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The Journal of Immunology, 2003, 170: 6125-6132.
Copyright © 2003 by The American Association of Immunologists

OX40 Ligation on Activated T Cells Enhances the Control of Cryptococcus neoformans and Reduces Pulmonary Eosinophilia

Ian R. Humphreys*, Lorna Edwards*, Gerhard Walzl*, Aaron J. Rae*, Gordon Dougan*, Sue Hill{dagger} and Tracy Hussell1,*

* Center for Molecular Microbiology and Infection, Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, United Kingdom; and {dagger} Xenova Research, Cambridge, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pulmonary eosinophilia induced in C57BL/6 mice after Cryptococcus neoformans infection is driven by CD4+ Th2 cells. The immunological mechanisms that protect against eosinophilia are not fully understood. Interaction of OX40 (CD134) and its ligand, OX40L, has been implicated in T cell activation and cell migration. Unlike CD28, OX40 is only expressed on T cells 1–2 days after Ag activation. Manipulation of this pathway would therefore target recently activated T cells, leaving the naive repertoire unaffected. In this study, we show that engagement of OX40 by an OX40L:Ig fusion protein drives IFN-{gamma} production by CD4+ T cells and reduces eosinophilia and C. neoformans burden in the lung. Using gene-depleted mice, we show that reduction of eosinophilia and pathogen burden requires IL-12 and/or IFN-{gamma}. C. neoformans infection itself only partially induces OX40L expression by APCs. Provision of exogenous OX40L reveals a critical role of this pathway in the prevention of C. neoformans-induced eosinophilia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Optimal activation of naive T cells requires two signals: TCR recognition of antigenic peptide presented by MHC class I or II and a second signal delivered from the APC via a costimulatory molecule. The most well-characterized costimulatory interaction is that between CD28, which is expressed constitutively by T cells, and B7-1 (CD80) or B7-2 (CD86) ligands on APCs (1). Recently, a number of other costimulatory molecules have been described (2, 3). OX40 (CD134) is a member of the TNF receptor superfamily and, unlike CD28, is not expressed by naive T cells. Rather, it is up-regulated on activated T cells 1–2 days after Ag encounter (4, 5, 6). Furthermore, it can be induced by TCR signaling in the absence of other costimulatory signals (7). Its ligand, OX40 ligand (OX40L),2 has homology to TNF, is expressed by APCs, and provides a costimulatory signal to CD4+ T cells (4, 8, 9). The importance of OX40 costimulation is revealed in OX40- and OX40L-deficient mice in which CD4+ T cell responses are abrogated (8, 10, 11, 12, 13). Conversely, engagement of OX40 prevents activation-induced cell death (14), breaks peripheral T cell tolerance (15), and induces cytokine production (16, 17).

Although the role of OX40 has been elucidated in a number of disease models, the impact of this costimulatory signal during infection-induced pathology is less clear. In the absence of OX40, immune responses to lymphocytic choriomeningitis virus and influenza are impaired (11). Similarly, anti-OX40L treatment reduces the immunopathology associated with Leishmania infection (18). The role of OX40 in infectious conditions associated with eosinophilia is currently not known.

Cryptococcus neoformans is a significant respiratory pathogen causing pulmonary eosinophilia in immunocompetent hosts (19) and summer-type hypersensitivity pneumonitis in Japan (20). In immune deficient patients, dissemination of the pathogen occurs due to the inability of the host to limit the infection (21, 22). Protection from C. neoformans infection depends on both CD4+ and CD8+ T cells (23, 24, 25, 26). Although both lymphocyte subsets may affect the pathogen directly, indirect immune activation of NK cells (27), neutrophils, and macrophages (28) may also confer host resistance.

In mice, protection from C. neoformans infection depends on the genetic background of the inbred strain used. Resistant mice (CBA, C.B-17, BALB/c) generally produce higher concentrations of type 1 cytokines in response to C. neoformans infection (29, 30, 31). In contrast, susceptible strains (C57BL/6, C3H, and B10.D2) develop a Th2-driven pulmonary eosinophilia, and at the peak of pathogen burden up to 40% of airway cells are eosinophils. This response is nonprotective and results in tissue damage resulting from degranulation and crystal deposition by eosinophilia (32). The role of eosinophils in antifungicidal activity is controversial. Although in vitro studies show that they are capable of phagocytosing C. neoformans (33), this event in vivo has been more difficult to elucidate (32). Eosinophils have been observed juxtaposed to C. neoformans and are thought to be involved in recruiting macrophages and T cells by the production of proinflammatory cytokines and chemokines (32, 34)

We now report the beneficial effect of administering OX40L fusion proteins (OX40L:Ig) to C. neoformans-infected mice. This represents a novel treatment strategy for infectious disease because the fusion protein binds to OX40 on the Ag-activated T cell and promotes its proliferation and survival (35). This strategy has previously been used to effectively enhance antitumor immunity (36). We now report that OX40L:Ig fusion protein enhances CD4+ T cells secreting IFN-{gamma}, reduces pulmonary eosinophilia, and promotes control of C. neoformans replication in C57BL/6 mice. The reduction of eosinophils and C. neoformans burden is IFN-{gamma} and IL-12 dependent because the fusion protein had no effect in mice genetically defective in these cytokines. This study clearly shows that manipulating a single, late costimulatory signal has promising therapeutic potential during certain eosinophil-inducing lung infections.


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

Eight- to 12-wk-old female C57BL/6 mice (Harlan Olac, Bichester, U.K.) were kept in pathogen-free conditions, according to Home Office guidelines. IFN-{gamma}-/- and IL-12-/-p40 mice (backcrossed to C57BL/6 background at least 10 times) were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained by homozygous matings under contract at Bantin & Kingman Universal (Fremont, CA). All mice came from specific pathogen-free colonies. C. neoformans strain 52 was obtained from the American Type Culture Collection (Manassas, VA) and for infection grown to stationary phase (48–72 h) at room temperature on a shaker in Sabouraud dextrose broth (1% neopeptone and 2% dextrose; Difco, Detroit, MI). The cultures were washed in saline, counted on a hemocytometer, and diluted in sterile nonpyrogenic saline to the required infective dose.

Preparation of OX40L fusion proteins

Murine OX40L:murine IgG1 fusion protein (OX40L:Ig) was constructed, as previously described (36), using a chimeric cDNA containing the C-terminal region of OX40L fused to the C region of murine IgG1. LPS contamination of fusion protein was analyzed by gas chromatography-mass spectrometry of fatty acid methyl esters. Briefly, samples were derivatized using methanolic HCl, and resulting fatty acid methyl esters were dissolved in hexanes before on column injection on a Stabilwax column (30 m x 0.25 mm internal diameter; Restek, Bellefonte, PA). Samples were analyzed using a temperature gradient of 150–250°C at a rate of 3°C/min. Characteristic retention times and spectra of known standards were used to identify any fatty acid methyl esters present in the samples. Results confirmed no LPS contamination in fusion protein samples.

Mouse infections and treatment

On day 0, mice were anesthetized with halothane and intranasally infected with 2 x 104 CFU C. neoformans in 50 µl sterile PBS. Some groups of mice were injected i.p. with 100 µg OX40L:Ig fusion proteins (Xenova Pharmaceuticals, Cambridge, U.K.) or mouse IgG (Sigma-Aldrich, Poole, Dorset, U.K.) on various days after infection, as indicated in the text. Mice were then sacrificed at various time points after C. neoformans infection by injection of 3 mg pentobarbitone and exsanguinated via the femoral vessels. Bronchoalveolar lavage (BAL) fluid, lung tissue, and sera were recovered using methods described previously (37).

Cell recovery

Briefly, the lungs of each mouse were inflated six times with 1 ml 1 mM EDTA in DMEM and placed in sterile tubes on ice. A total of 100 µl BAL fluid from each mouse was cytocentrifuged onto glass slides. The remainder was centrifuged, and the supernatant was removed and stored at -70°C in 200-µl aliquots for analysis of cytokines by ELISA. Cell viability was assessed using trypan blue exclusion, and the pellet was resuspended in RPMI containing 10% FCS, 2 mM/ml L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin (R10F) at a final concentration of 106 cells/ml. Eosinophils were enumerated as granulocytes by flow cytometry, using forward and side scatter. Identification was confirmed by counting eosinophils in H&E-stained cytocentrifuge preparations.

Flow cytometric analysis of intracellular and cell surface Ags

A total of 1 x 106 BAL- and lung-derived cells was stained using APC-conjugated anti-CD4, anti-CD8 PerCP, anti-CD44 PE (all BD PharMingen, Heidelberg, Germany), and anti-OX40 FITC (Serotec, Oxford, U.K.) for 30 min on ice. Cells were then fixed for 20 min at room temperature with 2% formaldehyde. To detect intracellular cytokines, 106 cells/ml were incubated with 50 ng/ml PMA (Sigma-Aldrich), 500 ng/ml ionomycin (Calbiochem, Nottingham, U.K.), and 10 mg/ml brefeldin A (Sigma-Aldrich) for 4 h at 37°C. Cells were then stained with anti-CD4 APC or anti-CD8 PerCP and fixed, as described above. After permeabilization with PBS containing 1% saponin/1% BSA/0.05% azide (saponin buffer) for 10 min, FITC-conjugated anti-IFN-{gamma} (BD PharMingen) and PE-conjugated anti-IL-5 (BD PharMingen) diluted 1/50 in saponin buffer were added. Thirty minutes later, cells were washed once in saponin buffer and once in PBS/1% BSA/0.1% azide. All data were acquired on a FACSCalibur and analyzed with CellQuest Pro software (BD Biosciences, Erembodegem, Belgium). To set limits of background fluorescence, appropriately labeled isotype-matched control Abs were used.

Lung histology

In some studies, lungs were inflated and fixed with 10% Formalin in PBS, excised, and embedded in paraffin wax by the histopathology department at Hammersmith Hospital (London, U.K.). Sections (4 µm) were stained with H&E. The lungs from four to five mice per group were analyzed.

Enumeration of C. neoformans from lung homogenates

Lungs were homogenized by passage through 100-µm cell strainers (BD Labware, Bedford, MA). A total of 100 µl of cell suspension was diluted in PBS and incubated at room temperature for 48 h on Sabouraud dextrose agar plates (Sigma-Aldrich). The total CFU per lung was then determined (number of colonies x dilution factor x original cell suspension volume).

C. neoformans-specific Ab ELISA

A total of 2 x 105 CFU/ml heat-killed C. neoformans in PBS was used to coat 96-well microtiter plates overnight at room temperature on a shaker. After blocking with 3% BSA/PBS for 2 h at 37°C, dilutions of sample sera were added for a further hour at room temperature. Bound Ab was detected using peroxidase-conjugated rabbit anti-mouse Ig and O-phenylene-diamine as a substrate. The reaction was stopped with 50 µl 2.5 M sulfuric acid. ODs were read at 490 nm, and mean blank values (ODs from normal mouse serum) were subtracted from the OD values of test samples.

Nasal IgE Ab ELISA

Ninety-six-well microtiter plates were coated overnight with nasal wash (200 µl PBS/mouse) at 4°C. After blocking with 3% BSA/PBS for 2 h at 37°C, bound Ab was detected using peroxidase-conjugated rat anti-mouse IgE (Serotec) and O-phenylene-diamine as a substrate. The reaction was stopped with 50 µl 2.5 M sulfuric acid. ODs were read at 490 nm, and mean blank values (ODs from normal mouse serum) were subtracted from the OD values of test samples

Immunohistochemical determination of cellular proliferation

In vivo incorporation of 5-bromo-2'-deoxyuridine (BrdU; Sigma-Aldrich) was determined to assess cellular proliferation in the lung (38). Mice were injected i.p. with 75 mg/kg BrdU 90 min before sacrifice. Lungs were inflated with 10% paraformaldehyde in PBS and embedded in paraffin. Sections (4 µm) were digested in 0.01% trypsin and then 1 M hydrochloric acid. BrdU incorporation was detected with overnight incubation with a biotin-labeled anti-BrdU mAb (Caltag Laboratories, South San Francisco, CA), followed by streptavidin peroxidase (Dako, Carpenteria, CA) for 1 h. Sections were then incubated with diaminobenzidine substrate (Vector Laboratories, Burlingame, CA) for 10 min. To compare proliferation between groups, sections were analyzed microscopically, and the percentage of positively stained cells was counted in the perivascular regions in each section. At least 400 cells were counted per section.

Statistics

Statistical significance was evaluated using the Student t test, two tailed, assuming unequal variance within the Minitab software program.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
C. neoformans induces OX40-expressing T cells in the lung and airway

C57BL/6 mice infected with C. neoformans develop extensive pulmonary eosinophilia that peaks ~11–14 days after infection. The kinetics of eosinophilia parallels that of C. neoformans CFU in the lung (Fig. 1A). Peak recruitment of CD4+ T cells and to a lesser extent CD8+ T cells also occurs at times of maximal eosinophilia and pathogen burden (Fig. 1B). As reported previously, C57BL/6 mice do not completely clear the infection, and low levels remained in the lung indefinitely.



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  		FIGURE 1. Time course of C. neoformans infection is accompanied by OX40 expression by CD4+ T cells in the lung and airways. The number of CFU/lung was determined by plating out lung homogenate in 10-fold serial dilutions on Sabouraud dextrose. BAL eosinophils were enumerated as granulocytes by flow cytometry and confirmed using H&E-stained cytocentrifuge preparations over a 45-day time course experiment (a). Cells from the lungs of C. neoformans-infected mice taken on days 0, 2, 7, 11, 14, 19, and 45 days postinfection were stained with anti-CD4 and anti-CD8 Ab and analyzed by flow cytometry. Total numbers were determined by multiplying the percentage of positive cells by flow cytometry with the total viable cells recovered from the lung (b). OX40 expression by CD4+ T cells in the BAL and lung was analyzed by flow cytometry (c). A representative plot of CD4 (y-axis) vs OX40 (x-axis) in total lymphocytes on day 11 postinfection in the BAL is shown in d and is representative of four to five mice per group in two individual experiments. In a–c, each point represents the mean of five mice per group with SD from the mean, and is representative of two to three separate experiments.

 
Surface expression of OX40 is up-regulated by T cells 1–2 days after Ag stimulation. The uninfected mouse lung and airway did not contain any CD4+ T cells expressing OX40. Of the CD4+ T cells recruited to the lung during infection, however, up to 15% express OX40 (Fig. 1C). Peak OX40 expression occurs first in the lung and then in the airway, which probably represents migration of T cells to the site of high pathogen burden. A representative dot plot of OX40 expression on CD4+ T cells is shown in Fig. 1D. This typical staining pattern shows that OX40 is expressed at various intensities on CD4+ T cells. Cells negative for CD4, but OX40+, are all CD8+ (data not shown). C. neoformans also induced the expression of OX40L (7.3 ± 2.83% on CD11b+ cells) in the BAL and lung. This induction, however, was not maximal. LPS treatment of lung samples induced a significantly higher proportion of OX40L-expressing cells (13.6 ± 4.86 after 24-h LPS stimulation).

OX40L:Ig fusion protein enhances CD4+ T cell responses during C. neoformans infection

After determining that OX40 was expressed by infiltrating T cells, we then determined whether T cell responses could be influenced by OX40L:Ig fusion protein administration. In uninfected mice, this fusion protein has no effect. We believe this is due to: 1) the low number of T cells in naive mice, but more importantly, 2) that those T cells present do not express OX40 until activated by Ag. In addition, administration of OX40L:Ig alone did not induce any noticeable change in the cellular composition in the BAL or lung (data not shown). Administration of OX40L:Ig to C. neoformans-infected mice, however, increased the kinetics of accumulation of T cells in the lung affecting both CD4+ (Fig. 2a) and CD8+ T cells (data not shown), which may represent increased survival and/or proliferation. Although the kinetics of CD8+ T cell accumulation in the presence of OX40L:Ig was identical with that of CD4+ T cells, the actual number was 1–2 logs lower (data not shown). Enhanced inflammatory infiltration can clearly be observed in H&E-stained sections of lung. In IgG-treated mice, a minimal infiltrate is observed around the airways and blood vessels 11 days postinfection (Fig. 2b). OX40L:Ig treatment significantly increases this inflammatory infiltration (Fig. 2c).



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  		FIGURE 2. OX40L:Ig enhances CD4+ T cell numbers in the lung during C. neoformans infection. C57BL/6 mice (n = 4 mice/time point) were infected with C. neoformans and treated with control Ig (•) or OX40L:Ig ({circ}) on days 0, 2, 5, 8, and 11 after infection. Lung cells were taken on days 0, 2, 7, 11, 14, and 19 postinfection and stained with anti-CD4 Ab. Total numbers were determined by multiplying the percentage of positive cells by flow cytometry with the total viable cells recovered from the lung. Each point represents the mean of five mice per group with SD of the mean, and is representative of two separate experiments. *, p < 0.05 for control Ig vs OX40L:Ig (a). Eleven days after infection, lungs from mice treated with either control Ig (b) or OX40L:Ig (c) were inflation fixed with 10% formaldehyde and paraffin embedded, and sections were stained with H&E (original magnification x200). Airways and blood vessels are indicated by arrows and triangles, respectively. These sections are representative of five mice per group in three independent experiments.

 
To determine the effect on cell proliferation, BrdU was administrated 90 min before termination of OX40L:Ig- and control-treated C. neoformans-infected mice. Lungs were then inflation fixed, removed, and embedded in paraffin, and 4-µm sections were stained with an anti-BrdU Ab. For 10 days after C. neoformans infection, there was no discernible difference in the number of lung cells expressing BrdU. By day 11, however, there were significantly more proliferating cells in the lungs of OX40L:Ig (Fig. 3b)- compared with control (Fig. 3a)-treated mice (Fig. 3c). This difference persisted throughout the remainder of the time course.



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  		FIGURE 3. OX40L:Ig enhances cellular proliferation in the lung. Cell proliferation 11 days postinfection in the lungs of C. neoformans-infected mice treated with isotype control Ig (a) and OX40L:Ig (b) was analyzed by injection of BrdU 90 min before sacrifice and stained with anti-BrdU mAb with a hematoxylin counterstain (original magnification x400). The proportion of cells staining positive for BrdU was calculated within the total nucleated population, including at least 500 cells per section (c). Results represent mean values of proliferating cells from four mice per group with SD of mean shown (three sections/mouse). **, p < 0.005 for control Ig vs OX40L:Ig.

 
OX40L:Ig controls pathogen burden and reduces associated eosinophilia

Because T cell numbers were increased, we next determined the effect of OX40L:Ig fusion protein treatment on pathogen burden and associated eosinophilia. Similar CFUs were recovered from infected mice during initial time points, regardless of the treatment. From day 12 onward, a significantly reduced pathogen burden was observed in OX40L:Ig-treated mice compared with controls in three independent experiments (Fig. 4a). The recruitment of eosinophils to the lung follows similar kinetics to that of the pathogen (i.e., both are minimal at early stages after infection and peak at about days 12–15). OX40L:Ig treatment reduced the percentage of eosinophilia in the lung (Fig. 4b).



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  		FIGURE 4. OX40L:Ig administration increases pathogen clearance and reduces associated pulmonary eosinophilia during C. neoformans infection. C. neoformans-infected mice were treated with either control Ig (•) or OX40L:Ig ({circ}) on days 0, 2, 5, 8, 11, and 14 days postinfection. At multiple time points (days 0, 2, 7, 11, 14, and 19), CFU/lung was determined from lung homogenate of five individual mice (a), and BAL eosinophils were enumerated in H&E-stained cytocentrifuge preparations (b). Results shown represent the mean and SD. *, p < 0.05 for control Ig vs OX40L:Ig.

 
OX40L:Ig treatment increases IFN-{gamma} in CD4+ T cells

Because eosinophilia in this model requires CD4+ T cells secreting type 2 cytokines, we next investigated the cytokine profile in treated mice by intracellular cytokine staining. Only a few lung CD4+ T cells expressed intracellular IFN-{gamma} in control-treated mice (Fig. 5a). OX40L:Ig, however, increased this relative production 2- to 3-fold (Fig. 5b), and total numbers of CD4+/IFN-{gamma}+ cells were increased from 1.61 ± 1.34 x 104 cells in control mice to 11.67 ± 3.4 x 104 cells in OX40L:Ig-treated mice (p < 0.05). Fig. 5b shows that OX40L:Ig treatment also increases IFN-{gamma} in CD4- cells. Although we observed a modest increase in CD8+ T cells secreting IFN-{gamma}, the results were not significant. It is possible that CD4- cells could represent T cells that have down-regulated the CD4 molecule or are NK cells. By calculating the ratio of IFN-{gamma}:IL-5 or IL-4-expressing cells, we can see that OX40L:Ig treatment significantly shifts the CD4+ T cell cytokines in the lung to a Th1 phenotype (Fig. 5c). The actual percentage of T cells expressing IL-4 or IL-5, however, was not affected.



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  		FIGURE 5. Enhanced OX40 signaling promotes IFN-{gamma} production by CD4+ T cells in the lung during C. neoformans infection. IFN-{gamma} production by CD4+ T cells 12 days postinfection was evaluated by intracellular cytokine staining in Cryptococcus-infected control (a)- and OX40L:Ig (b)-treated mice. These results are representative of three experiments containing five mice per group. The ratio of IFN-{gamma}/IL-5-producing CD4+ T cells was calculated by dividing percentage of IFN-{gamma}-expressing CD4+ T cells by the percentage producing IL-5, as detected by intracellular cytokine staining (c). Results represent mean values with SDs of the ratio IFN-{gamma}/IL-5 expression from each mouse with five mice per group. **, p < 0.005 for control Ig vs OX40L:Ig. The level of IgE in nasal washes was determined by ELISA. Each data point represents an individual mouse and is representative of three individual experiments (d).

 
Consistent with the alteration of the Th1/Th2 balance, we observed a reduction in IgE 7 days postinfection in nasal wash following OX40L:Ig treatment compared with control-treated mice (Fig. 5d, p < 0.05). IgG1 and IgG2a were both increased in OX40L:Ig-treated mice (4.9- and 2.4-fold, respectively) compared with control mice (data not shown).

IFN-{gamma} is required for the reduction in pathogen burden and eosinophilia by OX40L:Ig

The mechanism by which OX40L:Ig mediates its effect was then investigated in IFN-{gamma} and IL-12 knockout mice. Wild-type immunocompetent mice produced similar results to previous experiments by showing increased CD4+ T cells (Fig. 6a), but reduced pathogen burden (Fig. 6b) and eosinophils (Fig. 6c) after OX40L:Ig treatment. Experiments in the knockout mice produced surprising results. The absence of IFN-{gamma} or IL-12 did not affect the OX40L:Ig-induced increment in CD4+ T cell numbers (Fig. 6a) or their activation, as detected by CD45RBlow expression (data not shown). The ability of OX40L:Ig to reduce pathogen burden (Fig. 6b) and eosinophilia (Fig. 6c), however, was impaired and indistinguishable from control-treated wild-type groups. We originally thought that eosinophils might actually increase in both knockout mouse strains due to impaired IFN-{gamma} production. This was not the case, despite the low levels of IFN-{gamma} and the small, but significant, increase in intracellular IL-5 production by CD4+ T cells in IL-12 knockout mice (Table I).



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  		FIGURE 6. IFN-{gamma} and IL-12 mediate OX40L:Ig-dependent inhibition of eosinophilia and promotion of C. neoformans clearance, but not CD4+ T cell activation. Groups of five wild-type (C57BL/6), IFN-{gamma}-/-, and IL-12-/- mice (C57BL/6 background) were infected with C. neoformans and treated with either OX40L:Ig or control Ab on days 0, 2, 5, and 8. Mice were sacrificed 12 days postinfection. CD4+ T cells (a) in the lung of infected mice were determined by multiplying the percentage of positive cells by flow cytometry with the total viable cells recovered from the lung. The CFU/lung was measured from lung homogenate (b). Eosinophils were enumerated from H&E-stained cytocentrifuge preparations of BAL fluid (c). All results represent mean values and SDs of five mice per group. *, p < 0.05, and **, p < 0.005 for control Ig vs OX40L:Ig in either wild-type or IFN-{gamma}-/- mice.

 

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  		Table I. Intracellular cytokine production by lung homogenatea

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The OX40:OX40L axis presents a novel method of targeting activated T cells because OX40 is only induced after T cells receive two initial signals: TCR engagement of MHC class I- or II-bound peptide and binding of CD28 to B7 molecules. There are a number of infectious diseases in which protection is poor due to insufficient or transient immunity. The specific enhancement of T cells responding to infection or vaccination in situ may therefore afford better protective immunity without disturbing the naive T cell repertoire. We now report for the first time the beneficial effect of promoting T cells via OX40 engagement during C. neoformans infection using OX40L fusion protein (OX40L:Ig). Such immune stimulation promotes cell-mediated immunity and IFN-{gamma} production by CD4+ T cells, while reducing the pathogen burden in the lung and associated eosinophilia.

OX40L:Ig administration alone does not produce any visible effect in naive mice and yet has a profound influence when present during inflammation caused by C. neoformans. The critical trigger appears to be the level of OX40 expression on T cells, which is virtually absent in uninfected control mice, but increases on CD4+ T cells during infection. The next reasonable question to pose during C. neoformans infection would therefore be why the endogenous natural interaction between OX40 and OX40L does not result in the same effect in the absence of the fusion protein. OX40L expression, although constitutive on cardiac myocytes (39) and a subset of dendritic cells in SCID mice (40), is limiting in the absence of a danger signal. Studies detailing the ability of infections to provide a sufficient OX40L-inducing signal to the APC are limiting. We show that although C. neoformans induces OX40L expression, it is not to the same level as LPS. OX40L:Ig fusion protein administration in our model is therefore likely to provide increased engagement over and above that naturally provided in vivo. This hypothesis is supported by studies showing a similar enhancement of T cells in noninfectious models such as transgenic mice overexpressing OX40L on dendritic cells (41, 42) and immune responses to soluble and super Ags (14, 43). An alternative explanation is that enhanced signals via OX40 using OX40L:Ig override the negative influence of CTLA-4. C. neoformans induces T cell expression of CTLA-4 (44), and anti-CTLA-4 treatment has been shown to enhance pathogen clearance (45).

The increase of numbers of CD4+ T cells by OX40L:Ig in our study is likely to reflect enhanced proliferation and/or survival because signaling via OX40 up-regulates Bcl-2 and Bcl-xL (46). Enhanced BrdU uptake by infiltrating cells was clearly observed in lung sections. It is interesting to note that this effect was only observed 12 days after C. neoformans infection, which coincides with maximal pathogen burden and eosinophilia. There is therefore a lag phase during which T cells are recruited and Ag activated before OX40L:Ig can exert its effect. Enhanced T cell responses by OX40 engagement have also been reported in TCR transgenic mice in which responsiveness to Ag persisted 95 days after initial Ag exposure (47) and in vitro using OX40L-transfected fibroblasts (5, 8).

One concern during these studies was that OX40L:Ig would prevent extravasation of T cells into the lung because the ligand is also expressed by endothelial cells and mediates adhesion of activated T cells (48, 49). If T cells expressed OX40 before exiting the blood, then OX40L:Ig may bind and prevent interaction with the native ligand on endothelial cells. The increment in T cell numbers in the lung suggests that this did not occur and probably reflects the lack of OX40 expression on T cells before Ag activation in the lung itself.

The eosinophilic response during C. neoformans infection is dependent on T cells secreting type 2 cytokines. The reduction of eosinophils by OX40L:Ig treatment may therefore be a consequence of one or more of the following: 1) enhanced direct apoptosis (50) and/or recirculation out of the airway lumen (51) induced by IFN-{gamma}; 2) reduced IL-5; or 3) decreased T cell stimulation due to the lower pathogen burden. We believe option 1 is more likely because IFN-{gamma}-producing cells were increased 3-fold, T cell numbers in the lung were actually increased, and eosinophils were not reduced in IFN-{gamma} or IL-12 knockout mice infected with C. neoformans and treated with OX40L:Ig. Although we did not observe a change in intracellular IL-5 expression by CD4+ T cells, the cytokine balance is definitely in favor of a Th1 environment in wild-type mice following OX40L:Ig treatment. The induction of IFN-{gamma} by enhanced OX40 signaling has been described previously in recall responses to Ags (47, 52). CD8+ intraepithelial lymphocytes expressing OX40 and OX40L after CD3 stimulation also produce high levels of IFN-{gamma} (53). The cytokine response by T cells after engagement of OX40 appears to alter depending on the cell population and the Ag being studied because effects on Th1 and/or Th2 cytokines have been reported (5, 12, 17).

The reduction of C. neoformans burden by OX40L:Ig is also likely to be dependent on enhanced IFN-{gamma}, which activates macrophages and increases their fungicidal activity (54, 55, 56). CD4+ T cells play an important role in recruiting macrophages during virulent cryptococcal infection (57). In our study, we observed increased CD4+ T cells secreting IFN-{gamma} and an increase in the number of macrophages (data not shown). This therefore provides an environment more equipped to manage fungal clearance. This is supported by the inability of OX40L:Ig to decrease Cryptococcus CFUs in IL-12 and IFN-{gamma} knockout mice. CD4+ T cells expressing OX40 are also present in the lung earlier during OX40L:Ig treatment and may therefore assist macrophages in the early control of the pathogen. Previous studies have detailed the importance of IL-12 in protection from C. neoformans (29, 30, 31). Optimal production of IL-12 requires CD40 on T cells interacting with CD40L on APCs (58, 59). Blockade of this interaction decreases IFN-{gamma} and fungicidal activity (60). The presence of more T cells in OX40L:Ig-treated mice may therefore enhance CD40:CD40L interactions.

Because we have demonstrated an alteration in the cytokine environment, the next obvious question was what has happened to Ab production? OX40:OX40L interactions are thought to play a role in Ab production (12, 61). We initially thought that the OX40L:Ig treatment may block the signal to OX40L-expressing B cells. Total B cell numbers and IgE production in the BAL were not affected (data not shown), and both IgG1 and IgG2a were increased in OX40L:Ig-treated mice, presumably due to the presence of other B cell stimulatory signals such as CD40 and CD70 (7, 62). This is similar to the absence of any effect on B cell immunity reported in OX40 knockout mice (10, 11)

It is interesting to note that OX40L:Ig still increased T cell numbers in both IL-12 and IFN-{gamma} knockout mice. Such cytokine defects do not appear to prevent Ag presentation and T cell clonal expansion. IL-12 in addition to increasing IFN-{gamma} is also reported to induce TNF-{alpha}, GM-CSF, IL-8, and even IL-10 (63). These, however, obviously play no role in preventing eosinophilia in IFN-{gamma} knockout mice. The lack of a reduction of eosinophilia and C. neoformans burden in IL-12 knockouts by OX40L:Ig is likely to reflect reduced IFN-{gamma} in these mice.

This is the first study showing that OX40L activation of T cells alters the course of an infectious disease reducing pathogen burden and eosinophilia, an effect dependent on IFN-{gamma} and IL-12. Because OX40 is expressed on Ag-activated T cells, this represents a novel strategy to enhance immunity to infectious diseases (for a review, see Ref. 64). Enhancement in the number and function of Ag-specific T cells actually participating in an immune response has not previously been tested in an infectious disease. This strategy therefore holds significant promise for the plethora of infections that induce insufficient immunity or latency.


   Acknowledgments
 
We thank Paul Hitchen of Imperial College for help with determining the LPS content of OX40L:Ig fusion proteins; Lorraine Lawrence, Department of Leukocyte Biology, Imperial College, for processing of samples for analysis of BrdU; and Brigitte Askonas for invaluable advice.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Tracy Hussell, Center for Molecular Microbiology and Infection, Department of Biochemistry, Imperial College of Science, Technology and Medicine, Exhibition Road, London SW7 2AZ. Back

2 Abbreviations used in this paper: OX40L, OX40 ligand; BAL, bronchoalveolar lavage; BrdU, 5-bromo-2'-deoxyuridine. Back

Received for publication November 8, 2002. Accepted for publication April 3, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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