Effects of co-stimulation by CD58 on human T cell cytokine production: a selective cytokine pattern with induction of high IL-10 production

Dominique M. A. Bullens1,2, Khadija Rafiq1, Lydia Charitidou1, Xiaohui Peng1, Ahmad Kasran1, Petra A. M. Warmerdam3, Stefaan W. Van Gool1,2 and Jan L. Ceuppens1

1 Laboratory of Experimental Immunology, Department of Pathophysiology, Faculty of Medicine, Catholic University of Leuven, Herestraat 49, 3000 Leuven, Belgium
2 Division of Pediatrics, University Hospital Gasthuisberg, Catholic University of Leuven, Belgium
3 Center for Transgene Technology and Gene Therapy, Flemish Institute of Biotechnology, Catholic University of Leuven, Belgium

Correspondence to: Correspondence to: J. Ceuppens


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD58 is the ligand for the CD2 molecule on human T cells and has been shown to provide a co-stimulatory signal for T cell activation. However, its physiological role is still unclear. We studied the effects of co-stimulation by CD58 on the production of Th1-type (IL-2- and IFN-{gamma}) or Th2 type (IL-4, IL-5 and IL-10) cytokines in an in vitro culture system of purified human T cells with CD58-transfected P815 cells and with anti-CD3 as the primary stimulus. Co-stimulation of T cells by CD58 potently induced IL-10 and IFN-{gamma} production (at the protein and at the mRNA level), and transforming growth factor-ß production (at the mRNA level), comparable to what can be found in CD80 co-stimulated T cell cultures. In contrast, we found low to absent IL-2, IL-4, IL-5, IL-13 and tumor necrosis factor-{alpha} production after CD58 co-stimulation, and this was not due to suppressive effects of endogenously produced IL-10. CD80 co-stimulation strongly induced all these cytokines. Intracellular staining for cytokine expression revealed the existence of a T cell subpopulation induced by CD58 co-stimulation to produce both IFN-{gamma} and IL-10. We furthermore found that the selective cytokine profile induced by CD58 co-stimulation is further accentuated by rIL-12 and by rIFN-{alpha}. Using cyclosporin A as an inhibitor of the calcineurin enzyme, we could show that production of all cy tokines in this system is calcium dependent. CD58 co-stimulation thus induces a cytokine pattern corresponding to that described for T regulatory (Tr) 1 cells and to the pattern reported to be induced by the newly identified B7 family member, B7-H1.

Keywords: CD58, CD80, IFN-{gamma}, IL-10, Th1, Th2


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
It is widely accepted that physiological activation of T cells requires two signals. The first signal results from TCR–CD3 triggering by antigenic peptides presented on MHC molecules. The second signals are also called co-stimulatory signals. Many cell-bound receptor–ligand pairs have now been shown to be involved in T cell co-stimulation (1). Ligation of the T cell membrane molecule CD28 with its natural ligand CD80 and/or CD86 provides the most potent helper signal identified till now (2). The physiological role of CD58/CD2 or CD48/CD2 interaction is less clear. The CD2 molecule is a 50 kDa transmembrane glycoprotein (3). In humans, CD2 is expressed on all mature T lymphocytes, on 95% of thymocytes and on the majority of NK cells. CD2 can function as an adhesion molecule and as a co-stimulatory signal receptor (3). The physiological ligand for CD2 is the CD58 (LFA-3) molecule in humans and the CD48 molecule in mouse (4,5). The ligation of human CD2 by its natural ligand CD58 (LFA-3) has been studied with regard to its importance in T cell activation (3,68), T cell cytokine production (911) or generation of cytotoxicity (12). Stimulation by mAb against two functional epitopes on CD2 can also directly stimulate human T cells in the absence of TCR–CD3 triggering (13). Co-stimulation by human CD58 for IL-2 production by murine ovalbumin-specific T cell hybridomas, in which human CD2 complementary DNAs were introduced, has also been described (14). CD58 and CD80 induce distinct cytokine profiles by both CD4+ and CD8+ superantigen-stimulated human T cells (9,10).

In mice, the effect of CD48–CD2 interaction has been reported to be the enhancement of the anti-CD3-dependent T cell proliferation (5). CD48 has also been shown to enhance antigen-induced responses of CD4+ T cells in a CD2- dependent way (15). Moreover, T cells from CD2–/– mice are defective in proliferation and cytokine production, and CD48-deficient mice have a pronounced defect in CD4+ T cell activation (16,17). Some in vivo data suggest a role for CD2 triggering in Th2 development in mice (18,19).

Most studies on human T cell co-stimulation through CD2 have used mAb to the CD2 molecule. We therefore wanted to study the effects of co-stimulation by CD58. Here we report that CD58 in combination with TCR–CD3 triggering induces a selective cytokine pattern, which is further accentuated by rIL-12 and by rIFN-{alpha}. This cytokine pattern is similar to what has recently been described as typical for the so-called regulatory T regulatory (Tr) 1 cells (20) and corresponds to the cytokine profile that can be observed by T cells co-stimulated by B7-H1 (21).


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Isolation of peripheral blood cells
Peripheral blood mononuclear cells (PBMC) were obtained from healthy adult volunteers of both sexes. Heparinized venous blood was centrifuged on Ficoll-Hypaque density gradients (Pharmacia, Uppsala, Sweden). Interphase cells were washed 3 times with PBS (Boehringer Ingelheim, Heidelberg, Germany) and they were resuspended at a concentration of 5x106 cells/ml in complete medium, consisting of RPMI 1640 (Boehringer Ingelheim) supplemented with 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml) and 10% iron-supplemented bovine calf serum (Hyclone, Logan, UT). T cells were purified with the use of Lymphokwik-T (One Lambda, Los Angeles, CA). A mAb mixture, consisting of complement-fixing anti-Leu-11b (anti-CD16) (Becton Dickinson, Erembodegem, Belgium) and anti-NKH-1A (anti-CD56) (Coulter, Hialeah, FL) mAb, was added for 30 min at 4°C as reported (22) followed by a second Lymphokwik-T treatment; resulting T cells were >98% CD3+ and <1% CD64+.

In some experiments T cells were further separated in naive and memory T cells. For that purpose, T cells were incubated with either a mouse anti-human anti-CD45RO mAb, UCHL-1 (a gift from P. Beverley, Jenner Institute for vaccine Research, Compton, UK), or a mouse anti-human anti-CD45RA mAb, 2H4 (Coulter, Miami, FL). Goat anti-mouse IgG-coated Dynabeads (Dynal, Oslo, Norway) were added and after incubation cell separation was performed according to the manufacturer's instructions. Negatively selected T cells were >97% CD3+/CD45RA+ or >97% CD3+/CD45RO+ cells respectively. CD45RO+ and CD45RA+ double-positive cells were <5% in both isolations.

Cytokines, mAb and other reagents
Two different mouse anti-human CD3 mAb were used: UCHT-1 was a gift from Dr P. Beverley (Compton, UK) and OKT3 (mIgG2a) was from ATCC (Rockville, MD). mAb anti-human IL-4R antibody was purchased from R & D Systems (Abingdon, UK; clone 25463.11). Monoclonal mouse anti-human IL-10R (clone 37607.11) was from R & D Systems. Previous experiments in human PBMC demonstrated a blocking effect of this anti-IL-10R mAb on IL-10 consumption (our own unpublished results). rIL-12 was a gift from Genetics Institute (Cambridge, MA). rIFN-{alpha} was purchased from Roche (Nutley, NJ). Cyclosporin A (CsA) was purchased from Novartis Pharma (Basel, Switzerland).

Cell lines
The P815 cell line is an NK-resistant DBA/2-derived mouse mastocytoma cell line that expresses mouse Fc{gamma}RII and Fc{gamma}RIII. The P815 cell line was derived from ATCC; P815 cells transfected with human CD80, CD86 or CD58 were a gift from L. L. Lanier (DNAX, Palo Alto, CA).

Activation of T cells for cytokine production
Purified T cells (1x106) were cultured in 24-well culture plates in a 1 ml volume at 37°C in 5% CO2/95% air and supernatants were collected after different periods of time as indicated in the results. T cells were stimulated with soluble anti-CD3 mAb (UCHT-1) captured by mouse P815, P815/CD80 or P815/CD58 cells, at a T:P815 ratio of 1:1. P815 cells were mitomycin C treated and washed 5 times before use. We have shown earlier that IL-4 consumption by purified T cells can interfere with its in vitro measurements (23). To block this consumption, a blocking anti-IL-4R{alpha} mAb was added to the T cell cultures in which IL-4 production was measured. As our previous experiments in the absence or the presence of two IL-2R-blocking mAb had shown that IL-2 consumption occurs after the first 24 h of T cell cultures (data not shown), we measured IL-2 in the supernatants harvested at 24 h.

Cytokine measurements
The concentrations of IL-4, IL-5, IL-10, tumor necrosis factor (TNF)-{alpha}, IL-13 and IFN-{gamma} in culture supernatants were measured with a sandwich ELISA technique using combinations of unlabeled and biotin-coupled mAb to different epitopes of each cytokine. Coating mAb for these four assays respectively were 860A4B3 (Biosource Medgenix Diagnostics, Fleurus, Belgium), TRFK5 (PharMingen, San Diego, CA), JES3-9D7 (PharMingen), 68B2B3/68B6A3 (Biosource Medgenix Diagnostics), JES10-5A2 (PharMingen) and 350B10G6 (Biosource Medgenix Diagnostics). Biotinylated detection mAb were 860F10H12 (Biosource Medgenix Diagnostics), JES1-SA10 (PharMingen), JES3-12G8 (PharMingen), 68B3C5 (Biosource Medgenix Diagnostics), B69-2 (PharMingen) and 67F12A8 (Biosource Medgenix Diagnostics). The concentrations of IL-2 in culture supernatants were detected by human IL-2 Duoset ELISA (Genzyme, Cambridge, MA). Human recombinant IL-4, IL-5, IL-10, IL-2, TNF-{alpha}, IL-13 and IFN-{gamma} were used as standards. The lower detection limit for all cytokines was <20 pg/ml.

Intracellular staining
During the last 4 h of T cell cultures, 3 mM monensin (Sigma, St. Louis, MO) was added to the cultures together with phorbol myristate acetate (Sigma; 1 ng/ml) and ionomycin (Sigma; 0.5 µg/ml). Cells were collected and fixed with 500 µl paraformaldehyde (Janssen Pharmaceutica, Beerse, Belgium) 2% in PBS at 4°C for 10 min. For permeabilization, cells were washed with PBS containing 0.5% BSA (Sigma Diagnostics) and 0.2% saponin (Sigma Diagnostics). Cells were then resuspended in 100 µl of this permeabilization buffer and 10 µl normal mouse serum was added for 5 min at room temperature. Then, 10 µl of either PE-conjugated control IgG1 antibody (Becton Dickinson), FITC-conjugated IgG2a or IgG1 antibody (Becton Dickinson), phycoerythrin (PE)-labeled anti-human IL-10 (18555A) (PharMingen) or FITC-labeled anti-human-IFN-{gamma} (clone B-B1) (Innotest, Besancion, France) was added. Tubes were gently mixed for 30 min in the dark at room temperature. Cells were resuspended, washed twice with permeabilization buffer and resuspended in PBS. Cells were analyzed with a FACSort flow cytometer (Becton Dickinson). Calibration was done with CaliBRITE 3, unlabeled, FITC, PE and PerCP (Becton Dickinson).

Plots were generated with the CellQuest software.

Cytokine mRNA analysis by PCR
T cell cultures were harvested 24 h after initial stimulation as described above. Stimulated T cells (5x105) were pelleted and directly used for total RNA isolation, isolated as described (24). Total RNA (1.25 µg) was used for oligo(dT)-primed cDNA synthesis (Ready-to-go-kit; Pharmacia, Uppsala, Sweden). After 90 min at 37°C, the reverse transcriptase was inactivated by incubating the cDNA samples for 5 min at 95°C. The cDNA samples were then subjected to PCR analysis using the following primers: IFN-{gamma}: 5'-GCA TCG TTT TGG GTT CTC TTG GCT GTT ACT GC-3' and 5'-CTC CTT TTT CGC TTC CCT GTT TTA GCT GCT GG-3'; IL-2: 5'-ACT CAC CAG GAT GCT CAC AT-3' and 5'-AGG TAA TCC ATC TGT TCA GA-3'; IL-10: 5'-GAG TAC CAG GGG CAT GAT ATC-3' and 5'-AAA TTT GGT TCT AGG CCG GG-3'; transforming growth factor (TGF)-ß1: 5'-CAG AAA TAC AGC AAC AAT TCC TGG-3' and 5'-TTG CAG TGT GTT ATC CCT GCT GTC-3'; IL-4: 5'-CTT CCC CCT CTG TTC TTC CT-3' and 5'- TTC CTG TCG AGC CGT TTC AG-3'; and CD3{delta}: 5'-GTA CTG AGC ATC ATC TCG ATC-3' and 5'-CTG GAC CTG GGA AAA CGC ATC-3' as cDNA quality control. All primer pairs were purchased from Pharmacia (Sweden). Samples were amplified after 2 min at 95°C by Taq polymerase (Boehringer, Mannheim, Germany) at 33 cycles of denaturation at 94°C for 45 s, annealing at 60°C for 30 s and extension at 72°C for 30 s. After 33 cycles, samples were kept for 10 min at 72°C. PCR products were analysed by electrophoresis on 1.6% agarose gels and visualized by ethidium bromide staining.

Cytotoxicity assay
Cytotoxic T lymphocyte (CTL) activity was tested as the 4 h target cell lysis of P815 cells in the presence of anti-CD3 mAb OKT3 (2 µg/ml) as described (25). By bridging the effector CTL to the target cell Fc{gamma}R with saturating amounts of anti-CD3 mAb, this anti-CD3-redirected cytotoxicity system permits detection of CTL activity regardless of antigen specificity of the CTL. Anti-CD3-dependent total release was calculated as the difference between `total' lysis (in the presence of anti-CD3) and the `spontaneous' release by the target cells with the formula: [(total – spontaneous release)/(maximal – spontaneous release)]x100 (25).

Statistical analysis
Results were compared with two-tailed (or one-tailed where appropriate) paired Wilcoxon non-parametric tests.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD80 and CD58 both provide a potent co-stimulatory signal for T cells
We first compared the ability of CD80 and CD58 to provide a co-stimulatory signal for CTL generation. To study the effect of CD2 or CD28 ligation by its natural ligands, CD58 or CD80, purified T cells were cultured with anti-CD3 (UCHT1) captured on either P815-, P815/CD80- or P815/CD58-transfected cells. After 4 days, CTL activity generated in these cultures was tested as the 4 h target cell lysis of P815 cells coated with anti-CD3 mAb (anti-CD3 redirected CTL activity) as described (25). As shown in Fig. 1Go, CD80 and CD58 were equally potent to provide a co-stimulatory signal for CTL generation. Results were also comparable if CD86 was used as a co-stimulatory signal (data not shown).



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Fig. 1. Generation of CTL activity by co-stimulation with CD80 or CD58. T cells (1x106/ml) were cultured with either 1x106 P815, P815/CD80 or P815/CD58 cells with anti-CD3 (UCHT-1) (2 µg/ml) as the primary signal. After 4 days, cells were washed and cytotoxic T lymphocyte activity was tested as the 4 h target cell lysis of P815 cells in the presence of anti-CD3 mAb OKT3 (2 µg/ml). Results are expressed as percentage of total 51Cr release (%TR). Results are the mean ± SEM of three independent experiments on different donors.

 
Ligation of CD2 by CD58 provides a potent helper signal for IL-10 and IFN-{gamma} production
In the second set of experiments, we used the same in vitro system to study the effect of CD2 ligation by CD58 on T cell cytokine production. Compared to stimulation by P815 cells and anti-CD3 alone, CD58 co-stimulation significantly enhanced the production of IL-5, IFN-{gamma}, TNF-{alpha} and IL-10 (Fig. 2Go), whereas it did not induce a significant increase in the production of IL-4, IL-13 or IL-2 (Fig. 2Go). For IL-4 production, results are shown for memory T cell cultures, as we have previously demonstrated that adult naive T cells produce only very low levels of IL-4 (26). The production of IL-5 and TNF-{alpha} by T cells stimulated with anti-CD3 and CD58 was significantly lower than by T cells co-stimulated by CD80. In contrast, CD58 was as potent as CD80 to induce IL-10 and IFN-{gamma}. Both IL-10 and IFN-{gamma} production started within 24 h after stimulation. Kinetic analysis showed that the production of IL-10 and IFN-{gamma} by T cells co-stimulated by CD58 remained comparable to their production by T cells co-stimulated by CD80 (Fig. 3Go), although IL-10 production was slightly higher at 72 h of CD58 co-stimulation (P < 0.05).



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Fig. 2. Comparison of cytokine production by T cells when co-stimulated by CD58 or CD80. Either 1x106/ml either CD45RA T cells (A) or total T cells (B–G) were stimulated with anti-CD3 (UCHT1) (2 µg/ml) and (1x106/ml) mitomycin C-treated P815-, P815/CD80- or P815/CD58-transfected cells. Supernatants were collected after 24 (D) or 48 (A–C and E–G) h, and IL-4 (A), IL-5 (B), IL-13 (C), IL-2 (D), IFN-{gamma} (E), TNF-{alpha} (F) and IL-10 (G) concentrations in the supernatants were measured by ELISA. The box extends from the 25th to the 75th percentile, with a horizontal line at the median. Whiskers extend down to the smallest value and up to the largest. Results of n = 6 (A), 11 (B), 12 (E, G), 7 (C) or 8 (D and F) independent experiments on different donors are represented. *P < 0.05, **P < 0.01, ***P < 0.001.

 


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Fig. 3. Kinetics of IL-10 and IFN-{gamma} production by T cells. Purified T cells (1x106/ml) were stimulated with anti-CD3 (UCHT1) (2 µg/ml) and (1x106/ml) mitomycin C-treated P815-, P815/CD80- or P815/CD58-transfected cells for 24, 48 and 72 h. Supernatants were collected after 24, 48 and 72 h, and IL-10 and IFN-{gamma} concentrations in the supernatants were detected by ELISA. Results are the mean ± SEM of four independent experiments on different donors. *P < 0.05.

 
Analysis of cytokine mRNA expression in purified human T cells co-stimulated through CD80/CD28 or CD58/CD2 interaction
We then studied the induction of IFN-{gamma}, IL-10, IL-2 and IL-4 at the mRNA level with a semi-quantitative RT-PCR technique. As shown in Fig. 4Go(B–D), IL-10 and IFN-{gamma} mRNA are equally induced in anti-CD3-activated T cells when co-stimulated by either CD80 or CD58, whereas IL-2 mRNA was more prominent in T cells co-stimulated by CD80 as compared to CD58. IL-4 mRNA was only induced if T cells were co-stimulated by CD80 (data not shown). Media with serum contain TGF-ß, which interferes with the quantification of TGF-ß in T cell culture supernatants. We therefore studied the TGF-ß production only at the mRNA level. As indicated in Fig. 4Go(E), TGF-ß1 mRNA induction was similar whether T cells were co-stimulated by CD80 or by CD58.



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Fig. 4. Comparison of cytokine mRNA production by T cells when co-stimulated by CD58 or CD80. Purified T cells (5x105/ml) were incubated with 5x105 P815 (lane A2, B1–D1 and E2), P815/CD80 (lane A3, B2–D2 and E3) or P815/CD58 (lane A4, B3–D3 and E4) cells and anti-CD3 (2 µg/ml). Total RNA was isolated using the guanidium chloroform method and reverse-transcripted into cDNA. The cDNA was used as template for PCR using CD3{delta} primers as a control and IFN-{gamma}, IL-10, IL-2 and TGF-ß1 primers. PCR products were verified after gel electrophoresis and ethidium bromide staining. Lanes A1 and E1 are the mRNA transcripts for CD3{delta} and TGF-ß from freshly isolated T cells. Lanes A5, B4–D4 and E5 represent negative controls. One representative experiment out of two is shown.

 
Co-stimulation by CD58 thus signals T cells to produce high amounts of IL-10, IFN-{gamma} and TGF-ß, comparable to the amounts measured in T cell cultures co-stimulated by CD80. This co-stimulatory effect on cytokine production is at least partly at the transcriptional level.

Blocking IL-10 bioactivity does not influence the level of IL-10 and of other cytokines
IL-10 is a cytokine-synthesis inhibitory substance (27,28). To study the cytokine modulation by endogenously produced IL-10, we compared cytokine levels in T cell cultures performed in the presence or absence of a blocking anti-IL-10R mAb. Anti-IL-10R mAb did not significantly change the IL-10 levels in the culture supernatants, suggesting that there is little or no autoconsumption of IL-10 by T cells (data not shown). The use of this anti-IL-10R mAb also enabled us to demonstrate that the production of IL-4, IL-5, IL-13, IL-2 and TNF-{alpha} by T cells was not influenced by the bioactivity of IL-10 (data not shown). Thus, endogenously produced IL-10 is not responsible for the low levels of IL-4, IL-5, IL-2, IL-13 and TNF-{alpha} found in cultures of T cells co-stimulated by CD58.

Cytokine production by T cells co-stimulated by CD58 is calcineurin dependent
To study the role of calcineurin signaling for the induction of IFN-{gamma} and IL-10 production by T cells co-stimulated either by CD80 or CD58, we compared cytokine productions in the absence or presence of CsA. Blocking calcineurin activity almost totally blocked IFN-{gamma}, IL-10 and IL-5 production in CD58-co-stimulated T cell cultures (Fig. 5Go). In contrast, IL-5 and IL-10 production by T cells co-stimulated by CD80 was only partially suppressed by CsA, whereas IFN-{gamma} production by these cells was enhanced, as also reported earlier (29). Therefore, the co-stimulatory signal by CD58 is calcium dependent, which is in accordance with previous data on the importance of NF-AT in CD2 signal transduction (30).



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Fig. 5. Role of calcineurin activity for cytokine production by T cells. Purified T cells (1x106/ml) were stimulated with anti-CD3 (UCHT1) (2 µg/ml) and (1x106/ml) mitomycin C-treated P815/CD80- or P815/CD58-transfected cells in the presence or absence of CsA (400 ng/ml). Supernatants were collected after 96 h, and IFN-{gamma} (A), IL-10 (B) and IL-5 (C) concentrations in the supernatants were detected by ELISA. Results of four independent experiments on different donors are shown.

 
rIL-12 and rIFN-{alpha} enhance IL-10 and IFN-{gamma} production by T cells
Both IL-12 and IFN-{alpha} can enhance IL-10 and IFN-{gamma} production by human T cells (3134). As indicated in Fig. 6Go(A), IL-10 production by T cells co-stimulated by CD58 is further enhanced in a synergistic way by adding either exogenous IL-12 (P < 0.05) or IFN-{alpha} (P < 0.05). Adding rIFN-{alpha} did not influence the IFN-{gamma} production. Adding rIL-12 to T cells cultures, stimulated with anti-CD3 and P815 alone, also significantly enhanced their IFN-{gamma} production (P < 0.05), but the amounts of IFN-{gamma} produced remained low (see Fig. 6BGo). rIL-12 significantly enhanced the IFN-{gamma} production by CD58-stimulated T cells (P < 0.05) (Fig. 6BGo). Again, CD58 and IL-12 acted in a synergistic way. rIL-12 (but not IFN-{alpha}) also enhanced the TNF-{alpha} production (Fig. 6CGo). The production of the Th2 cytokine IL-5, on the other hand, was lowered by adding rIL-12 or rIFN-{alpha} to the T cell cultures (Fig. 6DGo), which is in accordance with the previously reported effects of these cytokines on IL-5 production (33).



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Fig. 6. Effect of rIL-12 and rIFN-{alpha} on T cell cytokine production. Resting T cells (1x106/ml) were cultured with mitomycin C-treated P815, P815/CD80 or P815/CD58 cells (1x106/ml) in the presence or absence of either rIL-12 (1 ng/ml) or rIFN-{alpha} (100 U/ml). Anti-CD3 mAb (UCHT1) was added at a concentration of 1 µg/ml. Supernatants were collected after 96 h of culture and the cytokine concentrations in the supernatants were determined by ELISA. Results of four independent experiments on different donors are shown.

 
We can conclude that both IFN-{alpha} and IL-12, on the one hand, and CD58 triggering, on the other hand, act synergistically in stimulating IL-10 productions by T cells. IL-12 also acts synergistically with CD58 to stimulate IFN-{gamma} production in T cell cultures, and both cytokines (IL-12 and IFN-{alpha}) thus accentuate the selective cytokine pattern induced by CD58 co-stimulation.

IL-10- and IFN-{gamma}-producing T cells are partly overlapping populations
Intracellular staining was used to study IL-10 and IFN-{gamma} production at the individual cell level. Naive and memory T cells (CD45RO and CD45RA T cells) were isolated, and stimulated for 48 h with anti-CD3 and either P815, P815/CD80 or P815/CD58 cells; intracellular staining for IFN-{gamma} and IL-10 was analyzed (35). Naive T cells stain for IFN-{gamma} if co-stimulation by CD80 or CD58 is provided, but they fail to stain for IL-10 (data not shown). As illustrated in Fig. 7Go, for memory T cells, co-stimulation by CD58 enhances IFN-{gamma} staining and IL-10 staining. Co-stimulation by CD80, on the other hand, enhances IFN-{gamma} staining but not IL-10 staining. Cytokine production studies have, however, demonstrated that CD80 co-stimulation does enhance IL-10 production in cultures of total T cells (Fig. 2Go) and of memory cells (not shown). The reason for this discrepancy between production and intracellular staining is unclear. The results that IL-10 production is restricted to memory T cells have previously been reported by Yssel et al. (36) and by ourselves (37).



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Fig. 7. Comparison of intracellular IL-10 and IFN-{gamma} productions by T cells when co-stimulated by CD58 or CD80. Purified memory (CD45RA) T cells (5x105/ml) were incubated with either P815, P815/CD80 or P815/CD58 cells and anti-CD3 (UCHT-1) (2 µg/ml). After 48 h, cells were harvested and permeabilized, and subsequently stained with either FITC-conjugated IgG2a and IgG1 control mAb or FITC-conjugated anti-IFN-{gamma} and either PE-conjugated control IgG1 mAb or PE-conjugated anti-IL-10. Cells were then analyzed on the FACS. The number of cells (out of total cell population) in each region is represented in percentage. R1 contains T cells only producing IL-10, R2 contains T cells only producing IFN-{gamma}, and R3 contains T cells producing both IL-10 and IFN-{gamma}. Gates are defined on the basis of the control experiments with either IL-10–PE mAb or IFN-{gamma}–FITC mAb alone. One representative experiment out of four is shown.

 
Figure 7Go also shows that approximately half of the IL-10-producing cells also produce IFN-{gamma}. The nature of this double-positive subpopulation has to be defined.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this paper we demonstrate that T cells stimulated with anti-CD3 and -CD58 express a selective cytokine pattern, consisting predominantly of IL-10 and IFN-{gamma}. These cells also produce TGF-ß. CD58 does not induce a typical Th2 pattern of cytokines, as there is no or only low IL-4 production, neither does it induce a typical Th1 pattern, as there is only low IL-2 production. Both IL-12 and IFN-{alpha} further enhance the production of IL-10 by T cells stimulated with anti-CD3 and CD58. Furthermore, the fact that only low levels of other T cell cytokines are produced (IL-4, IL-5, IL-13, IL-2 and TNF-{alpha}) is not due to an inhibitory effect of endogenously produced IL-10.

The signal transduction pathway involved in cytokine gene activation after CD2 ligation is not yet completely clarified. It has been shown that signal transduction after triggering with pairs of mAb to CD2 and CD2R epitopes is largely similar to that after triggering of the TCR–CD3 complex. Both pathways stimulate the tyrosine phosphorylation of polypeptides (38,39), and the formation of 1,2-diacylglycerol and inositol phosphates, causing protein kinase C activation and elevation of intracellular Ca2+ respectively (40). CD2 is functionally associated with the {zeta} chain of the CD3 complex, responsible for TCR–CD3 signal transduction (41). Furthermore, it has recently been shown that, similarly to the CD28-mediated co-stimulatory signal, the CD2 receptor appears capable of generating a signal that improves the level of T cell activation without affecting TCR down-regulation (11). CD2 triggering by mAb enhances p72syk tyrosine phosphorylation and its kinase activity (whereas co-stimulation with anti-CD28 does not), resulting in increased tyrosine phosphorylation of the downstream adapter molecules, Shc and Cbl (42). CD58 co-stimulation of T cells was also shown to induce NF-AT, AP-1 and NK-{kappa}B nuclear factors, with NF-AT being the key target nuclear factor for the CD2/CD58 pathway (43). Cooperation of NF-AT with AP-1 proteins has been noted in the promotor regions of certain cytokines, such as IL-2, IL-4 and IL-5 (30). Differences in the proteins induced and in their cooperation could contribute to the selective cytokine pattern observed in CD58 co-stimulated T cells. In our experiments, production of all cytokines by T cells stimulated with anti-CD3 and CD58 is almost totally blocked by CsA which blocks calcineurin activity. This is in agreement with the dominant role of NF-AT in CD58 signal transduction, as NF-AT is highly dependent on calcineurin phosphatase activity (30,43).

Observations on a selective cytokine induction depending on CD58 or CD80 co-stimulation have also been obtained with superantigen-stimulated CD4+ and CD8+ T cells (9,10). In an in vitro model with HLA-DR and either CD80 or CD58 double-transfected cells, Parra et al. showed that LFA-3 co-stimulation induced large amounts of IFN-{gamma} but only marginal amounts of IL-2 in staphylococcal enterotoxin A-stimulated T cells. This pattern is in agreement with our data, but we also found differences for several other cytokines. In previous experiments anti-CD2 mAb (9-1 and 9-6) in combination with anti-CD3 mAb induced high IL-4 and IL-5 production which is clearly different from our present data with CD58 (31). It is not clear whether or not these differences can be explained by stereotactic differences in the TCR after the combination of these three mAb have been added to the cultures.

There are more indications now that certain co-stimulatory molecules can induce very selective cytokine patterns. In recent experiments by other authors, distinct IL-10 and TNF-{alpha} profiles depending on either ICAM-1 (CD54), ICAM-2 or ICAM-3 (CD50) co-stimulation have been demonstrated in anti-CD3-stimulated human T cells (44). ICAM-1 co-stimulation induced 4 times higher IL-10 production and slightly enhanced IFN-{gamma} production as compared to co-stimulation by ICAM-2 or -3, whereas the TNF-{alpha} production was significantly lower in ICAM-1 co-stimulated T cells.

Both IL-12 and IFN-{alpha} further enhance CD58 induced IL-10 production. Other data in the literature also indicate that IL-12 and IFN-{alpha} promote IL-10 production. We ourselves have reported that IL-10 production is synergistically up-regulated by IL-12 and B7/CD28 interaction in human T cells in vitro (32). IL-12 and IL-2 can also synergize in inducing IL-10 production by human T cells (45). Upon stimulation with anti-CD2 mAb, IL-12 induces IL-10 production in human T cells (31,46). In vivo experiments in mice have demonstrated a large increase in the number of IL-10-secreting spleen cells, following injection of IL-12 (47). Furthermore, during primary and secondary Nippostrongylus brasiliensis infections, IL-12 has been shown to induce IFN-{gamma} and IL-10 gene expression (48). An increase of IL-10 induced by IL-12 was also observed in IFN-{gamma}-deficient mice (49,50). Although the influence of IFN-{alpha} on IL-10 production by T cells has been studied less intensively, IFN-{alpha} has been shown to stimulate both CD4+ T cells and monocytes to produce IL-10 (51). Schandené et al. have demonstrated that IL-10 production by T cells stimulated with anti-CD3 and CD28 triggering was up-regulated by IFN-{alpha}, whereas IL-5 production by these cells was down-regulated (33), as also observed in our experiments with CD58 co-stimulation. Most interestingly, we observed synergistic effects between CD58 and IFN-{alpha} and between CD58 and IL-12, suggesting that both signals represent a physiological mechanism of immunomodulation.

Using two-color immunofluorescence for intracellular cytokine staining, we could identify a subpopulation of cells producing both IFN-{gamma} and IL-10 after co-stimulation with CD58 or CD80. These are potentially Th1 cells, which also produce IL-10, but they can also belong to a separate subgroup of T cells. Indeed, the low IL-2 production in the cultures with CD58 co-stimulation rather points to another possibility. It has been shown recently that both human and murine CD4+ T cell clones stimulated in the presence of IL-10 can give rise to primed CD4+ T cell clones with low proliferative activity (20). These clones produce high levels of IL-10, intermediate levels of IFN-{gamma} and IL-5, and low levels of IL-2, and they do not produce IL-4. These antigen-specific T cell clones suppress the proliferation of CD4+ T cells in response to antigen and prevent experimental colitis induced in a SCID mice model by pathogenic CD4+CD45RBhigh splenic T cells. They are called Tr1 cells (20). This pattern corresponds to the selective cytokine pattern induced by co-stimulation with CD58. It is not impossible that in our in vitro system, stimulation by anti-CD3 and CD58 preferentially triggers this subgroup of regulatory T cells. On the other hand, this cytokine profile also corresponds to the cytokine pattern that has been recently reported after co-stimulation with B7-H1, a new member of the B7 family (21). T cells co-stimulated with B7-H1 produce high amounts of IL-10 and IFN-{gamma}, and low levels of IL-2, whereas IL-4 production is absent. The B7-H1 molecule is a third member of the B7 family that does not bind CD28, cytotoxic T lymphocyte A4 or inducible co-stimulator. The receptor for this molecule still has to be defined, but according to mouse data, could be the human ICOS analogue (52). It is tempting to speculate that both receptor/ligand pairs use similar intracellular signaling pathways. Whether T cells stimulated with anti-CD3 and -CD58 belong to a subgroup of so-called `regulatory' cells and whether these cells are also stimulated after co-stimulation with B7-H1 is the aim of future study.

If CD58 on the basis of these results represents a regulatory mechanism in T cell immune responses, the question arises whether or not CD58 co-stimulation can occur in vivo without CD80/86 co-stimulation. CD58 can be found on various cell types, in the absence of CD80 or CD86 expression, such as fibroblasts, endothelium and epithelia, but their function with regard to immune activation remains unclear. In contrast, resting CD58+ antigen-presenting cells (APC), such as monocytes, dendritic cells and B cells, also express low levels of CD80 and CD86, and these molecules are further induced after activation. The interaction and relative amount of these co-stimulatory molecules on the APC could be of importance in either inducing T cell activation or tolerance induction. Experiments with variable relative expressions of these co-stimulatory molecules on the APC can be of great interest in this regard.


    Acknowledgments
 
We want to thank Martine Adé for expert assistance with the ELISA techniques, Rik Lories for producing the photographs and Lieve Coorevits for general technical assistance. This work was supported by a grant from the `Onderzoeksfonds' of the Catholic University of Leuven (KUL, Belgium) and a grant from the Fund for Scientific Research (FWO, Vlaanderen) to J. L. C. D. M. A. B. and S. W. V. G are postdoctoral fellows supported by the FWO. We also thank P. Beverley (E. Jenner Institute for vaccine research, Compton, UK), L. Lanier (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA) and Genetics Institute (Cambridge, MA) for providing cells or reagents used in this study.


    Abbreviations
 
APC antigen-presenting cells
CsA cyclosporin A
CTL cytotoxic T lymphocyte
PE phycoerythrin
PBMC peripheral blood mononuclear cell
Tr1 T regulatory 1 cells
TGF transforming growth factor
TNF tumor necrosis factor

    Notes
 
Transmitting editor: H. Bazin

Received 3 May 2000, accepted 26 October 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. van Seventer, G. A., Shimizu, Y. and Shaw, S. 1991. Roles of multiple accessory molecules in T-cell activation. Curr. Opin. Immunol. 3:294.[ISI][Medline]
  2. Lenschow, D. J., Walunas, T. L. and Bluestone, J. A. 1996. CD28/B7 system of T cell co-stimulation. Annu. Rev. Immunol. 14:233.[ISI][Medline]
  3. Davis, S. J. and van der Merwe, A. P. 1996. The structure and ligand interactions of CD2: implications for T-cell function. Immunol. Today 17:177.[ISI][Medline]
  4. Selvaraj, P., Plunkett, M. L., Dustin, M., Sanders, M. E., Shaw, S. and Springer, T. A. 1987. The T lymphocyte glycoprotein CD2 binds the cell surface ligand LFA-3. Nature 326:400.[ISI][Medline]
  5. Kato, K., Koyanagi, M., Okada, H. 1992. CD48 is a counter-receptor for mouse CD2 and is involved in T cell activation. J. Exp. Med. 176:1241.[Abstract]
  6. Koyasu, S., Lawton, T., Novick, D. et al. 1990. Role of interaction of CD2 molecules with lymphocyte function-associated antigen 3 in T cell recognition of nominal antigen. Proc. Natl Acad. Sci. USA 87:2603.[Abstract]
  7. Bierer, B. E., Barbosa, J., Herrmann, S. and Burakoff, S. J. 1988. Interaction of CD2 with its ligand, LFA-3, in human T cell proliferation. J. Immunol. 140:3358.[Abstract/Free Full Text]
  8. Bierer, B. E., Peterson, A., Gorga, J. C., Herrmann, S. H. and Burakoff, S. J. 1988. Synergistic T cell activation via the physiological ligands for CD2 and the T cell receptor. J. Exp. Med. 168:1145.[Abstract]
  9. Parra, E., Wingren, A. G., Hedlund, G., Kalland, T. and Dohlsten, M. 1997. The role of B7-1 and LFA-3 in co-stimulation of CD8+ T cells. J. Immunol. 158:637.[Abstract]
  10. Parra, E., Wingren, A. G., Hedlund, G. 1994. Co-stimulation of human CD4+ T lymphocytes with B7 and lymphocyte function-asssociated antigen-3 results in distinct cell activation profiles. J. Immunol. 153:2479.[Abstract/Free Full Text]
  11. Le Guiner, S., Le Dréan, E., Labarrière, N. et al. 1998. LFA-3 co-stimulates cytokine secretion by cytotoxic T lymphocytes by providing a TCR-independent activation signal. Eur. J. Immunol. 28:1322.[ISI][Medline]
  12. Van de Wiel-van Kemenade, E., Te Velde, A. A., De Boer, A. J. 1992. Both LFA-1-positive and -deficient T cell clones require the CD2/LFA-3 interaction for specific cytolytic activation. Eur. J. Immunol. 22:1467.[ISI][Medline]
  13. Meuer, S. C., Hussey, R. E., Fabbi, M. et al. 1984. An alternative pathway of T-cell activation: a functional role for the 50 kd T11 sheep erythrocyte receptor protein. Cell 36:897.[ISI][Medline]
  14. Moingeon, P., Chang, C. H., Wallner, B. P., Stebbins, C., Frey, A. Z. and Reinherz, E. 1989. CD2-mediated adhesion facilitates T lymphocyte antigen recognition function. Nature 339:312.[ISI][Medline]
  15. Latchman, Y. and Reiser, H. 1998. Enhanced murine CD4+ T cell responses induced by the CD2 ligand CD48. Eur. J. Immunol. 28:4325.[ISI][Medline]
  16. Teh, S. J., Killeen, N., Tarakhovsky, A., Littman, D. R. and Teh, H. S. 1997. CD2 regulates the positive selection and function of antigen-specific CD4 CD8+ T cells. Blood 89:1308.[Abstract/Free Full Text]
  17. Gonzalez-Cabrero, J., Wise, C. J., Latchman, Y., Freeman, G. J., Sharpe, A. H. and Reiser, H. 1999. CD48-deficient mice have a pronounced defect in CD4+ T cell activation. Proc. Natl. Acad. Sci. USA 96:1019.[Abstract/Free Full Text]
  18. Biancone, L., Andres, G., Ahn, H. et al. 1996. Distinct regulatory roles of lymphocyte co-stimulatory pathways on T helper type 2-mediated autoimmune disease. J. Exp. Med. 183:1473.[Abstract]
  19. Chavin, K. D., Qin, L., Yon, R., Lin, J., Yagita, H. and Bromberg, J. S. 1994. Anti-CD2 mAbs suppress cytotoxic lymphocyte activity by the generation of Th2 suppressor cells and receptor blockade. J. Immunol. 152:3729.[Abstract/Free Full Text]
  20. Groux, H., O'Garra, A., Bigler, M. et al. 1997. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389:737.[ISI][Medline]
  21. Dong, H., Shu, G., Tamada, K. and Chen, L. 1999. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat. Med. 5:1365.[ISI][Medline]
  22. Ceuppens, J. L., Baroja, M. L., Lorre, K., Van Damme, J. and Billiau, A. 1988. Human T cell activation with phytohemagglutinin. The function of IL-6 as an accessory signal. J. Immunol. 141:3868.[Abstract/Free Full Text]
  23. Bullens, D. M. A., Kasran, A., Peng, X., Lorré, K. and Ceuppens, J. L. 1998. Effects of anti-interleukin-4-receptor monoclonal antibody on T cell cytokine levels in vitro: interleukin-4 production by T cells from non-atopic donors. Clin. Exp. Immunol. 113:320.[ISI][Medline]
  24. Peng, X., Kasran, A., Warmerdam, P. A. M., de Boer, M. and Ceuppens, J. L. 1996. Accessory signaling by CD40 for T cell activation: induction of Th1 and Th2 cytokines and synergy with interleukin-12 for interferon-{gamma} production. Eur. J. Immunol. 26:1621.[ISI][Medline]
  25. Van Gool, S. W., de Boer, M. and Ceuppens, J. L. 1993. CD28 ligation by mAb or B7/BB1 provides an accessory signal for the CsA-resistant generation of cytotoxic T cell activity. J. Immunol. 150:3254.[Abstract/Free Full Text]
  26. Bullens, D. M. A., Rafiq, K., Kasran, A., Van Gool, S. W. and Ceuppens, J. L. 1999. Naive human T cells can be a source of IL-4 during primary immune responses. Clin. Exp. Immunol. 118:384.[ISI][Medline]
  27. Taga, K. and Tosato, G. 1992. IL-10 inhibits human T cell proliferation and IL-2 production. J. Immunol. 148:1143.[Abstract/Free Full Text]
  28. Fiorentino, D. F., Zlotnik, A., Vieira, P. et al. 1991. IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. J. Immunol. 146:3444.[Abstract/Free Full Text]
  29. Rafiq, K., Kasran, A., Peng, X. et al. 1998. Cyclosporin A increases IFN-{gamma} production by T cells when co-stimulated through CD28. Eur. J. Immunol. 28:1481.[ISI][Medline]
  30. Rao, A., Luo, C. and Hogan, P. G. 1997. Transcription factors of the NFAT family: regulation and function. Annu. Rev. Immunol. 15:707.[ISI][Medline]
  31. Peng, X., Kasran, A., Bullens, D. and Ceuppens, J. L. 1997. Ligation of CD2 provides a strong helper signal for the production of type 2 cytokines interleukin-4 and -5 by memory T cells. Cell. Immunol. 181:76.[ISI][Medline]
  32. Peng, X., Kasran, A. and Ceuppens, J. L. 1997. Interleukin 12 and B7/CD28 interaction synergistically upregulate interleukin 10 production by human T cells. Cytokine 9:499.[ISI][Medline]
  33. Schandené, L., Del Prete, G. F., Cogan, E. et al. 1996. Recombinant interferon-alpha selectively inhibits the production of interleukin-5 by human CD4+ T cells. J. Clin. Invest. 97:309.[Abstract/Free Full Text]
  34. Schandené, L., Cogan, E., Crusiaux, A. and Goldman, M. 1997. Interferon-{alpha} upregulates both interleukin-10 and interferon-{gamma} production by human CD4+ T cells. Blood 89:1110.[Free Full Text]
  35. Rafiq, K., Bullens, D. M. A., Kasran, A., Lorré, K., Ceuppens, J. L. and Van Gool, S. W. et al. 2000. Differences in regulatory pathways identify subgroups of T cell-derived Th2 cytokines. Clin. Exp. Immunol. 121:86.[ISI][Medline]
  36. Yssel, H., de Waal Malefyt, R., Roncarolo, M. G. 1992. IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells. J. Immunol. 149:2378.[Abstract/Free Full Text]
  37. Rafiq, K., Charitidou, L., Bullens, D. M. A., Kasran, A., Lorré, K., Ceuppens, J. L. and Van Gool, S. W. 2000. Regulation of IL-10 production by human T cells. Scand. J. Immunol., in press.
  38. Kanner, S. B., Damle, N. K., Blake, J., Aruffo, A. and Ledbetter, J. A. 1992. CD2/LFA-3 ligation induces phospholipase-C{gamma}1 tyrosine phosphorylation and regulates CD3 signaling. J. Immunol. 148:2023.[Abstract/Free Full Text]
  39. Ley, S. C., Davies, A. A., Druker, B. and Crumpton, M. J. 1991. The T cell receptor/CD3 complex and CD2 stimulate the tyrosine phosphorylation of indistinguishable patterns of polypeptides in the human T leucemic cell line Jurkat. Eur. J. Immunol. 21:2203.[ISI][Medline]
  40. Pantaleo, G., Olive, D., Poggi, A., Kozumbo, W. J., Moretta, L. and Moretta, A. 1987. Transmembrane signaling via the T11-dependent pathway of human T cell activation: Evidence for the involvement of 1,2-diaglycerol and inositol phosphates. Eur. J. Immunol. 17:55.[ISI][Medline]
  41. Gassmann, M., Amrein, K. E., Flint, N. A., Schraven, B. and Burn, P. 1994. Identification of a signaling complex involving CD2, {zeta} chain and p59fyn in T lymphocytes. Eur. J. Immunol. 24:139.[ISI][Medline]
  42. Umehara, H., Huang, J.-Y., Kono, T. et al. 1998. Co-stimulation of T cells with CD2 augments TCR–CD3-mediated activation of protein tyrosine kinase p72syk, resulting in increased tyrosine phosphorylation of adapter proteins, Shc and Cbl. Int. Immunol. 10:833.[Abstract]
  43. Parra, E., Varga, M., Hedlund, G., Kalland, T. and Dohlsten, M. 1997. Co-stimulation by B7-1 and LFA-3 targets distinct nuclear factors that bind to the interleukin-2 promotor: B7-1 negatively regulates LFA-3-induced NF-AT DNA binding. Mol. Cell. Biol. 17:1314.[Abstract]
  44. Bleijs, D. A., de Waal-Malefyt, R., Figdor, C. G. and van Kooyk, Y. 1999. Co-stimulation of T cells results in distinct IL-10 and TNF-{alpha} cytokine profiles dependent on binding to ICAM-1, ICAM-2 or ICAM-3. Eur. J. Immunol. 29:2248.[ISI][Medline]
  45. Jeannin, P., Delneste, Y., Seveso, M., Life, P. and Bonnefoy, J. Y. 1996. IL-12 synergizes with IL-2 and other stimuli in inducing IL-10 production by human T cells. J. Immunol. 156:3159.[Abstract]
  46. Meyaard, L., Hovenkamp, E., Otto, S. A. and Miedema, F. 1996. IL-12-induced IL-10 production by human T cells as a negative feedback for IL-12-induced immune responses. J. Immunol. 156:2776.[Abstract]
  47. Morris, S. C., Madden, K. B., Adamovicz, J. J. et al. 1994. Effects of interleukin-12 on in vivo cytokine gene expression and Ig isotype selection. J. Immunol. 152:1047.[Abstract/Free Full Text]
  48. Finkelman, F. D., Madden, K. B., Cheever, A. W. et al. 1994. Effects of interleukin-12 on immune responses and host protection in mice infected with intestinal nematode parasites. J. Exp. Med. 179:1563.[Abstract]
  49. Wang, Z. E., Sheng, S., Corry, D. B. et al. 1994. Interferon-{gamma}-independent effects of interleukin-12 administered during acute or established infection due to Leishmania major. Proc. Natl Acad. Sci. USA 91:12932.[Abstract/Free Full Text]
  50. Wynn, T. A., Jankovic, S., Hieny, S. et al. 1995. IL-12 exacerbates rather than suppresses T helper 2-dependent pathology in the absence of exogenous IFN-{gamma}. Immunology 154:3999.
  51. Aman, J. A., Tretter, T., Eisenbeis, I. et al. 1996. Interferon-{alpha} stimulates production of interleukin-10 in activated CD4+ T cells and monocytes. Blood 87:4731.[Abstract/Free Full Text]
  52. Yoshinaga, S. K., Whorlskey, J. S., Khare, S. D. et al. 1999. T-cell co-stimulation through B7RP-1 and ICOS. Nature 402:827.[ISI][Medline]