The CD47 Ag, also named integrin-associated protein, was recently reported to regulate the production of IL-12 by human monocytes and dendritic cells. The present study shows that CD47 ligation by CD47 mAb in primary cultures of cord blood mononuclear cells inhibits IL-12-driven Th1 cell development, as revealed by the cytokine secretion profile at restimulation and IFN-γ production at the single-cell level. F(ab′)2 fragments of CD47 mAb or the synthetic peptide 4N1K, corresponding to the CD47 binding site of thrombospondin, display the same activity. CD47 engagement does not change the phenotype of IL-12-primed cells from Th1 to Th2 or affect IL-4-induced Th2 cell development. Moreover, CD47 mAb inhibits IL-12- but not IL-4-induced IL-2 production as well as IFN-γ in primary cultures, which was correlated with a decrease of the IL-12Rβ2 chain expression. Inclusion of exogenous IL-2 at priming corrects IL-12R expression as well as the inhibition of Th1 cell development. The data thus underline the role of IL-2 in Th1 cell development and further suggest that targeting IL-2 and IL-12 simultaneously may have some therapeutic advantage in Th1 autoimmune diseases.

Upon activation, naive CD4+ T cells can differentiate into distinct Th subsets differing by their lymphokine secretion pattern (1). Th1 cells produce high levels of IFN-γ, lymphotoxin-α (LT-α),3 and TNF-α but no or little IL-4 and IL-5; Th2 cells produce high levels of IL-4, IL-5, IL-9, IL-10, and IL-13 but no or little IFN-γ and LT-α. These subsets of T cells have distinct regulatory and effector functions, and the inappropriate selection of Th1 or Th2 phenotype results in ineffective or disease-promoting immune responses (2). As recently reviewed (3, 4, 5), several factors are implicated in the regulation of naive T cell differentiation into Th1 or Th2 effectors. These include 1) the intensity and the nature of TCR-mediated activation signal, 2) the strength and the nature of costimulatory signals, 3) the cytokine and hormonal milieu in which T cells are primed, and 4) the genetic background of the naive T cells. Among all of these factors, cytokines appear to exert the most important role, with IL-4 and IL-12 promoting Th2 and Th1 responses, respectively. The other factors may act by influencing the production of these two cytokines at priming.

IL-12 promotes Th1 cell development by signaling maturing T cells through a high affinity receptor consisting of two chains, IL-12Rβ1 and IL-12Rβ2, that are differentially regulated (review in Ref. 6). IL-12Rβ1 is constitutively expressed on several types of cells, including resting T and B cells, which are not responsive to IL-12 (7). The coexpression of these two chains is required for IL-12 responsiveness and Th1 cell development. Expression of IL-12Rβ2 on naive T cell activation is enhanced by Th1-inducing factors (IFN-γ, IFN-α, and IL-12 itself) and down-regulated by Th2-promoting factors (IL-4, PGE2, and glucocorticosteroids) (8, 9). Thus, IL-12 responsiveness increases during Th1 cell differentiation, whereas it disappears during Th2 cell development (10, 11, 12). The latter phenomenon is associated with, but perhaps not entirely dependent on, IL-12Rβ2 down-regulation. Indeed, forced expression of IL-12Rβ2 during Th cell differentiation does not prevent the development of Th2 cells or confer IL-12 responsiveness (13, 14).

CD47 Ag, also known as integrin-associated protein, is a widely expressed multispan transmembrane protein, which is physically and functionally associated with αvβ3 integrin, the vitronectin receptor (15, 16). Indeed, CD47 cell lines expressing αvβ3 do not bind vitronectin-coated beads, and CD47-deficient mice rapidly die of Escherichia coli peritonitis, a phenomenon directly associated with a reduction in leukocyte activation in response to β3, but not β2, integrin ligation (17). CD47 has also been implicated in leukocyte transendothelial migration (18, 19). Its natural ligand, thrombospondin (TSP), is a homotrimeric extracellular matrix protein that is produced not only by platelets but also by monocytes and alveolar macrophages. TSP is transiently expressed at high concentration in damaged and inflamed tissue (20). A potential role for TSP and CD47 in immune regulation was recently suggested by the finding that they regulate the in vitro production of IL-12 (21, 22). Engagement of CD47 by mAb, TSP, or 4N1K (a peptide of the C-terminal domain of TSP selectively binding CD47) inhibited IL-12 release by human monocytes. The suppression was selective for IL-12 and occurred after T cell-dependent or -independent stimulation of monocytes (21). More recently, ligation of CD47 on human monocyte-derived dendritic cells was shown to inhibit their activation, cytokine production, and maturation (22). Here we show that in addition to blocking IL-12 production, CD47 ligation also inhibits the responsiveness to exogenous IL-12 and the maturation of naive neonatal human T cells into Th1 effectors.

Anti-CD3 mAb (UCHT-1) was kindly provided by Dr. P. Beverley (University College Middlesex School of Medicine, London, U.K.). Neutralizing mouse anti-human IL-4 mAb (clone 8F12), rhIL-2, neutralizing anti-IL-12 Ab, and rhIL-12 were kindly provided by C. Heusser (Novartis, Basel, Switzerland), D. Bron (Bordet Institute, Brussels, Belgium), and F. K. Kahn and M. Gately (Hoffmann-La Roche, Nutley, NJ), respectively. rhIL-4 was received from Immunex (Seattle, WA). The CD32 and B7.1 double-transfected mouse L fibroblasts have been described (23). The 4N1K peptide (KRFYVVMWKK) corresponding to the C-terminal domain of TSP and the mutant 4NGG peptide (KRFYGGMWKK) were obtained from Genosys (The Woodlands, TX). Three anti-CD47 mAbs were used in soluble form: clones B6H12 (mouse IgG1; Biosource, Montreal, Quebec, Canada), 10G2 (described in Ref. 21) and 2D3, (received from E. Brown, Washington University, St. Louis, MO). Mouse IgG1 control mAb anti-Rye and anti-CD2 (OKT11) were produced in our laboratory and purchased from American Type Culture Collection (Manassas, VA), respectively. F(ab′)2 anti-CD47 mAb fragments were prepared using a commercial kit from Pierce (Brockville, Ontario, Canada).

Umbilical cord blood mononuclear cells (CBMCs) were isolated by density gradient centrifugation of heparinized umbilical cord blood from normal healthy volunteers using Lymphoprep (Nycomed, Oslo, Norway). CBMCs (1 × 106 cells/ml) were cultured in triplicate in 24-well culture plates in 1 ml of RPMI 1640 (BioWhittaker, Walkersville, MD) containing 5% FCS, 2 mM l-glutamine, 50 IU penicillin and 100 μg streptomycin, and PHA (2 μg). For the induction of Th1 and Th2 polarization, cells were cultured in the presence of rIL-12 (60 pM) and anti-IL-4 mAb (1 μg/ml) or a combination of rIL-4 (20 ng/ml) and anti-IL-12 Ab (5 μg/ml), respectively. After 3 days, cells were washed and cultured at a starting concentration of 0.5 × 106 cells/ml in culture medium supplemented with 50 U/ml rhIL-2, in 24-well plates. After 9–12 days of IL-2 culture, cellular expansion in control cultures was ∼35-fold; cells were then washed and 1 × 106 viable T cells/ml were restimulated for cytokine production with plastic-bound anti-CD3 mAb (10 μg/ml) alone, plastic-bound anti-CD3 mAb (1 μg/ml) together with soluble anti-CD28 mAb (clone CD28.2; PharMingen, Ontario, Canada; 2 μg/ml), or anti-CD3 (200 ng/ml) immobilized on irradiated CD32/B7.1 L cells (2.5 × 105 cells/ml).

Cells were stained with FITC- or PE-conjugated mAbs to CD3, CD25, CD45RA, CD45RO, or isotype-matched controls (Ancell, London, Ontario, Canada) and analyzed using a FACScan (Becton Dickinson, Mountain View, CA). To detect intracytoplasmic IFN-γ, T cells were stimulated with anti-CD3 mAb immobilized on irradiated CD32/B7.1 L cells for 6 h in the presence of Monensin (3 mM final concentration; Hornby, Ontario, Canada). Cells were stained with the ICScreen Intracellular Staining Kit from Medicorp (Montreal, Quebec, Canada). IL-12Rβ1 and IL-12Rβ2 surface expression was assessed using a three-step procedure. Briefly, cells were first incubated with anti-IL-12Rβ1 mAb (2B10), anti-IL-12Rβ2 mAb (2B6), or class-matched negative control mAb at 2.5 μg/ml in the presence of normal human IgG (150 μg/ml) for 1 h at 4°C. Cells were then incubated with biotinylated goat anti-rat IgG (Bio/Can Scientific, Mississauga, Ontario, Canada) for 1 h at 4°C followed by PE-labeled streptavidin (Ancell) for 30 min at 4°C. Fas (CD95) and FasL (CD95L) surface expression was assessed using a two-step procedure. Cells were first incubated with biotinylated anti-Fas (M3), biotinylated anti-FasL (NOK-1), or biotinylated class-matched negative control at 5 to 10 μg/ml in the presence of normal human IgG (150 μg/ml) for 1 h at 4°C. Cells were then incubated with PE-labeled streptavidin for 30 min at 4°C. Mean fluorescence intensity (MFI) was calculated as follows: MFI of sample – MFI of negative control.

Phosphatidylserine exposure was done by flow cytometry analysis using a FACScan. Cells were double-stained with 2 μg/ml of FITC-labeled annexin-V (Biodesign International, Kennebunkport, ME) and propidium iodide (PI; Sigma).

IL-2, IL-4, IL-5, IL-10, LT-α, TNF-α, and IFN-γ were measured by two-site sandwich ELISA or RIA exactly as described (24, 25). At priming, secretion of IL-2 and IFN-γ was assessed at 24 h and 72 h, respectively. Unless otherwise indicated, cytokines were measured after 24 h (IL-2 and IL-4) or 48 h (IFN-γ, TNF-α, LT-α, IL-5, and IL-10) of restimulation.

CBMCs were collected after 6 h of stimulation in Th1-polarizing conditions in the presence of anti-CD47 mAb or isotype-matched control mAb. Total RNA was prepared with RNeasy Total RNA kit (Qiagen, Chatsworth, CA). Exactly as described (24), 1 μg of RNA from each sample was reversed transcribed by GeneAmp RNA PCR kit from Perkin-Elmer/Cetus (Emeryville, CA) with oligo(dT)16 as the first-strand cDNA primer. Reverse-transcription product (1/20 vol) was mixed with known quantities of serially diluted competitive internal standards (PCR MIMICs; Clontech Laboratories, Palo Alto, CA) and was subjected to quantitative RT-PCR according to the manufacturer’s protocol. Target-specific primer pairs of IL-2 and G3PDH were also purchased from Clontech. After 36 cycles of amplification, the PCR products were resolved on a 1.8% agarose gel containing ethidium bromide. The intensities of competitor-generated bands and the cDNA sample-generated bands determined the quantity of target gene product. The amount of target cDNA was ascertained by determining the amount of competitor required to produce equal molar quantities of target and competitor products. The photographs of agarose gels were further analyzed by computer imaging (NIH Image, version 1.61; National Institutes of Health, Bethesda, MD), and the ratio of the mean histogram of target-generated to competitor-generated bands was calculated. The specificity of the amplified bands was validated by their predicted size.

Student’s paired t test was used to determine statistical significance of the data. Values of p < 0.05 were chosen for rejection of the null hypothesis.

Several positive or negative regulators of IL-12 production reportedly control IL-12 responsiveness (26, 27, 28, 29, 30). Our previous report that CD47 down-regulates IL-12 release by APCs (21, 22) prompted us to evaluate the effect of CD47 ligation on the response of CBMCs to IL-12. CBMCs were activated with PHA in Th1 (IL-12 + anti-IL-4 mAb) or Th2 (IL-4 + anti-IL-12 Ab) conditions, in the absence or presence of CD47 mAb. Inclusion of CD47 mAb markedly reduced both IFN-γ and IL-2 production in Th1 primary cultures without affecting IL-2 production in Th2 primary cultures (Fig. 1,A). Control cultures were supplemented with either normal mouse IgG1 or an irrelevant cell-binding mAb (anti-CD2 mAb). After 3 days of priming, cells were washed, expanded in IL-2-supplemented medium for 9 to 12 days, washed again, and examined for surface molecule expression and cytokine production. As depicted in Fig. 1 B, regardless of the priming conditions, virtually all of the cells recovered at the end of IL-2 expansion were T lymphocytes (CD3+) displaying the phenotype of newly generated effector cells in that they were CD45RA+/RO+ and CD25low. The proportion of CD4/CD8 T cells (1.6 ± 0.3, n = 4) in this homogenous T cell population was unaffected by treatment with CD47 mAb. The cellular viability was uniformly higher than 70% as determined by trypan blue dye exclusion.

FIGURE 1.

CD47 mAb inhibits Th1 development with no immune deviation toward Th2. CBMCs were primed at 1 × 106 cells/ml for 3 days in Th1 (IL-12 + anti-IL-4 mAb) or in Th2 (IL-4 + anti-IL-12 Ab) polarizing conditions in the absence (–) or in the presence of anti-CD47 mAb (10 μg/ml) or control mAb (10 μg/ml). A, IFN-γ was measured at the end of primary cultures. IL-2 was measured at 24 h. Shown is the mean ± SEM of five experiments. ∗∗∗, p < 0.005; ∗∗∗∗∗, p < 0.0001. B, Cells were then washed; counted with trypan blue; expanded at a starting concentration of 0.5 × 106 cells/ml in IL-2 (50 U/ml) for 9 to 12 days; and assessed for CD3, CD45RA, CD45RO, and CD25 surface expression by a direct staining procedure. Background staining is in dotted line. Shown is one representative experiment of three. C, After the expansion phase in IL-2, cells were washed, counted with trypan blue, adjusted to 1 × 106 viable cells/ml, and restimulated with anti-CD3 mAb immobilized on 2.5 × 105 irradiated CD32-B7.1-transfected L cells. Cytokines were measured at 24 h (IL-4) or 48 h (IFN-γ) of secondary cultures. Shown is the mean ± SEM of five experiments. ∗∗∗∗∗, p < 0.0001.

FIGURE 1.

CD47 mAb inhibits Th1 development with no immune deviation toward Th2. CBMCs were primed at 1 × 106 cells/ml for 3 days in Th1 (IL-12 + anti-IL-4 mAb) or in Th2 (IL-4 + anti-IL-12 Ab) polarizing conditions in the absence (–) or in the presence of anti-CD47 mAb (10 μg/ml) or control mAb (10 μg/ml). A, IFN-γ was measured at the end of primary cultures. IL-2 was measured at 24 h. Shown is the mean ± SEM of five experiments. ∗∗∗, p < 0.005; ∗∗∗∗∗, p < 0.0001. B, Cells were then washed; counted with trypan blue; expanded at a starting concentration of 0.5 × 106 cells/ml in IL-2 (50 U/ml) for 9 to 12 days; and assessed for CD3, CD45RA, CD45RO, and CD25 surface expression by a direct staining procedure. Background staining is in dotted line. Shown is one representative experiment of three. C, After the expansion phase in IL-2, cells were washed, counted with trypan blue, adjusted to 1 × 106 viable cells/ml, and restimulated with anti-CD3 mAb immobilized on 2.5 × 105 irradiated CD32-B7.1-transfected L cells. Cytokines were measured at 24 h (IL-4) or 48 h (IFN-γ) of secondary cultures. Shown is the mean ± SEM of five experiments. ∗∗∗∗∗, p < 0.0001.

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To examine cytokine production, 1 × 106 viable T cells were restimulated for 24 or 48 h with anti-CD3 mAb immobilized on CD32/B7.1 L transfectants. Cells that had been primed in Th1 conditions in the presence of CD47 mAb secreted much less IFN-γ and no IL-4 at restimulation (Fig. 1 C). Of note, similar levels of inhibition of IFN-γ production were observed after restimulation with plastic-coated anti-CD3 mAb (not shown). Most interestingly, CD47 ligation in Th2 priming conditions had no effect on Th2 cell development.

The decrease of IFN-γ production at priming and restimulation was CD47 specific and not Fc mediated. As shown in Fig. 2, a CD47-Fc fusion protein containing the extracellular domain of CD47 completely abrogated the CD47 mAb-mediated inhibition of IFN-γ production. Furthermore, F(ab′)2 fragments of CD47 mAb as well as the synthetic peptide 4N1K, corresponding to the CD47 binding site of TSP, decreased IFN-γ production almost as efficiently as intact mAb. Control mutant peptide of TSP had no inhibitory effect (not shown).

FIGURE 2.

CD47 mAb-mediated inhibition of Th1 development is CD47 specific and not Fc mediated. CBMCs were primed at 1 × 106 cells/ml for 3 days in Th1-polarizing conditions (IL-12 + anti-IL-4 mAb) in the absence (–) or in the presence of anti-CD47 mAb (10 μg/ml); CD47-Fc (10 μg/ml); F(ab′)2 anti-CD47 mAb (20 μg/ml); peptide 4N1K (50 μg/ml), which corresponds to the CD47 binding site of TSP; or control mAb (10 μg/ml). A, IFN-γ was measured at the end of primary cultures. Upper panel, One representative experiment of three. Lower panel, Mean ± SEM of five experiments. ***p < 0.005; ∗∗∗∗, p < 0.001. B, After the expansion phase in IL-2, cells were washed, counted with trypan blue, adjusted to 1 × 106 viable cells/ml, and restimulated with plastic-coated anti-CD3 mAb for 48 h. IFN-γ was measured at the end of secondary cultures. Top, One representative experiment of three; bottom, Mean ± SEM of five experiments. ∗, p < 0.05; ∗∗, p < 0.01.

FIGURE 2.

CD47 mAb-mediated inhibition of Th1 development is CD47 specific and not Fc mediated. CBMCs were primed at 1 × 106 cells/ml for 3 days in Th1-polarizing conditions (IL-12 + anti-IL-4 mAb) in the absence (–) or in the presence of anti-CD47 mAb (10 μg/ml); CD47-Fc (10 μg/ml); F(ab′)2 anti-CD47 mAb (20 μg/ml); peptide 4N1K (50 μg/ml), which corresponds to the CD47 binding site of TSP; or control mAb (10 μg/ml). A, IFN-γ was measured at the end of primary cultures. Upper panel, One representative experiment of three. Lower panel, Mean ± SEM of five experiments. ***p < 0.005; ∗∗∗∗, p < 0.001. B, After the expansion phase in IL-2, cells were washed, counted with trypan blue, adjusted to 1 × 106 viable cells/ml, and restimulated with plastic-coated anti-CD3 mAb for 48 h. IFN-γ was measured at the end of secondary cultures. Top, One representative experiment of three; bottom, Mean ± SEM of five experiments. ∗, p < 0.05; ∗∗, p < 0.01.

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We next evaluated the complete cytokine profile of effector cells generated after priming in the presence of CD47 mAb. As seen in Table I, CD47 mAb added at priming in Th1 conditions suppressed not only IFN-γ but also TNF-α and LT-α production at restimulation. Treatment with CD47 mAb did not affect the production of IL-4, IL-5, or IL-10, which remained undetectable in these Th1-promoting culture conditions. The suppressive effect on Th1 development contrasted with the lack of an effect on Th2 cell development after priming in the presence of exogenous IL-4. Similar results were obtained by examining cytokine production by CD4+ T or CD8+ T cells purified at the end of the IL-2 expansion culture (not shown).

Table I.

Cytokine profiles of CD47-primed cells in Th1 or Th2 conditionsa

IFN-γTNF-αLT-αIL-4IL-5IL-10
IL-12 + anti-IL-4       
Control mAb 9.8 ± 0.5 2.3 ± 0.9 5.6 ± 2.9 ND ND ND 
Anti-CD47 1.2 ± 0.4 0.0 ± 0.0 0.3 ± 0.2 ND ND ND 
IL-4+ anti-IL-12       
Control mAb ND ND ND 2.4 ± 0.3 18.0 ± 1.4 3.0 ± 0.8 
Anti-CD47 ND ND ND 2.3 ± 0.4 17.1 ± 2.8 3.4 ± 0.6 
IFN-γTNF-αLT-αIL-4IL-5IL-10
IL-12 + anti-IL-4       
Control mAb 9.8 ± 0.5 2.3 ± 0.9 5.6 ± 2.9 ND ND ND 
Anti-CD47 1.2 ± 0.4 0.0 ± 0.0 0.3 ± 0.2 ND ND ND 
IL-4+ anti-IL-12       
Control mAb ND ND ND 2.4 ± 0.3 18.0 ± 1.4 3.0 ± 0.8 
Anti-CD47 ND ND ND 2.3 ± 0.4 17.1 ± 2.8 3.4 ± 0.6 
a

CBMCs were primed at 1 × 106 cells/ml for 3 days in Th1 (IL-12 + anti-IL-4 mAb) or in Th2 (IL-4 + anti-IL-12 Ab) polarizing conditions in the presence of anti-CD47 mAb (10 μg/ml) or control mAb (10 μg/ml). Cells were then washed, counted with trypan blue, and expanded in IL-2 for 9–12 days. Viable cells (1 × 106/ml) were restimulated with anti-CD3 mAb immobilized on 2.5 × 105 irradiated CD32-B7.1-transfected L cells. Cytokines were measured at 24 h (IL-4) or 48 h (IFN-γ, TNF-α, LT-α, IL-5, and IL-10). Shown is the mean ± SEM of five experiments. All values are in nanograms per ml except for IL-2 which is in units/ml. ND, Not detectable.

Collectively, the above data suggested that engagement of CD47 by mAb or its natural ligand TSP on CBMCs inhibited the generation of IL-12-induced Th1 but not IL-4-induced Th2 effectors.

We next analyzed the possible mechanisms whereby CD47-treated cells secreted decreased levels of Th1 cytokines. First, we showed that it did not result from an increase of activation-induced cell death as measured by FACs analysis of FITC-annexin-and PI-stained cells before and after anti-CD3 stimulation (Fig. 3,A). Note that the 2-fold increase in annexin+/PI+ CD47-treated cells was not considered to be significant, because it was not consistently observed in three independent experiments in the course of a daily kinetic study performed during 9 days of cellular expansion in IL-2 (not shown). Also, the expression of Fas and FasL (albeit a low level of expression) was not significantly affected by CD47 mAb treatment (Fig. 3,B). Second, we examined whether CD47 ligation impaired IL-12 responsiveness at priming. The data in Fig. 4,A revealed that inclusion of CD47 mAb completely prevented the enhanced production of IFN-γ stimulated by IL-12 + anti-IL-4 mAb. Most importantly, the inhibition of Th1 cell development was confirmed by showing reduced IFN-γ production at the single-cell level (Fig. 4,B). Third, we examined whether the CD47-mediated inhibition of IL-12 responsiveness was correlated with down-regulation of IL-12R expression at the end of the primary cultures. In agreement with previous reports (9, 10), the expression of IL-12Rβ2 was increased in the presence of IL-12, whereas the constitutive expression of IL-12Rβ1 was not enhanced (Fig. 4 C). As depicted in the same figure, CD47 mAb markedly suppressed IL-12Rβ2 expression on CBMCs activated with PHA as well as the enhancing effect of IL-12.

FIGURE 3.

CD47 ligation impairs Th1 development without inducing cell death. CBMCs were primed at 1 × 106 cells/ml for 3 days in Th1-polarizing conditions (IL-12 + anti-IL-4 mAb) with anti-CD47 mAb (10 μg/ml) or control mAb (10 μg/ml). Cells were then washed, counted with trypan blue, and expanded in IL-2 for 9 to 12 days before restimulation. A, Percentage of dead cells was assessed by annexin/PI double staining at the end of the expansion in IL-2 (before restimulation) and 24 h after restimulation of 1 × 106 viable cells/ml with coated anti-CD3 mAb. Shown is one representative experiment of three. B, Fas and FasL surface expression was assessed, as in A, before and after restimulation by a double-staining procedure as described in Materials and Methods. Background staining is in dotted line. Shown is one representative experiment of three.

FIGURE 3.

CD47 ligation impairs Th1 development without inducing cell death. CBMCs were primed at 1 × 106 cells/ml for 3 days in Th1-polarizing conditions (IL-12 + anti-IL-4 mAb) with anti-CD47 mAb (10 μg/ml) or control mAb (10 μg/ml). Cells were then washed, counted with trypan blue, and expanded in IL-2 for 9 to 12 days before restimulation. A, Percentage of dead cells was assessed by annexin/PI double staining at the end of the expansion in IL-2 (before restimulation) and 24 h after restimulation of 1 × 106 viable cells/ml with coated anti-CD3 mAb. Shown is one representative experiment of three. B, Fas and FasL surface expression was assessed, as in A, before and after restimulation by a double-staining procedure as described in Materials and Methods. Background staining is in dotted line. Shown is one representative experiment of three.

Close modal
FIGURE 4.

CD47 ligation inhibits the response of naive T cells to IL-12 and their development into IFN-γ-producing cells. CBMCs were primed for 3 days in nonpolarizing (Medium) and in Th1-polarizing (IL-12 + anti-IL-4 mAb) conditions with anti-CD47 mAb (10 μg/ml) or control mAb (10 μg/ml). A, After the expansion phase in IL-2, cells were restimulated at 1 × 106 viable cells/ml with anti-CD3 mAb immobilized on 2.5 × 105 irradiated CD32-B7.1-transfected L cells for 48 h. IFN-γ was measured at the end of secondary cultures. Shown is the mean ± SEM of five experiments. ∗∗∗∗∗, p < 0.0001. B, After the expansion phase in IL-2, cells in Th1-polarizing conditions were restimulated at 1 × 106 viable cells/ml with anti-CD3 mAb immobilized on 2.5 × 105 CD32-B7.1-transfected L cells. Intracytoplasmic staining of IFN-γ was done 6 h after restimulation. Background staining is in dotted line. MFI was calculated as described in Materials and Methods. Shown is one representative experiment of three. C, IL-12Rβ1 and IL-12Rβ2 surface expression was assessed in nonpolarizing (Medium) and in Th1-polarizing conditions at the end of primary cultures by a triple-staining procedure as described in Materials and Methods. Background staining is in dotted line. Shown is one representative experiment of three.

FIGURE 4.

CD47 ligation inhibits the response of naive T cells to IL-12 and their development into IFN-γ-producing cells. CBMCs were primed for 3 days in nonpolarizing (Medium) and in Th1-polarizing (IL-12 + anti-IL-4 mAb) conditions with anti-CD47 mAb (10 μg/ml) or control mAb (10 μg/ml). A, After the expansion phase in IL-2, cells were restimulated at 1 × 106 viable cells/ml with anti-CD3 mAb immobilized on 2.5 × 105 irradiated CD32-B7.1-transfected L cells for 48 h. IFN-γ was measured at the end of secondary cultures. Shown is the mean ± SEM of five experiments. ∗∗∗∗∗, p < 0.0001. B, After the expansion phase in IL-2, cells in Th1-polarizing conditions were restimulated at 1 × 106 viable cells/ml with anti-CD3 mAb immobilized on 2.5 × 105 CD32-B7.1-transfected L cells. Intracytoplasmic staining of IFN-γ was done 6 h after restimulation. Background staining is in dotted line. MFI was calculated as described in Materials and Methods. Shown is one representative experiment of three. C, IL-12Rβ1 and IL-12Rβ2 surface expression was assessed in nonpolarizing (Medium) and in Th1-polarizing conditions at the end of primary cultures by a triple-staining procedure as described in Materials and Methods. Background staining is in dotted line. Shown is one representative experiment of three.

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These data indicated that CD47 ligation impaired IL-12 responsiveness of naive T cells as well as their development into Th1 effectors.

IL-2 reportedly up-regulated IL-12R expression on activated T cells (6) and, as indicated in Fig. 1,A, CD47 mAb suppressed IL-12- but not IL-4-induced IL-2 production in primary Th1 cultures of CBMCs. The reduced IL-2 secretion was confirmed at the mRNA level by RT-PCR (Fig. 5,A). We therefore examined whether addition of exogenous IL-2 at priming counteracted CD47 mAb-mediated inhibition of Th1 development. As depicted in Fig. 5, exogenous IL-2 strongly up-regulated IL-12Rβ2 expression and completely prevented CD47 mAb-mediated down-regulation of IL-12Rβ2 expression at the end of primary cultures (Fig. 5,B) and IFN-γ production at priming and restimulation. This effect was dose dependent (Fig. 5 C).

FIGURE 5.

Exogenous IL-2, added at priming, restores IL-12Rβ2 expression and Th1 development. CBMCs were primed for 3 days in Th1-polarizing conditions (IL-12 + anti-IL-4 mAb) with anti-CD47 mAb (10 μg/ml) or control mAb (10 μg/ml) in the absence or in the presence of exogenous IL-2 (20 U/ml). A, IL-2 mRNA and G3PDH mRNA were extracted and measured by competitive quantitative RT-PCR after 6 h of primary stimulation as described in Materials and Methods. Similar results were obtained in one additional experiment. B, IL-12Rβ2 surface expression was assessed with or without exogenous IL-2 at the end of primary cultures. Background staining is in dotted line. Shown is one representative experiment of three. C, Left, IFN-γ production at the end of primary cultures of cells stimulated with or without exogenous IL-2. Shown is one representative experiment of three. Cells were then washed, counted with trypan blue, and expanded in IL-2 for 9 to 12 days. Middle, IFN-γ production of cells restimulated at 1 × 106 viable cells/ml with coated anti-CD3 mAb for 48 h. Shown is one representative experiment of three. Right, IFN-γ production of cells primed without or with increasing concentrations of exogenous IL-2 and restimulated at 1 × 106 viable cells/ml with coated anti-CD3 mAb and soluble anti-CD28 mAb for 48 h. Shown is one representative experiment of three.

FIGURE 5.

Exogenous IL-2, added at priming, restores IL-12Rβ2 expression and Th1 development. CBMCs were primed for 3 days in Th1-polarizing conditions (IL-12 + anti-IL-4 mAb) with anti-CD47 mAb (10 μg/ml) or control mAb (10 μg/ml) in the absence or in the presence of exogenous IL-2 (20 U/ml). A, IL-2 mRNA and G3PDH mRNA were extracted and measured by competitive quantitative RT-PCR after 6 h of primary stimulation as described in Materials and Methods. Similar results were obtained in one additional experiment. B, IL-12Rβ2 surface expression was assessed with or without exogenous IL-2 at the end of primary cultures. Background staining is in dotted line. Shown is one representative experiment of three. C, Left, IFN-γ production at the end of primary cultures of cells stimulated with or without exogenous IL-2. Shown is one representative experiment of three. Cells were then washed, counted with trypan blue, and expanded in IL-2 for 9 to 12 days. Middle, IFN-γ production of cells restimulated at 1 × 106 viable cells/ml with coated anti-CD3 mAb for 48 h. Shown is one representative experiment of three. Right, IFN-γ production of cells primed without or with increasing concentrations of exogenous IL-2 and restimulated at 1 × 106 viable cells/ml with coated anti-CD3 mAb and soluble anti-CD28 mAb for 48 h. Shown is one representative experiment of three.

Close modal

Taking these findings together, we suggest that CD47 ligation induced a decrease of IL-12-induced IL-2 production at priming, which led to reduced IL-12Rβ2 expression, IL-12 responsiveness of naive T cells, and impaired Th1 development.

It has been established that IL-12 plays an important role not only in protective innate and adaptive immunity but also in chronic inflammatory diseases associated with a strong Th1 response (31, 32, 33). We recently reported that CD47 has potential anti-inflammatory activity in that its ligation by soluble mAbs or TSP, its natural ligand, inhibits IL-12 production by human monocytes and dendritic cells and prevented the maturation of the latter into potent T cell immunostimulators (21, 22). In the present study, we show that the same CD47 ligands also block the response of naive T cells to exogenous IL-12 and prevent their differentiation into Th1 effectors. The integrin-associated protein CD47 recently emerged from the literature as a potential immunoregulatory molecule, in addition to being involved in integrin signaling and cell migration. CD47 mAbs were reported to up- or down-regulate T cell activation as well as T cell and B cell death, depending on their epitope specificity, their physical form (immobilized or soluble), and the experimental conditions (34, 35, 36, 37, 38, 39, 40). Our unpublished results indicated that two additional soluble CD47 mAbs (clones 2D3 and 10G2), directed against different epitopes, similarly impaired Th1 development of naive T cells.

Interestingly, most of the molecules inhibiting IL-12 production also suppress IL-12 responsiveness, including IL-4, IL-10, TGF-β, glucocorticosteroids, PGE2, cholera toxin, adenosine, the compound lisofylline, and 1,25(OH)2 vitamin D3 (8, 26, 27, 28, 29, 30, 41, 42, 43). Ligation of CD47, like 1,25(OH)2 vitamin D3 (44), inhibits Th1 development without inducing a deviation toward Th2. There was no increase of endogenous IL-4 production in that 1) the experiments were conducted in the presence of anti-IL-4 neutralizing mAb and 2) IL-4 remained undetectable. Inhibition of Th1 development is often but not necessarily associated with an induction of Th2 phenotype. For instance, coactivation of naive T cells by anti-CD4 mAb inhibited Th1 differentiation and induced Th2 development (45). Also, IL-12-deficient BALB/c mice, when primed with Ag in CFA, mounted a Th2 instead of a Th1 response. By contrast, IL-12-deficient C57BL/6 mice failed to develop Th1 and Th2 responses, a phenomenon not mediated by endogenous IL-4 or IL-10 (46). A role for endogenous IL-10 in CD47 mAb-mediated inhibition of Th1 response is not supported by the observations that IL-10 was undetectable in primary and secondary cultures and that an anti-IL-10 mAb did not affect CD47 mAb-mediated suppression (data not shown). Our previous reports indicated that the suppression of IL-12 production by CD47 ligation was IL-10 independent (21, 22). Finally, the inhibition of Th1 development by CD47 mAb is selective, given that it has no effect at all on the Th2 response induced by exogenous IL-4.

Most importantly, the inhibition of Th1 development by CD47 mAb is evidenced by the suppressed production of IFN-γ at the single-cell level and a markedly reduced secretion of Th1 cytokines (IFN-γ, LT-α, and TNF-α) at restimulation of primed T cells that have been expanded for 9 to 12 days in IL-2. In agreement with Rogers et al. (47), IL-2 promoted the development of effectors as well as their expansion. Note that almost complete inhibition of Th1 cytokine production was also observed in the absence of intermediate culture in IL-2 (M.-N. Avice, unpublished data).

The inhibition of IL-12 responsiveness is often associated with down-regulation of IL-12Rβ2 and/or STAT-4 phosphorylation (26, 27). As mentioned, factors regulating the expression of the IL-12Rβ2 subunit play a critical role in Th1 cell development (6). After TCR-mediated activation, primary T cells transiently express low levels of IL-12Rβ2 that are sufficient to confer T cell responsiveness to IL-12. The latter up-regulates IL-12Rβ2 by itself and induces IFN -γ production, which in its turn induces long-term expression of this receptor at high levels (9, 10). It was recently demonstrated that the initial up-regulation of IL-12Rβ2 after TCR stimulation is strictly dependent on IL-2 production by the activated T cells (48). Thus, in the absence of this early IL-2 production, activated T cells fail to respond to IL-12 and subsequently IFN-γ and cannot acquire a Th1 phenotype, even when primed in the presence of exogenous IL-12 or IFN- γ (48).

We here show that ligation of CD47 in primary cultures of mitogen- and IL-12- activated CBMCs not only inhibits the expression of IL-12Rβ2 but also the production of IL-2 and IFN-γ. We therefore propose that the selective inhibition of Th1 response results from CD47-mediated suppression of IL-2 production by activated naive T cells costimulated with IL-12 but not IL-4 (Fig. 1). Indeed, IL-2 synthesis is suppressed in primary cultures as determined at the protein and mRNA levels, and exogenous IL-2 completely reverses all the effects of CD47 mAb, i.e., IL-12Rβ2 expression, IFN-γ production, and Th1 cell differentiation. Note that anti-IL-2 and anti-IL-2R mAbs suppress IL-12-induced IFN-γ production to the same extent as anti-CD47 mAb (M-N. Avice, unpublished observations). These results are in complete agreement with recent findings in the mouse demonstrating that early IL-2 production is required for in vitro and in vivo induction of Th1 response (48) and provide an explanation to the earlier observations on the critical role of IL-2 in Th1 cell development (49). Our unpublished data further indicate that IL-2 could be replaced by IL-15 that was shown to synergize with IL-12 in inducing IFN-γ production by T cells (25, 50). As mentioned, IL-4 completely overrides the CD47 mAb-mediated inhibitory effect. This is not surprising, given that IL-4 and IL-15 utilize the β and γ chains of the IL-2R. However, we cannot rule out the possibility that IL-2 recruits additional regulatory mechanisms that circumvent the CD47-dependent pathway of inhibition.

Taken collectively, the present findings underline the immunoregulatory role of CD47 and its natural ligand TSP. Together with their ability to inhibit IL-12 production and dendritic cell maturation, our data support the notion that this pair of receptor-ligand molecules may function as natural anti-inflammatory and immunosuppressive agents. TSP is a multifunctional molecule with distinct binding sites for αvβ3, α4β1, and α5β1 integrins; immature TGF-β; CD36; and CD47 (20, 51, 52, 53). It reportedly enhances the phagocytosis of apoptotic cells, a process that not only fails to induce inflammation but may even suppress it (54, 55). In addition, TSP transforms latent TGF-β into a very potent anti-inflammatory cytokine, and TSP-deficient animals display diffuse inflammatory lesions that are corrected by the administration of TGF-β (56, 57). Note that signal-regulatory protein (SIRP), selectively expressed on neuronal and myeloid cells, was recently described to serve as a novel ligand for CD47 (58), and we are currently investigating its role in the regulation of IL-12 response.

Agents capable of blocking IL-12 production and/or responsiveness are therefore potential candidates for the treatment or prevention of Th1 autoimmune diseases (31, 32, 33, 44). For instance, administration of anti-IL-12 mAb but not anti-CD40L mAb (which only decreased IL-12 secretion but not IL-12 responsiveness) completely abrogated established colitis of 4,4,6-trinitrobenzine sulfonic acid-treated mice. Also, 1,25(OH)2 vitamin D3, a potent inhibitor of Th1 response, is an effective treatment of chronic-relapsing experimental allergic encephalomyelitis. Thus, if applicable in vivo, the present findings suggest that inhibiting IL-12-induced IL-2 production and IL-12 responsiveness by targeting CD47 represents a novel and unexplored pathway for the management and prevention of disease-promoting or undesired Th1 responses.

We thank Norma Del Bosco for secretarial assistance.

1

This work is supported by Medical Research Canada Grants MT13311 and MT14432.

3

Abbreviations used in this paper: LT-α, lymphotoxin-α, TSP, thrombospondin; CBMC, umbilical cord blood mononuclear cell; MFI, mean fluorescence intensity; PI, propidium iodide; rh, recombinant human.

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