The CD226/CD155 Interaction Regulates the Proinflammatory (Th1/Th17)/Anti-Inflammatory (Th2) Balance in Humans

Ester Lozano, Nicole Joller, Yonghao Cao, Vijay K. Kuchroo and David A. Hafler
J Immunol October 1, 2013, 191 (7) 3673-3680; DOI: https://doi.org/10.4049/jimmunol.1300945

Abstract

CD226 costimulatory signals strongly promote Th1 differentiation, enhancing IFN-γ production by naive T cells. We recently reported that knockdown of CD226 on human T cells resulted in a decrease in T-bet and IFN-γ expression. However, the role of CD226 on Th2 and Th17 cells remains unknown. In this study, we found that CD226 and its ligand CD155 were decreased on Th2-polarized naive T cells, whereas both were highly expressed under Th17 conditions. Most IFN-γ– and IL-17–producing cells expressed high levels of CD226, but production of IL-13 did not correlate with CD226 expression. CD226 knockdown by lentiviral transduction resulted in increased STAT-6 phosphorylation, enhanced GATA3 expression, and consequently higher production of IL-4 and IL-13. Under Th17 conditions, CD226-depleted cells showed slightly impaired IL-17 secretion, suggesting that CD226 contributes, in part, to IL-17 production but is dispensable for Th17 cell generation. In line with these results, CD226 blockade with neutralizing Abs efficiently inhibited T cell activation and proliferation and production of IFN-γ and IL-17, whereas IL-13 secretion remained functional. Taken together, our results establish an important role for CD226 in differentially regulating the proinflammatory (Th1/Th17)/anti-inflammatory (Th2) balance, suggesting that the CD226/CD155 interaction could potentially be targeted in therapeutic approaches to human autoimmune diseases.

Introduction

Costimulatory molecules can promote or inhibit TCR-mediated activation, playing an important role in fine-tuning TCR-mediated T cell functions (1, 2). The prototypical CD28/B7 family has been intensively studied as crucial costimulatory molecules to achieve complete T cell activation (3). However, T cell function is ultimately dictated by an array of costimulatory signals, and it has become clear that the relative importance of a costimulatory pathway may vary depending on the T cell subset and the specific mechanism of pathogenesis (2, 4, 5). Given that the alternative costimulatory pathway comprising CD226 and its ligand CD155 has been associated with autoimmune diseases, including multiple sclerosis (MS) and type 1 diabetes (T1D) (6), we investigated the functional role of CD226 and the effects of specifically targeting CD226 on human T cell responses.

CD226 (also known as DNAM-1) is a glycoprotein belonging to the Ig superfamily that is expressed on the surface of NK cells, platelets, monocytes, and activated CD4+ T cells (7). CD226 binds to CD155 (also known as the poliovirus receptor) and CD112, both expressed on APCs (8), but only CD155 is induced in T cells upon their activation (9). CD226 mediates cellular adhesion and triggers NK cell effector functions. In human CD4+ T cells, CD226 associates with LFA-1 and contributes to LFA-1 costimulatory signals that promote Th1 cell differentiation (10). We previously demonstrated that CD226 also counteracts the activity of the T cell coinhibitory receptor, T cell Ig and ITIM domain (TIGIT), by competing for the same ligand CD155 (9). CD226 allelic variants have been defined as a genetic risk factor for developing MS and T1D (11, 12). This disease-associated single-nucleotide polymorphism is located in the coding region and generates an amino acid change Ser307Gly in the intracellular domain (11). Although these genomic data identify molecules involved in autoimmunity in an unbiased manner, the functional role of CD226 in human autoimmune diseases has not been defined.

CD155 is a glycoprotein composed of two Ig-like C2-type domains and one Ig-like V-type domain that is necessary for poliovirus binding and uptake. CD155 is a broadly expressed receptor that can interact with several ligands, such as CD226, TIGIT, CD96, vitronectin, integrin αvβ3, and PDGFR. The functions of CD155 include roles in cell adhesion, neural differentiation (13), and NK cell effector functions (14). TIGIT binds with high affinity to CD155 on the dendritic cell (DC) surface, which causes increased secretion of IL-10 and decreased secretion of IL-12, promoting the generation of mature immunoregulatory DCs (15). Furthermore, TIGIT is a coinhibitory receptor that can transduce a negative signal into activated T cells that attenuates T cell proliferation and cytokine production (16). In fact, ligation of TIGIT by agonistic Ab inhibits IL-2 production and T cell activation (9). In addition to its cell-intrinsic role, TIGIT may outcompete CD226 for binding to CD155 because of its higher affinity for their common ligand CD155 (15, 17). In support of this concept, TIGIT-depleted cells showed higher expression of T-bet and IFN-γ, and these effects were overcome with blockade of CD226/CD155 interaction, demonstrating that TIGIT and CD226 are indeed competing for CD155 binding (9).

In this article, we report that CD226 and CD155 are differentially expressed on different Th cell lineages, suggesting distinct roles for CD226 and CD155 in controlling T cell function. By silencing CD226 gene expression, we show that CD226 promotes Th1 responses and represses Th2 function. Thus, CD226 knockdown enhanced STAT-6 phosphorylation and GATA3 expression, leading to a significant increase in Th2 cytokines. Considering the association of the CD226/CD155 pathway with human autoimmune diseases, we also analyzed the effect of CD226 blockade on primary human T cells. Treatment with neutralizing anti-CD226 mAb efficiently inhibited T cell activation and proliferation. Blockade of CD226 signaling decreased IFN-γ and IL-17 production. In contrast, IL-13 expression was not affected under these experimental conditions. Finally, treatment with neutralizing anti-CD226 mAb decreased T cell activation and inhibited proliferation of CD4+ T cells isolated from untreated patients with relapsing remitting MS or T1D. Manipulation of the interaction between CD226 and CD155 could be a promising molecular target for therapeutic intervention in human autoimmune diseases.

Materials and Methods

T cell purification

Peripheral venous blood was obtained from healthy volunteers and patients in compliance with Institutional Review Board protocols at Yale University School of Medicine. Human naive CD4+ T cells were purified by pre-enrichment of CD4+ T cells from PBMCs by MACS using negative selection (CD4+ T Cell Isolation Kit II; Miltenyi Biotec, Auburn, CA) and sorted by flow cytometry for CD4+CD62L+CD25−CD45RO− naive T cells (FACSAria; BD Biosciences, San Jose, CA).

Differentiation assays

FACS-sorted naive T cells were stimulated in the presence of anti-CD3 (5 μg/ml; UCHT1) and anti-CD28 (1 μg/ml; 28.2; both from BD Biosciences) under the following conditions: Th1 induction, rIL-12 (10 ng/ml; R&D Systems); Th2 induction, rIL-4 (25 ng/ml); and Th17 induction, TGF-β (5 ng/ml), IL-1β, IL-21, and IL-23 (all 25 ng/ml; R&D Systems). Recombinant human IL-2 was obtained through the AIDS Research and Reference Reagent Program (Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health) and was used at 10 U/ml, except under Th17 conditions.

Intracellular cytokine staining

Before detection of intracellular cytokine production, T cells were restimulated with PMA (50 ng/ml) plus ionomycin (250 ng/ml) for 4 h in the presence of GolgiStop (BD Biosciences). Staining with LIVE/DEAD Fixable Dead Cell Stain Kit (Molecular Probes) was performed before fixation to allow gating on viable cells. After fixation and permeabilization with Foxp3 Staining Buffer (eBioscience), cells were stained with anti–IFN-γ (B27), anti–IL-13 (JES10-5A2), anti–IL-4 (8D4-8), anti–IL-9 (MH9A4), and anti–IL-17 (BL168) (BioLegend, San Diego, CA). Samples were acquired on a Fortessa flow cytometer (BD Biosciences), and FlowJo software (TreeStar, Ashland, OR) was used for data analysis.

Quantification of mRNA expression levels by real-time PCR

RNA was isolated using QIAGEN RNeasy Micro Kit (QIAGEN, Valencia, CA) and converted to cDNA by RT with random hexamers and Multiscribe RT (TaqMan Reverse Transcription Reagents; Applied Biosystems, Foster City, CA). For mRNA gene-expression assays, probes were purchased from Applied Biosystems, and the reactions were run on a StepOne Real-Time PCR System (Applied Biosystems). Values are represented as the difference in Ct values normalized to β2-microglobulin for each sample as per the following formula: relative RNA expression = (2−dCt) × 1000.

Gene silencing by lentiviral transduction

Lentiviruses expressing specific short hairpin RNAs (shRNAs) to silence CD226 gene expression were obtained from the library of The RNAi Consortium (18). As previously described (9), ex vivo human CD4+ T cells were plated on a 96-well plate at a concentration of 105 cells/well and activated with anti-CD3 and anti-CD28 at 1 μg/ml. After 24 h, the medium was carefully removed; cells were infected with viral supernatants and polybrene (Millipore) at 6 μg/ml and centrifuged at 2250 rpm for 30 min at room temperature. After the centrifugation, medium was removed, and fresh supplemented RPMI 1640 medium was added. After 48 h, cells were selected with 2 μg/ml puromycin (Invitrogen, Carlsbad, CA) and collected for analysis 72 h after selection. At various time points, cells were collected to analyze gene expression and cell surface marker expression by flow cytometry.

Proliferation assays

CD4+ T cells (104/well) labeled with CFSE were stimulated with anti-CD3, anti-CD28, and IL-2 (10 U/ml) and cultured in the presence of plate-bound blocking anti-human CD226 (clone DX11; Abcam, Cambridge, MA) at 10 μg/ml, LEAF-purified mouse IgG1 isotype control (BioLegend), or plate-bound CD155 (R&D Systems). CFSE dilution and expression of Ki67 and activation markers were assessed on day 4 by flow cytometry.

Measurement of cytokine production by ELISA

ELISA measurements of cytokines were performed with purified coating and biotinylated detection Abs, as follows: IFN-γ, clone 2G1 and human IFN-γ mAb biotin-labeled (Thermo Scientific, Rockford, IL); IL-13, P130E and M130B clone 5A2 (Pierce Biotechnology, Rockford, IL); IL4, clones 8D4-8 and MP4-25D2 (BD Biosciences); and IL9, clone MH9A4 (BD Biosciences). Production of IL-17 was measured with a DuoSet ELISA Development kit (R&D Systems). Samples were normalized by cell number assessed on a guava easyCyte HT Flow Cytometer (EMD Millipore, Billerica, MA).

Statistical analysis

Statistical significance was assessed by either the two-tailed Student t test (two groups) or ANOVA for multiple groups, with a posthoc Tukey test to determine significance; all analyses were performed using Prism Graph Pad 4.0 software.

Results

CD226 and CD155 are decreased under Th2 conditions and increased under Th17-polarizing conditions

CD226 and its ligand CD155 are rapidly induced on human naive CD4+ T cells upon TCR cross-linking (9). However, it is not known whether these coreceptors are uniformly expressed on Th subsets or whether polarizing cytokines can differentially modulate their expression. Therefore, we first measured gene expression of CD226 and its ligand CD155 in human FACS-sorted naive T cells differentiated under Th1-, Th2-, and Th17-polarizing conditions. Subset analysis of Th populations showed gene expression of CD226 and CD155 was lower under Th2 conditions but was significantly upregulated under Th17 conditions (Fig. 1A). We analyzed CD226 and CD155 cell surface expression by flow cytometry on day 7 upon activation, and the decreased mean fluorescence intensity (MFI) of CD226 and CD155 under Th2 conditions confirmed the reduced expression of these molecules (Fig. 1B–D). Consistent with our results at the mRNA level, naive T cells activated under Th17-polarizing conditions showed an increased expression of both receptors. Interestingly, although human naive T cells differentiated in vitro expressed low levels of TIGIT, we found that TIGIT expression was lower under Th17 conditions (Supplemental Fig. 1). Our findings indicate that members of the CD226/CD155 pathway are differentially expressed on human Th subsets, suggesting that they play distinct roles in Th function and cytokine production.

FIGURE 1.
FIGURE 1.

Differential expression of CD226 and its ligand CD155 in human CD4+ Th subsets. (A) FACS-sorted naive T cells were polarized under Th0, Th1, Th2, or Th17 conditions. At day 5, RNA was collected and used as a template to determine the relative levels of CD226, CD155 (Pvr), T-bet (Tbx21), GATA3, and RORc transcripts. Data are representative of three independent experiments. (B) Cell surface expression of CD226 and CD155 was determined in each Th subset by flow cytometry. Representative graphs of CD226 and CD155 expression under each Th polarizing condition at day 7. Numbers indicate MFI. (C) Pooled data for MFI of CD226 expression for each subset, as shown in (B) (n = 8). (D) Statistics for MFI of CD155 expression at day 7 (n = 8). *p < 0.05.

Differential expression of CD226 and CD155 in cytokine-producing cells

Under in vitro polarizing conditions, only a portion of naive T cells undergoes the differentiation process and produces detectable levels of cytokines. To characterize CD226 and CD155 expression only on cytokine-producing cells, naive T cells were differentiated under polarizing conditions for Th1, Th2, and Th17 for 7 d; restimulated with PMA/ionomycin; and analyzed for the production of cytokines (IFN-γ, IL-13, and IL-17). As expected, CD226 cell surface expression was elevated in all of the conditions upon activation. However, under Th2 conditions, cells showed a lower frequency of CD226+ cells compared with Th0 conditions. In contrast, cells that differentiated under Th17 conditions showed a significantly higher frequency of CD226+ cells (Fig. 2A). Under Th2 conditions, both CD226+ and CD226− cells were able to produce IL-13, whereas the ability to produce IFN-γ or IL-17 was restricted to CD226+ cells in the Th1 and Th17 lineage, respectively (Fig. 2B). Thus, the majority of IFN-γ–producing cells and all IL-17+ cells expressed CD226, whereas IL-13–producing cells did not show a bias in CD226 expression.

FIGURE 2.
FIGURE 2.

Expression of CD226 and CD155 on cytokine-producing T cells. (A) FACS-sorted naive T cells were cultured under polarizing conditions for 7 d and then restimulated (PMA/ionomycin) for 4 h. Cells were stained for surface CD226 or CD155, followed by intracellular staining for IFN-γ, IL-13, and IL-17. An isotype-control staining for CD226 and CD155 is depicted for reference (negative control). Data are representative of eight donors. Percentage of cytokine-producing cells was analyzed, gating on CD226+ and CD226− cells (B) and on CD155+ and CD155− cells (C) (n = 8). *p < 0.05.

Interestingly, intracellular staining showed that most IL-17–producing cells expressed CD155, whereas the majority of IFN-γ+ cells were negative for CD155 (Fig. 2C). Similar to CD226, Th2 cytokine production was independent of CD155 expression (Fig. 2C). The fact that human IL-17–producing T cells upregulated both CD226 and its ligand CD155 indicates that, in Th17 cells, this costimulatory signaling can occur in the absence of APCs, whereas in IFN-γ+ cells it would likely depend on DC costimulation.

Higher CD226 expression correlates with lower GATA3 expression in ex vivo T cells

We next analyzed whether the differential functional programs related to CD226 expression on polarized naive T cells can be observed on ex vivo CD4+ T cells. Human memory T cells (CD4+CD62L+CD25medCD45RO+) were sorted into CD226low, CD226med, and CD226high subsets that were then stimulated with PMA and ionomycin for 4 h and analyzed for gene expression. In line with our results from the polarization experiments, CD226high cells expressed higher levels of T-bet and RORc, the master transcription factors for Th1 and Th17, respectively. In contrast, cells expressing higher levels of CD226 showed a decrease in GATA3, the master transcription factor for Th2 cells, and decreased Th2 cytokines, such as IL-4 and IL-13 (Fig. 3A). In line with these results, flow cytometric analysis confirmed that ex vivo IL-4–producing cells expressed significantly lower levels of cell surface CD226 compared with IFN-γ–producing cells (Fig. 3B, 3C). These data raised the question of whether high expression of CD226 has an inhibitory role in regulating Th2 responses.

FIGURE 3.
FIGURE 3.

Ex vivo CD226high cells express less Th2-associated genes. (A) Ex vivo memory T cells were sorted, gating on high, intermediate, and low CD226 expression. Cells were stimulated (PMA/ionomycin) for 4 h, and mRNA was isolated for gene-expression analysis (n = 6). (B) Ex vivo memory T cells were stimulated with PMA/ionomycin for 4 h and stained for CD226, followed by intracellular staining for IFN-γ, IL-4, IL-13, and IL-17. CD226 expression (MFI) was analyzed, gating on cytokine-producing cells. Representative data of six independent donors. (C) Statistical analysis of CD226 expression in each cytokine-producing subset (n = 6). *p < 0.05, ANOVA.

Knockdown of CD226 increases GATA3 expression and Th2 cytokine production

To examine the functional role of CD226 on cytokine production by each human Th subset, we first generated stable CD226-deficient cells. We previously reported that silencing CD226 expression on human CD4+ T cells results in a decrease in T-bet expression and a subsequent reduction in IFN-γ–producing cells (9). To address the role of CD226 in repressing Th2 cytokine production, we selectively knocked down CD226 expression by lentiviral transduction of primary CD4+ T cells with specific shRNAs. After transfection with shCD226 or an empty vector (control), we selected transfected cells with puromycin and restimulated them with anti-CD3 and anti-CD28. Upon activation, CD226-depleted cells showed enhanced GATA3 expression without addition of exogenous cytokines (Fig. 4A). Consequently, we observed an increase in IL-13 mRNA levels in cells transfected with shCD226. To confirm these results at the protein level, we restimulated CD226-depleted cells with PMA/ionomycin for 4 h to analyze CD226 cell surface expression versus the production of IFN-γ and IL-13. Consistent with our previous findings, CD226 knockdown resulted in reduced levels of IFN-γ and, conversely, a significant increase in IL-13 production (Fig. 4B). Measurement of Th2 cytokine secretion on the supernatants from shCD226 cells confirmed that deletion of CD226 significantly increased the production of IL-13 and IL-4 (Fig. 4C). Collectively, our data strongly suggest that CD226 modulates the Th1/Th2 balance in humans.

FIGURE 4.
FIGURE 4.

Knockdown of CD226 in human T cells increases the expression of GATA3 and Th2 cytokines. (A) Quantitative real-time RT-PCR analysis of relative CD226, GATA3, and IL-13 gene expression in human CD4+ T cells infected with lentivirus containing CD226-specific shRNA sequence or an empty vector, assessed on day 3 after puromycin selection. (B) Flow cytometry of the surface expression of CD226 versus IFN-γ and IL-13 intracellular staining on shCD226 cells, selected with puromycin and stimulated with PMA and ionomycin for 4 h in the presence of GolgiStop. Cells were stained with LIVE/DEAD Fixable Dead Cell Stain Kit before fixation to allow gating on viable cells. (C) Measurement of IL-13 and IL-4 in the supernatants of cells transduced with shCD226 derived from the same cultures as in (A) quantified by ELISA. Data are mean ± SEM and are representative of three independent experiments, with three healthy donors/experiment. *p < 0.05.

CD226 is not required for IL-17 production

IL-17–producing cells expressed high levels of both CD226 and CD155, suggesting a possible functional role of CD226/CD155 in Th17 cells. To determine the potential role of CD226/CD155 in modulating Th17 cell function, we also silenced CD226 expression under Th17 conditions. Although CD226 knockdown resulted in a slight decrease in Il17a gene expression, these differences were not significant, suggesting that CD226 is not required for IL-17 production (Fig. 5A). We also restimulated CD226-depleted cells with PMA and ionomycin to evaluate cytokine production at the protein level. Lack of CD226 resulted in a trend toward decreased frequency of IL-9–producing and IL-17+ cells, but the results did not reach statistical significance (Fig. 5B, 5C). In line with these results, the reduction in IL-17 and IL-9 secreted by CD226-depleted cells was not significant (Fig. 5D). Therefore, our data indicated that CD226 contributes, in part, to IL-17 production but is dispensable for Th17 cell generation.

FIGURE 5.
FIGURE 5.

CD226 is not required for IL-17 production. (A) Quantitative real-time RT-PCR analysis of RORc and IL-17 gene expression in human CD4+ T cells infected with shCD226 under Th17 conditions, assessed on day 3 after puromycin selection. (B) Flow cytometry of the surface expression of CD226 versus IL-9 and IL-17 intracellular staining on viable shCD226 cells. (C) Frequency of cells producing IL-17 or IL-9 among shCD226 cells (n = 6). (D) Measurement of IL-17 and IL-9 in the supernatants from the same cultures as in (A) quantified by ELISA. Data mean ± SEM and are representative of three independent experiments, with two healthy donors/experiment.

CD226 differentially modulates STAT-signaling pathways

To determine the mechanism by which CD226 differentially regulates Th subsets, we first analyzed whether loss of CD226 was altering the expression of key receptors for T cell differentiation, such as IL-12Rb2, IL-4R, and IL-21R. No significant differences were found in the levels of the transcripts for these receptors in cells transduced with shCD226 (Fig. 6A). We also analyzed changes in the gene expression of STAT family members that are part of the signaling pathways downstream of these receptors. Knockdown of CD226 did not modify mRNA levels of STAT-4, STAT-6, or STAT-3 in primary human T cells (Fig. 6A).

FIGURE 6.
FIGURE 6.

Increased phosphorylation of STAT-6 in shCD226 cells in response to IL-4. (A) Analysis of expression of genes encoding cytokine receptors in shCD226 cells. (B) Flow analysis of shCD226 cells stimulated with IL-12, IFN-γ, IL-4, and IL-21 for 20 min and stained for CD226 and pSTAT-4, pSTAT-1, pSTAT-6, and pSTAT-3, respectively. (C) Statistical data summarizing flow data of the percentage of cells positive for pSTAT-4 (n = 5), pSTAT-1 (n = 7), pSTAT-6 (n = 7), and pSTAT-3 (n = 7). (D) CD226 expression (MFI) was assessed on control cells transduced with an empty vector; expression on cells positive for the indicated phosphorylated STATs is depicted. *p < 0.05, ANOVA.

We next measured phosphorylation of signal transducers STAT-4, STAT-1, STAT-6, and STAT-3 in response to IL-12, IFN-γ, IL-4, and IL-21, respectively. As expected, flow analysis showed that STAT-1 and STAT-4 phosphorylation was markedly decreased in CD226-deficient T cells (Fig. 6B, 6C). In contrast, IL-4–induced STAT-6 activation was significantly increased compared with control cells, suggesting that CD226 negatively regulates the IL-4R–activated Jak-STAT pathway (Fig. 6B, 6C). STAT-3 phosphorylation in response to IL-21 stimulation was not impaired in CD226-depleted cells. Interestingly, control cells positive for pSTAT-6 showed significantly lower CD226 MFI than did the other groups (Fig. 6D). Overall, CD226 promotes Th1-signaling pathways while repressing Th2 responses by inhibiting STAT-6 phosphorylation, GATA3 expression, and, consequently, Th2 cytokine production.

CD226 blockade inhibits T cell activation, proliferation, and cytokine production

Considering that CD226 polymorphism is associated with a higher risk for developing MS and T1D, we analyzed whether blockade of CD226 signaling would reduce proinflammatory cytokines involved in the pathogenesis of these autoimmune diseases. To evaluate the effect of CD226 blockade, we performed functional assays on primary T cells in the presence of neutralizing anti-CD226 mAb. After CD4+ T cell incubation on plate-bound blocking anti-CD226 mAb for 4 d, T cell proliferation was assessed by CFSE dilution assay after gating on viable cells. As shown in Fig. 7, treatment with anti-CD226 mAb efficiently abolished human CD4+ T cell proliferation. No significant differences were observed in the frequency of viable cells (data not shown). To address the mechanism by which CD226 blockade leads to inhibition of proliferation, we evaluated the expression of cell surface markers of activation by flow cytometry. We observed that neutralizing CD226 signaling led to decreased expression of CD25, CD69, and CD226 itself (Fig. 7A, 7B). Thus, CD226, possibly through its interaction with LFA-1, has a crucial role in T cell activation, and blockade of CD226 signaling efficiently inhibits T cell proliferation.

FIGURE 7.
FIGURE 7.

CD226 blockade decreases proliferation, activation, and IFN-γ and IL-17 production but not IL-13 production. (A) Proliferation of human CD4+ T cells labeled with CFSE and activated with anti-CD3 and anti-CD28 with plate-bound blocking anti-CD226 or isotype-control Ab. On day 4, CFSE dilution was analyzed by flow cytometry after gating on viable cells. (B) Flow cytometry of the surface expression of CD25 and CD69 as markers of activation of human CD4+ T cells activated in the presence of neutralizing anti-CD226 mAb or isotype-control Ab assessed on day 4 after activation. (C) Gene expression of cytokines in memory T cells stimulated under Th1, Th2, or Th17 conditions for 4 d, in the presence of isotype-control Ab or neutralizing anti-CD226 mAb at 5 and 10 μg/ml. (D) Measurement of IFN-γ, IL-13, and IL-17 in the supernatants derived from the same cultures as in (C) quantified by ELISA. Data are mean ± SEM and are representative of three independent experiments, with two healthy donors/experiment. *p < 0.05.

To further study the inhibitory effect of CD226 blockade on each Th subset, we investigated the effect of CD226 mAb on proliferation and cytokine production. Because naive T cells do not express CD226, we isolated human CD4+CD62L+CD25medCD45RO+ memory T cells to enrich for CD226 expression. These cells were incubated with plate-bound anti-CD226 mAb or isotype-control mAb and stimulated in Th1, Th2, or Th17 conditions for 4 d. Neutralizing anti-CD226 mAb inhibited proliferation under each of the Th conditions to a similar extent (Supplemental Fig. 2). Consistent with our CD226-knockdown results, CD226 blockade efficiently inhibited gene expression of IFN-γ and IL-17 (Fig. 7C). Similarly, IFN-γ and IL-17 secretion was significantly impaired at the protein level when T cells were cultured in the presence of the neutralizing anti-CD226 (Fig. 7D). Interestingly, treatment with blocking anti-CD226 did not modify IL-13 expression at the mRNA and protein levels (Fig. 7C, 7D). These data indicate that Th2 cells were less affected by CD226 blockade, possibly as a result of the lower expression of CD226 under Th2 conditions (Figs. 1, 2). Overall, blockade of CD226 signaling inhibits T cell proliferation and production of proinflammatory cytokines, whereas Th2 responses remain functional.

Finally, we investigated whether treatment with neutralizing anti-CD226 mAb decreases T cell activation on hyperactivated CD4+ T cells from untreated patients with relapsing remitting MS and T1D. In line with our previous results, T cells cultured in the presence of blocking anti-CD226 showed less expression of activation marker CD25 and CD226 (Supplemental Fig. 3). As a consequence, CD226 blockade abrogated T cell proliferation compared with cells incubated with an isotype control (Supplemental Fig. 3). Therefore, the inhibitory effects of blocking CD226 signaling are fully functional in T cells from patients with MS and T1D, suggesting that manipulation of CD226/CD155 signaling may represent an option for the treatment of human autoimmune diseases.

Discussion

Genome-wide association studies in patients with autoimmune diseases identified allelic variants in a number of T cell costimulatory molecular pathways as genetic risk factors for disease pathogenesis. Genetic variation in the CD226 gene region is associated with several autoimmune diseases, including T1D and MS (6, 11, 12). Although CD226 was shown to promote the development of Th1 responses (10), it is unknown whether CD226 influences Th2 or Th17 functions. Studies in rodents showed that CD226 is specifically upregulated in Th1 cells and is downregulated during Th2 differentiation (19). In humans, we recently showed that knockdown of CD226 in primary CD4+ T cells results in a decrease in T-bet expression and, consequently, an impaired production of IFN-γ (9). In this study, we report that expression of CD226 and its ligand CD155 are decreased under Th2 conditions. Additionally, higher CD226 expression correlates with lower GATA3 expression in ex vivo human T cells. Furthermore, we found that ex vivo IL-4–producing cells showed significantly lower expression of CD226 compared with IFN-γ–producing cells. In accordance with these observations, CD226 knockdown results in decreased T-bet expression and increases GATA3 expression, skewing these cells toward Th2 function and, consequently, the secretion of higher levels of IL-13 and IL-4. To address the mechanism by which CD226 differentially regulates Th1/Th2 responses, we studied the STAT-signaling pathways, demonstrating that CD226 knockdown impairs STAT-4 and STAT-1 signaling but increases STAT-6 phosphorylation. Our results demonstrate that CD226 plays a critical role in maintaining a Th1 phenotype while repressing Th2-associated genes.

We also investigated the role of CD226/CD155 interaction under Th17 conditions. Unlike IL-13– or IFN-γ–producing cells, Th17 cells express both receptors, suggesting that this costimulatory pathway may be engaged in the absence of APCs. Ex vivo T cells expressing higher levels of CD226 showed increased expression of Th17-associated genes, such as RORc and IL-17. Knockdown of CD226 under Th17 conditions did not result in a significant decrease in IL-17 secretion, indicating that CD226 costimulation is dispensable for Th17 differentiation. However, CD226 blockade with neutralizing Abs efficiently inhibited IL-17 secretion, suggesting that CD226 costimulatory signaling positively contributes to IL-17 production. Taking into account that CD226 activation is a two-step process that requires phosphorylation by TCR, followed by phosphorylation by Fyn, it is interesting that Fyn−/− mice show an increase in Th2 cytokines (20) and impaired Th17 responses (21), which are consistent with our findings in human CD226-depleted T cells.

CD226 blockade had inhibitory effects on T cell activation, proliferation, and secretion of proinflammatory cytokines, such as IFN-γ and IL-17. In contrast, IL-13 production was not affected by treatment with anti-CD226 mAb. This result was unexpected, considering that silencing CD226 expression led to increases in Th2 cytokines. This discrepancy may be due to the fact that cells in the neutralization experiments were incubated under Th2 conditions and, consequently, expressed low levels of CD226. Another difference between CD226 knockdown and CD226 blockade is that the blocking anti-CD226 mAb significantly decreased T cell activation and proliferation, whereas we did not observe impaired proliferation in shCD226 cells. Taken together, our data demonstrate that CD226 provides a positive costimulatory signal for T cell proliferation and enhances proinflammatory cytokine production, thereby shifting the balance between Th1/Th17 and Th2 cells toward a proinflammatory response. Therefore, CD226 blockade may represent an alternative approach for treatment of Th1- and Th17-driven autoimmune diseases.

An increased Th1/Th2 ratio has been identified as one important underlying mechanism in a variety of human autoimmune disorders, including MS, psoriasis, and T1D. Successful therapeutic approaches, such as glatiramer acetate (Copaxone) approved for MS treatment, have been designed to shift this balance toward Th2 responses (22). In this study, we report that CD226 can control the proinflammatory/anti-inflammatory balance. Thus, therapeutic strategies directed against CD226 would allow inhibition of T cell proliferation and proinflammatory cytokine production while sparing Th2 responses. In mouse studies, treatment with anti-CD226 significantly delayed disease onset and reduced the severity of the initial phase of EAE (19). This therapeutic approach also was examined for the therapy of graft-versus-host disease, demonstrating that blockade of CD226 in vivo efficiently suppressed its development in mice (23).

Given that CD155 and CD112 are overexpressed in certain human tumors, including colorectal carcinomas (24), gastric cancer (25), and neuroblastomas (26), CD226 and TIGIT mechanisms of T cell regulation may also have implications in tumor immunology. CD226 and TIGIT have been implicated as major regulators for NK cell cytotoxicity (17, 27). However, the roles for CD226 and TIGIT on CD4+ T cells in tumors remain unclear. Of note, human CD4+ T cells may express both receptors CD226 and TIGIT, but their proliferation is strongly inhibited in the presence of CD155, confirming that TIGIT-negative signaling outcompetes CD226 costimulation (Supplemental Fig. 2C). Although activation of costimulatory pathways against tumor cells might represent a viable therapeutic option, the most effective immunotherapies are based on neutralizing coinhibitory-signaling pathways, such as anti-CTLA4 (28) and anti-PD-1 (29). In this regard, therapeutic interventions to neutralize the TIGIT pathway would simultaneously block an intrinsic negative signal on effector T cells and promote CD226 signaling (i.e., a positive costimulatory signal for T cell proliferation and proinflammatory cytokine [IFN-γ, IL-17, IL-9] production), leading to activation of immune effector T cells in the tumor microenvironment.

In summary, we demonstrate that CD226 is a negative regulator of Th2 cells. Knockdown of CD226 induces GATA3 by increasing STAT-6 phosphorylation. Accordingly, blockade of CD226/CD155 interaction with neutralizing Abs prevents T cell activation, leading to a significant decrease in T cell proliferation and proinflammatory cytokine production. Unlike the costimulatory molecule CD28, naive T cells do not express CD226 and would not be affected by treatments targeting CD226 signaling. Therefore, CD226 targeting would exclusively target proinflammatory Th1 and Th17 cells. Furthermore, treatment with neutralizing anti-CD226 mAb efficiently inhibits activation and proliferation of T cells from patients with autoimmune diseases. Overall, targeting this costimulatory molecule may provide a therapeutic approach that specifically modulates the proinflammatory (Th1/Th17)/anti-inflammatory (Th2) balance in a wide range of inflammatory diseases.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank the blood donors and patients with MS and T1D for their participation.

Footnotes

  • This work was supported by a National Multiple Sclerosis Society Collaborative Research Center Award (CA1061-A-18); National Institutes of Health Grants P01 AI045757, U19AI046130, U19 AI070352, and P01 AI039671; a Jacob Javits Merit award (NS2427) from the National Institute of Neurological Disorders and Stroke; and the Nancy Taylor Foundation for Chronic Diseases Inc. (to D.A.H.). E.L. was supported by Ministerio de Educación y Ciencia-Becas MEC/Fulbright y Cátedras Príncipe de Asturias-Plan Nacional de I-D+I 2008-2011 and is the recipient of a Postdoctoral Scholarship in the Beatriu de Pinós Programme (2011–2013) (Agència de Gestió d'Ajuts Universitaris i de Recerca-Government of Catalonia, Spain).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    DC
    dendritic cell
    MFI
    mean fluorescence intensity
    MS
    multiple sclerosis
    shRNA
    short hairpin RNA
    T1D
    type 1 diabetes
    TIGIT
    T cell Ig and ITIM domain.

  • Received April 9, 2013.
  • Accepted August 2, 2013.

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