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The Journal of Immunology, 2008, 180, 8073 -8082
Copyright © 2008 by The American Association of Immunologists, Inc.
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CD47 Expression on T Cell Is a Self-Control Negative Regulator of Type 1 Immune Response1

Salim Bouguermouh, Vu Quang Van, Julie Martel, Patrick Gautier, Manuel Rubio and Marika Sarfati2

Immunoregulation, Centre Hospitalier de l’Université de Montréal, Research Center, Hospital Notre-Dame, Montréal, Quebec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The cytokine milieu and dendritic cells (DCs) direct Th1 development. Yet, the control of Th1 polarization by T cell surface molecules remains ill-defined. We here report that CD47 expression on T cells serves as a self-control mechanism to negatively regulate type 1 cellular and humoral immune responses in vivo. Th2-prone BALB/c mice that lack CD47 (CD47–/–) displayed a Th1-biased Ab profile at steady state and after immunization with soluble Ag. CD47–/– mice mounted a T cell-mediated exacerbated and sustained contact hypersensitivity (CHS) response. After their adoptive transfer to naive CD47-deficient hosts 1 day before immunization with soluble Ag, CD47–/– as compared with CD47+/+CD4+ transgenic (Tg) T cells promoted the deviation of Ag-specific T cell responses toward Th1 that were characterized by a high IFN-{gamma}:IL-4 cytokine ratio. Although selective CD47 deficiency on DCs led to increased IL-12p70 production, CD47–/–Tg T cells produced more IFN-{gamma} and displayed higher T-bet expression than CD47+/+ Tg T cells in response to OVA-loaded CD47–/– DCs. CD47 as part of the host environment has no major contribution to the Th1 polarization responses. We thus identify the CD47 molecule as a T cell-negative regulator of type 1 responses that may limit unwanted collateral damage to maximize protection and minimize host injury.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In adaptive immunity, CD4+ T cells differentiate into Th1 and Th2 cells in response to antigenic stimulation (1, 2, 3, 4). Th1 cells produce mainly IL-2, IFN-{gamma}, TNF-{alpha}, and TNF-β, elicit delayed-type hypersensitivity responses, and participate in cellular immune responses against intracellular pathogens. Th1 cells are also involved in transplant rejection and protection from neoplasms (5). Th2 cells produce IL-4, IL-5, IL-10, and IL-13, mediate IgG1 and IgE production, eosinophilic inflammation, and are important for elimination of certain extracellular microbes and parasites (6). Overactivated Th1 responses cause organ-specific autoimmune diseases, such as type I diabetes. Thus, elucidation of the molecular mechanisms controlling Th1 effectors has both biological and clinical importance.

Several factors, that include costimulatory molecules, the extracellular cytokine environment, the nature of the APCs, and the method of Ag delivery may influence the outcome of naive T cell polarization (3, 7, 8). CD4+ T cell differentiation is regulated by multiple transcription factors, although T-bet and GATA-3 are considered as primary regulators of Th1 and Th2 development, respectively (9). T-bet is a Th1-specific transcription factor activated by IFN-{gamma} signaling via STAT1 (10). T-bet induces Runx3 and both transcription factors are required for maximal production of IFN-{gamma}, silencing of the gene encoding for IL-4 and up-regulating IL-12Rβ2 to reinforce Th1 development (11). Regulatory feedback mechanisms are taking place in the course of a microbial aggression to dampen Th1 responses and limit the damage to the host while maintaining an effector response. WSX-1 (IL-27R) down-regulates protective Th1 cell responses against Toxoplasma gondii (12). Similarly, expression of the branching β1,6-N-acetylglucosaminyltransferase V (Mgat5) N-linked glycans increases after T cell activation and selectively inhibits Th1 cell differentiation (13). Other regulatory pathways include the generation of Th1 cells expressing T-bet and producing regulatory IL-10 that serves as antagonist of T cell-mediated immune responses to intracellular protozoan infection (14, 15). Despite the progress in the molecular understanding of Th development, whether surface molecules negatively control Th1-mediated immune responses remains ill- defined. Galectin-9/Tim-3, expressed on repeatedly activated Th1 effectors, represents such a pathway that evolved to ensure the termination of undesired Th1 responses (16, 17).

CD47, considered as a marker of self on hematopoietic cells, has been shown to trigger a wide variety of cellular functions. Biological consequences of CD47 ligation vary according to the mode of CD47 engagement (soluble vs immobilized ligand), its association with different molecules in cis- or in trans, and its localization on the cell surface (18, 19, 20, 21). Although CD47 ligation appears to costimulate T cell proliferation (22) and induce their arrest and spreading on inflamed vascular endothelium (23), several lines of evidence suggest that CD47 expressed on T cells primarily functions as an inhibitory molecule. First, CD47 inhibits early T cell activation (24) and allogenic MLR (25). Second, CD47 ligation by CD47 mAb or its natural ligand thrombospondin (TSP)3 in primary cultures of human cord blood mononuclear cells induces a state of T cell unresponsiveness characterized by reduced cell proliferation and cytokine expression and favors the conversion of naive and memory CD4+CD25– T cells into Foxp3+ regulatory T cells (26, 27). Because CD47 ligation impairs human naive Th1 development in vitro with no immune deviation toward Th2 (28), we thought to evaluate whether CD47 contributes to these developmental changes of CD4+ T cells in vivo. To address the question as to whether the Ag-specific Th1 immune response is controlled by CD47, we used an adoptive transfer model system of DO11.10 TCR-transgenic (Tg) mice specific for OVA that were backcrossed onto CD47–/– BALB/c background, yielding CD47–/– Tg mice and showed that CD47–/– Tg T cell polarization was biased toward Th1 at the single-cell level as assessed in vivo and in vitro. The enhanced Th1 response was associated with an increase expression of T-bet transcription factor in CD47–/– Tg T cells. Finally, CD47-deficient mice displayed a biased Th1 Ab profile and an exacerbated T cell- mediated contact hypersensitivity response.

These results provide evidence in vivo that CD47 is a T cell surface inhibitory molecule that may have evolved to downgrade excessive or unwanted type 1 effector responses for the maintenance of homeostasis.


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

The CD47 (integrin-associated protein, IAP) null IAP+/– 129sv/eg mice were backcrossed into BALB/c for 16 to 18 generations and were a generous gift from Dr. P. A. Oldenborg (Department of Integrative Medical Biology, Section for Histology and Cell Biology, Umeå University, Umeå, Sweden). Mice expressing the DO11.10 TCR transgene, which is specific for the peptide residues 323–339 of chicken OVA in the context of I-Ad (BALB/c, CD47+/+ Tg) were purchased from Charles River Laboratories and backcrossed into CD47–/– mice (CD47–/– Tg). All 8- to 12-wk-old mice were housed in our breeding colony and care facilities under specific pathogen-free conditions. All experimental protocols were approved by the Centre de Recherche du Centre Hospitalier de Montreal and Canadian Council on Animal Care.

Culture medium, Abs, and reagents

Mouse cells were cultured in complete RPMI 1640 medium (Wisent) supplemented with 10% FBS, 500 U/ml penicillin, 500 µg/ml streptomycin, HEPES buffer (10 mM), and 2-ME (1 mM; Life Technologies). OVA323–339 peptide was purchased from Peptides International and OVA protein and LPS (Escherichia coli) were obtained from Sigma-Aldrich. Recombinant mouse cytokines IL-2, IL-4, IL-12, and rGM-CSF were purchased from PeproTech. Mitomycin-C was purchased from EMD Chemicals. Soluble CD40 ligand was a gift from Amgen. The following clones were purchased from American Type Culture Collection and purified in our laboratory: 145-2C11 (anti-CD3), GK1.5 (anti-CD4), R46A2 (anti-IFN-{gamma}), 11B11 (anti-IL-4), anti-IL-12 (C15.6), and N418 (anti-CD11c). PE- or allophycocyanin-conjugated anti-mouse KJ1.26 to identify DO11.10 T cells were purchased from Caltag Laboratories. HM40-3 and 3/23 (anti-CD40), allophycocyanin- or PE-conjugated anti-mouse CD4, PerCP-Cy5.5 anti-mouse CD3e, PE-labeled anti-mouse CD8, and biotinylated anti-CD4 (GK1.5) from BD Biosciences. IFA and CFA was obtained from MP Biomedicals and alum from Pierce. CFSE and PKH-26 red fluorescent cell linker were purchased from Sigma-Aldrich.

Adoptive transfer

Single-cell suspensions were prepared from spleens and lymph nodes (LN) harvested from naive CD47+/+ or CD47–/– Tg mice. CD4+ T cells were purified by positive selection using immunomagnetic beads (Easy Sep; StemCell Technologies). Purity was >98% as determined by flow cytometry. Syngeneic CD47+/+ and CD47–/– naive recipients were injected i.v. with 3 x 106 CFSE-labeled CD47+/+ and CD47–/– Tg T cells, respectively. One day later, the mice were immunized s.c. in the footpad with 50 µg of OVA peptide dissolved in 20 µl of PBS and emulsified in IFA. The draining LN (popliteal) were extracted 7 days after immunization to assess cell proliferation (CFSE dilution). Popliteal LN were minced in 3 ml of RPMI 1640 with EDTA and passed through a 70-µm nylon cell strainer (BD Biosciences). LN cells were restimulated with OVA323–339 peptide in vitro. Proliferative responses were measured by culturing 2 x 106 total LN cells with 0–10 µg/ml OVA323–339 peptide for 4 days. Cells were pulsed with 1 µCi of [3H]thymidine (Amersham Biosciences) for the last 12 h and then harvested for liquid scintillation counting. The number of KJ1.26+CD4+ T cells for each group of recipients was determined by FACS analysis, and proliferation was normalized to 104 KJ1.26+CD4+ input. Culture supernatants were collected on day 4. Data represent the mean ± SD of quadruplicates for each peptide concentration.

In some experiments, CD47–/– or CD47+/+ BALB/c mice were adoptively transferred with 10 x 106 CD47+/+ Tg T cells and immunized 1 day later s.c. with 0.25 x 106 activated OVA-pulsed CD47+/+ bone marrow-derived dendritic cells (BMDCs). Three days later, T cell-specific responses were examined in the draining LN after a 72-h in vitro restimulation with OVA peptide. For competitive migration, CFSE-labeled CD47–/– Tg T cells and PKH-26 red fluorescent cell linker-labeled CD47+/+ Tg T cells were adoptively transferred at the same time at a 1:1 ratio into CD47+/+ or CD47–/– BALB/c recipients. On day 1, mice were left untreated (noninflammatory) or immunized in the footpad with OVA323–339 peptide (inflammatory conditions). The draining LN were recovered at day 7. CFSE- and PKH-26-positive cells were retraced after gating on KJ1.26+CD4+ T cells.

In vivo immunization of unmanipulated mice

In vivo Ab response. Total and OVA-specific Ig serum level was quantified by ELISA in unmanipulated CD47+/+ and CD47–/– deficient mice at steady state and after immunization. For primary and secondary responses, 50 µg of OVA protein, in CFA or alum, had been injected i.p. at days 0 and 35, respectively. The IgG1 and IgG2b OVA-specific Ab response was quantified by ELISA in collected serum 35 days after the first immunization (primary response) and 8 days after boost (secondary response).

In vitro Ab production. For in vitro Ig production, B cells were obtained from CD47+/+ and CD47–/– mice spleens by CD43-depletion (EasySep) and stimulated for 7 days in complete RPMI 1640 medium by LPS (10 µg/ml) or IL-4 (20 ng/ml) and soluble CD40 ligand (1 µg/ml) or IL-4 and anti-CD40 mAb. Total IgG1 and IgG2a production was quantified in the culture supernatant by ELISA.

In vitro IFN-{gamma} production. BALB/c or CD47–/– mice were immunized s.c. with 25 µg of OVA protein and 1 µg of LPS (E. coli) in IFA. On day 7, the popliteal draining LN were recovered. Total LN cells were CFSE labeled and 106 cells were stimulated with OVA protein (2 mg/ml) in 96-well flat-bottom plates for 48–72 h. GolgiStop was added for the last 6 h. The cells were stained with CD4 mAb and fixed in 2% paraformaldehyde and permeabilized with 0.5% saponin (BD Biosciences) before staining with allophycocyanin-labeled Abs to IFN-{gamma} (BD Biosciences) in the presence of Fc blocker (clone 24G2; BD Biosciences).

Contact hypersensitivity response

Mice were painted on the shaved abdomens with 20 µl of 0.5% and 0.2% 2,4-dinitrofluorobenzebe (DNFB) dissolved in acetone/olive oil (4:1) on days 0 and 5, respectively. On day 19, mice were challenged with 20 µl of 0.2% DNFB on each side of the right ear. The left ear was painted with an identical amount of vehicle and used as control. Ear thickness was measured at four locations with a microgauge meter (Mitutoyo) 24 h after elicitation. Hapten-specific ear swelling was calculated as follows: T – T0, where T0 and T represent the values of ear thickness before and after the challenge, respectively. After 38 days, some mice were further boosted with 0.5% DNFB on the shaved abdomen. One day later, cells were recovered from draining LN of DNFB-sensitized CD47–/– and CD47+/+ mice and 3 x 106 purified CD4+ and CD8+ T cells using anti-CD4 and anti-CD8 PE-labeled mouse mAb and immunomagnetic beads (EasySep). T cells were passively transferred in naive CD47–/– recipients. The ear was challenged with 0.5% DNFB 18 h after T cell transfer and contact hypersensitivity response (CHS) response was measured on the next day. Results are expressed as the mean ear swelling (µm).

Preparation of BMDCs

BMDCs were generated as described previously (29). GM-CSF (5 ng/ml) was added for 10–14 days to 1 x 106 cells to induce dendritic cell (DC) differentiation. The culture medium was renewed every 3 days. Cell purity was >98% CD11c+ cells. In some experiments, BMDCs were activated overnight with 1 µg/ml LPS and 1 µg/ml OVA323–339 peptide (Peptide International).

In vitro Th differentiation/activation

Th1/Th2 differentiation. Isolated CD47+/+ or CD47–/–CD4+ T cells (1 x 106/ml) were cocultured with mitomycin-C-treated CD47+/+ T cell-depleted splenocytes (APCs, 2 x 106/ml). Cells were stimulated 3 days in complete RPMI 1640 medium by graded concentrations of anti-CD3 (1–10 µg/ml). For Th1/Th2 differentiation, CD47+/+ or CD47–/–CD4+ T cells (5 x 104/ml) were stimulated with soluble anti-CD3 (1 µg/ml) in the presence of CD11c+-purified splenic DCs (25 x 103/ml) and IL-12 (10 ng/ml) and anti-IL-4 (10 µg/ml; Th1 condition) or IL-4 (20 ng/ml) and anti-IFN-{gamma} (10 µg/ml; Th2 condition). After 7 days, T cells were restimulated for 2 days with plastic-coated anti-CD3 (1 µg/ml) and cytokine production was measured in the culture supernatant by ELISA. CD11c+ cells were purified from splenocytes by positive selection using (N418) anti-CD11c-biotinylated mAb and immunomagnetic beads (EasySep).

Primary and secondary Ag-specific responses. For primary responses, 2 x 105 Tg T cells were cultured in 96-well flat-bottom plates with 2 x 106 mitomycin-C-treated APCs or 1 x 105 Tg T cells were cultured in 96-well round-bottom plates with 5 x 104 BMDCs for 4 days in the presence of 0–10 µg/ml OVA323–339 peptide. For secondary response, CD4+ T cells primed with OVA peptide (10 µg/ml) were rested overnight with 50 U/ml IL-2, then restimulated in vitro under the same conditions as in the primary response. To measure cell proliferation, cells were pulsed with 1 µCi of [3H]thymidine on day 4 (primary response) and day 3 (secondary response) for 12 h and then harvested for liquid scintillation counting. Data reflect the mean ± SD of quadruplicates for each peptide concentration. Culture supernatants were collected on days 3 and 4 and cytokine levels were determined by ELISA.

ELISA

ELISA kits (BD Pharmingen) were used according to the manufacturer’s protocols (IL-4, IL-10, IL-12p70, and IFN-{gamma}). Assays were done in quadruplicate. Error bars in ELISA figures indicate mean ± SEM.

Intracellular cytokine staining

Cytokine production was analyzed after stimulating 2 x 106 draining LN cells with OVA peptide (10 µg/ml) in 24-well plates, either for 6 h with GolgiStop or until day 4, in the presence of GolgiStop (BD Biosciences and BD Pharmingen) the last 6 h. The cells were fixed in 2% paraformaldehyde and permeabilized with 0.5% saponin (BD Biosciences) before staining with allophycocyanin-labeled Abs to IFN-{gamma} and PE-labeled Abs to IL-4 (all from BD Biosciences) in the presence of Fc blocker (clone 24G2; BD Biosciences). All samples were washed with PBS with 1% BSA (Wisent) and 0.01% sodium azide (Fisher Scientific) and analyzed on a BD Biosciences FACSCalibur machine and CellQuest software.

Western blot

T cells were lysed in ice-cold lysis buffer, containing 1x TNE buffer (50 mM Tris (pH 8.0), 0.1% IGEPAL CA-630, and 2 mM EDTA (pH 8.0)) and 10 µg/ml each of the protease inhibitors leupeptin, aprotinin, N-tosyl-L-phenylalanine chloromethyl ketone, and PMSF as well as the phosphatase inhibitors sodium fluoride (50 mM) and sodium orthovanadate (1 mM). Lysates were left on ice for 10 min and centrifuged for 10 min at 13,000 x g in a microcentrifuge at 4°C to remove nuclei membranes. Proteins were separated on 8.5% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dried milk in TBST (1% Tween 20) and incubated with Ab against mouse T-bet (4B10; Santa Cruz Biotechnology) diluted in TBST and 5% BSA, followed by goat anti-mouse IgG Ab (H + L) conjugated to HRP (Jackson ImmunoResearch Laboratories). Immunolabeling was detected using an ECL detection system (GE Healthcare). For normalization, anti-β-actin mAb (Sigma-Aldrich) was used as a control.

Statistical analysis

Statistical analyses were performed using the GraphPad Instat program.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The lack of CD47 on T cells enhances in vitro Th1 differentiation

CD47 ligation decreases IL-12 responsiveness by neonatal and adult human CD4+ T cells and impairs their in vitro development into Th1 effectors (28, 30). In this study, we examined whether the absence of CD47 on murine CD4+ T cells affects their in vitro capacity to produce IFN-{gamma} in neutral conditions. Polyclonal activation of purified CD47–/–CD4+ T cells induced large amounts of IFN-{gamma} when compared with CD47+/+CD4+ T cells. In fact, lack of CD47 significantly lowered the threshold of TCR cell activation for IFN-{gamma} production. CD47–/–CD4+ T cells stimulated by anti-CD3 (1 µg/ml) secreted similar quantities of IFN-{gamma} as CD47+/+CD4+ T cells activated by anti-CD3 (10 µg/ml). Under the same conditions, cell proliferation and IL-4 production were comparable in CD47–/– and CD47+/+ T cells (Fig. 1A). We next examined whether the increased IFN-{gamma} production seen in CD47–/– T cells was directly related to an altered CD3{epsilon} expression and found that the density of CD3{epsilon} was unchanged in both groups of mice (Fig. 1B). We next showed that CD4+ T cells deviated their cytokine profile toward Th1 under polarizing conditions. CD4+ T cells were activated with anti-CD3 in the presence of APC and anti-IL-4 plus IL-12 (Th1) or anti- IFN-{gamma} plus IL-4 (Th2) for 7 days and then restimulated with anti-CD3. The Th1-induced bias seen in CD47–/–CD4+ T cells was reflected by a high IFN-{gamma}:IL-4 vs low IL-4:IFN-{gamma} ratio in Th1- and Th2-polarizing conditions, respectively (Fig. 1C). Notably, CD47–/– CD4+ T cells produced significantly more IFN-{gamma} as compared with CD47+/+CD4+ T cells and IL-4 production was not influenced by CD47 ablation (data not shown). These in vitro data indicate that CD47 on mouse CD4+ T cells negatively regulates the generation of Th1 effectors, confirming results obtained with human naive T cells.


Figure 1
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  		FIGURE 1. CD47 on T cells negatively regulates Th1 development in vitro. A, CD47+/+ or CD47–/– CD4+ T cells (1 x 106/ml) were stimulated with graded concentrations of anti-CD3 mAb in the presence of mitomycin-C-treated T cell-depleted splenocytes (APC; 2 x 106/ml) for 3 days. Proliferation was measured by [3H]thymidine incorporation for the last 6 h of the culture period and IFN-{gamma} and IL-4 production was detected by ELISA (Student’s paired t test, *, p < 0.05, **, p < 0.01, ***, p < 0.001 n = 5 independent experiments). B. Flow cytometric analysis of TCR/CD3 (CD3{epsilon}) on CD47+/+ and CD47–/–CD4+ T cells. Representative result from six mice. C, CD47+/+ or CD47–/–CD4+ T cells (5 x 104/ml) were primed with soluble anti-CD3 mAb (1 µg/ml) in the presence of CD11c+-purified splenic DCs (25 x 103/ml) for 7 days in Th1 (IL-12 (10 ng/ml) and anti-IL-4 (10 µg/ml) mAb) or in Th2 (IL-4 (20 ng/ml) and anti-IFN-{gamma} (10 µg/ml) mAb)-polarizing conditions. Cells were washed and restimulated with plastic-coated anti-CD3 mAb (1 µg/ml) for 2 days. IFN-{gamma} and IL-4 were measured by ELISA at the end of the restimulation (Mann-Whitney U test, **, p < 0.01 n = 5 independent experiments).

 
CD47 deficiency in BALB/c mice drives Th1 polarization in vivo

To directly assess the in vivo role of CD47 in Th1 differentiation in response to soluble Ag, DO11.10 TCR-Tg mice which is specific for chicken OVA peptide (aa 323–329) presented on the MHC class II molecule I-Ad were backcrossed to CD47–/– BALB/c mice to generate CD47–/– Tg mice. We first examined the cell proliferation and cytokine profile of CFSE-labeled CD47–/– and CD47+/+ Tg T cells that were passively transferred into naive CD47–/– or BALB/c hosts, respectively, 1 day before their s.c. immunization with OVA peptide in IFA (Fig. 2). After 7 days, draining LN were collected and the proportion of clonotypic CD4+ T cells, hereafter referred as CD47+/+ and CD47–/– Tg T cells, that underwent cell division (CFSE dilution) was comparable in the two hosts (mean ± SEM of eight mice from eight separate experiments, 81.5 ± 3.3% and 76.0 ± 4.9% for CD47+/+ and CD47–/– Tg T cells, respectively, p > 0.05 (Fig. 2A, left panels). By contrast, CD47–/– Tg T cells transferred into CD47–/– mice produced significantly more IFN-{gamma} on a per cell basis as compared with CD47+/+ Tg T cells in CD47+/+ mice. Notably, the frequency of IFN-{gamma}-producing cells among CD47–/– Tg T cells was increased when assessed at the end of the 7 days of in vivo priming (Fig. 2A, right panels) and after 4 days of in vitro secondary stimulation with OVA peptide (Fig. 2C). The cytokine production was mainly restricted to T cells that divided in response to Ag stimulation. As for the ex vivo cell proliferation, the extent of Tg T cell proliferation after in vitro restimulation was comparable regardless of the presence of CD47 on T cells and the peptide concentration. In contrast to in vitro polyclonal activation of purified CD4+ T cells, IL-4 secretion was reduced in CD47–/– LN cells after in vivo immunization with soluble Ag (Fig. 2B). Collectively, these data support the conclusion that CD47 deficiency in Th2-prone BALB/c mice alters Th1/Th2 balance in favor of the Th1 subset.


Figure 2
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  		FIGURE 2. CD47 deficiency favors Th1 differentiation in vivo. CFSE-labeled CD47+/+ and CD47–/– Tg T cells were adoptively transferred to CD47+/+ or CD47–/– naive mice, respectively, 1day before s.c. immunization with 50 µg of OVA peptide in IFA. On day 7, the draining LN were recovered: A, CFSE dilution profiles of responding Tg T cells after gating on clonotypic KJ1.26+CD4+ T cells (left and middle panels). IFN-{gamma} and IL-4 production among KJ1.26+CD4+ T cell was analyzed by intracellular cytokine staining following OVA peptide stimulation for 6 h in the presence of GolgiStop (right panel). Data are representative of three to six mice per group. Numbers indicate the percentage of positive cells in each quadrant and the mean fluorescence intensity. B, In vitro recall responses to OVA of CD47–/– or CD47+/+ Tg T cells. LN cells (2 x 106/ml) were cultured with increasing concentrations of OVA peptide. Cell proliferation and IFN-{gamma} and IL-4 secretion were measured after 72 h. Data represent the mean ± SEM of five to eight individually analyzed LN from separate mice. C, Intracellular cytokine production gated on KJ1.26+CD4+ T cells. Draining LN cells were stimulated with OVA (10 µg/ml) for 4 days and GolgiStop was added for the last 6 h. Numbers indicate percentage of cytokine-producing KJ1.26+CD4+ T cells and mean fluorescence intensity. One representative experiment of three mice per group.

 
Absence of CD47 on CD4+ T cells favors in vivo Th1 responses and T-bet expression

We next thought to delineate whether Th1-induced bias was caused simply by the lack of CD47 on Tg T cells or more generally by CD47 deficiency in the host. Indeed, CD47 is expressed on all hematopoietic and nonhematopoietic cells. To directly address this question, it is desirable to compare the Th profile of CD47–/– Tg T cells to that of CD47+/+ Tg T cells in a CD47+/+ host. However, this is not feasible because CD47 is considered as a marker of self on live cells and as such, several studies report the immediate clearance of CD47-deficient cells in wild-type hosts as a result of the lack of an inhibitory signal delivered through signal-regulatory protein (SIRP)-{alpha} (19). To ascertain these observations, CFSE-labeled CD47–/– and PKH26-labeled CD47+/+ Tg T cells were coinjected i.v. at 1:1 ratio in the two types of hosts and retraced after 7 days in the draining LN. One day after adoptive T cell transfer, mice were either left untreated (noninflammatory conditions) or immunized s.c. with OVA peptide in IFA (inflammatory conditions). As clearly depicted in Fig. 3A, CD47–/– Tg T cells were readily eliminated in CD47+/+recipients under both conditions, confirming and extending published reports (31, 32). In contrast to their clearance from a BALB/c host, CD47–/– Tg T cells survived as well as CD47+/+ Tg T cells in a CD47–/– recipient.


Figure 3
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  		FIGURE 3. Impact of CD47-deficient environment on in vivo CD4+ Tg T cell responses. A, CFSE-labeled CD47–/– Tg T cells were eliminated from the draining LN of CD47+/+ recipients in homeostatic and inflammatory conditions. CFSE-labeled CD47–/– Tg T cells and PKH 26-labeled CD47+/+ Tg T cells were coinjected at a 1:1 ratio into naive CD47+/+ or CD47–/– BALB/c recipients. On day 1, some mice were immunized s.c. with OVA peptide in IFA (inflammatory condition). Shown are CFSE and PKH-26 profiles on gated clonotypic KJ1.26+CD4+ T cells. Data are representative of at least two mice per group. B, CD47+/+ Tg T cells were passively transferred into CD47+/+ or CD47–/– syngeneic recipients. One day later, mice were immunized s.c. with 0.25 x 106 activated OVA-pulsed CD47+/+ BMDCs. After 72 h, the draining LN were extracted and LN cells were stimulated with 10 µg/ml OVA peptide. [3H]Thymidine incorporation and IFN-{gamma} production were measured on day 3. Data represent mean ± SEM of three to five individually analyzed LN from separate mice.

 
We first evaluated the role of CD47 as part of the environment in the deviation toward Th1. CD47+/+ Tg T cells were adoptively transferred into a CD47–/– or CD47+/+ host 1 day before s.c. immunization with OVA-loaded bone marrow-derived CD47+/+ DCs (Fig. 3B). After 3 days, we collected the draining LN and found a comparable level of Tg T cell proliferation and IFN-{gamma} secretion in the two recipients. These results strongly suggest that absence of CD47 in the host has no major contribution to the quality of the Ag-specific CD47+/+ T cell response to CD47+/+ APC, not ruling out a potential facilitating role for CD47-deficient APC on T cell responses. Because of the in vivo elimination of CD47–/– APC from the CD47+/+ host, we next performed in vitro coculture experiments to assess the impact of CD47 ablation on DCs on T cell responses (Fig. 4A). CD47–/– APC were superior to CD47+/+ at eliciting IFN-{gamma} production by CD47+/+ or CD47–/– Tg T cells (Fig. 4A). CD47–/– APC produced more IL-12p70 and equal amounts of IL-10 than CD47+/+ APC. Nonetheless, CD47 ablation on CD4+ T cells led to an increase in IFN-{gamma} secretion in the presence of either APC. These data point toward a relative contribution of DC-derived IL-12 to the Th1 bias seen in CD47–/– mice (Fig. 4B).


Figure 4
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  		FIGURE 4. CD47 deficiency on DCs and T cells enhances IL-12, IFN-{gamma}, and T-bet expression. A, Purified CD47+/+ or CD47–/– Tg T (1 x 105/ml) were cocultured for 4 days with CD47+/+ or CD47–/– BMDCs (5 x 104/ml) loaded with increasing concentrations of OVA peptide. Cells were restimulated with mitomycin-C-treated APC with increasing doses of OVA peptide for 3 days and IFN-{gamma} secretion was detected by ELISA. One representative experiment of three. B, CD47–/– or CD47+/+ BMDC (0.5 x 106) were stimulated with LPS (1 µg/ml) and IFN-{gamma} (20 ng/ml). Supernatants were harvested at 24 h and assayed for IL-12p70 and IL-10 by ELISA. Mean ± SEM of five independent experiments. C, Naive CD47+/+ or CD47–/– Tg T cells were stimulated with OVA peptide (10 µg/ml) in the presence of CD47–/– mitomycin-C-treated APC for 5 days. Cell lysates were analyzed by Western blot for T-bet expression. As positive control, CD47+/+ Tg T cells were activated in Th1-polarizing conditions. One representative experiment of three. D, Naive CD47+/+ or CD47–/– Tg T cells were primed in vitro with CD47–/– mitomycin-C-treated APC with OVA peptide (10 µg/ml) in the presence or absence of neutralizing Abs to IL-12. After 4 days of priming, CD4+ T cells were collected and expanded in IL-2 for another 3 days. CD4+ T cells were then restimulated for 6 h with PMA and ionomycin, and T-bet expression was determined by intracellular staining. One representative experiment of two.

 
T-bet expression is known as a Th1-lineage commitment transcription factor. IFN-{gamma} transactivates T-bet in Ag-activated T cells (33). We thus examined whether the increased IFN-{gamma} production in CD47–/– Tg T cells was correlated with an enhancement in T-bet expression. Activated CD47–/– Tg T cells in the presence of OVA-loaded CD47–/– APC expressed more T-bet (Fig. 4C) as compared with CD47+/+ Tg T cells. Increased T-bet expression and IFN-{gamma} production was still observed after IL-12 neutralization (Fig. 4D and data not shown), suggesting that the Th1 bias of CD47–/– mice was not strictly dependent on paracrine IL-12/IFN-{gamma} production.

Finally, to directly examine the function of CD47 in vivo on T cells, CD47–/– recipients were immunized with OVA in IFA s.c. 1day after adoptive transfer of Tg T cells expressing or not CD47 (Fig. 5). Both types of Tg T cells were recovered in comparable numbers after transfer in a CD47–/– host (6.4 x 105 ± 1.4 and 6.9 x 105 ± 1.4 for CD47+/+ and CD47–/– Tg T cells, respectively, mean ± SEM of seven mice from separate experiments). Our data revealed that CD47–/– Tg T cells deviated their response toward Th1 as compared with CD47+/+ Tg T cells. Indeed, we found a significant increase in the frequency of IFN-{gamma}-producing cells and a slight decrease in the proportion of IL-4-positive cells among CD47–/– Tg T cells as compared with CD47+/+ Tg T cells (Fig. 5, A and B). This was reflected by an augmented IFN-{gamma}:IL-4 ratio (Fig. 5C), demonstrating that CD47 on T cells negatively regulates IFN-{gamma} and Th1 development in CD47–/– mice. The absence of CD47 in the environment altered the CD47+/+ Tg T cell proliferation without reducing the proportion of IFN-{gamma}-positive cells, which in fact remained as low in CD47–/– (6.33 ± 1.5%) and CD47+/+ (6.35 ± 2.2%) hosts. Furthermore, we noticed a reduction in T cell recovery in CD47-deficient mice. As previously reported (29), this might result from the defective CD47–/– DC migration across lymphatic vessels and a subsequent impaired T cell priming. Collectively, these results demonstrated that CD47 expression on CD4+ T cells negatively regulates IFN-{gamma} production and T-bet expression as well as in vivo Th1 development in response to soluble Ag. CD47 expression on APC may influence the Th1/Th2 balance in vivo.


Figure 5
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  		FIGURE 5. Absence of CD47 on CD4+ T cells favors Th1 polarization in vivo. A, CFSE-labeled CD4+ Tg T cells lacking or not CD47 were adoptively transferred into naive CD47–/– or CD47+/+ hosts. At day 1, mice were immunized s.c. with OVA peptide in IFA. After 7 days, draining LN were recovered, and cell proliferation was assessed by CFSE dilution profiles (gated on CD4+ and KJ1.26+ T cells) and numbers indicate the total cell recovery (middle and right panels). LN were stimulated with OVA peptide for 6 h in the presence of GolgiStop. Shown are the frequencies of IFN-{gamma}- and IL-4-producing cells and mean fluorescence intensity among KJ1.26+CD4+ T cells (left panel). Data are representative of three mice per group. B and C, Shown are the proportion of KJ1.26+IFN-{gamma}+CD4+ T cells (B) and the IFN-{gamma}:IL-4 ratio in draining LN (C). Each individually analyzed LN is represented by a single dot for separate experiments. The average value for each group is represented by a horizontal bar. (Wilcoxon-matched pairs signed-rank test, *, p < 0.05).

 
CD47 deficiency promotes a Th1-biased Ab profile

We next thought to evaluate whether the humoral immune responses were biased toward Th1 in CD47-deficient mice. We examined the serum Ab isotypes in unmanipulated CD47–/– mice and observed a prominent increase in Ig isotypes associated with a Th1 phenotype, IgG2a and IgG2b, with no significant effect on IgG1 serum levels, which was associated to the Th2 profile (Fig. 6A). The strong Th1 Ab isotype profile observed in CD47–/– mice at steady state was maintained after primary and secondary antigenic challenge. Indeed, upon immunization with OVA in CFA, IgG2a OVA-specific Abs were at least 10-fold higher in CD47–/– mice when compared with BALB/c mice (Fig. 6B). Moreover, CD47–/– mice still showed a strong Th1 bias when using alum, a Th2 adjuvant. In contrast, the IgG1 Ag-specific primary response was not significantly modified in the CD47–/– mice at 35 days postimmunization. Thus, in vivo, in the absence of CD47, primary immune responses to soluble Ag are strongly biased toward a Th1 phenotype, with a slight delay in Th2 Ab isotype production. Upon secondary challenge in IFA, not only were OVA-specific IgG2a Abs increased but IgG1 Abs now showed a significant decrease (Fig. 6C). Therefore, using CFA followed by IFA as an adjuvant, the Th1 Ab response was enhanced while Th2 was abated. Similarly, with alum, the Th1 secondary antigenic response was enhanced, as observed by an increase in OVA-specific IgG2a Abs (Fig. 6C). In contrast, the Th2 Ab response was not reduced as alum drives strong Th2 responses.


Figure 6
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  		FIGURE 6. Th1-biased Ab profile in CD47–/– mice. A, CD47–/– mice showed enhanced secretion of IgG2a and IgG2b Ab at steady state and upon immunization. Ig isotype from the sera of 6- to 9-wk-old female mice were quantified by ELISA. Of note, similar data were obtained with male mice. B, Female mice were immunized i.p. with 50 µg of OVA either in CFA (favor Th1/Th2 mixed response) or in alum (Th2 response). Thirty-five days after the injection, the mice were bled and OVA-specific Abs were quantified by ELISA (C). The mice were rechallenged i.p. on day 35 with 50 µg of OVA, either in alum or for CFA previously immunized mice, in IFA. Seven days after the second injection, the mice were bled and OVA-specific Abs were quantified by ELISA. ***, p < 0.001; **, p < 0.01.

 
We next examined whether the Th1 Ab response in nonmanipulated CD47–/– mice was caused by the Th1-biased cytokine profile (enhanced IFN-{gamma} production) and/or attributed to a B cell intrinsic defect. To this end, BALB/c- and CD47-deficient mice were immunized s.c. with OVA protein in IFA plus LPS and Ag-specific T cell response was examined in the draining LN. The frequency of proliferating IFN-{gamma}-producing CD4+ T cells was significantly increased in CD47–/– when compared with CD47+/+ mice (Fig. 7A). However, we found no significant difference in the quality and the quantity of IgG1 and IgG2a produced in vitro by LPS-, IL-4-, and CD40–activated purified B cells expressing or lacking CD47 (Fig. 7B).


Figure 7
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  		FIGURE 7. Enhanced Ag-specific Th1 responses with no B cell-intrinsic defect in non-Tg CD47–/– mice. A, Intracellular IFN-{gamma} production gated on CFSE+CD4+ T cells. BALB/c or CD47–/– mice were immunized s.c. with 25 µg of OVA protein and 1 µg of LPS (E. coli) in IFA. On day 7, the draining LN cells were labeled with CFSE and stimulated with OVA protein (2 mg/ml) for 48–72 h. GolgiStop was added for the last 6 h. Histograms indicate a percentage of CFSE+ IFN-{gamma}-producing CD4+ T cells. Data represent mean ± SEM of seven to eight individually analyzed LN from separate mice (unpaired t test; *, p < 0.05). B, Absence of B cell-intrinsic defect in CD47-deficient mice. B cells were purified from spleen by anti-CD43-negative selection and cultured in vitro for 5–7 days with LPS (10 µg/ml) or IL-4 (20 ng/ml) plus either CD40 ligand (1 µg/ml) or anti-CD40 mAb (1 µg/ml). Ab production was measured by ELISA. Data represent mean ± SD of four mice per group.

 
Taken together, CD47 ablation in a Th2-prone BALB/c background is sufficient to promote a Th1-biased cellular and humoral response both at steady state and following in vivo immunization with soluble Ag.

CD47 deficiency exacerbates contact hypersensitivity response

It is generally believed that CD8+ T cells play a role in CHS with a significant contribution of CD4+ T cells and IFN-{gamma}. CHS are mediated by hapten-specific T cells generated in response to a chemical skin irritant exposure such as DNFB. We therefore assessed the development of CHS in CD47–/– mice. BALB/c and CD47–/– mice were sensitized with DNFB at day 0 with a boost at day 5 and next challenged on the ear on day 19. As depicted in Fig. 8A, the ear swelling response was significantly increased in CD47–/– as compared with BALB/c mice. The enhancement was more prominent after a 24-h challenge but still present at 48 h. In the next series of experiments, BALB/c and CD47–/– mice were hyperimmunized and 18 h after the last DNFB immunization, sensitized T cells were purified from draining LN and passively transferred i.v. into naive CD47–/– mice that were challenged with DNFB on the ear the next day (Fig. 8B). Ear swelling was measured from day 1 after DNFB challenge. We here showed that purified CD47–/– T cells from hyperimmunized mice were capable of transferring exacerbated CHS to CD47–/– mice, demonstrating that CD47–/– T cells mediated the sustained local inflammatory process. However, our unpublished observations indicated that CHS was exclusively observed after transfer of CD4+ and CD8+ T cells (1:1 ratio) mixture and not using purified CD4+ T cells alone.


Figure 8
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  		FIGURE 8. Exacerbated DNFB-induced CHS response in CD47–/– mice. A, Mice were sensitized by painting the shaved abdomen with 0.5% DNFB and boosted 5 days later. On day 19, the right ear was challenged with 0.2% DNFB and the left ear was painted with vehicle (acetone). Ear thickness was measured 24 h later. Results are expressed as the mean ± SEM ear swelling (µm). B, CHS response after the T cell adoptive transfer. Mice were immunized and challenged as in A. On day 38, hyperimmunized CD47+/+ and CD47–/– mice were boosted with 0.5% DNFB on the shaved abdomen. On day 40, T cells were recovered from draining LN and 3 x 106 cells were passively transferred in naive CD47–/– recipients. Ear challenge with 0.5% DNFB was performed 18 h after adoptive T cell transfer and the CHS response was measured on the next day. A and B, Results are expressed as the mean ± SEM of ear swelling (µm). Hapten-specific ear swelling was calculated as follows: [treated ear thickness – control ear thickness] – background swelling (paired t test; *, p < 0.05; **, p < 0.01, n = at least 6 mice/group). Data are one representative experiment of two.

 
Taken all, our data demonstrate that CD47 negatively regulates in vivo type 1 cellular immune responses and thus may serve to limit unwanted inflammatory reactions.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Type 1 immune responses and the effector protein IFN-{gamma} play a critical role in the control of the inflammatory process, the protection against pathogens and tumors (4, 5). In this study, we demonstrated that CD47 negatively regulated type 1 humoral and cellular immune responses in vivo. By ablating CD47 in mice, we found that Th2-prone BALB/c mice developed a Th1-biased cytokine and Ab profile at steady state and after immunization. More specifically, the CD47–/– Tg T cells were poised to deviate toward the Th1 phenotype in response to soluble Ag in primary and recall responses in vitro and in vivo. Thus, our results identify the T cell surface CD47 molecule as a self-control negative regulator of Th1 responses.

Th1 vs Th2 polarization and helper function for Ab production is dictated by rather complex interactions. DCs that have been activated by pattern recognition receptors instruct naive T cell differentiation into Th1 effectors (34). A selective combination of two TLR signals synergizes for the production of biological IL-12 by DCs and strongly favors Th1 response (35). Similarly, a cooperative stimulation between Nod-like receptor (Nod1) and TLR is essential for successful elicitation of adjuvant-induced adaptive cellular and humoral immune responses (36). CD47–/– mice immunized with soluble Ag in CFA or alum mounted a Th1-biased cellular and humoral response. IFN-{gamma} production was increased in the draining LN and serum Ab profile showed elevated IgG2a and IgG2b levels after immunization with soluble OVA. How this conforms with the generalized (IgG1 and IgG2) blunted immune response to particulate Ag reported in CD47–/– Th1-prone C57BL/6 mice is unclear (37). The present study provided evidence that CD47–/– BALB/c not only displayed a Th1-biased Ab profile but also developed an exacerbated type 1-mediated CHS response to DNFB. Our data revealed that CD47–/– T cells from DNFB-hyperimmunized mice transferred the enhanced CHS response to naive CD47–/– mice when compared with CD47+/+T cells. Also, CD4+Tg T cells lacking CD47 up-regulated their T-bet expression. Hence, T-bet-transgenic mice develop contact dermatitis mediated by IFN-{gamma} produced by type 1 cytotoxic T and CD4+ Th cells (38). Activated NK cells produce large amounts of IFN-{gamma}, potentiate Th1 priming, and also participate in the CHS reaction (39, 40). Our unpublished data showed that CD47–/– mice displayed a higher proportion of NK cells when compared with BALB/c and the CD47-deficient NK contribution to the Th1-biased phenotype, and the CHS response remains to be determined. Epidermal and dermal DCs control the induction of CHS and the absence of Langerhans cells even augments CHS (41). In fact, the lack of CD47 expression on myeloid DCs negatively regulates their transendothelial migration. As a consequence, the number of skin-derived DCs is reduced in the LN at steady state and after FITC sensitization and the splenic marginal zone are quasi-depleted of CD4+ DCs (29). Of interest, IFN-{gamma} negatively regulates DC migration (42).

Taken all, the Th1 bias reported in the present study combined to the defective skin-derived DC migration in CD47–/– mice may contribute to the exacerbated CHS response observed in CD47–/– mice. Our present data are in apparent contradiction with the decreased DNFB response observed in CD47–/– C57BL/6 mice (37). However, our results concurred with the prolonged inflammatory response to oxazolone reported in the latter strain of mice (43). Local deficiency in T cell apoptosis in CD47–/– mice provides an additional mechanism for the augmented CHS response (44). Hence, CD47 ligation by TSP or a CD47 mAb induces caspase-independent cell death in human primary B and T cells and CD47–/– T cells are resistant to Fas-mediated apoptosis (20, 44, 45).

IL-12 is a dominant player to drive Th1 polarization. The TSP/CD47 pathway is an autocrine-negative regulator of IL-12 secretion in human DCs and impairs Th1 development (46). Likewise, bone marrow-derived CD47–/– DCs secreted more IL-12p70 than wild-type DCs and facilitated in vitro IFN-{gamma} production by CD47–/– as compared with CD47+/+ Tg T cells. Enhanced T-bet expression may still be observed in CD47–/–CD4+ T cells in the presence of neutralizing anti-IL-12 mAb, suggesting a minor contribution of paracrine IL-12. In vivo, Ag-pulsed DEC205+CD8+ DCs, which are the main source of IL-12, induce the development of Th1, whereas CD4+ myeloid DCs drive Th2 when injected into animals (47). However, the fine tune of the Th1 response may be regulated by signals other than IL-12 delivered by DCs to T cells. Among others, expression of CD70 on DEC205+CD8+ DCs and Delta4 Notch-like ligand on CD4+ DCs promote an IL-12-independent Th1 response in vivo (48, 49). SIRP family members, which include SIRP-{alpha} and {gamma}, represent the other CD47-binding partners that are selectively expressed by myeloid and T cells, respectively, and negatively regulate the IL-12 response (30, 50). SIRP-{alpha}/CD47 interactions in trans impair IL-12 but not IL-2 T cell responsiveness (30). Thus, both T cells and DCs coexpress CD47 and its two ligands (SIRP and TSP) (50). The respective and predominant contribution of CD47/TSP vs CD47-SIRP interactions through competition for CD47 access in cis- and its potential impact on Th1-driven responses definitely need to be addressed.

A strong TCR signal ensures a robust IFN-{gamma} response by purified T cells, which in turns promotes IL-12Rβ2 expression (51). We here showed that absence of CD47 on CD4+ T cells lowered the threshold of CD3 activation for IFN-{gamma} secretion but no alteration in CD3{epsilon} expression was found in CD47–/– mice (52).

Interestingly, low quantities of NO are reported to promote Th1 differentiation by up-regulating IL-12Rβ2 expression via an increase in cGMP (53), and TSP-CD47 interactions on vascular endothelial cells impair the NO response by inhibiting cGMP synthesis (54). This suggests that interference with the NO pathway may represent another potential mechanism for CD47 to alter the Th1 response. Notably, endogenous TSP is rapidly expressed on the T cell surface following CD3 stimulation, provided T cells established a contact with the extracellular matrix protein fibronectin (55).

Despite their Th1 bias, CD47-deficient mice did not develop spontaneously autoimmune diseases. Paradoxically, enhanced IFN-{gamma} does not necessarily translates into an inflammatory or autoimmune phenotype (56, 57). IFN-{gamma} is reported to induce Treg development and oppose the development of the newly described proinflammatory Th17 subset (58, 59). However, T-bet–/– mice, lacking Th1 effectors, are resistant to the development of experimental autoimmune encephalomyelitis despite the presence of Th17 producers, underlying the potential benefit of interrupting a Th1 response (60).

Nonetheless, IFN-{gamma} plays a critical role in the protective response against intracellular pathogens that include parasites, mycobacteria, Salmonella, and Listeria (5, 10, 61). Whether CD47 decreases pathogen-driven Th1 responses remains to be evaluated. In that regard, a CD47-like protein (M128L) serves as a potent immunomodulatory virulence factor expressed by myxoma virus to provide the virus with a selective advantage in vivo. This likely correlates to the ability of M128L to inhibit myeloid-lineage cell activation (62).

We here propose that CD47 is a T cell-negative sensor that may serve to dampen unchecked proinflammatory Th1 responses. Thus, manipulating CD47 signaling pathway may reduce the intensity of undesired collateral tissue damage to maximize protection and minimize host injury.


   Acknowledgments
 
We thank S. Lesage for comments and suggestions.


   Disclosures
Top
 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 Canadian Institutes for Health Research (Grant MOP-53152). S.B. and V.Q.V. are recipients of the Canadian Institutes Health Research/Canada Graduate Scholarships Doctoral Award. Back

2 Address correspondence and reprint requests to Dr. Marika Sarfati, Immunoregulation Laboratory, Centre Hospitalier de l’Université de Montréal, Hôpital Notre-Dame (Pavillon Mailloux, M4211K), 1560 Sherbrooke Street East, Montreal, Quebec H2L 4M1, Canada. E-mail address: m.sarfati{at}umontreal.ca Back

3 Abbreviations used in this paper: TSP, thrombospondin; Tg, transgenic; LN, lymph node; BMDC, bone marrow-derived dendritic cell; DNFB, 2,4-dinitrofluorobenzene; CHS, contact hypersensitivity response; DC, dendritic cell; SIRP, signal-regulatory protein. Back

Received for publication September 6, 2007. Accepted for publication April 10, 2008.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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