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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 722-730, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0220OC

Evaluation of Inducible Costimulator/B7-Related Protein-1 as a Therapeutic Target in a Murine Model of Allergic Airway Inflammation

Ryan E. Wiley, Susanna Goncharova, Theresa Shea, Jill R. Johnson, Anthony J. Coyle and Manel Jordana

Division of Respiratory Diseases and Allergy, Centre for Gene Therapeutics, and Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada; and Department of Experimental Medicine, Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts

Address correspondence to: Dr. Manel Jordana, Department of Pathology and Molecular Medicine, Room 4H17, Health Sciences Centre, McMaster University, 1200 Main Street West, Hamilton, ON, L8N 3Z5 Canada. E-mail: jordanam{at}mcmaster.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Given its primary role in the execution of T cell, and especially Th2, effector activity, the inducible costimulator (ICOS)/B7-related protein (RP)-1 costimulatory pathway is currently being heralded as a promising therapeutic target for immune-inflammatory disorders such as asthma. This study investigates the merits of ICOS blockade in a murine model of experimental asthma in which mice are sensitized to ovalbumin (OVA) through the respiratory mucosa. Intraperitoneal treatment of mice with anti-ICOS neutralizing antibody during sensitization resulted in a marked reduction in airway eosinophilia and IL-5 in bronchoalveolar lavage, but had no effect on interleukin (IL)-4, IL-13, and eotaxin content in bronchoalveolar lavage or the production of OVA-specific immunoglobulin E in serum. Cultured splenocytes from mice sensitized to OVA in the context of ICOS ablation produced enhanced levels of IL-4 and IL-5 upon stimulation with OVA, and this correlated with elevated inflammation and immunoglobulin E secretion upon long-term in vivo OVA recall; the deleterious effects ICOS blockade, however, were not associated with reduced IL-10 production by splenocytes. Peculiarly, anti-ICOS intervention during OVA rechallenge had no effect on airway inflammation or immunoglobulin production, despite high levels of ICOS expression on infiltrating CD4+ T cells. This study provides in vivo evidence of an exacerbated long-term immune-inflammatory response following acute ICOS blockade, and suggests that ICOS costimulation is functionally redundant in established allergic disease.

Abbreviations: adenovirus, Ad • B7-related protein-1, B7RP-1 • bronchoalveolar lavage, BAL • enzyme-linked immunosorbent assay, ELISA • granulocyte-macrophage colony-stimulating factor, GM-CSF • Hanks' balanced salt solution, HBSS • inducible costimulator, ICOS • immunoglobulin E, IgE • interleukin, IL • monoclonal antibody, mAb • ovalbumin, OVA • phosphate-buffered saline, PBS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The premise that activation and differentiation of naive T cells critically require the delivery of two signals—cognate interaction between the T cell receptor and the peptide:MHC complex on antigen-presenting cells (APC), and engagement of CD28 by B7 molcules—has been instrumental to our understanding of the initiation and regulation of immune responses. The therapeutic applications of this paradigm have been vigorously investigated, with intervention strategies proposed for transplantation (1–3), cancer (4, 5), and some immune-inflammatory disorders (3, 6, 7). However, CD28:CD80/86-mediated costimulation appears to be essentially redundant for T cell effector function, suggesting that CD28 blockade may have limited value in established disease (8). It is for this reason that a number of recently-described costimulatory pathways, including inducible costimulator (ICOS)/B7-related protein (RP)-1 (9–15) and PD-1/B7H-1 (16, 17), have received considerable attention as a potential avenue to ameliorate established, T cell–mediated inflammatory disorders.

In this regard, ICOS, the third member of the CD28/CTLA-4 superfamily and the receptor for B7RP-1, has emerged as a costimulatory pathway with compelling therapeutic promise (18–20). ICOS/B7RP-1 has been characterized primarily as a costimulatory pathway that orchestrates events downstream of T cell activation (11, 21–23), including humoral immunity (24–27). ICOS is distinguished in particular by its association with Th2 effector activity (11, 21, 27–29) and, as such, ICOS has received attention in models of asthma and allergic airway inflammation (30). Indeed, inhibition of ICOS function during antigen challenge has been shown to attenuate eosinophil accumulation in the airway, and immunoglobulin (Ig)E production and cytokine content in bronchoalveolar lavage (BAL) in models of antigen-induced allergic airway inflammation (31, 32). Importantly, ICOS blockade does not alter the course of allergic sensitization in these models, indicative of the putative role of ICOS in the elaboration of effector activity in previously differentiated T cells.

Sperling and colleagues (19) have speculated that ICOS/B7RP-1 may represent a costimulatory pathway with unique therapeutic advantages. Unlike CD28/B7 costimulation, whose inhibition may lead to generalized immunosuppression, ICOS/B7RP-1 activity is apparently restricted to activitated, differentiated T cells; by extension, its suppression may preferentially modulate the established responses characteristic of immune-inflammatory disorders. However, although intriguing, this therapeutic postulate has not been investigated comprehensively, as most studies to date have examined the effects of ICOS inhibition during incipient stages of experimental disease. We therefore executed this study of ICOS function in two models of antigen-induced airway inflammation in which mice were mucosally sensitized to aerosolized ovalbumin (OVA) in the context of a granulocyte macrophage–colony-inducing factor (GM-CSF)– or GM-CSF and interleukin (IL)-12–enriched airway microenvironment, which elicit prototypic Th2- (33) and Th1-polarized (34) immune-inflammatory responses, respectively. We report three key findings: (i) ICOS blockade during acute OVA expsoure inhibits Th2- but not Th1-associated inflammation in the airway; however, (ii) inhibition of ICOS does not suppress Th2-polarized sensitization to OVA and ultimately exacerbates the response to OVA recall in vivo; and (iii) therapeutic neutralization of ICOS during OVA recall in mice with established allergic disease does not ameliorate allergic airway inflammation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female Balb/c mice (6–8 wk old) were purchased from Charles River Laboratories (Wilmington, MA). The mice were housed under specific pathogen–free conditions and maintained on a 12 h light-dark schedule. All experiments described in this study were approved by the Animal Research Ethics Board of McMaster University (Hamilton, ON, Canada). A total of 322 mice were killed during the course of these experiments.

Models of Antigen-Induced Airway Inflammation
Mice were subjected to our Th1- or Th2-polarizing protocols as previously described (33, 34). Briefly, mice were exposed to aerosolized ovalbumin in the context of an airway microenvironment conditioned by GM-CSF (Th2 model) or GM-CSF and IL-12 (Th1 model). To elicit local expression of GM-CSF or IL-12, a replication-deficient human type 5 adenoviral (Ad) construct encoding murine GM-CSF or IL-12 cDNA in the E1 region of the viral genome was delivered intranasally to isoflurane-anesthetized animals on Day -1, 24 h before the first exposure to OVA. Ad/GM-CSF and Ad/IL-12 were administered at doses of 3 x 107 and 1 x 107 pfu, respectively, in a total volume of 30 µl of phosphate-buffered saline (PBS) vehicle. Over a period of seven consecutive days (Days 2–8), mice were placed in a Plexiglas chamber (10 cm x 15 cm x 25 cm) and exposed for 20 min daily to aerosolized OVA (1% wt/vol in 0.9% saline; Sigma-Aldrich, Oakville, ON). The OVA aerosol was generated by a Bennett nebulizer at a flow rate of 10 liters/min. For the long-term in vivo rechallenge experiments, sensitized mice were allowed to recover from acute inflammation ({bsim} 4 wk) and were then exposed to a 1% OVA aerosol for 20 min on three consecutive days (Days 0–2).

In Vivo Neutralization of ICOS Activity
ICOS fusion protein (ICOS-Ig) (11) or anti-ICOS neutralizing monoclonal antibody (mAb) 12A8 (31, 36) were administered intravenously or intraperitoneally at a dose of 100 µg in 100 µl or 500 µl PBS, respectively, on Days 2, 4, 6, and 8 of the aerosolization protocol. To assess the therapeutic potential of ICOS ablation in sensitized mice, 100 µg anti-ICOS in 500 µl PBS was administered intraperitoneally on each of Days 0, 2, and 4 of the long-term in vivo rechallenge protocol. An equivalent dose of human IgG (control Ig) (Sigma-Aldrich) was used as a control for all experiments.

Collection and Measurement of Specimens
Twenty-four hours after the seventh OVA exposure (Day 9), or 72 h after the third OVA exposure during long-term in vivo recall (Day 5 of the rechallenge protocol), mice were killed and BAL was obtained as previously described (35). In brief, the lungs were dissected and the trachea cannulated with a polyethylene tube (Becton Dickinson, Sparks, MD). The lungs were lavaged twice with PBS (0.25 ml followed by 0.2 ml). Approximately 0.3 ml of the instilled fluid was consistently recovered. Total cell counts were determined using a hemocytometer. After centrifugation, supernatants were stored at -20°C for measurement of cytokines by enzyme-linked immunosorbent assay (ELISA); cell pellets were resuspended in PBS and smears were prepared by cytocentrifugation (Shandon, Pittsburgh, PA) at 300 rpm for 2 min. Diff-Quik (Baxter, McGraw Park, IL) was used to stain all smears. Differentiation of leukoctye subsets in BAL was determined by counting at least 500 white blood cells using standard hemocytologic procedures to classify the cells as neutrophils, eosinophils, lymphocytes, or macrophages/monocytes. Additionally, blood was collected by retro-orbital bleeding. Serum was obtained by centrifugation after incubating whole blood for 30 min at 37°C. Blood smears were prepared from peripheral blood collected in heparinized capillary tubes; leukocytes were differentiated by counting at least 300 white blood cells.

Cytokine and Immunoglobulin Measurement
ELISA kits for IL-4, IL-10, IL-13, and eotaxin were purchased from R&D Systems (Minneapolis, MN), and the kit for IL-5 was obtained from Amersham (Buckinghamshire, UK); each of these systems has a threshold of detection of 1.5 to 5 pg/ml. Levels of OVA-specific IgE were detected using an antigen-capture (biotinylated OVA) ELISA method as described (35); anti-mouse IgE antibodies were obtained from Southern Biotechnology Associates (Birmingham, AL). This ELISA was standardized with serum obtained from mice sensitized to OVA according to a conventional intraperitoneal sensitization model and bled 7 d following the second sensitization (35); immunglobulin levels, therefore, are expressed in units (U)/ml relative to this standard serum.

Splenocyte Culture
Spleens were triturated between the frosted ends of glass slides to disperse mononuclear cells; the resulting cell suspension was filtered through nylon mesh. Red blood cells (RBC) were lysed by resuspending dispersed cells from each spleen in 1 ml ACK lysis buffer for 1 min. Splenocytes were then washed twice in supplemented RPMI (containing 10% FBS, 1% penicillin/streptomycin, 1% L-glutamine, and 0.1% mercaptoethanol) and cultured in 96-well plates at a density of 8 x 105 cells/well in a total volume of 200 µl RPMI. Cells were stimulated with OVA at a concentration of 40 µg/well for 5 d, at which point supernatants were harvested and stored at -20°C for detection of cytokines.

Lung Cell Isolation and Flow Cytometric Analysis of Lung Cell Subsets
Lungs were perfused with 10 ml Hanks' balanced salt solution (HBSS) through the right ventricle, cut into small ({bsim} 2 mm diameter) pieces and agitated at 37°C for 1 h in 15 ml collagenase III (Life Technologies, Rockville, MD) at a concentration of 150 U/ml in HBSS. Using the plunger from a 5-ml syringe, the lung pieces were triturated through a metal screen into HBSS, and the resulting cell suspension was filtered through nylon membrane. Mononuclear cells were isolated at the interphase between layers of 30% and 60% Percoll following density gradient centifugation. Cells were washed twice and stained for flow cytometric analysis. For each antibody combination, 1.0 x 106 cells were incubated with monoclonal antibodies at 0–4°C for 30 min; the cells were then washed and treated with second stage reagents. Data were collected using a FACScan (Becton Dickinson, Sunnyvale, CA) for three-color flow cytometry or a FACSCalibur (Becton Dickinson, Sunnyvale, CA) for four-color flow cytometry, and were analyzed using WinMDI software (Scripps Research Institute, La Jolla, CA). The following antibodies and reagents were used: hamster IgG anti-mouse CD3{epsilon}, PE-conjugated and Cy-Chrome–conjugated (145–2C11); rat IgG2a anti-mouse CD4, FITC-conjugated, biotinylated, and APC-conjugated (RM4–5); rat IgG2b anti-mouse CD44, FITC-conjugated (IM7); rat IgG2a anti-mouse CD62L, PE-conjugated (MEL-14) (all purchased from BD PharMingen, San Diego, CA); rat IgG1 anti-mouse T1/ST2, PE-conjugated; rat IgG2b anti-mouse ICOS, biotinylated (both produced in-house by Millennium Pharmaceuticals, Cambridge, MA); all appropriate isotype control antibodies, Streptavidin PerCP, and Streptavidin Cy-Chrome (BD PharMingen). The antibodies were titrated to determine optimal concentration.

Data Analysis
Data are expressed as mean ± SEM, unless otherwise indicated. Results were interpreted using Student's t test. Differences were considered statistically significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ICOS Ablation during Sensitization Inhibits Th2- but Not Th1-Polarized Inflammation in BAL, but Has No Effect on Production of OVA-Specific IgE
To investigate the function of ICOS in antigen-induced airway inflammation, and to ascertain the T helper specificity of ICOS activity, we administered ICOS fusion protein (ICOS-Ig), anti-ICOS neutralizing antibody or control Ig systemically to mice undergoing Th1- or Th2-polarized sensitization to aerosolized OVA; as previously reported, the anti-ICOS (12A8) mAb antagonizes the association of ICOS with its ligand and does not deplete ICOS+ cells (36). On Day -1, mice were injected intranasally with 3 x 107 pfu Ad/GM-CSF with (Th1 model) or without (Th2 model) concurrent instillation of 1 x 107 pfu Ad/IL-12; from Days 2–8 inclusive, mice were exposed daily to aerosolized OVA, and then killed on Day 9. In untreated mice (i.e., sensitized in the absence of concurrent control Ig or ICOS intervention; data not shown), and as previously reported (33, 34), this regimen resulted in robust accumulation of mononuclear cells and eosinophils (Th2 model) or neutrophils (Th1 model) in BAL—an inflammatory profile that was unaltered in mice treated intravenously (Figures 1A and 1B) or intraperitoneally (Figure 1C) with control Ig. In the Th2 model, neutralization of ICOS—either through intravenous administration of ICOS-Ig (Figure 1B) or intraperitoneal injection of anti-ICOS (Figure 1C)—attenuated the accumulation of eosinophils in BAL by 50–75% compared with control Ig–treated mice, a result consistent with the documented participation of ICOS in Th2 effector function and allergic airway inflammation. In contrast, ablation of ICOS activity in mice undergoing Th1-privileged sensitization to OVA had no effect on mononuclear cell or neutrophil accumulation in BAL (Figure 1A); this confirmed the putative association of ICOS activity with Th2-dominant processes, and justified the exclusion of the Th1 model from further investigation. No consistent changes were observed following ICOS intervention with respect to neutrophils in the Th2 model, in which neutrophil accumulation is comparably small (33), or eosinophils in the Th1 model, in which eosinophils are virtually absent (34). It should be noted that, to be exhaustive, we have presented data in Figure 1 from both ICOS-Ig– and anti-ICOS–treated mice, thereby validating the similar effects of these two well-documented reagents in our model; wishing to avoid redundancy in our experimental design, we included only anti-ICOS intervention in subsequent studies.



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  		Figure 1. Effect of ICOS neutralization on Th1- or Th2-polarized inflammation in BAL at Day 9 of the aerosolization protocol. Mice were exposed repeatedly to OVA in the context of a GM-CSF (B and C) or GM-CSF/IL-12 (A) airway milieu to elicit Th2- or Th1-polarized immune-inflammatory responses, respectively. One hundred micrograms of ICOS-Ig, anti-ICOS, or Ig control were administered intravenously (ICOS-Ig) or intraperitoneally (anti-ICOS) on Days 2, 4, 6, and 8 of the aerosolization protocol; mice were killed at Day 9. Graphs describe mononuclear cell, neutrophil (A), and eosinophil (B and C) accumulation in BAL. Data are expressed as mean ± SEM; n = 3–4 (A and B) or 7–10 (C) per group; *P < 0.05 by Student's t test compared with Ig control.

 
The effects of anti-ICOS intervention on local inflammatory phenomena can, in part, be understood in terms of local cytokine production and systemic changes in peripheral blood. Table 1 displays the leukocyte profile in peripheral blood, OVA-specific IgE in serum, and levels of key cytokines and chemokines in BAL at Day 9 of the aerosolization protocol following intraperitoneal treatment of mice with anti-ICOS neutralizing antibody. Whereas concentrations of IL-4, IL-13, and the IL-4/IL-13–inducible chemokine eotaxin remain unaltered in mice treated with anti-ICOS, levels of IL-5 have been reduced by {bsim} 50%—a degree of inhibition comparable to that observed for eosinophil accumulation in BAL, and indicative of impaired activation or mobilization of eosinophils from bone marrow. It should be noted, however, that ICOS blockade had no statistically significant effect on eosinophil or mononuclear cell content in peripheral blood, suggesting a deficiency in leukocyte migration across the vascular endothelium into the lung parenchyma rather than attenuated bone marrow eosinopoeisis.


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  		TABLE 1 Peripheral blood leukocytes, OVA-specific IgE in serum, and cytokines/chemokines in BAL following inhibition of ICOS during sensitization (Day 9)

 
ICOS Ablation Inhibits the Recruitment of Th2 Cells to the Lung but Does Not Impair the Maturation of a Th2 Phenotype In Vivo
To examine the effects of anti-ICOS intervention on CD4+ T cells, flow cytometric analysis was performed on dispersed cells from the lungs of mice killed on Day 9 of the aerosolization protocol. That ICOS is upregulated in our model of allergic airway inflammation, and that intraperitoneal administration of anti-ICOS extinguishes endogenous ICOS expression, are documented in the upper panel of Figure 2 ; indeed, although ICOS was detected on {bsim} 19% of CD3+CD4+ cells from the lungs of Ig-treated and untreated (data not shown) mice, ICOS was expressed by only {bsim} 6% of CD4+ T cells following anti-ICOS intervention—a level comparable to that observed in naive mice. Consistent with the putative role of ICOS in Th2 effector function, it is noteworthy that the fraction of CD4+ T cells expressing ICOS in Ig-treated mice is similar to that of T1/ST2 (16%), a prototypic marker of Th2 effector cells (37–39). In this regard, a modest reduction in expression of T1/ST2 by lung-derived T helper cells in anti-ICOS–treated mice suggests impaired influx, rather than differentiation, of effector Th2 cells, as four-color flow cytometric analysis of CD4+ T cells indicates a preferential amplification of the CD44loCD62Lhi (naive) phenotype, and a concomitant depression of CD44hiCD62Llo (memory/effector) T cell infiltration (Table 2). Moreover, analysis of ICOS expression on naive and memory/effector CD4+ T cells is instructive: although {bsim} 46% of CD4+CD44hiCD62Llo cells coexpress ICOS, it is virtually undetectable on CD4+CD44loCD62Lhi cells, entirely consistent with the proposed role of ICOS in the elaboration of T cell effector activity (Table 2).



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  		Figure 2. Flow cytometric analysis of T cells from the lungs of anti-ICOS–treated mice acutely exposed to OVA. Mice were sensitized to OVA according to the Th2-polarizing regimen, treated with anti-ICOS or control Ig intraperitoneally between Days 2 and 8, and killed at Day 9. Enriched mononuclear cells from lung tissue were pooled from each group and stained for flow cytometry. Data depict ICOS (upper panels) or T1/ST2 (lower panels) expression on gated CD3+CD4+ cells from naive (untreated, unsensitized) mice and mice treated with control Ig or anti-ICOS concurrent with OVA exposure. 50,000 events were collected in the mononuclear cell gate; n = 5 per group.

 

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  		TABLE 2 Four-color flow cytometric analysis of ICOS expression on naive and memory/effector T cell populations in lung tissue at Day 9 of the aerosolization protocol

 
That Th2 differentiation is intact following anti-ICOS intervention is illustrated in Figure 3 , which documents spontaneous and OVA-stimulated IL-4 and IL-5 production by cultured splenocytes from mice killed on Day 9 of the aerosolization protocol. Indeed, splenocytes from mice treated with anti-ICOS in vivo liberate significantly higher levels of IL-4 and IL-5 than cells from Ig-treated mice, both in the context of OVA and, in the case of IL-5, constitutively (medium alone). Similar trends were observed for IL-13 in culture supernatants (data not shown). These results corroborate previous reports of preserved Th2 differentiation in the absence of ICOS activity, and suggest that ablation of ICOS may in fact exaggerate Th2 polarization in vivo.



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  		Figure 3. IL-4 and IL-5 production by cultured splenocytes from mice acutely exposed to aerosolized OVA in the context of ICOS ablation. Mice were sensitized to OVA under Th2-polarizing conditions, treated with anti-ICOS or control Ig intraperitoneally between Days 2 and 8, and killed at Day 9 of the aerosolization protocol. Splenocytes were cultured for 5 d in medium alone (med) or with OVA stimulation (OVA); IL-4 and IL-5 content in supernatants was detected by ELISA. Cells were plated at a density of 8 x 105 cells/well. Data are expressed as mean ± SEM; n = 7–12 per group; *P < 0.05 by Student's t test compared with Ig control.

 
ICOS Impairment during Sensitization Exacerbates the Long-Term Immune-Inflammatory Response to OVA In Vivo
To date, reports of ICOS function have generally examined events during antigen sensitization and acute antigen exposure, and have therefore not addressed the long-term consequences of ICOS/B7RP-1 intervention in vivo. To elucidate the long-term impact of anti-ICOS delivery during sensitization to OVA, groups of mice were permitted to recover from acute inflammation in the lungs ({bsim} 4 wk) and were then rechallenged with aerosolized OVA. In untreated (data not shown) and Ig-treated mice, in vivo recall elicited robust mononuclear cell and eosinophil infiltration into the airway at Day 5 of the rechallenge protocol (Figure 4) ; there was also evidence of a matured humoral response, with elevated levels of OVA-specific IgE detected in serum ({bsim} 10-fold higher than levels at Day 9) (Figure 4). Not unexpectedly, these phenomena were not impaired in mice sensitized to OVA in the context of ICOS ablation—unequivocal evidence that ICOS is not required for Th2 differentiation (Figure 4). On the contrary, and consistent with the trend toward higher spontaneous and OVA-stimulated cytokine production by splenocytes from anti-ICOS–treated mice both at Day 9 (Figure 3) and following in vivo rechallenge (Table 3), intervention with anti-ICOS during sensitization actually exacerbated mononuclear and eosinophilic inflammation and IgE production upon in vivo recall, resulting in levels that were 50–100% higher than those observed in Ig-treated mice (Figure 4).



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  		Figure 4. Effect of ICOS neutralization during sensitization on inflammation in BAL and IgE in serum upon long-term antigen rechallenge. Mice were exposed repeatedly to OVA in the context of a Th2-polarizing airway microenvironment and were concurrently treated with 100 µg anti-ICOS or control Ig intraperitoneally on each of Days 2, 4, 6, and 8 of the aerosolization protocol. Acute inflammatory phenomena were allowed to resolve ({bsim} 4 wk) before mice were rechallenged with OVA. Graphs depict mononuclear cell and eosinophil accumulation in BAL, and OVA-specific IgE content in serum 72 h after the final OVA recall challenge (Day 5). Data are expressed as mean ± SEM; n = 7–9 per group; *P < 0.05 by Student's t test compared with Ig control.

 

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  		TABLE 3 IL-5 and IL-13 production by cultured splenocytes from mice treated with anti-ICOS during sensitization and rechallenged with OVA following the resolution of acute inflammatory events

 
To investigate whether the deleterious long-term effects of ICOS blockade were related to impaired T cell immunoregulatory function, IL-10 was measured in supernatants from cultured splenocytes harvested either at Day 9 of the aerosolization protocol or following in vivo rechallenge (Table 4). Interestingly, spontaneous and OVA-specific IL-10 production was generally enhanced in splenocytes from mice treated with anti-ICOS during sensitization, irrespective of the time of harvesting.


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  		TABLE 4 IL-10 production by cultured splenocytes from mice treated with anti-ICOS during sensitization

 
Therapeutic Delivery of Anti-ICOS to Mice with Established Allergic Airway Disease Has No Effect on Airway Inflammation or IgE Production During OVA Rechallenge
We next evaluated the therapeutic potential of ICOS attenuation in mice with an established allergic phenotype. We therefore sensitized mice to OVA in the absence of anti-ICOS intervention and, following the resolution of acute inflammation, rechallenged mice in the context of ICOS blockade. Whether anti-ICOS was administered intraperitoneally on two occasions (the first and last challenges on Days 0 and 2, respectively) or on three occasions (Days 0, 2 and 4), mice mounted an equivalent inflammatory response in BAL and produced similar levels of OVA-specific IgE in serum compared with untreated (data not shown) and Ig-treated mice (Figure 5) . This observation is particularly striking given the robust degree ({bsim} 39%) of ICOS expression on CD4+ T cells from the lungs of control mice, and its virtual nondetection in anti-ICOS–treated mice, according to flow cytometric analysis (Figure 6) . This observation, coupled with the unaltered distribution of T1/ST2 on T helper cells from mice rechallenged in the context of anti-ICOS intervention, would suggest that ICOS expression is redundant for both the recruitment and function of T cells in mice with established allergic airway disease.



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  		Figure 5. Effect of therapeutic anti-ICOS intervention on inflammation in BAL and IgE in serum during long-term antigen rechallenge of mice with established allergic airway disease. Mice were sensitized to aerosolized OVA in the context of a Th2-polarizing airway microenvironment. Acute inflammatory phenomena were allowed to resolve ({bsim} 4 wk) before mice were rechallenged with OVA on Days 0–2 and concurrently treated with anti-ICOS or control Ig on Days 0 and 2 (2x) or Days 0, 2, and 4 (3x) of the in vivo recall protocol. Graphs display mononuclear cell and eosinophil accumulation in BAL, and OVA-specific IgE content in serum 72 h after the final OVA recall challenge (Day 5). Data are expressed as mean ± SEM; n = 7–10 per group.

 


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  		Figure 6. Flow cytometric analysis of T cells from the lungs of sensitized mice following long-term OVA rechallenge in the context of ICOS ablation. Mice were sensitized to OVA according to the Th2-polarizing regimen and rechallenged with OVA while receiving treatment with anti-ICOS or control Ig. Mice were killed at Day 5 of the in vivo recall protocol; lungs were homogenized and enriched mononuclear cells were pooled from each group and stained for flow cytometry. Data depict ICOS (upper panels) or T1/ST2 (lower panels) expression on gated CD3+CD4+ cells from mice treated with control Ig or anti-ICOS at the time of OVA rechallenge. 50,000 events were collected in the mononuclear cell gate; n = 5 per group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The recent identification of new costimulatory pathways with specialized effector or inhibitory activities has afforded novel, prospective therapeutics for a variety of immunologic disorders. The ICOS/B7RP-1 axis, in particular, has received attention based on its putative role in T cell effector function (11, 21–23), which may predispose it to modulation that selectively blocks the expression of disease rather than indiscriminately impairing the development of an adaptive response generally. In this vein, ICOS/B7RP-1 represents a costimulatory target of therapeutic relevance to established disease; unlike the CD28/CD80/CD86 system, which is critically engaged during the initial stages of antigen presentaton and T cell activation, ICOS/B7RP-1 may be indispensable to the execution of an immunologic program during periods of antigen challenge (19). Here we have appraised this hypothesis in a murine model of allergic airway inflammation in which ICOS/B7RP-1 costimulation has been discretely impaired during mucosal allergic sensitization or during in vivo antigen recall in mice with established allergic disease.

Consistent with the observations of others (31, 32), we have shown that blockade of the ICOS/B7RP-1 pathway in mice during initial encounter with antigen (OVA) attenuates Th2-polarized allergic airway inflammation, but does not alter systemic features of sensitization. In this model of mucosal allergic sensitization, mice are exposed to an OVA aerosol in the context of airway GM-CSF expression; this results in a marked eosinophilic infiltrate in the airway after {bsim} 10 d, accompanied by Th2 cytokines in the BAL, OVA-specific IgE in serum, and the production of prototypic cytokines by splenocytes stimulated with OVA (33). Compared to untreated or Ig-injected controls, concurrent intraperitoneal or intravenous treatment of mice with either anti-ICOS antibody or ICOS fusion protein during sensitization significantly reduced airway eosinophilia, and, in a complementary manner, IL-5 content in BAL at Day 9 of the protocol, but did not impair OVA-specific IgE or the capacity of splenocytes to yield Th2 cytokines. These findings argue persuasively for the preferential involvement of ICOS/B7RP-1 costimulation in the expression, rather than the genesis, of T helper phenotype, and generally concur with published findings in conventional murine models of antigen-induced allergic airway inflammation (which typically involve intraperitoneal delivery of OVA in the context of an adjuvant (31, 32)). Moreover, flow cytometric analysis of T cells at Day 9 demonstrates that ICOS is expressed in our model of antigen-induced airway inflammation, and that the neutralizing antibody used in our studies clearly affected its expression/detection. Whereas 15–20% of CD4+ T cells expressed ICOS in OVA-exposed control mice—an expression profile comparable to the distribution of the Th2 marker T1/ST2 (37–39)—ICOS was virtually undetectable on an equivalent population of cells in anti-ICOS–treated mice; as shown by Özkaynak and coworkers (36), who used the same antibody in a murine model of allograft rejection, this is indicative of receptor neutralization in vivo rather than depletion of ICOS+ cells. Interestingly, that ICOS was detected on {bsim} 50% of CD44hi CD62LloCD4+ cells of the memory/effector T cell compartment, but only on < 5% of CD44loCD62LhiCD4+ naive cells, in OVA-exposed mice at Day 9 is commensurate with its putative role in the effector activity of differentiated T cells.

Mechanistically, the effects of ICOS neutralization on acute eosinophilic inflammation in BAL reflect a diminution in the capacity of activated, polarized Th2 cells to execute their effector program upon cognate recognition of OVA in the airway. Indeed, that ICOS blockade failed to modify the magnitude or phenotype of airway inflammation in our model of Th1-polarized sensitization to OVA illustrates the Th2 selectivity of the ICOS pathway (34). Moreover, the {bsim} 50% reduction in IL-5 in the BAL of anti-ICOS–treated mice, coupled with unaltered eosinophil content in peripheral blood, suggests that the downstream effects of ICOS ablation may include reduced expression of cell-adhesion molecules or chemokine receptors by eosinophils, rather than impaired mobilization of eosinophils from the bone marrow. Of note, however—and in contrast to evidence of modest IL-5 inhibition—anti-ICOS–treated mice exhibited unaltered levels of IL-4, IL-13, and eotaxin, the prototypic eosinophil-recruiting chemokine, in BAL. It would seem, then, that the activity of ICOS in this model does not extend to all Th2 processes, but is restricted to the potentiation of particular proinflammatory phenomena.

That neutralization of ICOS attenuates allergic airway inflammation during sensitization and initial respiratory exposure to antigen, however, does not necessarily bear on the long-term consequences of ICOS ablation in vivo, nor on the potential of the ICOS/B7RP-1 pathway as a therapeutic target in established disease. To address this question, we examined the effects of anti-ICOS delivery on the long-term response (i.e., after the resolution of acute inflammation) to in vivo rechallenge with aerosolized OVA. As expected, intraperitoneal treatment with anti-ICOS during mucosal sensitization to OVA (acute exposure) did not inhibit airway eosinophilia upon OVA rechallenge (chronic exposure). This observation extends our in vitro findings, which demonstrate that ICOS blockade in vivo did not compromise T cell sensitization, and provides novel in vivo verification that the anti-inflammatory effects of ICOS/B7RP-1 neutralization are both transient and secondary to the development and maturation of an adaptive Th2-polarized response. In fact, and peculiarly, mice treated with anti-ICOS during sensitization presented evidence of an exacerbated allergic phenotype upon long-term rechallenge: airway eosinophilia and OVA-specific IgE in serum were both elevated (on the order of 50–100%) compared with Ig-treated controls. The mechanism by which impairment of ICOS during sensitization might magnify immune-inflammatory phenomena upon in vivo recall merits further study. However, that splenocytes from mice treated with anti-ICOS in vivo produce higher levels of IL-4 and IL-5 when stimulated with OVA is consistent with an hypothesis of T cell hyperpolarization or dysregulation in the absence of ICOS signaling. Likewise, reports of exacerbated clinical manifestations and accelerated mortality in mice treated with anti-ICOS during the sensitization phase of EAE (29) lend credence to the notion that ICOS function may not be restricted to the expression of an effector program in differentiated T cells, and may extend more broadly to T cell activation, education, and regulation. Indeed, as Akbari and colleagues (40) have recently demonstrated, ICOS–ICOS ligand interactions may be critically involved in the development of the IL-10–producing regulatory T cells that mediate inhalation tolerance and attenuate the expression of an asthma phenotype in mice. Although we found that the capacity of splenocytes to produce IL-10 was not impaired—and was generally enhanced—in mice treated with anti-ICOS during sensitization, suggesting that IL-10 deficiency is not responsible for the long-term effects of ICOS neutralization in our model, the possibility that the immunoregulatory activities of particular T cell subsets fail to mature in the absence of ICOS signaling is an hypothesis under active investigation in our laboratory.

Of particular therapeutic interest, and in striking contrast to observations in acutely-exposed mice, our studies unexpectedly show that intraperitoneal administration of anti-ICOS during long-term rechallenge of sensitized mice does not alter the magnitude or phenotype of lung inflammation, or the content of OVA-specific IgE in serum. The inefficacy of this treatment, moreover, cannot be attributed to kinetics of ICOS expression: {bsim} 40% of CD4+ T cells isolated from the lungs of control mice co-expressed ICOS during in vivo OVA recall—a marked amplification of levels observed at Day 9 of the protocol and consonant with the presumed expansion of memory lymphocytes at rechallenge. Paradoxically, then, ICOS expression, although robustly identifying lymphocytes mobilized during a memory response, may be functionally irrelevant to the elaboration of the memory response. It is not clear why ICOS should exhibit this biological redundancy, nor why its activity during antigen sensitization and acute exposure should differ from its role in established disease. However, it may be that ICOS operates in a narrow window between CD28/B7-mediated T cell activation and irrevocable commitment of cells to a memory/effector phenotype. That is, ICOS ligation may modulate (positively or negatively depending, for example, on the strength of the activation signal) the differentiation of activated T cells into Th subsets either directly or by regulating the secretion of Th-polarizing cytokines. Once the memory/effector T cell pool has expanded and matured—a protracted process that may take several rounds of stimulation, and which may explain the apparently discordant conclusions in more compressed models of antigen sensitization and challenge (31, 32)—cells become hyporesponsive to further modulation by ICOS. This explanation is somewhat reminiscent of Sperling and Bluestone's "strength of signal" hypothesis of ICOS function (41): as the strength of the TCR/B7 activation signal increases (or, in the case of a memory response, the requirements for activation decrease), the need for ICOS costimulation diminishes. Additionally, there may also exist a subset of Th2-polarized memory cells that do not express ICOS and whose activity compensates for the functional deficiency of ICOS-dependent lymphocytes.

Our study, therefore, challenges the notion that the ICOS/B7RP-1 costimulatory axis represents a promising therapeutic target for immune-inflammatory disorders such as asthma. Although neutralization of ICOS during sensitization and acute antigen exposure attenuated airway inflammation, this intervention ultimately exacerbated immune-inflammatory phenomena upon long-term in vivo recall. Moreover, blockade of ICOS signaling during rechallenge of mice with established allergic disease elicited no therapeutic effect, intimating that ICOS may be functionally redundant in mature memory responses.


   Acknowledgments
 
The secretarial assistance of Mary Kiriakopoulos is gratefully acknowledged. This research has been funded by a CIHR operating grant. R.E.W. is supported by a CIHR Doctoral Research Award, J.R.J. is supported by an Ontario Graduate Scholarship, and M.J. holds a Canada Research Chair.

Received in original form October 18, 2002

Received in final form December 11, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Benhamou, P. Y. 2002. Immunomodulation with CTLA4-Ig in islet transplantation. Transplantation 73:S40–S42.[CrossRef][Medline]
  2. Rifle, G., and C. Mousson. 2002. Dendritic cells and second signal blockade: a step toward allograft tolerance? Transplantation 73:S1–S2.
  3. Salomon, B., and J. A. Bluestone. 2001. Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation. Annu. Rev. Immunol. 19:225–252.[CrossRef][Medline]
  4. Hurwitz, A. A., E. D. Kwon, and A. van Elsas. 2000. Costimulatory wars: the tumor menace. Curr. Opin. Immunol. 12:589–596.[CrossRef][Medline]
  5. Schlom, J., and J. W. Hodge. 1999. The diversity of T-cell co-stimulation in the induction of antitumor immunity. Immunol. Rev. 170:73–84.[CrossRef][Medline]
  6. Anderson, D. E., A. H. Sharpe, and D. A. Hafler. 1999. The B7–CD28/CTLA-4 costimulatory pathways in autoimmune disease of the central nervous system. Curr. Opin. Immunol. 11:677–683.[CrossRef][Medline]
  7. Daikh, D. I., and D. Wofsy. 2001. Treatment of autoimmunity by inhibition of T cell costimulation. Adv. Exp. Med. Biol. 490:113–117.[Medline]
  8. Schweitzer, A. N., and A. H. Sharpe. 1998. Studies using antigen-presenting cells lacking expression of both B7–1 (CD80) and B7–2 (CD86) show distinct requirements for B7 molecules during priming versus restimulation of Th2 but not Th1 cytokine production. J. Immunol. 161:2762–2771.[Abstract/Free Full Text]
  9. Aicher, A., M. Hayden-Ledbetter, W. A. Brady, A. Pezzutto, G. Richter, D. Magaletti, S. Buckwalter, J. A. Ledbetter, and E. A. Clark. 2000. Characterization of human inducible costimulator ligand expression and function. J. Immunol. 164:4689–4696.[Abstract/Free Full Text]
  10. Brodie, D., A. V. Collins, A. Iaboni, J. A. Fennelly, L. M. Sparks, X. N. Xu, P. A. van der Merwe, and S. J. Davis. 2000. LICOS, a primordial costimulatory ligand? Curr. Biol. 10:333–336.[CrossRef][Medline]
  11. Coyle, A. J., S. Lehar, C. Lloyd, J. Tian, T. Delaney, S. Manning, T. Nguyen, T. Burwell, H. Schneider, J. A. Gonzalo, M. Gosselin, L. R. Owen, C. E. Rudd, and J. C. Gutierrez-Ramos. 2000. The CD28-related molecule ICOS is required for effective T cell- dependent immune responses. Immunity 13:95–105.[CrossRef][Medline]
  12. Hutloff, A., A. M. Dittrich, K. C. Beier, B. Eljaschewitsch, R. Kraft, I. Anagnostopoulos, and R. A. Kroczek. 1999. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28. Nature 397:263–266.[CrossRef][Medline]
  13. Mages, H. W., A. Hutloff, C. Heuck, K. Buchner, H. Himmelbauer, F. Oliveri, and R. A. Kroczek. 2000. Molecular cloning and characterization of murine ICOS and identification of B7h as ICOS ligand. Eur. J. Immunol. 30:1040–1047.[CrossRef][Medline]
  14. Yoshinaga, S. K., J. S. Whoriskey, S. D. Khare, U. Sarmiento, J. Guo, T. Horan, G. Shih, M. Zhang, M. A. Coccia, T. Kohno, A. Tafuri-Bladt, D. Brankow, P. Campbell, D. Chang, L. Chiu, T. Dai, G. Duncan, G. S. Elliott, A. Hui, S. M. McCabe, S. Scully, A. Shahinian, C. L. Shaklee, G. Van, T. W. Mak, and G. Senaldi. 1999. T-cell co-stimulation through B7RP-1 and ICOS. Nature 402:827–832.[CrossRef][Medline]
  15. Yoshinaga, S. K., M. Zhang, J. Pistillo, T. Horan, S. D. Khare, K. Miner, M. Sonnenberg, T. Boone, D. Brankow, T. Dai, J. Delaney, H. Han, A. Hui, T. Kohno, R. Manoukian, J. S. Whoriskey, and M. A. Coccia. 2000. Characterization of a new human B7-related protein: B7RP-1 is the ligand to the co-stimulatory protein ICOS. Int. Immunol. 12:1439–1447.[Abstract/Free Full Text]
  16. Dong, H., G. Zhu, K. Tamada, and L. Chen. 1999. B7–H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat. Med. 5:1365–1369.[CrossRef][Medline]
  17. Tamura, H., H. Dong, G. Zhu, G. L. Sica, D. B. Flies, K. Tamada, and L. Chen. 2001. B7–H1 costimulation preferentially enhances CD28-independent T-helper cell function. Blood 97:1809–1816.[Abstract/Free Full Text]
  18. Chambers, C. A. 2001. The expanding world of co-stimulation: the two-signal model revisited. Trends Immunol. 22:217–223.[CrossRef][Medline]
  19. Sperling, A. I. 2001. ICOS costimulation: is it the key to selective immunotherapy? Clin. Immunol. 100:261–262.[CrossRef][Medline]
  20. Sporici, R. A., and P. J. Perrin. 2001. Costimulation of memory T-cells by ICOS: a potential therapeutic target for autoimmunity? Clin. Immunol. 100:263–269.[CrossRef][Medline]
  21. Dong, C., A. E. Juedes, U. A. Temann, S. Shresta, J. P. Allison, N. H. Ruddle, and R. A. Flavell. 2001. ICOS co-stimulatory receptor is essential for T-cell activation and function. Nature 409:97–101.[CrossRef][Medline]
  22. Gonzalo, J. A., T. Delaney, J. Corcoran, A. Goodearl, J. C. Gutierrez-Ramos, and A. J. Coyle. 2001. Cutting edge: the related molecules CD28 and inducible costimulator deliver both unique and complementary signals required for optimal T cell activation. J. Immunol. 166:1–5.[Abstract/Free Full Text]
  23. Ogawa, S., G. Nagamatsu, M. Watanabe, S. Watanabe, T. Hayashi, S. Horita, K. Nitta, H. Nihei, K. Tezuka, and R. Abe. 2001. Opposing effects of anti-activation-inducible lymphocyte-immunomodulatory molecule/inducible costimulator antibody on the development of acute versus chronic graft-versus-host disease. J. Immunol. 167:5741–5748.[Abstract/Free Full Text]
  24. Dong, C., U. A. Temann, and R. A. Flavell. 2001. Cutting edge: critical role of inducible costimulator in germinal center reactions. J. Immunol. 166:3659–3662.[Abstract/Free Full Text]
  25. Guo, J., M. Stolina, J. V. Bready, S. Yin, T. Horan, S. K. Yoshinaga, and G. Senaldi. 2001. Stimulatory effects of B7-related protein-1 on cellular and humoral immune responses in mice. J. Immunol. 166:5578–5584.[Abstract/Free Full Text]
  26. McAdam, A. J., R. J. Greenwald, M. A. Levin, T. Chernova, N. Malenkovich, V. Ling, G. J. Freeman, and A. H. Sharpe. 2001. ICOS is critical for CD40-mediated antibody class switching. Nature 409:102–105.[CrossRef][Medline]
  27. Tafuri, A., A. Shahinian, F. Bladt, S. K. Yoshinaga, M. Jordana, A. Wakeham, L. M. Boucher, D. Bouchard, V. S. Chan, G. Duncan, B. Odermatt, A. Ho, A. Itie, T. Horan, J. S. Whoriskey, T. Pawson, J. M. Penninger, P. S. Ohashi, and T. W. Mak. 2001. ICOS is essential for effective T-helper-cell responses. Nature 409:105–109.[CrossRef][Medline]
  28. McAdam, A. J., T. T. Chang, A. E. Lumelsky, E. A. Greenfield, V. A. Boussiotis, J. S. Duke-Cohan, T. Chernova, N. Malenkovich, C. Jabs, V. K. Kuchroo, V. Ling, M. Collins, A. H. Sharpe, and G. J. Freeman. 2000. Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells. J. Immunol. 165:5035–5040.[Abstract/Free Full Text]
  29. Rottman, J. B., T. Smith, J. R. Tonra, K. Ganley, T. Bloom, R. Silva, B. Pierce, J. C. Gutierrez-Ramos, E. Ozkaynak, and A. J. Coyle. 2001. The costimulatory molecule ICOS plays an important role in the immunopathogenesis of EAE. Nat. Immunol. 2:605–611.[CrossRef][Medline]
  30. O'Neill, L. 2001. Co-stimulating allergy. Trends Immunol. 22:183.
  31. Gonzalo J. A., J. Tian, T. Delaney, J. Corcoran, J. B. Rottman, J. Lora, A. Al-garawi, R. Kroczek, J. C. Gutierrez-Ramos, and A. J. Coyle. 2001. ICOS is critical for T helper cell-mediated lung mucosal inflammatory responses. Nat. Immunol. 2:597–604.[CrossRef][Medline]
  32. Tesciuba, A. G., S. Subudhi, R. P. Rother, S. J. Faas, A. M. Frantz, D. Elliot, J. Weinstock, L. A. Matis, J. A. Bluestone, and A. I. Sperling. 2001. Inducible costimulator regulates Th2-mediated inflammation, but not Th2 differentiation, in a model of allergic airway disease. J. Immunol. 167:1996–2003.[Abstract/Free Full Text]
  33. Stämpfli, M. R., R. E. Wiley, G. S. Neigh, B. U. Gajewska, X. F. Lei, D. P. Snider, Z. Xing, and M. Jordana. 1998. GM-CSF transgene expression in the airway allows aerosolized ovalbumin to induce allergic sensitization in mice. J. Clin. Invest. 102:1704–1714.[Medline]
  34. Stämpfli, M. R., G. Scott Neigh, R. E. Wiley, M. Cwiartka, S. A. Ritz, M. M. Hitt, Z. Xing, and M. Jordana. 1999. Regulation of allergic mucosal sensitization by interleukin-12 gene transfer to the airway. Am. J. Respir. Cell Mol. Biol. 21:317–326.[Abstract/Free Full Text]
  35. Ohkawara, Y., X. F. Lei, M. R. Stampfli, J. S. Marshall, Z. Xing, and M. Jordana. 1997. Cytokine and eosinophil responses in the lung, peripheral blood, and bone marrow compartments in a murine model of allergen-induced airways inflammation. Am. J. Respir. Cell Mol. Biol. 16:510–520.[Abstract]
  36. Özkaynak, E., W. Gao, N. Shemmeri, C. Wang, J. C. Gutierrez-Ramos, J. Amaral, S. Qin, J. B. Rottman, A. J. Coyle, and W. W. Hancock. 2001. Importance of ICOS-B7RP-1 costimulation in acute and chronic allograft rejection. Nat. Immunol. 2:591–596.[CrossRef][Medline]
  37. Coyle, A. J., C. Lloyd, J. Tian, T. Nguyen, C. Erikkson, L. Wang, P. Ottoson, P. Persson, T. Delaney, S. Lehar, S. Lin, L. Poisson, C. Meisel, T. Kamradt, T. Bjerke, D. Levinson, and J. C. Gutierrez-Ramos. 1999. Crucial role of the interleukin 1 receptor family member T1/ST2 in T helper cell type 2-mediated lung mucosal immune responses. J. Exp. Med. 190:895–902.[Abstract/Free Full Text]
  38. Hoshino, K., S. Kashiwamura, K. Kuribayashi, T. Kodama, T. Tsujimura, K. Nakanishi, T. Matsuyama, K. Takeda, and S. Akira. 1999. The absence of interleukin 1 receptor-related T1/ST2 does not affect T helper cell type 2 development and its effector function. J. Exp. Med. 190:1541–1548.[Abstract/Free Full Text]
  39. Townsend, M. J., P. G. Fallon, D. J. Matthews, H. E. Jolin, and A. N. McKenzie. 2000. T1/ST2-deficient mice demonstrate the importance of T1/ST2 in developing primary T helper cell type 2 responses. J. Exp. Med. 191:1069–1076.[Abstract/Free Full Text]
  40. Akbari, O., G. J. Freeman, E. H. Meyer, E. A. Greenfield, T. T. Chang, A. H. Sharpe, G. Berry, R. H. DeKruyff, and D. T. Umetsu. 2002. Antigen-specific regulatory T cells develop via the ICOS ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity. Nat. Med. 29:1024–1032.
  41. Sperling, A. I., and J. A. Bluestone. 2001. ICOS costimulation: It's not just for Th2 cells anymore. Nat. Immunol. 2:573–574.[CrossRef][Medline]



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