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The Journal of Immunology, 1999, 162: 6708-6715.
Copyright © 1999 by The American Association of Immunologists

Expression and Contribution of B7-1 (CD80) and B7-2 (CD86) in the Early Immune Response to Leishmania major Infection1

M. Merle Elloso and Phillip Scott2

Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD28 interactions promote T cell responses, and whether B7-1 or B7-2 is utilized may influence Th cell subset development. CD28 blockade by CTLA-4Ig treatment or by targeted gene disruption has yielded different conclusions regarding the role of CD28 in the development of Th1 and Th2 cells following Leishmania major infection. In this study, we demonstrate that B7-mediated costimulation is required for the development of the early immune response following infection of resistant or susceptible mice. In contrast, CD28-/- BALB/c mice infected with L. major produce cytokines comparable to those of infected wild-type mice. Treatment of CD28-/- mice with CTLA-4Ig did not diminish this response, suggesting that a B7-independent pathway(s) contributes to the early immune response in these mice. In conventional BALB/c or C3H mice, B7-2 functions as the dominant costimulatory molecule in the initiation of early T cell activation following L. major infection, leading to IL-4 or IFN-{gamma} production, respectively. The preferential interaction of B7-2 with its ligand(s) in the induction of these responses correlates with its constitutive expression relative to that of B7-1. However, B7-1 can equally mediate costimulation for the production of either IL-4 or IFN-{gamma} when expressed at high levels. Thus, in leishmaniasis, costimulation involving B7-1 or B7-2 can result in the production of either Th1 or Th2 cytokines, rather than a preferential induction of one type of response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
For optimal activation, T cells require a costimulatory signal in addition to TCR engagement (1). Costimulation delivered through the CD28 molecule on T cells results in efficient activation and proliferation of T cells induced by Ag, as well as prolonged cell survival and cytokine production (reviewed in 2). While it is widely recognized that the interaction of either B7-1 (CD80) or B7-2 (CD86) with CD28 can provide costimulation for T cell activation (3, 4, 5, 6, 7, 8, 9), the question still remains whether B7-1 and B7-2 mediate distinct signals.

Studies both in vivo and in vitro have suggested that B7-1 and B7-2 have distinct roles in Th cell differentiation (10, 11). For example, during experimental allergic encephalomyelitis, a disease mediated by a dominant Th1 response, treatment of mice with anti-B7-1 mAb ameliorated disease, whereas anti-B7-2 mAb treatment resulted in exacerbated disease (10). These results suggested that while B7-1 signaling leads to the induction of Th1 responses, B7-2-mediated costimulation promotes Th2 development. In contrast, in vivo blocking studies demonstrated that B7-2 is the dominant molecule functioning in the induction of diabetes in nonobese diabetic mice, another Th1-mediated disease (12). Other studies using B7-1- or B7-2-expressing cell lines have provided evidence that neither B7-1 nor B7-2 differentially regulate Th cell development (13, 14, 15). Moreover, studies in which TCR transgenic cells were stimulated with Ag in the presence of B7-1-/- or B7-2-/- APCs demonstrate that either B7-1 or B7-2 could contribute to IFN-{gamma} and IL-4 production (16).

Experimental infection with the intracellular parasite Leishmania major is a well-established model for studying Th1 and Th2 development. In experimental murine leishmaniasis, infections in resistant mice (C3H, C57BL/6) are associated with Th1 responses, whereas infections in susceptible mice (BALB/c) are associated with a Th2 response (17, 18, 19, 20). It is well documented that the early production of IL-12 and IFN-{gamma} after L. major infection promotes Th1 differentiation in resistant mice (19, 21, 22, 23). In BALB/c mice, the early IL-4 burst after infection promotes Th2 development and prevents IL-12 responsiveness (20, 24, 25, 26). Furthermore, immunotherapies that promote Th1 development in susceptible mice (e.g., neutralization of IL-4, treatment with IL-12) are only effective if administered before the development of an established Th2 response (<7–14 days postinfection) (20, 25, 27, 28, 29). Thus, the early immune response after infection with L. major is critical for the development of Th1 and Th2 cells.

The effects of B7 blockade on Th1 and Th2 development during infection with L. major were initially examined using CTLA-4Ig, a soluble form of CTLA-4 that binds to both B7-1 and B7-2 (30, 31). CTLA-4Ig treatment of resistant C57BL/6 mice had no effect on the outcome of disease (30). In contrast, treatment of BALB/c mice with a single dose of CTLA-4Ig at the time of infection promoted resistance in these mice, which was associated with decreased levels of IL-4 mRNA. These results suggested that B7, presumably through its interaction with CD28, was required for the development of a Th2, but not a Th1, response during leishmaniasis. More recently, blockade of B7-2 (but not B7-1) in WT3 BALB/c mice was shown to affect disease outcome by decreasing parasite burden and the production of Th2 cytokines measured 4 wk postinfection with L. major (32). Anti-B7-2 treatment of C57BL/6 mice decreased the parasite load, but did not alter cytokine production. B7-1 blockade did not affect the resistant phenotype. These results led to the conclusion that B7-2 was critical for Th2 differentiation, but that during L. major infection, a Th1 response does not depend upon B7-1 signaling. An alternative interpretation of these results is that the differential effects of anti-B7-1 and anti-B7-2 mAbs reflect the differences in the kinetics or relative levels of tissue expression of these molecules. Thus, instead of differentially promoting Th1 and Th2 development, it has been proposed that B7-1 and B7-2 may differentially contribute to immune responses due to the nature of their expression patterns (33). Whether B7-1 and B7-2 preferentially promote Th1 and Th2 development, respectively, or are differentially expressed during L. major infection has not been determined. Furthermore, while the studies described above demonstrated the effects of B7 blockade on disease outcome, the effect of B7 blockade on the early immune response has not been examined.

Despite these findings that demonstrate a role for B7-mediated costimulation in the development of a Th2 response, subsequent findings using CD28-/- BALB/c mice showed that CD28 was not an absolute requirement for Th2 development after L. major infection (34). These observations raised the possibilities that either alternative ligands for B7 or compensatory costimulation pathways may exist that promote Th2 development in the absence of CD28. Therefore, it remains to be determined whether CD28-/- mice, in addition to their capacity to mount a Th2 response, exhibit an early IL-4 response during L. major infection that is independent of B7-mediated costimulation. In this study, we investigated: 1) whether B7-1 and B7-2 differentially contribute to Th cell development during the early (day 3) immune response to L. major infection, 2) whether the relative contribution of each molecule reflects levels of their expression, and 3) if B7-independent costimulation contributes to the early IL-4 response after L. major infection of CD28-/- BALB mice.


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

L. major (MHOM/IL/80/Friedlin) parasites were maintained in Grace’s insect cell culture medium (Life Technologies, Grand Island, NY) containing 20% FBS (HyClone, Logan, UT), 100 U/ml penicillin (Sigma, St. Louis, MO), 100 µg/ml streptomycin sulfate (Sigma), and 2 mM L-glutamine (Sigma). Stationary phase metacyclic promastigotes were isolated by negative selection using peanut agglutinin (Sigma), as described previously (35). Female BALB/cByJ and C3HeB/FeJ mice (obtained between 5 and 6 wk of age from The Jackson Laboratory (Bar Harbor, ME)) were infected in the hind footpads with 2 x 106 metacyclic parasites. CD28-/- BALB/c breeder pairs (backcrossed 6 times with BALB/c WT mice) were obtained from The Jackson Laboratory and bred and maintained in pathogen-free facilities.

Reagents

Human and murine CTLA-4Ig, a B7-1-specific mutant CTLA-4Ig (Y100F), and the control L6 fusion protein were generously provided by Drs. R. Peach and P. Linsley (Bristol-Myers Squibb Research Institute, Seattle, WA). Rat anti-mouse B7-2 (GL1) was protein G-purified from ascites by Harlan Bioproducts for Science (Madison, WI). Purified anti-mouse B7-1 was purchased from PharMingen (San Diego, CA). Control rat IgG was purchased from Sigma. Control human IgG and hamster IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Animals were injected with 200 µg of reagent i.p. and with 200 µg in the footpads mixed together in the parasite inoculum in a volume of 50 µl at the time of infection. In some experiments, a single dose (200 µg) of reagent was given i.p. at the time of infection.

Flow cytometry

Popliteal lymph nodes (LNs) were isolated from normal or infected mice 3 days postinfection, and single cell suspensions were prepared. Cells (5 x 105) were transferred to 12 x 75-mm polystyrene tubes and washed with 2 ml FACs buffer (PBS containing 0.1% BSA and 0.1% sodium azide). Pelleted cells were resuspended in the residual volume of FACs buffer and incubated with Fc block (10 µg anti-FcR{gamma}III/II (2.4G2) and 10 µg rat IgG) for 5–10 min to block nonspecific binding of Ab to FcR. Cells were then stained with fluorochrome-labeled mAbs for 25 min on ice in the dark in the presence of the Fc block, then washed in FACs buffer. Abs used were FITC-labeled anti-mouse Mac-1, CD4, B220, or rat IgG isotype control, and PE-labeled anti-mouse B7-1, B7-2, or rat or hamster IgG isotype control (all from PharMingen). Propidium iodide (Sigma) was added just before acquisition to allow the exclusion of dead cells. Acquisition of live, gated Mac-1+, CD4+, or B220+ cells was performed using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). A minimum of 10,000 live/CD4+ or live/B220+ events was acquired for analysis of B7 expression. Fewer events (2500–5000 live/Mac-1+) representative of the Mac-1+ population were acquired due to the lower frequency of these cells in the draining LNs. Expression of B7-1 or B7-2 on gated cell populations was analyzed with CELLQUEST software (Becton Dickinson).

In vitro recall responses

Single cell suspensions were prepared from the draining popliteal LNs of infected mice at 3 days postinfection. In some experiments, LNs were harvested at 14 days postinfection. Cells were resuspended in complete tissue culture medium (DMEM containing 10% FBS (HyClone), 100 U/ml penicillin, and 100 µg/ml streptomycin, 2 mM L-glutamine (Sigma), 25 mM HEPES (Sigma), and 5 x 10-5 M 2-ME (Sigma)) and cultured in 96-well tissue culture plates at 8 x 105 cells/well, in the presence or absence of 50 µg/ml soluble leishmanial Ag (SLA) (prepared as described previously; 36). Cells were cultured with or without 2.5 µg/ml anti-IL-4R mAb (M1; generously provided by Dr. F. Finkelman (University of Cincinnati, Cincinnati, OH)) to block consumption of IL-4. Blocking reagents (anti-B7-1, anti-B7-2, CTLA-4Ig, Y100F, or controls) were added at a final concentration of 5–20 µg/ml. Supernatants were collected 3 days later, and IL-4 and IFN-{gamma} were measured by ELISA.

Proliferation assays

Cell division was assessed by flow cytometry using the dye carboxyfluorescein diacetate succinimydlester (CFSE; Molecular Probes, Eugene, OR), as described previously (37). Briefly, LN cells were washed in PBS, resuspended in PBS at 8 x 106 cells/ml, and incubated at room temperature with an equal volume of 2.5 µM CFSE diluted in PBS. After 5 min, 0.5 ml of FBS was added to stop the labeling, and the cells were washed once in PBS followed by one wash in complete tissue culture medium. Cells were cultured in 96-well U-bottom plates in the presence or absence of SLA as described above. After 3 days, the cells were harvested from each well, washed in FACs buffer, and surface stained with PE-labeled anti-CD4 (PharMingen). Assessment of cell division within the CD4+ population was performed with a FACScalibur flow cytometer on 20,000 propidium iodide-/CD4+ events.

ELISAs

IFN-{gamma} and IL-4 were measured by ELISA, as previously described (20, 38), using R46A2 and polyclonal rabbit anti-IFN-{gamma} as the coating and detecting Abs, respectively, for IFN-{gamma}, and 11B11 and BVD6 for IL-4. IFN-{gamma} produced from a Th1 clone stimulated with plate-bound anti-CD3 was used to generate a standard curve, from which the levels of IFN-{gamma} in culture supernatants were calculated. IL-4 levels were calculated from a standard curve using murine rIL-4 (generously provided by Dr. F. Finkelman).

CD4+ T cell purification

CD4+ T cells were purified by negative selection using magnetic cell separation (MACS) (Miltenyi Biotec, Sunnyvale, CA), according to the manufacturer’s instructions. Cells were incubated with biotinylated anti-FcRIII/II, -CD8, -B220, and -MHC class II, followed by washing in PBS and subsequent incubation with MACS streptavidin microbeads. Labeled cells were applied to the separation column and the CD4-enriched cell population collected. Purity was assessed by flow cytometry (>=97% CD4+).

Stimulation of CD4+ T cells using B7-transfected P815 cell lines

CD4+ T cells were incubated with irradiated (100 Gy) B7-1-transfected P815 cells, B7-2-transfected P815 cells, or nontransfected parent P815 cells (Dr. L. Lanier, DNAX, Palo Alto, CA; and Dr. M. Azuma, National Children’s Medical Research Center, Tokyo, Japan) at a 1:2 ratio (T cell:transfectant). Anti-CD3 (145-2C11) was added at a final concentration of 100 ng/ml, along with M1 (2.5 µg/ml final concentration). Supernatants were harvested 48 h later and assayed for IFN-{gamma} and IL-4 by ELISA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vivo treatment with CTLA-4Ig results in the inhibition of early cytokine production and T cell proliferation after L. major infection

To determine the effect of B7 blockade on the early immune response to L. major infection, BALB/c and C3H mice were injected with CTLA-4Ig or control Ig at the time of infection. At 3 days postinfection, cells isolated from the draining LN of infected BALB/c mice treated with control Ig produced IL-4 when restimulated with SLA in vitro (Fig. 1GoA). In contrast, IL-4 production by LN cells from CTLA-4Ig-treated mice was markedly reduced. In a total of four experiments, the average reduction in IL-4 after CTLA-4Ig treatment of BALB/c mice was 71 ± 19.8%. IFN-{gamma} production was also reduced as a result of CTLA4-Ig treatment. Similarly, IFN-{gamma} production by LN cells from infected C3H was reduced when these mice were injected with CTLA-4Ig compared with infected control mice (Fig. 1GoA). In six experiments, IFN-{gamma} production was reduced by an average of 59.8 ± 39.7% when C3H mice were treated with CTLA-4Ig.



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  		FIGURE 1. Effect of CTLA-4Ig treatment on the early immune response to L. major infection. BALB/c and C3H mice were injected with CTLA-4Ig or control Ig i.p. at the time of infection. A, Draining LN cells were obtained at 3 days postinfection or from uninfected mice, and cultured in medium alone (med) or with 50 µg/ml SLA. Supernatants were collected 72 h later and measured for IFN-{gamma} and IL-4 by ELISA. B, LN cells were similarly cultured as in A after staining the cells with CFSE. After 72 h, the cells were harvested and surface-stained for CD4, and cell division was assessed by flow cytometry. Shown are the percentages of CD4+ cells that divided. Data shown is representative of three experiments for BALB/c mice and five experiments for C3H mice.

 
To examine the effect of B7 blockade on proliferative responses, we utilized a flow cytometric method, in which cell division can be detected by decreases in fluorescence intensity of cells labeled with the fluorescent dye CFSE (37). This analysis allowed us to determine that the majority (60–80%) of the LN cell population that responded to SLA were CD4+ T cells. Analysis of the CD4+ cells from infected BALB/c or C3H mice demonstrated that 12% of these cells had divided when restimulated with SLA (Fig. 1GoB) and >40% when stimulated with soluble anti-CD3. In contrast, few (<=2%) dividing CD4+ cells were detected when cells from CTLA-4Ig-injected mice were restimulated with SLA. This percentage was similar to that observed when cells from infected control animals were cultured in medium alone, or when cells from uninfected animals were cultured in the presence of SLA (Fig. 1GoB).

Effect of B7 blockade after infection of BALB/c CD28-/- mice

CTLA-4Ig inhibited both T cell proliferation and cytokine production in infected BALB/c and C3H mice, which suggests that CD28/B7 interactions are critical in the early immune response during Leishmania infection. However, the requirement for CD28 in generating a Th2 response has been questioned recently since CD28-/- BALB/c mice are still susceptible to leishmaniasis (34). These results seemingly contradict previous studies in which treatment of WT BALB/c mice with CTLA-4Ig or anti-B7-2 significantly affected disease outcome (30, 32). One explanation for these divergent results could be that B7 binds to CTLA-4 (or another ligand) in CD28-/- mice and promotes T cell activation (39). To assess this possibility, we tested whether CTLA-4Ig treatment would have the same effect in CD28-/- mice as it did in conventional animals. As shown above, treatment of BALB/c WT mice with CTLA-4Ig resulted in a marked reduction of IL-4 (and IFN-{gamma}) production (Fig. 2GoA), as well as proliferation by LN cells isolated 3 days postinfection (Fig. 2GoB). In contrast, CTLA-4Ig did not block cytokine production or proliferation of SLA-stimulated LN cells from infected CD28-/- mice (Fig. 2Go, A and B). Thus, it appears that CD28-/- mice may have developed a compensatory costimulatory pathway, which we are investigating currently. However, in normal mice, this compensatory pathway does not seem to play a significant role in establishing early immune responses, since CTLA-4Ig treatment blocks T cell responses in WT mice (Figs. 1Go and 2Go).



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  		FIGURE 2. Effect of B7 blockade on the early immune response after infection of CD28-/- BALB/c mice. BALB/c WT and CD28-/- mice were injected with murine CTLA-4Ig or control L6 fusion protein i.p., as well as in the parasite inoculum, at the time of infection. A, Draining LN cells were obtained at 3 days postinfection and restimulated with 50 µg/ml SLA. Supernatants were collected 72 h later and measured for IFN-{gamma} and IL-4 by ELISA. Results represent the mean ± SD of three to five mice per group. CTLA-4Ig had no effect on cytokine production by CD28-/- LN cells in three of three experiments. B, LN cells were stained with CFSE and cultured as in A. The cells were harvested after 72 h and surface-stained for CD4, and cell division was assessed by flow cytometry. Results represent the mean ± SD of three to five mice per group. C, LNs were harvested and pooled at 3 days postinfection, and cells were stimulated with SLA in the presence or absence of anti-B7-1, anti-B7-2, both, Y100F, or CTLA-4Ig. Supernatants were collected after 72 h and measured for IL-4. Shown are the average results of two experiments.

 
Effect of B7-1- vs B7-2-blockade on early cytokine production

Next, we examined the relative contribution of B7-1 and B7-2 to cytokine production following L. major infection. LN cells were isolated from WT or CD28-/- BALB/c mice 3 days after infection and restimulated in vitro with SLA in the absence or presence of blocking mAbs specific for B7-1, B7-2, or both. Additional wells contained SLA with CTLA-4Ig, Y100F (a B7-1-specific CTLA-4Ig mutant; 40), or the control fusion protein, L6. Anti-B7-1 mAb had little effect on IL-4 production compared with cells that were cultured in the presence of control Ab (Fig. 2GoC). Y100F also had no effect on IL-4 production by SLA-stimulated LN cells. In contrast, blocking B7-2 alone, or blocking both B7-1 and B7-2, resulted in a marked decrease in the production of IL-4 by WT BALB/c LN cells. While CTLA-4Ig, anti-B7-2, or both anti-B7-1 and anti-B7-2 reduced IL-4 production by LN cells from WT mice, all of the treatments shown had the same apparent lack of effect on cytokine production by LN cells from CD28-/- mice (Fig. 2GoC). These results are consistent with those obtained after in vivo B7 blockade (Fig. 2Go, A and B).

It has been suggested previously that B7-1 may function to prolong primary T cell responses, since it has been found to be up-regulated later during an immune response (33, 41). Therefore, we examined whether B7-1 contributed to IL-4 production in WT or CD28-/- mice by measuring recall responses by LN cells isolated from mice 14 days after infection (Fig. 3Go). IL-4 production by WT LN cells was decreased in the presence of anti-B7-2, a combination of anti-B7-1/anti-B7-2, or CTLA-4Ig (Fig. 3Go). In contrast, B7-1 blockade had no effect on IL-4 production. Similar to previous findings by Brown et al. (34), IL-4 production in CD28-/- mice several weeks after L. major infection is markedly reduced compared with that of WT mice. Nevertheless, blocking B7-1, B7-2, or both combined had no effect on Th2 responses in CD28-/- mice up to 14 days postinfection.



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  		FIGURE 3. Effect of B7-1 and B7-2 blockade on cytokine production at 14 days postinfection with L. major. WT and CD28-/- mice were infected, and LNs were harvested and pooled at 14 days postinfection. Cells were stimulated with SLA in the presence of blocking reagents indicated. Supernatants were collected after 72 h and measured for IL-4. Shown is one representative of two experiments.

 
The relative contribution of B7-1 and B7-2 to early cytokine production after infection of C3H mice was also examined to determine whether B7-2 is also the primary molecule that contributes to IFN-{gamma} production. Similar to the effects of B7-2 blockade on IL-4 production by BALB/c LN cells, IFN-{gamma} production by LN cells from C3H mice infected for 3 days was inhibited by B7-2 blockade (Fig. 4GoA). In contrast, anti-B7-1 mAb had little or no effect on either IFN-{gamma} or IL-4 production. In the presence of CTLA-4Ig, or both anti-B7-1 and anti-B7-2, cytokine production by BALB/c and C3H LN cells was further reduced compared with cultures that contained anti-B7-2 mAbs only. Furthermore, B7-2 blockade did not result in a concomitant increase in IFN-{gamma} production by LN cells from BALB/c mice, but instead resulted in a marked reduction of IFN-{gamma} as well (data not shown). Similar results were obtained with LN cells obtained from infected C57BL/6 mice (data not shown).



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  		FIGURE 4. Effect of B7-1 and B7-2 blockade on the early response to L. major infection of BALB/c and C3H mice. A, BALB/c and C3H mice (three to five mice per group) were infected, and draining LN cells were harvested, pooled, and restimulated with SLA in vitro, in the presence of control Ig, or blocking mAbs specific for B7-1, B7-2, both, or CTLA-4Ig. Supernatants were collected after 72 h and measured for IL-4 (BALB/c) and IFN-{gamma} (C3H) by ELISA. Shown is one representative of three experiments. B, BALB/c and C3H mice (three to five mice per group) were injected with anti-B7-2, Y100F, or control Ig at the time of infection. After 3 days, the cells from draining LNs were harvested and restimulated with Ag (SLA). Supernatants were collected after 72h and measured for IL-4 (BALB/c) and IFN-{gamma} (C3H) by ELISA. Shown are the mean and SD of individual mice in each group (*, p < 0.05 compared with control Ig). This experiment was performed three times using LN cells from BALB/c mice and four times using LN cells from C3H mice.

 
To determine whether B7-2 was also the principal costimulatory molecule in vivo, we tested whether blockade of B7-1 or B7-2 in vivo would affect T cell priming and therefore alter the early immune response during infection. Treatment of BALB/c mice with anti-B7-2 at the time of infection resulted in decreased IL-4 production by LN cells upon restimulation with SLA in vitro (Fig. 4GoB). Similarly, blockade of B7-2 in C3H mice resulted in decreased IFN-{gamma} production when LN cells were restimulated with SLA (Fig. 4Go). In contrast, blockade of B7-1 with Y100F had little or no effect on cytokine production by LN cells from both infected BALB/c and C3H mice.

Expression of B7-1 and B7-2 during L. major infection

To determine whether the differential effects of B7-1 vs B7-2 blockade on early cytokine responses reflected the relative levels of expression of these molecules, we examined B7-1 and B7-2 expression in the draining LN cells of uninfected, as well as infected, BALB/c and C3H mice at 3 days postinfection. Flow cytometric analysis of gated Mac-1+, B220+, and CD4+ cell populations revealed that, consistent with previous findings (42), B7-2 is constitutively expressed on all three cell populations (Fig. 5Go, A and B). The level of expression is increased on B220+ cells in both BALB/c and C3H mice during infection and on CD4+ cells in C3H mice. In contrast, little or no detectable B7-1 is present on B220+ or CD4+ cells from uninfected or infected mice. B7-1 is expressed on Mac-1+ cells from both BALB/c and C3H mice, although expression of this molecule does not appear to increase during infection. Similar patterns of expression were observed on LN cell populations from infected and uninfected C57BL/6 mice (data not shown).



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  		FIGURE 5. B7-1 and B7-2 expression in the draining LN during L. major infection. BALB/c (A) and C3H (B) mice were infected, and draining LN cells were harvested and assessed for B7-1 and B7-2 expression on Mac-1+, B220+, or CD4+ cells by two-color flow cytometric analysis. B7-1 and B7-2 expression on gated cell populations from infected (heavy solid line) and uninfected (dashed line) mice are shown relative to staining with PE-labeled isotype control mAbs (shaded histograms). Shown is one representative of five experiments.

 
B7-1 and B7-2 can provide similar signals for cytokine production by LN cells from infected mice

Our data suggest that B7-2 is the dominant ligand for CD28/CTLA-4 during early immune responses to L. major infection. Since its function appears to correlate with its expression, we wanted to determine whether B7-1 could provide equal costimulation for Leishmania-reactive cells if expressed at levels comparable with B7-2. To address this question, CD4+ T cells were purified from infected BALB/c and C3H mice and stimulated with anti-CD3 in the presence of P815 cells transfected with B7-1 or B7-2. Flow cytometric analysis confirmed similar levels of B7 expression in these transfectants (Ref. 13, and data not shown). Coculturing anti-CD3-stimulated CD4+ T cells from infected BALB/c mice with B7-1 transfectants resulted in increased IL-4 production when compared with anti-CD3-stimulated CD4+ T cells cultured in the presence of nontransfected P815 cells (Fig. 6Go). Costimulation provided by the transfectants was mediated by B7, because cytokine production was inhibited by the addition of B7-specific mAbs. Both B7-1 and B7-2 transfectants provided costimulation for IL-4 production by BALB/c CD4+ T cells; the amount of cytokine produced in the presence of B7-2 transfectants was comparable to that obtained with the B7-1 transfectants. Low levels of cytokines were measured in cultures that contained transfectants and T cells without anti-CD3 (Fig. 6Go), or in cultures that contained anti-CD3 and transfectants without T cells (data not shown). Similarly, either B7-1- or B7-2-transfected cells were capable of costimulating anti-CD3-induced IFN-{gamma} production by CD4+ T cells from C3H mice (Fig. 6Go).



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  		FIGURE 6. B7-1 and B7-2 provide costimulation for both IL-4 and IFN-{gamma} production by Leishmania-primed cells. CD4+ T cells were purified from infected BALB/c and C3H mice and cultured with or without anti-CD3 in the presence of P815 cells, or P815 cells transfected with B7-1 or B7-2. Blocking reagents (anti-B7-1 or anti-B7-2) were added at 10 µg/ml. Supernatants were collected after 72 h and measured for cytokine production. Shown is one representative of two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results demonstrate that B7 interactions with its ligand(s) are critical for early immune responses following L. major infection, and that B7-2 is the dominant costimulatory molecule involved. CTLA-4Ig or anti-B7-2 treatment blocked early proliferation and cytokine production in both C3H and BALB/c mice. In contrast, minimal effects were obtained with anti-B7-1 (or Y100F). In addition, B7-2 blockade resulted in a marked reduction of both IL-4 and IFN-{gamma} produced by BALB/c LN cells, rather than a concomitant increase of IFN-{gamma}. These observations indicate that blocking CD28/B7-2 interactions prevents efficient T cell priming and activation rather than resulting in immune deviation.

Flow cytometric analysis suggested that the differential effects of B7-1 vs B7-2 blockade reflect the different patterns of expression of these molecules on LN cell populations. Although both B7-1 and B7-2 are expressed at high levels on Mac-1+ cells, these cells constitute a small percentage (~1–3%) of the cells present in the draining LN. The vast majority of the remaining cell types (B220+, CD4+, collectively representing >=75% of an uninfected LN and >=90% of an infected LN) express constitutive levels of B7-2, but little to no detectable levels of B7-1. While not all of these cells are capable of presenting Ag, it has been shown that CD4+ T cells are capable of responding to B7 costimulatory signals delivered in "trans" (i.e., signals provided by cells other than the APC, or bystander cells) (43). Considering the relative expression of B7-2 and B7-1 on LN cell populations, our observation that B7-2, but not B7-1, blockade affects early immune responses induced by Leishmania infection would suggest that B7-2-expressing bystander cells may contribute to the induction of these responses.

Although B7-1 appears to play a minor role in promoting early immune responses in L. major infected BALB/c or C3H mice, B7-1 can provide sufficient costimulation for cytokine production in the absence of B7-2 or if expressed at high levels. Maximal inhibition of cytokine production was obtained with CTLA-4Ig or in the presence of both anti-B7-1 and anti-B7-2 mAbs compared with either mAb alone. Moreover, B7-1-transfected cell lines induced comparable levels of cytokines as B7-2 transfectants. Furthermore, since costimulation provided by B7-1 or B7-2 transfectants led to both IFN-{gamma} and IL-4 production by C3H and BALB/c LN cells, respectively, it appears that during the immune response to infection with L. major, signaling by B7-2 does not preferentially promote Th2 responses, nor does signaling by B7-1 preferentially promote Th1 responses.

Whereas our studies are focused on the role of B7-mediated costimulation during the early immune response after L. major infection, previous findings have demonstrated the effects of B7 blockade on disease outcome (30, 32). Our observations that B7 blockade inhibited the early immune responses in BALB/c mice are consistent with the findings that B7 blockade promotes a resistant phenotype in BALB/c mice that is associated with decreased IL-4 production and a decrease in parasite burden (30, 32). Therefore, it would appear that B7 (B7-2) blockade affects both early immune responses and Th2 development following L. major infection of susceptible mice. In contrast, while our results suggest that B7-mediated costimulation is important during the early immune response in C3H mice, the absence of B7-2-mediated costimulation does not appear to influence either the capacity of resistant mice to resolve L. major infection or Th1 development (30, 32). Thus, although B7 blockade affects early immune responses in both resistant and susceptible mice, subsequent Th1 and Th2 development are differentially affected in the absence of B7-mediated costimulation.

Several studies have demonstrated that the role for CD28 in primary T cell activation is to prolong cell survival and sustain clonal expansion (44, 45, 46). Therefore, blocking CD28/B7 interactions results in unresponsiveness, or clonal anergy, that ultimately limits the size of the developing Th cell population. Previous reports have shown that Th2 development after L. major infection is influenced by the magnitude of the early immune response. Treatments that diminish the magnitude of the initial T cell response after infection of BALB/c mice (e.g., sublethal irradiation (47), treatment with anti-CD4 (27), anti-IL-2 (48), or anti-IL-4 (20, 28) Abs) affect disease outcome and result in the development of a Th1, rather than a Th2, response. It is conceivable that B7 blockade early after L. major infection would decrease the magnitude of the developing T cell response, thereby affecting Th2 development. In contrast, altering the level of B7-mediated costimulation, which can affect clonal expansion and the magnitude of the early immune response, would have less impact on Th1 development. This contention is consistent with previous findings where relatively fewer numbers of responding cells during infection are necessary to promote Th1 development (49). Thus, although clonal expansion of T cells in infected C3H mice may be limited in the absence of CD28, the development of a Th1 response will proceed largely unaffected. These observations may be further explained by recent findings by Malvey et al. (50), which demonstrated that the induction of unresponsiveness to Ag limits the clonal expansion of responding cells, but does not prevent effector cell function. Although anergy induction resulted in a lack of clonal expansion, differentiation into Th1 cells capable of mediating delayed-type hypersensitivity responses and providing help for IgG2a production was unaffected.

While CD28/B7 interactions appear to be critical to Th2 responses in WT mice, BALB/c mice lacking CD28 exhibit no demonstrable defect in Th2 development after infection with L. major (34). It has been speculated that this discrepancy may be due to developmental compensatory mechanisms that have arisen in the CD28-/- BALB/c mice as a result of their genetic defect (34). Gause and his colleagues (51, 52, 53) have suggested that B7-mediated costimulation in the absence of CD28 contributes to IL-4 production, since B7 blockade in WT mice inhibits the Th2 response after infection with the nematode Heligmosomoides polygyrus, yet CD28-/- mice infected with H. polygyrus develop a Th2 response. Similarly, in leishmaniasis, CTLA-4 may be signaling in a positive fashion (54). However, the inability of CTLA-4Ig or anti-B7-1/anti-B7-2 mAb to inhibit IL-4 production in CD28-/- mice suggests that a B7-independent compensatory pathway is operating in these animals. Moreover, previous reports have demonstrated a role for CTLA-4 in negatively regulating immune responses during leishmaniasis (55, 56). Recently, it has been shown that heat stable Ag (HSA) can provide costimulation for IL-4 production in response to keyhole limpet hemocyanin in the absence of CD28 (57). IL-4 production, however, is ablated in mice that are deficient in both CD28 and HSA. Whether this and/or other costimulatory pathways compensate in CD28-/- mice during leishmaniasis is currently under investigation.

In summary, this study demonstrates that B7-mediated costimulation is critical in promoting the early immune responses after L. major infection in both resistant and susceptible mice. However, this observation does not appear to be true in CD28-deficient mice; these mice have developed B7-independent pathways that enable the development of early IL-4 responses after L. major infection. Consistent with its level of expression, B7-2 is the dominant molecule that contributes to early immune responses in BALB/c and C3H mice. However, B7-1 or B7-2, when expressed at comparable levels, can provide equal costimulation leading to either early IFN-{gamma} or IL-4 production, which suggests that B7-1 and B7-2 do not mediate distinct signaling pathways that promote early immune responses during Leishmania. Immunoprophylaxis designed to preferentially promote a particular Th response for controlling leishmaniasis therefore may not necessitate the exclusive use of B7-1 vs B7-2 for providing costimulation.


   Acknowledgments
 
We thank A. Park and B. Hondowicz for excellent technical assistance; Drs. R. Peach, P. Linsley, F. Finkelman, and W. Gause for the generous supply of reagents; Drs. L. Lanier and M. Azuma for providing the B7 transfectants; and Drs. A. Wells, C. Hunter, J. Farrell, and S. Reiner for helpful discussions.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI35914 and AI07518-01. Back

2 Address correspondence and reprint requests to Dr. Phillip Scott, School of Veterinary Medicine, Department of Pathobiology, 216 Rosenthal Building, 3800 Spruce Street, Philadelphia, PA 19104. E-mail address: Back

3 Abbreviations used in this paper: WT, wild-type; LN, lymph node; SLA, soluble leishmanial Ag; CFSE, carboxyfluorescein diacetate succinimydlester. Back

Received for publication September 11, 1998. Accepted for publication March 11, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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