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The Journal of Immunology, 2000, 165: 6908-6914.
Copyright © 2000 by The American Association of Immunologists

Costimulation Light: Activation of CD4+ T Cells with CD80 or CD86 Rather Than Anti-CD28 Leads to a Th2 Cytokine Profile1

Chris P. M. Broeren2,*, Gary S. Gray{dagger}, Beatriz M. Carreno{dagger} and Carl H. June3,{ddagger}

* Naval Medical Research Institute, Bethesda, MD 20814; {dagger} Genetics Institute, Cambridge, MA 02140; and {ddagger} Department of Molecular and Cellular Engineering, University of Pennsylvania, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To examine the role of CD28 and CTLA-4 in Th cell differentiation, we used a novel microsphere-based system to compare the effects of CD28 ligation by Ab or CD80/CD86. One set of beads was prepared by coating with anti-CD3 and anti-CD28 Ab. Another set of beads was prepared by immobilizing anti-CD3 and murine CD80-Ig fusion protein or murine CD86-Ig fusion protein on the beads. The three sets of beads were compared in their effects on the ability to activate and differentiate splenic CD4 T cells. When purified naive CD4+ cells were stimulated in vitro, robust proliferation of similar magnitude was induced by all three sets of beads. When cytokine secretion was examined, all bead preparations induced an equivalent accumulation of IL-2. In contrast, there was a marked difference in the cytokine secretion pattern of the Th2 cytokines IL-4, IL-10, and IL-13. The B7-Ig-stimulated cultures had high concentrations of Th2 cytokines, whereas there were low or undetectable concentrations in the anti-CD28-stimulated cultures. Addition of anti-CTLA-4 Fab augmented B7-mediated IL-4 secretion. These studies demonstrate that B7 is a critical and potent stimulator of Th2 differentiation, and that anti-CD28 prevents this effect.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tcells require at least two signals for efficient proliferation and cytokine production: an Ag-specific signal via the TCR complex and a second, also called a costimulatory, signal via a receptor-ligand interaction distinct from the TCR. A number of different pathways have been described to be able to deliver this costimulatory signal, including CD28/B7 (1), CD2/LFA-3 (2), OX2 (3), 4-1BB/4-1BBl (4), OX40 (5), ICAM-1/LFA-1 (6), and inducible costimulatory molecule (ICOS)4/B7h (7), of which the interaction of CD28 with the B7 molecules (CD80 and CD86) is best understood (8). Costimulation via CD28 has been shown to increase the IL-2 production of the T cell by stabilization of its mRNA (9) and by enhanced gene transcription due to a CD28-responsive element in the enhancer of the IL-2 gene (10). Besides interaction with CD28, CD80 and CD86 can also interact with CTLA-4 (CD152) on the T cell (11). This interaction has been shown to deliver a negative signal to the T cell by blocking IL-2 production and aborting cell cycle progression (12, 13).

To study the difference in activation of T cells in the presence or absence of the potentially negative interaction of CD80 or CD86 with CD152, we have developed a system in which we coupled anti-CD3 in combination with anti-CD28 mAb or the natural ligands CD80 or CD86 on cell-sized beads and used them as artificial APCs. In this way, signal 1 is delivered by the anti-CD3 mAb on the bead, while signal 2 is delivered by a mAb directed to CD28, which does not interact with CD152, or by a murine CD80-Ig or CD86-Ig fusion protein, which can interact with both CD28 and CD152 expressed on the T cell. Furthermore, the beads provide the ability to study the effects of anti-CD28 and B7 ligation in isolation and in the absence of cytokines such as IL-12 that are secreted by APCs. Using this model, we have discovered substantial differences between the effects of immobilized anti-CD28 and B7. Both forms of costimulation were able to induce comparable levels of proliferation; however, CD4 cells driven by B7-coated beads displayed a Th2 profile, whereas anti-CD28 resulted in a population of cells with a Th0/Th1 pattern of cytokine secretion. The effect did not appear to be due to selection based on experiments crossing cells over to the other form of stimulation resulting in switching of the cytokine secretion pattern.


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

Female BALB/cJ and C57BL/6 mice (6–12 wk of age) were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed under pathogen-free conditions at our institute according to institutional guidelines.

Antibodies

Anti-CD3 (145-2C11) (14) and anti-CTLA4 (UC10-4F10-11) (15) mAbs were kindly provided by Dr. J. A. Bluestone (University of Chicago, Chicago, IL). Anti-CD28 (PV-1) was generated (16) and purified in our laboratory. Anti-CD45R (B220/RA3-6B2), anti-CD8 (53-6.7), and anti-CD4 (GK1.5) were all obtained from the American Type Culture Collection (Manassas, VA) and purified in our laboratory. Anti-mCD80-PE (16-10A1), anti-mCD86-FITC (GL1), and anti-CD4-PE (GK1.5) were obtained from PharMingen (San Diego, CA). Anti-CD44-FITC (IM7.8.1) was obtained from Caltag (Burlingame, CA). hCTLA-4-Ig was produced at Genetics Institute (Cambridge, MA) and conjugated to FITC in our laboratory.

Preparation of mB7-Ig fusion proteins: mCD80-Ig and mCD86-Ig

Murine CD80-Ig and CD86-Ig fusion proteins were expressed in the pHTOP expression vector and constructed from cDNA encoding the signal and extracellular domains of murine CD80 or CD86 joined to the genomic DNA encoding the hinge, CH2, and CH3 regions of mouse IgG2a. The Ab hinge cysteines remained intact such that the expressed mCD80-Ig or mCD86-Ig was dimeric. Recombinant Chinese hamster ovary (CHO) cell lines, 74-18/0.02 and 73-15/0.02, expressing mCD80-Ig and mCD86-Ig, respectively, were grown in DMEM/Ham’s F-12 medium containing 10% FBS, 0.02 mM methotrexate, and 1.0 mg/ml G418. The cell lines were grown to confluence in roller bottles, and growth medium was replaced with serum-free medium. After 24 h, the medium was removed and clarified through 0.22-µm filters. This medium, containing the mCD80-Ig or mCD86-Ig, was passed over a recombinant protein A-Sepharose Fast Flow column (Pharmacia, Piscataway, NJ). The column was eluted with 20 mM citrate, pH 3.0; neutralized with 1 M Tris, pH 8.0; and reformulated in PBS, pH 7.2, by buffer exchange. Concentrations of mCD80-Ig and mCD86-Ig were determined by spectrophotometry (280 nm). Relative fusion protein concentrations, m.w. values, and purity were confirmed by SDS-PAGE.

Preparation of Ab-coated beads

Combinations of anti-CD3 (145-2C11) and anti-CD28 (PV-1) mAb, mCD80-Ig, or mCD86-Ig were covalently attached at 1:1, 1:5, or 1:10 molar ratios to polyurethane-coated tosyl-activated Dynabeads (Dynal, Lake Success, NY) as described earlier (13). To ensure equal loading during preparation of the anti-CD3-and/or anti-CD28-coated beads, the total amount of Abs was kept constant during loading using hamster Ig. Similarly, mouse IgG2a was used as a filler to assure equal loading during preparation of mCD80-Ig- or mCD86-Ig-coated beads.

Flow cytometric analysis and CFSE labeling

The beads were characterized after conjugation by flow cytometry to quantify the amounts of reagents that were bound. Beads were washed in cold PBS containing 5% FCS and 2 x 105 beads per sample were stained with pretitered amounts of FITC- or PE-conjugated anti-CD3, anti-CD28, anti-CD80, or anti-CD86 mAbs or with FITC-conjugated hCTLA-4-Ig as shown in Fig. 1Go. Single-color flow cytometry was performed on a FACScan (Becton Dickinson, Mountain View, CA) cytometer using standard acquisition/analysis software.



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  		FIGURE 1. Flow cytometric analysis of anti-CD3/anti-CD28 and anti-CD3/mB7-Ig-coated beads using goat anti-mouse-FITC (A) or hCTLA-4-Ig-FITC (B). Filled histogram represents anti-CD3/anti-CD28-coated beads. Dashed line represents anti-CD3/mCD80-Ig-coated beads. Solid line represents anti-CD3/mCD86-Ig-coated beads. There are three plots in B, with the dotted and dashed lines nearly overlapping. C, anti-CD80-PE; D, anti-CD86-FITC. Filled histogram represents anti-CD3/mCD80-Ig; open histogram represents anti-CD3/mCD86-Ig-coated beads.

 
Cells were labeled with the fluorescent dye CFSE using 1 µM CFDASE (Molecular Probes, Eugene, OR). CFSE labeling of lymphocytes was achieved as described (17) with some modifications (18).

Cell purification and activation

Single-cell suspensions were prepared from harvested spleens. After lysis of erythrocytes by use of ACK lysing solution (Biofluids, Rockville, MD), splenocytes were layered on 50% Percoll (Pharmacia) and centrifuged for 20 min at 1500 rpm at 4°C. Upon centrifugation, the pellet fraction was further purified by positive selection of the CD4 T cells using anti-CD4/L3T4-coated beads according to the manufacturer’s description (Dynal). Purification was monitored by FACS staining and routinely resulted in >95% CD4+ T cells, with <1% contaminating CD8+ or B220+ cells. For a number of experiments, naive and memory CD4+ T cells were isolated based on their expression levels of CD44. To do so, splenocytes were double-stained with CD4-PE and CD44-FITC and sorted on CD4+CD44dim (naive) and CD4+CD44bright (memory) cells using a FACSvantage (Becton Dickinson, EremBodegem-Aalst, Belgium), resulting in >99% pure cell populations. Cells were resuspended at 2 x 106 cells per ml in RPMI 1640 medium containing 5% heat-inactivated FCS (HyClone, Logan, UT), 25 mM HEPES (BioWhittaker, Walkersville, MD), 2 mM L-glutamine (Biofluids), 100 IU/ml of penicillin (Biofluids), 100 µg/ml streptomycin (Biofluids), and 5 µM ß-2-ME. Activation of the T cells was achieved by the addition of an equal number of coated beads. For secondary stimulation, live T cells were isolated from 7-day primary cell cultures using Ficoll-Hypaque density gradient centrifugation, and cells were washed and reseeded at 2 x 106 cells per ml in the presence of an equal number of fresh beads.

[3H]Thymidine incorporation assays

Purified T cells were plated at 5 x 104 cells/well in 96-well U-bottom plates (Costar, Gaithersburg, MD) at a final volume of 200 µl/well. Cells were activated with mAb-coated beads at a 1:1 cell/bead ratio. Cultures were pulsed at various time points with 1 µCi of [3H]thymidine and harvested 16 h later onto glass filters using a 96-well plate harvester. Incorporated radioactivity was measured using a liquid scintillation counter (Wallac, Gaithersburg, MD).

Cytokine-specific ELISA

The amount of IL-2, IL-4, IL-10, IL-13, or IFN-{gamma} in culture supernatants was determined by a cytokine-specific two-site ELISA according to the manufacturer’s description (PharMingen), using reference standard curves prepared with known amounts of recombinant cytokines.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
mCD80-Ig and mCD86-Ig retain their binding capacity for anti-mB7 mAbs and soluble hCTLA4-Ig after conjugation to beads

To compare effects of costimulation using anti-CD28 stimulation to ligation with the natural CD28 ligands CD80 and CD86, beads were prepared with covalently attached anti-CD3 and anti-CD28, or with anti-CD3 and mCD80-Ig or mCD86-Ig. The nominal composition of beads was a 1:10 ratio of anti-CD3 and anti-CD28 or anti-CD3 and either mCD80-Ig or mCD86-Ig. Anti-CD3 was coupled at a suboptimal ratio to the costimulatory ligand on the beads to more closely mimic the physiologic interaction of limiting MHC/TCR interaction with higher amounts of costimulation provided by APC. This approach also makes the test system more dependent on costimulatory signals. The effects of similar beads containing anti-CD3 and anti-CD28 on mouse T cells have been previously reported (12) and we have reported previously the effects of homologous beads on human T cells (13).

To test for retention of proper folding of the recombinant mB7-Ig fusion proteins after coupling them to the beads, binding of mAbs to the Ig-tail region of the mB7-Ig fusion proteins or to the mB7 molecule itself was analyzed by flow cytometry. Staining with CTLA-4-Ig was used to ensure that the CTLA-4/CD28 binding activity was retained after coupling the mCD80-Ig and mCD86-Ig fusion proteins to the beads. As indicated in Fig. 1GoA, the mIgG2a Fc regions of the mCD80-Ig and mCD86-Ig fusion protein-coated beads had equivalent staining intensities using a goat anti-mouse IgG2a mAb, confirming that similar amounts of mCD80-Ig and mCD86-Ig were incorporated into the bead preparations. Because both anti-CD3 and anti-CD28 are hamster mAbs, anti-CD3/anti-CD28-coupled beads served as a negative control in this FACS assay. Using hCTLA-4-Ig-FITC as a detection reagent, both mB7-Ig-coated beads showed positive staining that was equivalent (Fig. 1GoB). Fig. 1Go, C and D, shows staining with specific anti-CD80 (Fig. 1GoC) or anti-CD86 mAbs (Fig. 1GoD) of mCD80-Ig- and mCD86-Ig-coupled beads. These mAbs specifically stained the respective mCD80-Ig- and mCD86-Ig-coated beads as was expected. Together, the above results indicated that similar amounts of mCD80-Ig and mCD86-Ig were bound to the beads, and that similar amounts had retained binding for CTLA-4. However, given the present assay system, and given the different affinities of CD80 and CD86 for CTLA-4 (19), we do not expect to detect small differences in the relative amounts of reagents bound.

mCD80-Ig and mCD86-Ig coupled to beads can costimulate T cells

Previous reports have studied the functional effects of B7 expressed ectopically on cells (20, 21) or after adherence of B7-Ig to plastic plates (22, 23). The effects of B7 on CD4+ T cells in an APC-free system have not been reported, with the exception where B7 was tested in "trans" with the TCR signal being delivered by soluble Ab (23). To test the effects of B7 in "cis," we asked whether anti-CD3/mB7-Ig-coupled beads could costimulate the activation of CD4+ T cells and compared this to beads coupled with anti-CD3/anti-CD28. As indicated in Fig. 2Go, both mB7-Igs were able to enhance proliferation of purified CD4 T cells as thymidine incorporation was enhanced compared with cells stimulated with beads coupled with anti-CD3 only. At 72 h after activation, the anti-CD3/mB7-Ig-coated beads induced similar proliferation as measured by [3H]thymidine incorporation as did the anti-CD3/CD28-coated beads. However, the mCD86-Ig-induced proliferation was not as sustained because after 96 h of activation, the CD4+ T cells activated using either anti-CD3/mCD80-Ig or anti-CD3/anti-CD28-coated beads still showed a strong proliferative response, whereas the anti-CD3/mCD86-Ig-stimulated cells showed a diminished proliferative response equal to CD4+ T cells stimulated with beads coated with anti-CD3 only.



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  		FIGURE 2. Immobilized mCD80-Ig and mCD86-Ig induce CD4 cell proliferation similar to immobilized anti-CD28. [3H]thymidine incorporation (mean ± SE) of CD4+ T cells obtained from BALB/cJ mice after activation with beads coated with anti-CD3 plus control hamster Ig (ctrl-Ig), anti-CD3/CD28, anti-CD3/mCD80-Ig, or anti-CD3/mCD86-Ig. Filled columns indicate incorporation after 72 h; open columns indicate incorporation 96 h after activation. Cells cultured in medium only had <300 cpm thymidine incorporation (data not shown).

 
To further assess the relative abilities of the bead preparations to induce T cell division, we used CFSE-labeled T cells. CFSE segregates equally between daughter cells upon cell division, resulting in sequential halving of fluorescence intensity with each generation. T cells were labeled with CFSE and examined by flow cytometry after 48 h of culture (Fig. 3Go). A similar fraction of daughter and granddaughter cells was present in cultures after stimulation with anti-CD3/CD28 or anti-CD3/B7-Ig. In contrast, in cultures of cells stimulated with CD3 only, few viable cells were recovered, and few of those cells had divided twice. Thus, anti-CD28 and B7-Ig have quite similar abilities to induce the initial activation and proliferation of CD4+ T cells.



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  		FIGURE 3. CFSE-labeled splenocytes were cultured for 48 h in the presence of anti-CD3-coated beads (top left), anti-CD3/CD28-coated beads (bottom left), or beads coated with anti-CD3/mCD80-Ig or CD3/mCD86-Ig (right). The cells from individual wells were harvested, stained with PE-conjugated anti-CD4, and analyzed by flow cytometry. Histograms show the CFSE fluorescence profile of the live, CD4+ subset of the CFSE-labeled splenocytes.

 
Anti-CD3/mB7-Ig- and anti-CD3/anti-CD28-coupled beads induce distinct cytokine profiles

The role of CD28 costimulation in T cell differentiation remains controversial (24, 25, 26, 27, 28). To further address this issue, purified CD4+ T cells were activated using anti-CD3/anti-CD28- or anti-CD3/mB7-Ig-coated beads. Supernatants were collected daily and stored at -80°C, and cytokine-specific ELISAs were used to determine the amounts of cytokine produced. We were surprised to find that the production of Th2-like cytokines (IL-4, IL-10, and IL-13) was virtually completely restricted to CD4+ T cells stimulated with the mB7-Ig molecules (Fig. 4Go). The difference is most apparent in the case of IL-4, where high levels accumulated in the B7-Ig-stimulated cultures, whereas no measurable IL-4 was present in the anti-CD28-stimulated culture. In contrast, all three types of beads induced the secretion of comparable amounts of IL-2 and IFN-{gamma}. Interestingly, the production of IFN-{gamma} by anti-CD3/mCD86-Ig bead-activated cells did not subside at later time points as was seen for both anti-CD3/anti-CD28- and anti-CD3/mCD80-Ig bead-activated cells. Cells activated using beads coupled with only anti-CD3 did not have measurable cytokine secretion (data not shown).



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  		FIGURE 4. Cytokine production of T cells activated using anti-CD3/anti-CD28-coated beads ({blacksquare}), anti-CD3/mCD80-Ig-coated beads ({blacktriangleup}), anti-CD3/mCD86-Ig-coated beads (•), or anti-CD3 only. Cultures of cells stimulated with anti-CD3 only did not have any cytokine production and are therefore not depicted. Results are representative of three experiments.

 
It is possible that the differences in cytokine secretion could reflect differential consumption. To address this issue, we performed IL-4 mRNA PCR. We could readily detect IL-4 mRNA levels in the anti-CD3/B7-activated T cells but not in the anti-CD3/CD28-stimulated T cell populations (data not shown). Therefore, we feel it is unlikely that differential IL-4 consumption by the anti-CD3/CD28-stimulated cells can account for the observed differences. However, it is possible that differential rates of consumption contribute to the distinct levels of cytokines that we have observed.

The experiments shown in this paper are from experiments using beads coupled with a 1:10 ratio of anti-CD3 to anti-CD28 or B7-Ig (see Materials and Methods). Titration experiments were also performed using beads prepared at 1:1 and 1:5 ratios, and qualitatively similar results were obtained as IL-4 secretion was only observed when anti-CD3/B7-coated beads were used (data not shown). Thus the distinct cytokine profile induced by B7-Ig and anti-CD28 stimulation is observed at optimal and suboptimal levels of CD3 stimulation and is not the result of suboptimal anti-CD28 or B7-Ig stimulation.

We next determined whether the B7-mediated induction of IL-4 secretion was a reversible or stable phenotype. CD4+ T cells were first stimulated for 1 wk in the presence of anti-CD3/CD28, anti-CD3/mCD80-Ig, or anti-CD3/mCD86-Ig. The cells were then harvested, the beads removed, and the cells restimulated with a fresh preparation of the original beads or crossed over to stimulation with the alternative beads for an additional 4 days (Fig. 5Go). Cells stimulated with anti-CD28 for the first and second cycles of stimulation had low levels of IL-4 secretion and, as was expected, cells stimulated and restimulated with anti-CD3/B7-Ig had high levels of IL-4 secretion. In contrast, restimulation of T cells with anti-CD3/mB7-Ig-coated beads that had been previously activated using anti-CD3/anti-CD28-coated beads induced high level secretion of IL-4. Conversely, restimulation of T cells originally activated with anti-CD3/mB7-Ig with anti-CD3/anti-CD28-coated beads resulted in greatly reduced IL-4 production. Finally, in separate experiments, we found that B7-Ig-mediated production of the Th2-like cytokines was not restricted to CD4+ T cells derived from BALB/cJ mice. Experiments using C57BL/6 CD4+ T cells showed similar results (data not shown).



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  		FIGURE 5. Anti-CD28-mediated reversal of IL-4 secretion after CD80 and CD86 stimulation. CD4 cells were first stimulated using the indicated type of coated beads for 7 days and then restimulated as indicated (second stimulation). IL-4 production as measured by ELISA 4 days after restimulation. A representative experiment of three experiments is shown.

 
Interaction of mB7-Ig with CTLA-4 is not crucial for IL-4 production

One obvious explanation for the functional differences between the mB7-Ig fusion proteins and anti-CD28 is the possibility of the fusion proteins to interact with CTLA-4 on the activated T cell. In general, CTLA-4 provides negative effects on T cell activation (29); however, in some instances, CTLA-4 can provide a positive signal, resulting tumor rejection (30), and recent data indicates that CTLA-4 can limit Th2 cell differentiation (31). Therefore, we reasoned that CTLA-4 engagement could explain the differential cytokine secretion profile that we observed in our system. We blocked B7-CTLA-4 interaction on the T cell side by the addition of Fab of anti-CTLA-4 mAb 4F10. As shown in Fig. 6Go, this did not result in a decreased production of IL-4 by the CD4+ T cells, but rather in a further increase in the amount of IL-4 produced by the T cells. CD4+ T cells activated using anti-CD3/mCD80-Ig-coupled beads in the presence of anti-CTLA-4 Fab had a modest but reproducible 2-fold enhancement of IL-4 secretion. Addition of anti-CTLA-4 Fab to the anti-CD3/mCD86-Ig bead-activated T cells did not result in a significant increase in the amount of IL-4 produced. The proliferation of the anti-CD3/mCD80-Ig or anti-CD3/mCD86-Ig bead-activated CD4+ T cells was not changed significantly by the addition of the anti-CTLA-4 Fab preparation (data not shown).



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  		FIGURE 6. IL-4 production as measured by IL-4-specific ELISA 4 days after stimulation with anti-CD3/mCD80-Ig- or anti-CD3/mCD86-Ig-coated beads in the presence or absence of 50 µg/ml anti-CTLA-4 Fab mAb 4F10 or control hamster IgG. Representative experiment of three experiments is shown.

 
Anti-CD3/anti-CD28/mB7-Ig-coated beads do not induce IL-4 production

To test whether the Th2-like cytokine profile induced by the mB7-Ig fusion proteins was dominant over anti-CD28 (or vice versa), beads were prepared by coupling anti-CD3, anti-CD28, and mB7-Ig together on a single bead. CD4+ T cells stimulated in primary culture with these ‘trivalent’ beads proliferated equivalently to the cells stimulated with ‘bivalent’ beads (data not shown). Supernatants from cultures of CD4+ T cells thus activated were tested for the production of IL-4 after 4 days of activation. As shown in Fig. 7Go, stimulation of cells with anti-CD3/m80-Ig or mCD86-Ig resulted in substantial IL-4 production, whereas cells stimulated with anti-CD3/CD28 had the expected low level secretion. Interestingly, the addition of anti-CD28 to anti-CD3/mCD80-Ig- or anti-CD3/mCD86-Ig-coated beads abrogated IL-4 production, decreasing the IL-4 levels to that observed after stimulation by anti-CD3/28 only.



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  		FIGURE 7. Effect of simultaneous stimulation with anti-CD28 and CD80- or CD86-Ig. CD4+ T cells were cultured with beads coated with anti-CD3 and hamster Ig; anti-CD3 plus anti-CD28; anti-CD3 plus anti-mCD86-Ig or anti-CD3/CD28/mCD80- or mCD86-Ig. The ratio of anti-CD3 to anti-CD28 to mCD80 or mCD86-Ig was 3.75:3.75:30. Similar results were also obtained when beads were prepared with anti-CD28 and B7-Ig titrated in the range from 3.75 to 30, whereby total amounts were kept at 37.5. Hamster Ig or mouse IgG2a was used as a filler Ab as described in Materials and Methods to ensure that equivalent amounts of anti-CD28 and B7-Ig were coupled to the beads. IL-4 secretion was measured by IL-4-specific ELISA 4 days after stimulation with the indicated beads. Representative experiment from two experiments is shown.

 
Distinct cytokine profiles of highly purified naive and memory T cells both show similar cytokine profiles after activation with anti-CD3/CD28- vs anti-CD3-B7-Ig-coated beads

To exclude that the observed differences in cytokine response after activation of CD4 T cells with anti-CD3/CD28 or anti-CD3/B7-Ig coated beads are due to a different responsiveness of naive and previously activated/memory T cells, activation studies were performed using highly purified, FACS-sorted CD4+CD44dim (naive) and CD44bright (memory) T cells. As indicated in Fig. 8Go, both populations of T cells showed a differential IL-4 production after activation. As expected, the amounts of cytokine produced by the CD44bright T cells were higher but, still, anti-CD3/CD28-activated T cell cultures (if at all) contained IL-4, whereas both anti-CD3/CD80-Ig and CD86-Ig showed increased levels of IL-4 in naive and memory CD4 cells.



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  		FIGURE 8. IL-4 production by FACS-sorted CD4+CD44 dim and bright cells as measured by IL-4-specific ELISA 4 days after stimulation with anti-CD3/mCD80-Ig- or anti-CD3/mCD86-Ig-coated beads. Representative experiment of three experiments is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The role of B7 molecules in the activation and differentiation of T cells has been widely studied (35). Some of these studies have indicated that CD80 and CD86 have differential effects on T cell differentiation (24, 36), whereas others have not been able to lend support to this finding (20, 25, 37). To begin to address this controversy, and to uncover differences in using anti-CD28 vs the natural ligands CD80 and CD86, we have developed an in vitro system in which purified CD4+ T cells were activated using cell-sized beads coated with mAbs directed to CD3 and CD28 or the mB7-Ig fusion proteins mCD80-Ig or mCD86-Ig. In theory this APC-free system permits the ability to distinguish between effects due to cytokine secretion and other accessory molecules on APC from the concomitant B7 costimulation. Apart from the above-mentioned benefits, the use of recombinant mB7-Ig fusion proteins on beads also has some intrinsic differences with the natural activation. First of all, attachment of the reagents to the bead surface might be different from the interactions of the ligands freely moving in a lipid bilayer. However, recent studies by Viola et al. (38) have indicated that raft migration, which is widely recognized as an early and important step in T cell activation, can be observed using anti-CD3 and anti-CD28 coupled to beads. Secondly, the native B7 molecules on cells are believed to act as monomers and thus differ from the dimeric recombinant B7-Igs we use. However, recent crystallographic studies have indicated that soluble forms of CD80 readily form homodimers (39) and have implied that this dimeric is the naturally preferred complex. All bead preparations containing anti-CD3 plus anti-CD28 or B7-Ig induced similar levels of CD4+ T cell proliferation. Thus the beads coated with mB7-Ig fusion proteins appear to be a useful model to uncover functional differences between anti-CD28 and CD80 and CD86 in the presence of anti-CD3.

Analysis of the cytokine profiles of the anti-CD3/mB7-Ig-coupled beads compared with the anti-CD3/CD28-coupled beads revealed a striking difference in the production of the Th2-like cytokines IL-4, IL-10, and IL-13. Thus, IL-4 and IL-10 were hardly detectable, whereas IL-13 was produced at only low amounts after anti-CD28 costimulation. This was in marked contrast to the large amounts of IL-4, IL-10, and IL-13 secreted by T cells activated using anti-CD3/mB7-Ig-coated beads. This difference did not simply reflect differences in the level of activation, as rates of T cell division and the secretion of IL-2 was similar after anti-CD28 and B7 stimulation. These differences are particularly notable in that they were observed during the first cycle of stimulation and did not require multiple rounds of stimulation that are often required to observe distinct cytokine profiles.

In contrast to the striking differences between anti-CD28 and mB7-Ig costimulation in our in vitro system, the differential effects between mCD80-Ig and mCD86-Ig were modest. In all three individual experiments, we have seen a higher production of IL-4, IL-10, and IL-13 using anti-CD3/mCD86-Ig, which would be consistent with a stronger skewing toward Th2 after activation with mCD86-Ig. In a recent study using mB7-Ig to study differentiation of naive CD8+ T cells, no intrinsic differences between mCD80 and mCD86-Ig were revealed (23). However, we also observed a rather atypical production of IFN-{gamma} after the activation of T cells with anti-CD3/mCD86-Ig-coated beads, which does not fit with the normal Th1/Th2 phenotype. However, this late production of IFN-{gamma} might explain the reduced proliferation observed in Fig. 2Go, where mCD86-Ig-stimulated T cells showed decreased proliferation after 4 days, whereas mCD80-Ig-stimulated cells had sustained proliferation.

There are several explanations possible for the different cytokine response seen after activation of T cells with anti-CD28 or mB7-Ig. One mechanism would be the differential binding and signaling of CTLA-4 by the mB7-Ig and not anti-CD28 beads. Although some studies have described a costimulatory role for CTLA-4 (30), most reports have implicated CTLA-4 in dampening of the immune response. Blocking of CTLA-4 signaling via the mB7-Ig with anti-CTLA-4 Fab did not decrease, but rather further increased the production of IL-4. This is consistent with a recent study describing increased IL-4 production by CD4+ T cells after in vivo treatment with anti-CTLA4 mAbs in a staphylococcal enterotoxin B (SEB) model (40).

A second explanation would be that apart from CD28 and CTLA-4, CD4+ T cells express a third ligand for B7. Recently, Hutloff et al. described ICOS (41), a molecule structurally and functionally related to CD28. However, as indicated by the authors, it is unlikely that ICOS binds to B7 because of the absence of the MYPPPY binding motif, which has been described earlier to be of prime importance for the recognition of CD28 and CTLA4 by CD80 and CD86 (19). Recent studies indicate that the ICOS ligand does not bind to CD28 or CTLA-4 (7, 42) However, the existence of still another B7 ligand cannot be formally excluded based on these results.

A third and, in our opinion, the most favored explanation would be that anti-CD28 has a higher affinity than B7-Ig for CD28 and therefore induces more sustained cross-linking of CD28 than ligation of CD28 by the natural B7 ligands. The observation that beads coupled with anti-CD3/anti-CD28 and mB7-Ig did not induce a Th2 response fits best with this latter explanation of "overstimulation" of T cells with anti-CD28. Thus our data are most in accord with a "strength of signal" hypothesis, whereby the strength and/or duration of signal 2 contributes to differentiation toward Th1 or Th2. Indeed, our previous data in human CD4 cells is consistent with this hypothesis where we found that soluble anti-CD28 and anti-CD28 immobilized on separate surfaces led to IL-4 and IL-5 secretion, whereas anti-CD28 coimmobilized to the same bead resulted in Th0/Th1 cytokine secretion that was maintained over multiple cycles of restimulation (25, 26). There is precedence for this in other systems where the strength of signal 1 can affect the development of a Th2 profile (43, 44). Interestingly, Nunes et al. have described earlier a difference in p21ras activation after triggering of CD28 with Abs as compared with the natural ligand B7-1, in that anti-CD28 and not B7-1 was able to activate p21ras (45). Although this was in a different in vitro system using anti-human CD28 mAbs and hB7-1-transfected L cells, a similar explanation could apply to our model system as well.

The physiologic implications of our findings at present are unclear. It is possible that in vivo equivalents of "strong" and "weak" CD28 ligation could exist. For example, high level expression of B7 on mature dendritic cells would be expected to induce stronger and/or more prolonged CD28 ligation, whereas immature dendritic cells and nonprofessional APC have lower levels of B7 expression (46, 47).

The mechanisms involved in the regulation of Th1 and Th2 differentiation are complex and appear to involve a requirement for cell division, the cytokine milieu, and signal strength (reviewed in Ref. 48). A surprising finding in this study was in Fig. 5Go, where the apparent polarization toward the Th1 or Th2 cytokine pattern could be reversed, depending on the nature of lymphocyte restimulation. It is likely that we have not observed a switch in the commitment of cells toward one cytokine pattern or another, but rather a selection upon restimulation that favors the growth or death of subsets of cells that are present in the still heterogeneous population of CD4+ T cells. However, the data presented in Fig. 8Go, where highly purified naive or memory cells were used, rule out a naive/memory subset division as an explanation for the observed differences.

In conclusion, we have shown that recombinant mB7-Ig fusion proteins can be used as costimulatory molecules when coupled to cell-sized beads in the presence of anti-CD3 mAbs. In addition, we have shown that these mB7-Ig-coated beads induce a rapid polarization toward a Th2 type of T cell response, whereas anti-CD3/anti-CD28-coated beads induce or maintain a Th0/Th1 type of response. Given that the differentiation of CD4+ T cells into a Th1 vs Th2 phenotype profoundly influences the outcome of autoimmune and infectious diseases, these results have important implications for understanding how B7-CD28/CTLA4 blockade or stimulation can be effectively used to manipulate cytokine production in vivo.


   Acknowledgments
 
We thank Doug Smoot for help with flow cytometry, Nancy Craighead and Al Black for help with reagent preparation, Corlinda ten Brink for technical assistance, Drs. Patrick Blair and David Harlan for support and helpful comments, and Dr. Steve Reiner for review of the manuscript.


   Footnotes
 
1 The Naval Medical Research and Development Command sponsored this work. C.P.M.B. was sponsored by a TALENT fellowship from the Dutch organization for scientific research (N.W.O.). Back

2 Current address: University of Utrecht, Institute of Infectious Diseases and Immunology, Yalelaan 1, 3584 CL Utrecht, The Netherlands. Back

3 Address correspondence and reprint requests to Dr. Carl H. June, Biomedical Research Building II/III, Room 554, 421 Curie Boulevard, Philadelphia, PA 19104-6160. Back

4 Abbreviation used in this paper: ICOS, inducible costimulatory molecule. Back

Received for publication October 15, 1999. Accepted for publication September 1, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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