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The Journal of Immunology, 1999, 163: 4819-4825.
Copyright © 1999 by The American Association of Immunologists

Mutual Regulation Between B7-1 (CD80) Expressed on T Cells and IL-41

A. Nicola Schweitzer2 and Arlene H. Sharpe

Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have used T cells from B7-1-deficient TCR transgenic DO11.10 mice to demonstrate a functional role for B7-1 on T cells. B7-1-deficient DO11.10 T cells produce more IL-4 than wild-type DO11.10 T cells, suggesting that B7-1 expressed by T cells regulates the differentiation of IL-4-producing cells. In addition, we found that IL-4 inhibits B7-1 expression by wild-type DO11.10 T cells. Our results suggest that there is a reciprocal relationship between B7-1 expressed on T cells and IL-4 production, which results in a modulatory feedback loop. When high levels of IL-4 are produced by T cells, B7-1 expression by T cells is inhibited, which allows amplification of IL-4 production by these T cells. When low levels of IL-4 are produced by T cells, B7-1 expression by these T cells is increased, and a further reduction in IL-4 production follows. However, in addition to being influenced by IL-4, B7-1 expression by T cells is affected by peptide concentration and by B7 costimulation from APCs. The studies presented here demonstrate that B7-1 on T cells as well as on APCs regulates IL-4 production. However, whereas B7-1 expression on APCs can promote IL-4 production, IL-4 production is inhibited by B7-1 on T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Related molecules B7-1 (CD80) and B7-2 (CD86), when expressed on APCs, deliver an important costimulatory signal through the T cell surface molecule CD28 (1, 2, 3, 4, 5) which can permit the optimal Ag-specific activation of T cells and prevent the induction of anergy (6, 7, 8, 9, 10, 11, 12, 13, 14, 15). More recently, it has emerged that interaction between B7 molecules on APCs and CD28 on the T cell is essential for high levels of IL-4 production and Th2 differentiation (16, 17, 18). B7-1 and B7-2 also bind CTLA-4 (3) (CD152), an Ig superfamily member with homology to CD28. CTLA-4 is rapidly up-regulated following T cell activation (1, 19) and can deliver a negative signal to the activated T cell (19, 20, 21, 22).

B7 molecules are also expressed on T cells. B7-2 is constitutively expressed at low levels on murine T cells (23, 24) and is up-regulated upon T cell stimulation (24). Following repetitive stimulation of T cells or stimulation of T cell clones, B7-1 is expressed (23, 25, 26, 27), and B7-2 is down-regulated (23). High levels of B7 expression on T cells are observed in a number of autoimmune and infectious states (28, 29, 30, 31). However, the functional significance of B7 on T cells is unclear. An APC role has been suggested, where B7 expressed on T cells could enhance ongoing T cell responses (28, 29) or inhibit responses through induction of MHC class I-restricted counter-regulatory T cells (23). Alternatively, B7 on T cells may deliver a negative signal through CTLA-4 (21). Transgenic mice that constitutively express B7-1 or B7-2 on T cells show no overt differences from wild-type mice following immunization in vivo, although the ability to deliver costimulation through CD28 has been demonstrated during primary stimulation with anti-CD3 mAb in vitro (32). However, these mice show anomalies in B cell development as a result of transgene expression (32, 33) and may therefore not be a suitable model for functional studies.

T cells from B7-deficient (KO)3 mice (34, 35) provide a means to directly investigate the role of B7 on T cells during Ag-specific T cell responses. Anti-B7 mAbs cannot be used for this purpose because they will bind to B7 on APCs as well as T cells. For the studies presented here, we have intercrossed BALB/c B7-1 KO mice with DO11.10 TCR transgenic mice, which express the {alpha} and ß genes of a TCR recognizing OVA peptide 323–339 (pOVA) in the context of I-Ad, to generate a source of B7-1 KO as well as wild-type TCR transgenic T cells. We have compared T cells isolated from B7-1 KO DO11.10 mice with T cells from wild-type DO11.10 mice during stimulation with wild-type APCs to determine whether B7-1 on T cells is functional.

We report here that B7-1 KO DO11.10 T cells produce more IL-4 than wild-type DO11.10 T cells. In addition, we observe that IL-4 inhibits the expression of B7-1 by wild-type DO11.10 T cells. Consistent with this, the difference in IL-4 production between B7-1 KO and wild-type T cells diminishes under conditions where high levels of IL-4 are induced in wild-type T cells. These results suggest that B7-1 on T cells may be part of a feedback loop whereby conditions promoting high levels of IL-4 production would inhibit B7-1 expression on T cells and thereby amplify IL-4 production, while conditions unfavorable for IL-4 production would promote T cell expression of B7-1, further inhibiting IL-4 production.


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

Mice were maintained in a pathogen-free facility and were used at 8–15 wk of age. DO11.10 TCR transgenic mice (wild-type DO11.10 mice), which recognize OVA peptide 323–339 (see below) in association with I-Ad (36), were provided by Dr. Dennis Loh (Hoffmann-La Roche, Nutley, NJ) and maintained in our facility by breeding with BALB/c mice. BALB/c mice were originally obtained from Taconic Farms (Germantown, NY) and bred within the facility. Mice lacking B7-1 (B7-1 KO) were derived on a 129 background (34) and then bred for at least 10 generations onto BALB/c. BALB/c B7-1 KO mice were intercrossed with DO11.10 mice, and B7-1 heterozygous DO11.10 mice were backcrossed with B7-1 KO BALB/c mice to generate B7-1 KO DO11.10 mice. Pooled spleen cell and lymph node cell suspensions prepared from wild-type or B7-1 KO DO11.10 mice were stimulated for 4 days with 10 µg/ml LPS (Sigma, St. Louis, MO) and 20 µg/ml dextran sulfate, and B7-1 expression was analyzed by FACS to confirm the genotype. Mice lacking both B7-1 and B7-2 (B7dKO) were derived on a 129 background (35) and were backcrossed for three generations with BALB/c mice. At the F2 generation, mice were typed for expression of the MHC H-2d allele and lack of expression of the H-2b allele, by FACS, and intercrossed. Wild-type mice derived from parallel backcrosses of 129 with BALB/c were used as controls in these experiments. Stat6 KO (37) DO11.10 mice were provided by Dr. M. Grusby (Harvard School of Public Health, Boston, MA).

Peptide

pOVA was obtained, HPLC purified, from the Beckman Center, Stanford University Medical Center (Palo Alto, CA). The amino acid sequence is as follows: Ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu-Ala-Gly-Arg-COOH.

Abs and recombinant cytokines for culture

mAb used in the purification of APCs and T cells were produced from hybridomas obtained from American Type Culture Collection (Manassas, VA; GK1.5 (anti-CD4), ADH4 (anti-CD8), M5/114 (anti-class II I-Ab,d, I-Ed,k)). 11B11 (anti-IL-4) and rIL-4 were provided by Dr. Abul Abbas (Harvard Medical School), and anti-Thy1.2 was purchased from Serotec (Oxford, U.K.).

APC preparation

APCs were prepared from either whole spleen cells or whole spleen cells depleted of T cells by treating with anti-CD4 (GK1.5), anti-CD8 (ADH4), and anti-Thy1.2 mAb followed by Rabbit Low-Tox complement (Accurate Chemical & Scientific, Westbury, NY), and APC proliferation was inhibited by treating with 50 µg/ml mitomycin C at 37°C for 40 min (Sigma).

T cell preparation

Naive CD4 T cells were prepared by either negative or positive selection, as indicated in the figure legends, from pooled spleen and lymph node cell suspensions from DO11.10 mice. For negative selection, adherent cells were removed by passage over nylon wool columns. Remaining APCs and CD8+ T cells were removed by treating recovered cells with anti-CD8 (ADH4) and anti-class II (M5/114) mAb followed by complement. For positive selection, CD4 T cells were purified using Dynabeads conjugated to an anti-CD4 mAb, according to the manufacturer’s instructions (Dynal, Oslo, Norway). Briefly, cells were incubated with Dynabeads on a rocking platform for 45 min at 4°C. Bead-cell conjugates were washed using magnetic separation, then resuspended vigorously in the presence of Detachabead and incubated for an additional 45 min on a rocking platform at 4°C, and purified cells were separated magnetically from the remaining beads and washed.

Cell culture

T cells (2 x 105/ml unless otherwise stated) were incubated with mitomycin C-treated APCs (106/ml unless otherwise stated) with or without pOVA (concentration as indicated in figure legends) in RPMI 1640 supplemented with 10% heat-inactivated FCS (Sigma), 5 x 10-5 M 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin B, 10 mM HEPES (all from Life Technologies, Grand Island, New York), and 15 µg/ml gentamicin (BioWhittaker, Walkersville, MD). Cultures were established in 24-well plates at 2 ml/well or in 96-well plates at 200 µl/well. For restimulation of primed T cells, dead cells were removed by density gradient separation over Ficoll Hypaque (Organon Teknika, Durham, NC) after 4 days, and remaining T cells were rested overnight in medium before restimulating in 24- or 96-well plates at 106/ml (final concentration) with 106/ml (final concentration) fresh APCs obtained from wild-type BALB/c mice in the presence of 1 µg/ml pOVA. In some experiments, as indicated, T cells were primed or differentiated into Th1 or Th2 phenotypes by incubating pooled spleen and lymph node cell suspensions (at a final cell concentration of 106/ml) with peptide (1–10 µg/ml) alone or with either IL-4-neutralizing mAb 11B11 (a dilution of hybridoma ascites determined to be optimal for blocking IL-4 production and Th1 differentiation in these cultures, or 10 µg/ml purified ascites) for Th1 differentiation or rIL-4 (4000 U/ml final concentration) for Th2 differentiation. Primed T cells were prepared by recovering cells after 3 days of culture and treating with anti-class II I-Ad (M5/114) and anti-CD8 (ADH4) mAbs, followed by Rabbit Low-Tox complement (Accurate Chemical & Scientific) and rested overnight in medium before restimulating.

Cytokine analysis

Cytokine levels in culture supernatants collected 48 h after the initiation of either primary or secondary culture were analyzed by ELISA. mAbs and recombinant cytokine standards used in the ELISAs were obtained from PharMingen (San Diego, CA) for IL-2, IL-4, and IFN-{gamma} analysis or from Genzyme (Cambridge, MA) for some IFN-{gamma} analyses. Lower limits of detection, as determined using a standard curve, were as follows: for IL-2, 20–40 pg/ml; for IL-4, <20 pg/ml; and for IFN-{gamma}, 30–120 pg/ml.

FACS staining and analysis

To evaluate B7-1 expression on T cells, cell suspensions were stained with directly conjugated mAb (anti-CD4.CyC and either hamster IgG-FITC or anti-B7-1.FITC (clone 16-10A1) obtained from PharMingen (San Diego, CA) at a final concentration of 5 µg/ml in PBS containing 1% BSA (Sigma fraction V) and 0.02% sodium azide. The proportion of CD4 T cells expressing B7-1 was determined by gating on CD4 T cells and assessing the percentage of CD4 T cells lying within a gate that excluded all but 0.5–1.5% (depending on the experiment, but standardized between samples within an experiment) of cells stained with the isotype control mAb. Stained cells were analyzed on a FACStarPlus machine (Becton Dickinson, Mountain View, CA) using CellQuest software for the Macintosh. Before staining activated cells, dead cells were removed by density gradient centrifugation on Ficoll Hypaque (Organon Teknika).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Secondary production of IL-4 by B7-1 KO T cells is enhanced compared with that produced by wild-type T cells

We have previously identified conditions under which purified naive DO11.10 T cells primed with wild-type APCs and pOVA and rested overnight produce IL-2, IL-4, and IFN-{gamma} upon restimulation with fresh wild-type APCs and pOVA (17, 18). To determine whether B7-1 on T cells influences T cell differentiation, we primed naive wild-type or B7-1 KO DO11.10 T cells according to this protocol and analyzed secondary cytokine production. In three separate experiments, we observed that B7-1 KO T cells produced more IL-4 than wild-type T cells (Fig. 1Go and Table IGo). In experiments in which high levels of IL-4 were produced by wild-type T cells, the enhancement of IL-4 production by B7-1 KO T cells was less striking than in experiments in which wild-type T cells produced lower levels of IL-4. Similar results were obtained when DO11.10 T cells were stimulated with two different peptide concentrations within a single experiment. Priming with 1 and 10 µg/ml pOVA induced 1328 and 2100 pg/ml of IL-4 production, respectively, from wild-type T cells. IL-4 production by B7-1 KO T cells primed at these two peptide concentrations was 2.2- and 1.3-fold greater, respectively, than that produced by wild-type T cells. Thus, higher levels of IL-4 production by wild-type T cells are, indeed, associated with a less striking increase in the production of IL-4 by B7-1 KO T cells.



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  		FIGURE 1. Cytokine production following secondary stimulation of B7-1 DO11.10 T cells. CD4 T cells were purified from wild-type or B7-1 KO DO11.10 mice by negative selection using nylon wool columns followed by Ab/complement depletion. T cells (2 x 105/ml) were primed for 4 days with 106/ml T-depleted mitomycin C-treated spleen cells and 2.5 µg/ml pOVA, recovered by density gradient centrifugation, rested overnight, and restimulated at 105/ml with 106/ml fresh mitomycin C-treated T-depleted spleen cells and 1 µg/ml pOVA. Cytokine production was measured from supernatants obtained on day 2 of secondary culture. Similar experiments were performed four times as detailed in Table IGo and the text.

 

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  		Table I. Cytokine production by B7-1 KO DO11.10 T cells1

 
B7-1 KO DO11.10 T cells often produced less IFN-{gamma} than wild-type DO11.10 T cells, although the magnitude of the difference was less striking than that for IL-4 production (Fig. 1Go and Table IGo). Production of IL-2 was consistently similar between wild-type and B7-1 KO T cells (Fig. 1Go and Table IGo). Proliferation of wild-type and B7-1 KO DO11.10 T cells was also compared during primary and secondary stimulation. There was only a modest (up to 2-fold) and not reproducible increase in B7-1 KO over wild-type proliferation (data not shown).

Expression of B7-1 during CD4 T cell activation is regulated by IL-4

Because the difference in IL-4 production by wild-type and B7-1 KO DO11.10 T cells was less striking when the concentration of IL-4 produced by wild-type T cells was higher, we examined whether IL-4 affected B7-1 expression on activated T cells.

Under stimulatory conditions similar to those used in the functional assays above, B7-1 expression was maximal on days 3–4 of a 4-day culture (data not shown). Of note, expression was markedly abrogated when culture was performed in the presence of rIL-4. Conversely, expression was enhanced when anti-IL-4 mAb was added during culture (Fig. 2Goa). A similar effect was observed when T cells were primed in the presence of rIL-4 or anti-IL-4 mAb, then primed T cells were recovered after 3 days, rested overnight, and restimulated in the presence of fresh APCs (Fig. 2Gob). Priming under neutral conditions led to moderate levels of B7-1 expression on day 3 of secondary culture. Priming under the Th2-promoting (high levels of secondary IL-4 production, no IFN-{gamma}) condition of rIL-4 addition abrogated expression of B7-1. Priming under the Th1-promoting (no IL-4 production, high levels of IFN-{gamma} production) condition of anti-IL-4 addition dramatically enhanced B7-1 expression on T cells. We saw no evidence of B7-1 expression in the absence of pOVA stimulation under any of these priming conditions.



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  		FIGURE 2. Effect of IL-4 on B7-1 expression by wild-type DO11.10 T cells. a, CD4 T cells were purified from wild-type DO11.10 mice by positive selection using magnetic beads, and 105/ml CD4 T cells were stimulated with 10 µg/ml pOVA, 106/ml mitomycin C-treated spleen cells, and rIL-4 at 4000 U/ml or anti-IL-4 mAb 11B11 at an optimal dilution of hybridoma ascites, as indicated. On day 4 of culture, live cells were recovered by density gradient centrifugation and surface stained with anti-B7-1-FITC (thick lines) or a hamster isotype control (thin lines) in relation to anti-CD4-CyC. b, Primed T cells were recovered by mAb/complement depletion after stimulation of whole spleen and lymph node cells (106/ml) from wild-type DO11.10 mice for 3 days with 1 µg/ml pOVA and the indicated addition (as in a but anti-IL-4 mAb as purified ascites at 10 µg/ml), rested overnight, and restimulated at 105/ml with 1 µg/ml pOVA and 106/ml mitomycin C-treated spleen cells. B7-1 staining was assessed as described in a. No B7-1 was detected in the absence of stimulation. Data presented in this figure are representative of at least two comparable experiments.

 
These data indicate that B7-1 expression is inhibited by IL-4, whether that IL-4 is exogenously added or produced by the stimulated T cells. Consequently, when the concentration of IL-4 within a culture is high, wild-type T cells will appear similar to B7-1 KO T cells, in that B7-1 expression will be low or absent. As a result, wild-type and B7-1 KO T cells would be expected to produce similar amounts of IL-4. Conversely, when the concentration of IL-4 in the culture is low, expression of B7-1 on wild-type T cells will be high, and a more marked inhibition of IL-4 production by B7-1 expressed on the wild-type, but not B7-1 KO, T cells would be expected. This is consistent with our functional observations.

B7-1 expression on T cells decreases with increasing density of Ag-specific T cells during stimulation

We often observed that B7-1 expression on T cells was lower during primary culture of whole spleen cell or mixed spleen and lymph node cell suspensions from DO11.10 mice than when purified CD4 T cells from DO11.10 mice were stimulated in the presence of mitomycin C-treated BALB/c spleen cells. A major difference between the two protocols was in the ratio of DO11.10 T cells to APCs. Among whole spleen/lymph node cell preparations, T cells from DO11.10 mice represented 35–40% of all cells in the culture, in contrast to only 10–20% of cells in cultures containing purified subpopulations. Because the total number of B220 cells was similar under the two protocols, we tested the hypothesis that the density of Ag-specific T cells influences B7-1 expression during stimulation by diluting spleen and lymph node cells from DO11.10 mice with spleen and lymph node cells from BALB/c mice. Thus, the total APC:T cell ratio remained the same, but the fraction of T cells that were DO11.10 derived decreased.

The results presented in Fig. 3Goa show that B7-1 expression was, indeed, enhanced as the density of DO11.10 T cells decreased. Assessment of cytokine production under the three conditions examined demonstrated that IL-4 production decreased with decreasing density of DO11.10 T cells, consistent with previous observations (17). In this situation IFN-{gamma} production also diminished with decreasing DO11.10 T cell density (7708, 2414, and 820 pg/ml, respectively), supporting the role for IL-4, rather than the absence of IFN-{gamma}, in influencing B7-1 expression. Indeed, treating whole lymph node/spleen cell suspensions from DO11.10 mice with anti-IL-4 mAb enhanced B7-1 expression on stimulated T cells (Fig. 3Gob), confirming an inhibitory role for IL-4 under these conditions. This result demonstrates that parameters (in this case the number of peptide-specific T cells) markedly affecting the endogenous level of IL-4 production can influence the level of B7-1 expressed by stimulated T cells.



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  		FIGURE 3. B7-1 expression on CD4 T cells in relation to density of Ag-specific T cells. In a and b, a suspension of unfractionated spleen and lymph node cells was stimulated as indicated below, at 106 cells/ml. On day 4 (B7-1 expression) or day 2 (B7-2 expression) of culture, live cells were recovered by density gradient centrifugation, and the level of B7-1 or B7-2 staining on CD4 T cells (thick lines) was determined in relation to staining with the isotype control mAb (thin lines) as in Fig. 2Go. a, DO11.10 cells (top histogram) or DO11.10 cells diluted with a suspension of unfractionated spleen and lymph node cells from BALB/c mice as indicated (1:2 or 1:9) were stimulated with 1 µg/ml pOVA (top histogram). The total and relative numbers of B220+ vs CD3+ T cells remained the same (36 vs 64%), but the proportion of CD3 T cells that was KJ1–26+ (i.e., clonotype positive) decreased as the dilution increased. b, DO11.10 cells were stimulated with 10 µg/ml (B7-1 data) or 1 µg/ml (B7-2 data) pOVA with or without anti-IL-4 mAb (10 µg/ml purified ascites) or rIL-4 (4000 U/ml) as indicated. Data are representative of at least two comparable experiments.

 
Expression of B7-2 on CD4 T cells peaked on day 2 during stimulation of whole spleen/lymph node cells from DO11.10 mice. B7-2 expression on CD4 T cells was not reproducibly affected by the presence of IL-4 during culture (Fig. 3Gob). Thus, the effects of IL-4 are distinct for B7-1 vs B7-2 molecules expressed on T cells. IL-4 also differentially affects B7-2 expression on APCs and T cells, because IL-4 promotes B7-2 expression on APCs, but not T cells, even in the absence of stimulation (38 ; data not shown).

T cells from DO11.10 mice that lack Stat6 express enhanced levels of B7-1 on T cells during stimulation

Mice genetically deficient in Stat6 (Stat6 KO) fail to generate Th2 responses, because Stat6 is necessary for the up-regulation of IL-4R in response to IL-4, and its absence therefore prevents the critical autocrine promotion of Th2-driving IL-4 production (37, 39, 40). During priming with pOVA, T cells from Stat6 KO mice that had been bred to express the DO11.10 transgene expressed markedly enhanced levels of B7-1 at all peptide concentrations tested (Fig. 4Go). Addition of rIL-4 had little effect on the level of B7-1 expression by Stat6 KO DO11.10 T cells, indicative of the defect in the Stat6-IL-4 axis.



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  		FIGURE 4. CD4 T cells deficient in the IL-4 response element Stat6 express increased levels of B7-1 upon activation. CD4 T cells from wild-type or Stat6 KO DO11.10 mice were purified by positive selection using magnetic beads and 105/ml T cells primed with 106/ml mitomycin C-treated spleen cells and 0.1–10 µg/ml pOVA, with or without rIL-4 (4000 U/ml) as indicated (Priming). No significant B7-1 expression was detectable on either wild-type or Stat6 KO T cells (with or without rIL-4) in the absence of stimulation. After 4 days, live, primed T cells were recovered by density gradient centrifugation, rested overnight, and restimulated at 105/ml with 106/ml fresh mitomycin C-treated spleen cells and 1 µg/ml pOVA (Restimulation). FACS analysis of B7-1 expression on CD4 T cells was performed on day 4 of priming and day 2 of restimulation as described in Fig. 2Go. No B7-1 was detected in the absence of stimulation.

 
When Stat6 KO DO11.10 T cells were recovered after 4 days of primary culture, rested overnight, and restimulated with fresh APCs and peptide (1 µg/ml), expression of B7-1 was uniformly high, and higher than that on wild-type T cells for all priming conditions tested (Fig. 4Go, right). Whereas IL-4 was detectable in supernatants from each of the previously activated wild-type T cell cultures, no IL-4 was detectable during restimulation of either panel of Stat6 T cells (data not shown). This result is consistent with the data shown in Fig. 2Gob, in that the level of B7-1 expression is influenced by endogenous IL-4 production during restimulation of previously activated T cells.

B7-1 expression during stimulation of naive DO11.10 T cells is peptide dose dependent

The level of B7-1 expressed on CD4 T cells during primary culture increased with increasing peptide concentration during priming of both wild-type and Stat6 KO DO11.10 T cells (Fig. 4Go). Stat6 KO T cells expressed higher levels of B7-1 than wild-type T cells for all peptide concentrations, suggesting that despite IL-4 production being barely detectable during primary culture of purified wild-type DO11.10 T cells with mitomycin C-treated APCs (17, 18), endogenous IL-4 may nevertheless be dampening B7-1 expression. Although IL-4 production by wild-type DO11.10 T cells increases with increasing peptide concentration (17), the dose-dependent increase in B7-1 expression in wild-type as well as Stat6 KO T cells suggests that the B7-1-promoting effects of peptide concentration dominate over the B7-1-inhibiting effects of IL-4 production during primary stimulation.

B7 on APCs is required for optimal B7-1 expression on T cells

We have previously shown that differentiation of IL-4-producing T cells is impaired when wild-type DO11.10 T cells are primed with APCs from B7dKO mice (18). We therefore investigated whether the deficit in IL-4 production would be accompanied by enhanced B7-1 expression on the responding T cell. The data summarized in Table IIGo indicate that B7-1 expression was slightly reduced on wild-type DO11.10 T cells primed with B7dKO rather than wild-type APCs. When anti-IL-4 mAb was included in the culture, B7-1 expression was enhanced on T cells primed with B7dKO APCs, but remained lower than that on T cells primed with wild-type APCs and anti-IL-4 mAb (Table IIGo). As with wild-type APCs, addition of rIL-4 during priming with B7dKO APCs inhibited B7-1 expression on T cells (data not shown).


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  		Table II. B7-1 expression on T cells stimulated with wild-type or B7-deficient APCs1

 
These results demonstrate that B7 costimulation by APCs enhances B7-1 expression during primary stimulation of T cells. The deficit in IL-4 production does not result in enhanced B7-1 expression in the absence of B7 costimulation from APCs. Results also confirm the observation that although Th2 differentiation is impaired when naive T cells are primed with B7dKO APCs, low levels of IL-4 are nevertheless induced (18).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies using B7-1 KO DO11.10 T cells demonstrate for the first time under physiological conditions a functional role for B7-1 expressed on CD4 T cells. T cells from B7-1 KO DO11.10 mice produce more IL-4 than wild-type DO11.10 T cells, suggesting that B7-1 expressed on T cells regulates the differentiation of IL-4-producing T cells. In addition, IL-4 affects the expression of B7-1 on CD4 T cells. B7-1 expression is inhibited by IL-4 and enhanced when IL-4 is neutralized or when T cells are unable to respond to the differentiating signals of IL-4. The level of IL-4 production induced as well as the level of B7-1 expressed on T cells are also influenced by peptide concentration, density of Ag-specific T cells, and B7 costimulation by APCs. Thus, the reciprocal interaction between B7-1 on T cells and IL-4 production could skew T cell differentiation toward either high or low IL-4 production, depending upon the influence of stimulatory conditions on B7-1 expression by T cells as well as on the initial level of IL-4 production.

The percentage of T cells expressing B7-1 increased with increasing peptide concentration and when B7 costimulation was present during primary stimulation. Both these conditions also promote IL-4 production by responding T cells (17, 18), which would be expected to inhibit expression of B7-1 on T cells. However, during priming of naive T cells, the level of stimulation (peptide concentration, costimulation) appears to dominate for B7-1 expression, perhaps because primary IL-4 production is relatively low. Nevertheless, a modulatory effect of endogenous IL-4 is evident during a primary response. Neutralizing IL-4 production enhances B7-1 expression during priming of wild-type T cells with either wild-type or B7dKO APCs. Moreover, primary Stat6 KO DO11.10 T cells, which are unable to respond effectively to IL-4 (37, 39, 40), express enhanced levels of B7-1 compared with wild-type T cells in a peptide dose-dependent manner. (It is also possible that B7-1 expression on Stat6 KO T cells could be influenced by additional Stat6-dependent pathways.) Following restimulation of primed T cells, a clear influence of the level of IL-4 present during restimulation is evident (Fig. 2Gob and Fig. 4Go).

Taken together with the effects of IL-4 on B7-1 expression by T cells, functional data are consistent with a mutual regulatory interaction between IL-4 and B7-1. Although secondary IL-4 production by B7-1 KO DO11.10 T cells was always enhanced compared with that by wild-type DO11.10 T cells, the difference decreased as the level of IL-4 produced by the wild-type T cells increased. Under these conditions, wild-type T cells would be expected to express less B7-1 during restimulation and consequently appear more similar to B7-1 KO DO11.10 T cells. However, we were able to both increase the level of IL-4 production, and decrease the difference in IL-4 production between wild-type and B7-1 KO T cells during restimulation by increasing the peptide concentration during priming. The peptide dose dependence of B7-1 expression on T cells should lead to a greater rather than smaller difference in IL-4 production between wild-type and B7-1 KO T cells primed at the higher dose. This apparent paradox could be explained if B7-1 expression during restimulation, rather than priming, of T cells was critical for fine-tuning the ongoing level of IL-4 production. The peptide concentration during priming would determine the extent of Th2 differentiation and secondary IL-4 production, independently of B7-1 expression during priming, and secondary IL-4 production would, in turn, determine the level of secondary B7-1 expression, because the high levels of IL-4 produced during secondary stimulation are potently inhibitory for B7-1 expression by T cells (see Fig. 4Go). Alternatively, the higher level of peptide-driven primary IL-4 production induced by the higher peptide concentration could have a more potent inhibitory effect on B7-1 function (namely negative regulation of IL-4 production) than on B7-1 expression in T cells.

The mechanism by which B7-1 influences IL-4 production by T cells is not known. One possibility is that the subtle increases we detected in primary proliferation by B7-1 KO DO11.10 T cells indirectly result in the accumulation of increased levels of IL-4 during priming and, consequently, increased Th2 differentiation. Alternatively, B7-1 may deliver a signal to the T cell, either directly or via a counter-receptor on the T cell (CD28 or CTLA-4), which inhibits the production of IL-4. This latter possibility is particularly intriguing, since we have recently found that CTLA-4-deficient DO11.10 T cells stimulated in vitro produce markedly enhanced levels of IL-4 (41). This would be consistent with the absence of B7-1:CTLA-4 interactions in the present model. The less striking bias in the present system could indicate some degree of compensation for B7:CTLA-4 binding by B7-2 on T cells, the expression of which does not appear to be influenced by IL-4, or by B7 molecules on APCs.

The apparent role for B7-1 on T cells in inhibiting IL-4 production contrasts with the IL-4-promoting effects of B7-1 on APCs as detected in the same experimental system (17). Distinct functional properties of B7 molecules expressed on T cells as opposed to APCs have been reported previously, where T cell B7-2 binds CTLA-4 but not CD28, whereas APC B7-2 binds both ligands (42, 43). Data presented here also lend further support to the idea that the regulation of B7 expression by cytokines is highly specific for both the B7 molecule and the cell type on which it is expressed. Whereas IL-4 inhibited the Ag-specific induction of B7-1 expression on T cells, expression of B7-2 during T cell activation was not reproducibly affected by IL-4. The IL-4 independence of B7-2 expression on T cells contrasts with the striking ability of IL-4 to promote B7-2 expression on B cells even in the absence of an additional stimulus (38 ; data not shown). To date, we have detected only very low levels of B7-1 expression on APCs during peptide-specific stimulation (for example, see Ref. 17) and are therefore unable to comment on the effects of IL-4 on B7-1 expression by APCs during stimulation of DO11.10 T cells. Other investigators have demonstrated that IL-4 can induce low levels of B7-1 (as well as high levels of B7-2) on B cells in the absence of additional stimuli (38).

The results presented here lead us to predict that under Th2-biased conditions, no B7-1 expression would be detected on T cells, whereas under Th1-biased conditions, high levels of B7-1 on T cells would be expected. This is consistent with reports that B7-1 expression on T cells (as well as APCs) increases strikingly over the course of Th1-mediated experimental autoimmune encephalomyelitis (30, 31). Indeed, enhanced B7-1 expression occurred when a Th1 response was induced in response to foreign as well as self Ag (31). Functional data also support this prediction, because treatment of experimental autoimmune encephalomyelitis with anti-B7-1 mAbs or Fab can protect against disease (30, 44), consistent with our suggestion that blockade of the interaction between B7 and its ligand (perhaps CTLA-4) enhances the production of IL-4 and the development of protective Th2 responses.

Our results demonstrate a reciprocal relationship between B7-1 expressed on T cells and IL-4. This relationship suggests that B7-1 on chronically stimulated T cells could be either causative or predictive of an inhibited Th2 response. The inhibition of IL-4 production by B7-1 on T cells contrasts with our previous demonstration that B7-1 on APCs contributes positively to the production of IL-4. In view of the functional effect of B7-1 on T cells, our results emphasize the need for a better understanding of the regulation of expression of B7 molecules on T cells as well as APCs under specific pathological conditions, so that the outcome of therapeutic blockade of B7 molecules can be reliably predicted.


   Acknowledgments
 
We thank Sumi Scott for excellent technical assistance.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants RO1AI38310, PO1AI35297, and PO1AI35335. Back

2 Address correspondence and reprint requests to: Dr. A. Nicola Schweitzer, Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital, 221 Longwood Avenue, LMRC 521, Boston, MA 02115. E-mail address: Back

3 Abbreviations used in this paper: KO, knockout; pOVA, OVA peptide 323–339; B7dKO, lacking B7-1 and B7-2. Back

Received for publication May 20, 1999. Accepted for publication August 23, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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