to affect the APC function. One possible explanation is that 10E5 mAb induced T cells to alter macrophage function, either by inducing inhibitory pathways or by lowering APC stimulatory functions. This hypothesis was supported by the fact that CD11b+ cells derived from unimmunized, 10E5-treated mice did not suppress T cell proliferation (preliminary data; data not shown) suggesting that an active immune response was required to induce the inhibitory functions of the macrophages. This explanation was also supported by the fact that the addition of 10E5 mAb to in vitro T cell proliferation assays did not give strong or consistent inhibition of T cell response in contrast to the strong, highly reproducible effects of 10E5 mAb observed during in vivo immune responses. 10E5 mAb may compete with the natural ligands of CD226 and prevent macrophage activation required for efficient Ag presentation and T cell activation.

In agreement with our observations, Shibuya et al. (33, 34), also showed that CD226 is functional only upon T cell activation. Activation of human CD8+ T cells induces phosphorylation of the Ser329 residue of CD226. This phosphorylation event is required for CD226 to associate with LFA-1, which allows the Fyn protein tyrosine kinase to phosphorylate the Tyr322 of CD226 (33, 34). Because LFA-1 itself lacks a signaling domain, this raises a question as to whether CD226 acts as an adaptor molecule for costimulatory signals initiated by LFA-1 ligation. Because it is currently not known whether 10E5 mAb is an agonist or antagonist mAb, we cannot exclude the possibility that it has a depleting effect in vivo. If so, this could be a possible explanation for the reduced expansion of PLP 139–151-specific cells in mice treated with 10E5 mAb. However, our observations revealed a reduced number of CD8+ T cells but no significant differences in total number of CD4+ T cells or CD11b+ cells between mice treated with 10E5 mAb and isotype control. Furthermore, using equivalent numbers of CD4+ T cells during in vitro proliferation assays, CD4+ T cells from 10E5 mAb-treated mice consistently showed a lower proliferative response as compared with the control. Altogether, further characterization of 10E5 mAb will help us to understand the role of CD226 in effector Th1 T cells.

In vivo, we confirmed the suppressive effect of 10E5 mAb on the effector phase of the immune response in a Th1-driven disease model, PLP 139–151-induced EAE. We transferred activated, myelin PLP 139–151-specific Th1 effector cells into RAG2-deficient recipient mice and tested the effect of 10E5 mAb on the disease course. The 10E5 mAb treatment delayed the onset of EAE and decreased the clinical and histologic severity of the disease. This effect was also tested in wild-type mice by inducing EAE, but in this system suppression of disease severity was longer lasting, and cessation of the treatment with 10E5 mAb did not lead to an exacerbation of the disease (data not shown). Based on these observations, we propose that the 10E5 mAb prevented the optimal activation of macrophages and T cells during the effector phase of the immune response in vivo in the wild-type mice, resulting in long lasting protection. It has been shown in rats that reprogramming of transferred T cells is required for homing to the CNS and initiation of EAE (35). Therefore, a defect in this process could interfere with the development of autoimmune disease. Th1 cells are more prone to activation-induced cell death than Th2 cells (36, 37). Because the percentage of Ag-specific Th1 cells was reduced in 10E5 mAb-treated mice, and CD226 is specifically expressed on Th1 cells, the 10E5 mAb may selectively induce cell death of Th1 cells. Alternatively, the 10E5 mAb may directly inhibit the migration of inflammatory cells (T cells or macrophages) to the CNS. This hypothesis is supported by the structure of CD226 and by the ligands to which it binds. Two ligands of human CD226 have been identified that have also recently been shown to serve as ligands for murine CD226 (38): nectin-2 (CD112) and poliovirus receptor (CD155), closely related molecules expressed on endothelial and epithelial cells with known adhesive functions (39). Binding of CD226 to its ligands could thus regulate migration of effector cells. In a human system, CD226 was shown to regulate monocyte migration and, more specifically, extravasation via the interaction of CD226 on monocytes with poliovirus receptor expressed at endothelial cell tight junctions (40).

Emerging data also suggest that CD226 may play an important role as an adaptor molecule for costimulatory signals initiated by LFA-1 ligation because LFA-1 itself lacks a signaling domain. It was previously demonstrated that LFA-1/ICAM-1 stimulation is implicated in Th differentiation, (41, 42) and the blockade of this pathway alters transendothelial migration of Th1 cells (43). The recent report showing that a dominant negative mutation in the signaling domain of CD226 modifies Th cell differentiation and other LFA-1-mediated functions suggests a major role for CD226 in LFA-1-mediated costimulatory function (33). Our data are the first to demonstrate that CD226 is specifically expressed on murine Th1 cells and it regulates T cell expansion and modulates macrophage function, possibly by regulating the function of LFA-1.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the National Multiple Sclerosis Society Grants RG-2571-D-9 and FG-1478-A-1 and the National Institutes of Health Grants 1RO1NS045937-01, 2R01NS35685-06, 2R37NS30843-11, 1RO1AI44880-03, 2PO1AI39671-07, 1PO1NS38037-04, and 1F31GM20927-01. V.D. was funded by European Molecular Biology Organization Fellowship award ALTF 117-2002. Back

2 V.D. and A.S.S. contributed equally to this work. Back

3 Current address: Westborough Eye Care, Westborough, MA 01581 Back

4 Address correspondence and reprint requests to Dr. Vijay K. Kuchroo, Professor of Neurology, Center for Neurologic Diseases, Room 786, Harvard Institutes of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: vkuchroo{at}rics.bwh.harvard.edu Back

5 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; PLP, proteolipid protein; TMEV, Theiler’s murine encephalomyelitis virus. Back

Received for publication March 14, 2005. Accepted for publication May 16, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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