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DOI: 10.1002/eji.201445163
Katie E. Burrows et al.
Eur. J. Immunol. 2015. 45: 1116–1128
OX40 blockade inhibits house dust mite driven allergic lung inflammation in mice and in vitro allergic responses in humans Katie E. Burrows ∗ , Celine Dumont ∗ , Clare L. Thompson, Matthew C. Catley ∗ , Kate L. Dixon and Diane Marshall Immunology Therapeutic Area, UCB Pharma, Slough, Berkshire, UK The costimulatory receptor OX40 is expressed on activated T cells and regulates T-cell responses. Here, we show the efficacy and mechanism of action of an OX40 blocking antibody using the chronic house dust mite (HDM) mouse model of lung inflammation and in vitro HDM stimulation of cells from HDM allergic human donors. We have demonstrated that OX40 blockade leads to a reduction in the number of eosinophils and neutrophils in the lavage fluid and lung tissue of HDM sensitized mice. This was accompanied by a decrease in activated and memory CD4+ T cells in the lungs and further analysis revealed that both the Th2 and Th17 populations were inhibited. Improved lung function and decreased HDM-specific antibody responses were also noted. Significantly, efficacy was observed even when anti-OX40 treatment was delayed until after inflammation was established. OX40 blockade also inhibited the release of the Th2 cytokines IL-5 and IL-13 from cells isolated from HDM allergic human donors. Altogether, our data provide evidence of a role of the OX40/OX40L pathway in ongoing allergic lung inflammation and support clinical studies of a blocking OX40 antibody in Th2 high severe asthma patients.
Keywords: Allergic asthma r House dust mite
r
Lung inflammation r Memory T cells
r
OX40
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction OX40 (also known as CD134, TNFRSF4, ACT35, or TXGP1L) is a membrane-bound receptor belonging to the TNF receptor superfamily, which includes 4-1BB, CD27, CD30, and CD40. This costimulatory receptor is not expressed on resting T cells, but is transiently expressed on activated T cells after ligation of the TCR, with sequential engagement of CD28 and OX40 being required for optimal T-cell proliferation and survival [1]. Ligation of OX40 on activated T cells leads to enhanced cytokine production and proliferation of both CD4+ and CD8+ T cells [2, 3] and can contribute to both ongoing Th1 and Th2 responses [4, 5]. OX40 costimulation
Correspondence: Diane Marshall e-mail: [email protected] C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
prolongs T-cell survival beyond the initial effector phase of the immune response and increases the number of memory T cells through inhibition of effector T-cell death. This long-term survival of T cells via OX40 is thought to be a consequence of maintaining high levels of antiapoptotic proteins, such as Bcl-2, Bcl-xL, and Bfl-1 [6, 7]. The ligand for OX40, OX40L, is a member of the TNF family and is expressed on activated APC, including B cells, macrophages, Langerhans cells, and dendritic cells (DCs). Other cell types that are reported to express OX40L include mast cells, NK cells, airway smooth muscle cells, endothelial cells, and platelets [8].
∗
These authors are co-first authors.
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Eur. J. Immunol. 2015. 45: 1116–1128
Asthma is a chronic inflammatory disease of the airways in which many cells and mediators are involved. It is now clear that asthma is a heterogenic disease and patients can be stratified into different groups based on the drivers of their disease. One increasingly characterized group is the Th2-driven severe asthmatics. These patients are defined in a number of ways including high circulating periostin, elevated sputum eosinophils, and high circulating IgE despite treatment with high doses of inhaled corticosteroids. In addition, these patients respond to therapies such as anti-IL-5, anti-IL-13, and anti-IgE. Targeting Th2-driven inflammation is therefore clinically validated in human disease and indeed therapies targeting multiple facets of the Th2 pathway may be more effective than targeting single mediators [9–12]. The direct inhibition of T cells via inhibition of T-cell costimulation through interruption of CD28 signaling, utilizing CLTA4Ig, previously failed to modulate the response to allergen challenge or asthma symptoms in mild atopic asthma [13]. However, it has been postulated that T-cell inhibition via this mechanism would have a greater effect on the initiation of an immune response rather than inhibiting effector memory T cells [14, 15]. Chronic exposure to allergen is known to drive the development of antigenspecific memory effector T cells and therefore specifically targeting these pathogenic activated memory T cells may provide a selective novel treatment for severe Th2-driven asthma. Several studies have provided evidence for a role of OX40 in lung inflammation and asthma. OX40 KO mice primed and challenged with OVA exhibit diminished lung inflammation (80–90% reduction in eosinophilia), reduced mucus production, and significantly attenuated airway hyper-reactivity [16]. Monoclonal antibodies to OX40 ligand have also shown beneficial effects in murine models of lung inflammation [17, 18]. In addition, an antibody to human OX40L reduced levels of the Th2 mediators, IL-5 and IL-13, and effector memory T cells in bronchoalveolar lavage fluid (BALF) after allergen challenge in a model of lung inflammation in rhesus monkeys [19]. In patients, an increase in the number of OX40- and OX40L-positive cells in mild asthmatic patients has been shown to correlate with the number of eosinophils and IL-4+ cells in the bronchial submucosa [20] and serum. Similarly, OX40L has been noted to be increased in asthmatic children and adults and was associated with increased severity and persistence of disease [21, 22]. We have sought to expand the current knowledge surrounding the role of OX40 in allergic lung inflammation by using in vivo and in vitro models driven by house dust mite (HDM) allergen, known to be a common driver of asthmatic disease [23]. Chronic exposure to inhaled allergens induces an inflammatory response in the lung resulting in clinical symptoms of asthma. The chronic HDM model in mice, involving mucosal sensitization to inhaled HDM, is now considered to be an improved preclinical in vivo model to determine the efficacy and mechanism of action of potential novel therapeutics for asthmatic disease and was recently shown to be sensitive to anti-IL-13 treatment [24]. We have used the chronic HDM model of allergic lung inflammation to investigate the efficacy of an anti-OX40 blocking antibody. Using this in vivo model and correlation with data from an in vitro human model of HDM-driven C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Immunomodulation
allergic inflammation, we have investigated the potential mechanism of action of OX40 blockade in allergic inflammation, in particular determining the effect on specific T-cell subpopulations. Our studies have revealed a reduction in the number of activated and memory T cells and also both Th2 and Th17 cells after OX40 blockade. Importantly, efficacy was also noted when dosing of the OX40 antibody was delayed until after lung inflammation was already established.
Results Prophylactic anti-OX40 mAb prevents the development of allergic airways disease in mice Chronic exposure to HDM causes cellular infiltration of the airways by immune and inflammatory cells. Routinely, significant numbers of eosinophils, T cells, and neutrophils are found in BALF at week 5. To determine the effect of anti-OX40 blocking antibodies on the infiltration of leukocytes in the lung, we exposed mice to HDM extract for 5 weeks, during concomitant treatment with anti-OX40 antibody or saline vehicle at the indicated doses. As shown in Figure 1, chronic exposure to HDM resulted in cellular infiltration of the airways and lung parenchyma. Administration of anti-OX40 antibody at the time of initial exposure and throughout HDM exposure completely inhibited the infiltration of the lung tissue and airways by eosinophils (Fig. 1A and C) and resulted in a significant decrease in the number of infiltrated neutrophils in the BALF (by 81, 90, and 80% at 100, 30, and 10 mg/kg, respectively, relative to vehicle control (Fig. 1D)). The effects of anti-OX40 treatment on neutrophilia were more apparent in lung tissue than BALF as neutrophils in lung tissue were reduced to baseline levels and low-dose treatment (1 mg/kg) was also efficacious (Fig. 1B). Moreover, anti-OX40 prophylactic treatment demonstrated a significant inhibition of total CD4+ T-cell infiltration in the lung (by 60, 65, and 65% at 100, 30, and 10 mg/kg, respectively, relative to vehicle control) and in the BALF (by 90, 84, and 79% at 100, 30, and 10 mg/kg, respectively, relative to vehicle control; Fig. 2A and B). The number of CD8+ T cells was also significantly reduced (data not shown), although in this model of airway inflammation, CD4+ T cells are the most prominent population found in the inflamed lung. It has been shown that OX40–OX40L interaction contributes to the generation and/or survival of CD4+ effector memory cells. To further characterize the effect of OX40 blockade, we analyzed the phenotype of the CD4+ cells. Most of the CD4+ cells present in the airways after exposure to HDM were activated (CD4+ CD44hi CD25+ ) or displayed an effector memory like phenotype (CD4+ CD44hi CD25− CD62Llo ). Interestingly, the number of activated T cells and effector memory T cells was significantly reduced in the lung and BALF of anti-OX40 treated mice as compared to control, (Fig. 2C–F) while the na¨ıve CD4+ T cell numbers were unchanged (data not shown). These results are compatible with previous reports. The majority of CD4+ T cells in the lungs following HDM exposure were Th2 polarized as indicated by their expression of T1ST2 www.eji-journal.eu
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Figure 1. Effect of prophylactic treatment with anti-OX40 antibody on granulocytes in the lung and BALF. Cells in the lung after exposure to HDM for 5 weeks along with continuous administration of antiOX40 antibody were analyzed by multicolor flow cytometry. The number of (A, C) eosinophils and (B, D) neutrophils in the (A and B) lung and (C and D) in BALF. Data are presented as mean + SEM and are from one experiment, with n = 7–8 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control.
with some IL-17-producing cells also present. The number of IFN-γ-producing cells (Th1) is minimal in this model. As shown in Figure 3, the number of IL-13- or IL-17-producing CD4+ cells was significantly reduced in the lung and BALF of mice treated with prophylactic dosing of the OX40 blocking antibody (by at least 81% for Th2 cells (Fig. 3A and C) and by 88, 87, and 86% for IL-17-producing cells (Fig. 3E) at 100, 30, and 10 mg/kg, respectively, in the lung, relative to vehicle control). The level of inhibition of the number of Th2 cells and IL-17-producing cells in the BALF reaches over 90% in mice treated with 100 and 30 mg/kg of blocking antibody and 80% at 10 mg/kg (Fig. 3B, D, and F). In addition, a reduction in the number of IL-4- and IL-10producing cells was noted as is shown in Supporting Information Figure 6. In order to determine whether airway inflammation correlated with changes in lung function in our model, plethysmography was used to monitor changes in respiratory dynamics following exposure to methacholine. HDM exposure resulted in increased sensitivity to methacholine challenge shown by a significantly higher Penh measurement compared to sham-treated controls (Fig. 4A). This hyper-responsiveness to methacholine was abrogated in mice treated with anti-OX40 antibody. Inhibition of lung disease by anti-OX40 treatment was also confirmed by histology. Airway wall remodeling is observed in this model [24] and the anti-OX40 antibody inhibited subepithelial collagen deposition, as shown in a representative tissue section (Fig. 4C) compared with the mice exposed to HDM and treated with vehicle (Fig. 4B). Similarly, fewer mucus-producing goblet cells were also noted in mice treated with anti-OX40 antibody (Fig. 4E) compared to vehicletreated mice (Fig. 4D).
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Th2 immune responses are often associated with IgG1 and IgE antibody production. Analysis of the antibody response to HDM confirmed that intranasal administration of HDM resulted in the generation of HDM-specific IgG1 and IgE in plasma. Treatment with anti-OX40 antibody significantly reduced both IgG1 and IgE HDM-specific antibody levels (Fig. 5). Thus, these data indicate that blocking OX40 can reduce immune inflammatory cells and T-cell numbers in the airways and lung tissue leading to a reduction in the chronic inflammation of the airways, which is believed to drive the physiological features of asthma. The reduction in Th2 and Th17 cells provides evidence that this may be beneficial in asthmatics with Th2-driven disease. In summary, prophylactic administration of an anti-OX40 mAb prevented the development of HDM-induced inflammation. These effects were apparent within the airways, lung parenchyma, and systemically, thus verifying that the interaction between OX40 and OX40L is critical for the initiation of allergic airways disease.
Therapeutic treatment with anti-OX40 mAb inhibits lung inflammation in mice In order to determine whether OX40 blockade could modulate established allergic airway inflammation, a therapeutic dosing regimen was employed. BALB/c mice were exposed to HDM for 5 weeks to induce chronic pathology mimicking clinical asthma. Vehicle (saline), 100 or 30 mg/kg of anti-OX40 antibody, was
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Immunomodulation
Figure 2. Effect of prophylactic treatment with anti-OX40 antibody on CD4+ T cells in the lung and BALF. Cells in the lung after exposure to HDM for 5 weeks along with continuous administration of antiOX40 antibody were analyzed by multicolor flow cytometry. Total numbers of (A, B) CD4+ T cells, (C, D) activated CD4+ T cells (CD4+ CD44hi CD25+ CD62Llo ), and (E, F) effector memory like CD4+ T cells (CD4+ CD44hi CD25− CD62Llo ) in the (A, C, E) lung and (B, D, F) BALF. Data are presented as mean + SEM and are from one experiment, with n = 8 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control.
injected at week 6 (from day 35) and continued twice weekly for a further 3 weeks during concomitant HDM exposure. Lung and BALF samples were then collected and analyzed by multicolor flow cytometry after the 8th week of HDM exposure. As shown in Figure 6, therapeutic treatment of the mice with 100 mg/kg anti-OX40 antibody resulted in a significant reduction in the number of eosinophils and neutrophils in BALF. Moreover, the total number of CD4+ T cells in the lung (Fig. 7A) and BALF (Fig. 7B) was reduced in mice treated with 100 mg/kg anti-OX40 antibody, and to a lesser extent with 30 mg/kg (90 and 34% reduction (relative to vehicle control) in the lung, respectively). In concurrence with our prophylactic data, the CD4+ T-cell subsets most susceptible to inhibition by OX40 blockade were acti-
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
vated CD4+ T cells and effector memory like CD4+ T cells with a respective reduction in the lung of 90 and 91% (relative to vehicle control) with 100 mg/kg anti-OX40 antibody (Fig. 7C and E). This effect was mirrored in the airways, albeit to a lesser extent (Fig. 7D and F). OX40 blockade also resulted in a significant decrease of Th2 and Th17 cell numbers in the lung (Fig. 8A and B; 50 and 40%, respectively, at 100 mg/kg, relative to vehicle control) and in the BALF (Fig. 8C and D; 55 and 57% for Th2and IL-17-producing cells, respectively, at 100 mg/kg relative to vehicle control). A reduction in IL-10-positive cells was also noted and is shown in Supporting Information Figure 7. Altogether these data show that blockade of OX40 attenuates established inflammation of the lung and airways by reducing
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Figure 3. Effect of prophylactic treatment with anti-OX40 antibody on Th2 and Th17 cells in the lung and BALF. Cytokine production by CD4+ T cells in the lung after exposure to HDM for 5 weeks along with continuous administration of anti-OX40 antibody was analyzed by intracellular cytokine staining by multicolor flow cytometry. The number of (A, B) Th2 cells (CD4+ T1ST2+ ), (C, D) IL-13+ CD4+ T cells, and (E, F) Th17 cells in the (A, C, E) lung and (B, D, F) BALF. Data are presented as mean ± SEM and are from one experiment, with n = 8 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control.
the number of activated and effector memory CD4+ T cells, thus implicating the interaction between OX40 and OX40L in the perpetuation of allergic airways disease.
Blocking OX40 reduces Th2 cytokine production by PBMCs from HDM allergic donors Allergens such as those derived from HDM can induce proliferative responses and Th2 cytokine production in sensitized individuals. PMBCs from HDM allergic donors, when stimulated in vitro with HDM extract, produce high levels of the Th2 cytokines IL-5 and IL-13 compared to nonallergic donors (Fig. 9B and C). This is in contrast to the levels of Th1 cytokines, which are comparable between allergic and nonallergic donors (Fig. 9A). To understand whether the efficacy observed in murine models would translate to atopy in man, we used HDM allergic donors to investigate the effect of OX40 blockade on Th2 cytokine production following activation of PBMCs with HDM extract. PBMCs were stimulated with C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
HDM extract for 6 days in the presence of 50 μg/mL anti-OX40 or control antibody. Since large variations in response were found across donors and experiments, the data were pooled and represented as percent inhibition. (Data from an individual representative donor are shown in Supporting Information Fig. 1.) Treatment with the OX40 antibody significantly inhibited IL-5 and IL-13 production by 53 and 71%, respectively, compared to PBMCs cultured without antibody (Fig. 10). The control antibody did not inhibit Th2 cytokine production indicating that anti-OX40 treatment may be an effective treatment for allergic Th2-driven asthma.
Blocking OX40 inhibits both primary and secondary CD4+ T-cell responses to HDM antigens in vitro OX40 plays a central role in the generation of memory T-cell responses and in the promotion of effector T-cell survival after www.eji-journal.eu
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Figure 4. Effect of prophylactic treatment with anti-OX40 antibody on respiratory dynamics induced by methacholine, airway remodeling, and goblet cell hyperplasia. (A) Airway response of vehicle control or anti-OX40-treated mice to methacholine (24 h after exposure to HDM). Data presented as mean Penh + SEM and are from one experiment, with n = 7–8 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control. Representative photomicrographs of airway sections stained with (B, C) Masson’s trichrome or (D, E) AB/PAS from mice exposed to HDM and treated with (B, D) vehicle or (C, E) anti-OX40 antibody. Scale bar: 100 μm. Images are representative of one experiment.
antigen priming. We therefore hypothesized that treatment of human CD4+ T cells with an anti-OX40 antibody would not only inhibit a primary antigen response in vitro through costimulation blockade, but may also inhibit a secondary response to the same antigen due to a reduction in survival and effector function of antigen-specific memory T cells. To investigate this, CD4+ T cells from HDM allergic donors were stimulated with autologous APCs pulsed with HDM extract in the presence of 10 μg/mL anti-OX40 or control antibody for 7 days. At day 7, cytokine production was analyzed to evaluate the primary in vitro T-cell response. Since large vari-
ations in response were found across donors and experiments, the data were pooled and represented as percent inhibition. (Data from an individual representative donor are shown in Supporting Information Fig. 2.) Following the primary stimulation, the anti-OX40 antibody inhibited IL-5 production by 48% compared to cells cultured with no antibody (Fig. 11A). The CD4+ T cells were then washed and rested for 3 days without stimulation before being cultured with fresh autologous APCs pulsed with HDM for 3 days. No further antibodies were added during the secondary in vitro stimulation. After 3 days, the magnitude of the secondary immune response was measured by
Figure 5. Effect of prophylactic treatment with anti-OX40 antibody on levels of HDMspecific IgG1 and IgE. Levels of (A) HDMspecific IgG1 and (B) IgE were measured by ELISA in plasma taken from mice treated with vehicle or anti-OX40 antibody in a chronic HDM model of allergic airway inflammation. Data presented as mean ± SEM and are from one experiment, with n = 7–8 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control.
Figure 6. Effect of therapeutic treatment with anti-OX40 antibody on granulocytes in the BALF. Cells in the lung after exposure to HDM for 8 weeks with administration of anti-OX40 antibody from week 6 were analyzed by multicolor flow cytometry. Graphs show the numbers of (A) eosinophils and (B) neutrophils in the BALF. Data presented as mean + SEM and are from one experiment, with n = 9–10 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 7. Effect of anti-OX40 antibody therapeutic treatment on CD4+ cells in the lung and BALF. Mice were exposed to HDM for 8 weeks and dosed with anti-OX40 antibody from week 6. Cells in the lung were analyzed by multicolor flow cytometry. Graphs show the number of (A, B) total CD4+ T cells, (C, D) activated CD4+ T cells (CD4+ CD25+ CD44hi CD62Llo ), and (E, F) effector memory like CD4+ T cells (CD4+ CD25− CD44hi CD62Llo ) in the (A, C, E) lung and (B, D, F) BALF after therapeutic treatment with anti-OX40. Data presented as mean + SEM and are from one experiment, with n = 10 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control.
cytokine analysis and the number of activated CD4+ T cells was assessed by flow cytometry. Cells cultured with the anti-OX40 antibody during the primary stimulation produced less IL-5 following secondary stimulation compared to those treated with control antibody (Fig. 11A; 75% reduction compared to untreated cells). Flow cytometry analysis of the T cells treated with the antiOX40 antibody showed a decrease in the proportion of activated CD4+ CD25+ T cells after the secondary stimulation compared to cells treated with control antibody (15 and 38%, respectively; Fig. 11B). Since there was no antibody present in the culture during the secondary stimulation, these data suggest that an anti-OX40 antibody may be able to modulate antigen-specific human T cells and alter their potential to respond to the same antigen by reducing activation and effector function. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Discussion In this study, we have examined the role of the OX40/OX40L pathway in allergic asthma by utilizing a blocking OX40 antibody in a chronic HDM model of allergic lung inflammation in conjunction with human in vitro systems using cells isolated from allergic individuals. We have sought to understand the potential mechanism of action of OX40 blockade through detailed analysis of different T-cell populations that accumulate in the lung during the ongoing disease process. First, our data demonstrate that OX40 blockade inhibits both eosinophil and neutrophil infiltration into the airways and importantly into the lung tissue. This finding is relevant since it is clear that there is a group of patients with persistent eosinophilic inflammation who fail to respond to current therapies such as inhaled corticosteroids. Moreover, it is postulated
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Eur. J. Immunol. 2015. 45: 1116–1128
Immunomodulation
Figure 8. Effect of anti-OX40 antibody therapeutic treatment on Th2 and Th17 cells in the lung and BALF. Mice were exposed to HDM for 8 weeks and dosed with anti-OX40 antibody from week 6. Cytokine production by CD4+ T cells in the lung and BALF was analyzed by intracellular cytokine staining by flow cytometry. Graphs show the number of (A, C) Th2 (CD4+ T1ST2+ ) and (B, D) Th17 cells in the (A, B) lung and (C, D) BALF. Data presented as mean + SEM and are from one experiment, with n = 10 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control.
Figure 10. Effect of an anti-OX40 antibody on Th2 cytokine production from human PBMCs. PBMCs from HDM allergic donors were isolated and stimulated with HDM in the presence of anti-OX40 or control antibody for 6 days. Levels of Th2 cytokines were measured using MSD. Data are shown as mean + SEM (n = 3 replicates) and are pooled from four donors. Percent inhibition was calculated relative to cells cultured with no antibody. *p
DOI: 10.1002/eji.201445163
Katie E. Burrows et al.
Eur. J. Immunol. 2015. 45: 1116–1128
OX40 blockade inhibits house dust mite driven allergic lung inflammation in mice and in vitro allergic responses in humans Katie E. Burrows ∗ , Celine Dumont ∗ , Clare L. Thompson, Matthew C. Catley ∗ , Kate L. Dixon and Diane Marshall Immunology Therapeutic Area, UCB Pharma, Slough, Berkshire, UK The costimulatory receptor OX40 is expressed on activated T cells and regulates T-cell responses. Here, we show the efficacy and mechanism of action of an OX40 blocking antibody using the chronic house dust mite (HDM) mouse model of lung inflammation and in vitro HDM stimulation of cells from HDM allergic human donors. We have demonstrated that OX40 blockade leads to a reduction in the number of eosinophils and neutrophils in the lavage fluid and lung tissue of HDM sensitized mice. This was accompanied by a decrease in activated and memory CD4+ T cells in the lungs and further analysis revealed that both the Th2 and Th17 populations were inhibited. Improved lung function and decreased HDM-specific antibody responses were also noted. Significantly, efficacy was observed even when anti-OX40 treatment was delayed until after inflammation was established. OX40 blockade also inhibited the release of the Th2 cytokines IL-5 and IL-13 from cells isolated from HDM allergic human donors. Altogether, our data provide evidence of a role of the OX40/OX40L pathway in ongoing allergic lung inflammation and support clinical studies of a blocking OX40 antibody in Th2 high severe asthma patients.
Keywords: Allergic asthma r House dust mite
r
Lung inflammation r Memory T cells
r
OX40
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction OX40 (also known as CD134, TNFRSF4, ACT35, or TXGP1L) is a membrane-bound receptor belonging to the TNF receptor superfamily, which includes 4-1BB, CD27, CD30, and CD40. This costimulatory receptor is not expressed on resting T cells, but is transiently expressed on activated T cells after ligation of the TCR, with sequential engagement of CD28 and OX40 being required for optimal T-cell proliferation and survival [1]. Ligation of OX40 on activated T cells leads to enhanced cytokine production and proliferation of both CD4+ and CD8+ T cells [2, 3] and can contribute to both ongoing Th1 and Th2 responses [4, 5]. OX40 costimulation
Correspondence: Diane Marshall e-mail: [email protected] C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
prolongs T-cell survival beyond the initial effector phase of the immune response and increases the number of memory T cells through inhibition of effector T-cell death. This long-term survival of T cells via OX40 is thought to be a consequence of maintaining high levels of antiapoptotic proteins, such as Bcl-2, Bcl-xL, and Bfl-1 [6, 7]. The ligand for OX40, OX40L, is a member of the TNF family and is expressed on activated APC, including B cells, macrophages, Langerhans cells, and dendritic cells (DCs). Other cell types that are reported to express OX40L include mast cells, NK cells, airway smooth muscle cells, endothelial cells, and platelets [8].
∗
These authors are co-first authors.
www.eji-journal.eu
Eur. J. Immunol. 2015. 45: 1116–1128
Asthma is a chronic inflammatory disease of the airways in which many cells and mediators are involved. It is now clear that asthma is a heterogenic disease and patients can be stratified into different groups based on the drivers of their disease. One increasingly characterized group is the Th2-driven severe asthmatics. These patients are defined in a number of ways including high circulating periostin, elevated sputum eosinophils, and high circulating IgE despite treatment with high doses of inhaled corticosteroids. In addition, these patients respond to therapies such as anti-IL-5, anti-IL-13, and anti-IgE. Targeting Th2-driven inflammation is therefore clinically validated in human disease and indeed therapies targeting multiple facets of the Th2 pathway may be more effective than targeting single mediators [9–12]. The direct inhibition of T cells via inhibition of T-cell costimulation through interruption of CD28 signaling, utilizing CLTA4Ig, previously failed to modulate the response to allergen challenge or asthma symptoms in mild atopic asthma [13]. However, it has been postulated that T-cell inhibition via this mechanism would have a greater effect on the initiation of an immune response rather than inhibiting effector memory T cells [14, 15]. Chronic exposure to allergen is known to drive the development of antigenspecific memory effector T cells and therefore specifically targeting these pathogenic activated memory T cells may provide a selective novel treatment for severe Th2-driven asthma. Several studies have provided evidence for a role of OX40 in lung inflammation and asthma. OX40 KO mice primed and challenged with OVA exhibit diminished lung inflammation (80–90% reduction in eosinophilia), reduced mucus production, and significantly attenuated airway hyper-reactivity [16]. Monoclonal antibodies to OX40 ligand have also shown beneficial effects in murine models of lung inflammation [17, 18]. In addition, an antibody to human OX40L reduced levels of the Th2 mediators, IL-5 and IL-13, and effector memory T cells in bronchoalveolar lavage fluid (BALF) after allergen challenge in a model of lung inflammation in rhesus monkeys [19]. In patients, an increase in the number of OX40- and OX40L-positive cells in mild asthmatic patients has been shown to correlate with the number of eosinophils and IL-4+ cells in the bronchial submucosa [20] and serum. Similarly, OX40L has been noted to be increased in asthmatic children and adults and was associated with increased severity and persistence of disease [21, 22]. We have sought to expand the current knowledge surrounding the role of OX40 in allergic lung inflammation by using in vivo and in vitro models driven by house dust mite (HDM) allergen, known to be a common driver of asthmatic disease [23]. Chronic exposure to inhaled allergens induces an inflammatory response in the lung resulting in clinical symptoms of asthma. The chronic HDM model in mice, involving mucosal sensitization to inhaled HDM, is now considered to be an improved preclinical in vivo model to determine the efficacy and mechanism of action of potential novel therapeutics for asthmatic disease and was recently shown to be sensitive to anti-IL-13 treatment [24]. We have used the chronic HDM model of allergic lung inflammation to investigate the efficacy of an anti-OX40 blocking antibody. Using this in vivo model and correlation with data from an in vitro human model of HDM-driven C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Immunomodulation
allergic inflammation, we have investigated the potential mechanism of action of OX40 blockade in allergic inflammation, in particular determining the effect on specific T-cell subpopulations. Our studies have revealed a reduction in the number of activated and memory T cells and also both Th2 and Th17 cells after OX40 blockade. Importantly, efficacy was also noted when dosing of the OX40 antibody was delayed until after lung inflammation was already established.
Results Prophylactic anti-OX40 mAb prevents the development of allergic airways disease in mice Chronic exposure to HDM causes cellular infiltration of the airways by immune and inflammatory cells. Routinely, significant numbers of eosinophils, T cells, and neutrophils are found in BALF at week 5. To determine the effect of anti-OX40 blocking antibodies on the infiltration of leukocytes in the lung, we exposed mice to HDM extract for 5 weeks, during concomitant treatment with anti-OX40 antibody or saline vehicle at the indicated doses. As shown in Figure 1, chronic exposure to HDM resulted in cellular infiltration of the airways and lung parenchyma. Administration of anti-OX40 antibody at the time of initial exposure and throughout HDM exposure completely inhibited the infiltration of the lung tissue and airways by eosinophils (Fig. 1A and C) and resulted in a significant decrease in the number of infiltrated neutrophils in the BALF (by 81, 90, and 80% at 100, 30, and 10 mg/kg, respectively, relative to vehicle control (Fig. 1D)). The effects of anti-OX40 treatment on neutrophilia were more apparent in lung tissue than BALF as neutrophils in lung tissue were reduced to baseline levels and low-dose treatment (1 mg/kg) was also efficacious (Fig. 1B). Moreover, anti-OX40 prophylactic treatment demonstrated a significant inhibition of total CD4+ T-cell infiltration in the lung (by 60, 65, and 65% at 100, 30, and 10 mg/kg, respectively, relative to vehicle control) and in the BALF (by 90, 84, and 79% at 100, 30, and 10 mg/kg, respectively, relative to vehicle control; Fig. 2A and B). The number of CD8+ T cells was also significantly reduced (data not shown), although in this model of airway inflammation, CD4+ T cells are the most prominent population found in the inflamed lung. It has been shown that OX40–OX40L interaction contributes to the generation and/or survival of CD4+ effector memory cells. To further characterize the effect of OX40 blockade, we analyzed the phenotype of the CD4+ cells. Most of the CD4+ cells present in the airways after exposure to HDM were activated (CD4+ CD44hi CD25+ ) or displayed an effector memory like phenotype (CD4+ CD44hi CD25− CD62Llo ). Interestingly, the number of activated T cells and effector memory T cells was significantly reduced in the lung and BALF of anti-OX40 treated mice as compared to control, (Fig. 2C–F) while the na¨ıve CD4+ T cell numbers were unchanged (data not shown). These results are compatible with previous reports. The majority of CD4+ T cells in the lungs following HDM exposure were Th2 polarized as indicated by their expression of T1ST2 www.eji-journal.eu
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Figure 1. Effect of prophylactic treatment with anti-OX40 antibody on granulocytes in the lung and BALF. Cells in the lung after exposure to HDM for 5 weeks along with continuous administration of antiOX40 antibody were analyzed by multicolor flow cytometry. The number of (A, C) eosinophils and (B, D) neutrophils in the (A and B) lung and (C and D) in BALF. Data are presented as mean + SEM and are from one experiment, with n = 7–8 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control.
with some IL-17-producing cells also present. The number of IFN-γ-producing cells (Th1) is minimal in this model. As shown in Figure 3, the number of IL-13- or IL-17-producing CD4+ cells was significantly reduced in the lung and BALF of mice treated with prophylactic dosing of the OX40 blocking antibody (by at least 81% for Th2 cells (Fig. 3A and C) and by 88, 87, and 86% for IL-17-producing cells (Fig. 3E) at 100, 30, and 10 mg/kg, respectively, in the lung, relative to vehicle control). The level of inhibition of the number of Th2 cells and IL-17-producing cells in the BALF reaches over 90% in mice treated with 100 and 30 mg/kg of blocking antibody and 80% at 10 mg/kg (Fig. 3B, D, and F). In addition, a reduction in the number of IL-4- and IL-10producing cells was noted as is shown in Supporting Information Figure 6. In order to determine whether airway inflammation correlated with changes in lung function in our model, plethysmography was used to monitor changes in respiratory dynamics following exposure to methacholine. HDM exposure resulted in increased sensitivity to methacholine challenge shown by a significantly higher Penh measurement compared to sham-treated controls (Fig. 4A). This hyper-responsiveness to methacholine was abrogated in mice treated with anti-OX40 antibody. Inhibition of lung disease by anti-OX40 treatment was also confirmed by histology. Airway wall remodeling is observed in this model [24] and the anti-OX40 antibody inhibited subepithelial collagen deposition, as shown in a representative tissue section (Fig. 4C) compared with the mice exposed to HDM and treated with vehicle (Fig. 4B). Similarly, fewer mucus-producing goblet cells were also noted in mice treated with anti-OX40 antibody (Fig. 4E) compared to vehicletreated mice (Fig. 4D).
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Th2 immune responses are often associated with IgG1 and IgE antibody production. Analysis of the antibody response to HDM confirmed that intranasal administration of HDM resulted in the generation of HDM-specific IgG1 and IgE in plasma. Treatment with anti-OX40 antibody significantly reduced both IgG1 and IgE HDM-specific antibody levels (Fig. 5). Thus, these data indicate that blocking OX40 can reduce immune inflammatory cells and T-cell numbers in the airways and lung tissue leading to a reduction in the chronic inflammation of the airways, which is believed to drive the physiological features of asthma. The reduction in Th2 and Th17 cells provides evidence that this may be beneficial in asthmatics with Th2-driven disease. In summary, prophylactic administration of an anti-OX40 mAb prevented the development of HDM-induced inflammation. These effects were apparent within the airways, lung parenchyma, and systemically, thus verifying that the interaction between OX40 and OX40L is critical for the initiation of allergic airways disease.
Therapeutic treatment with anti-OX40 mAb inhibits lung inflammation in mice In order to determine whether OX40 blockade could modulate established allergic airway inflammation, a therapeutic dosing regimen was employed. BALB/c mice were exposed to HDM for 5 weeks to induce chronic pathology mimicking clinical asthma. Vehicle (saline), 100 or 30 mg/kg of anti-OX40 antibody, was
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Immunomodulation
Figure 2. Effect of prophylactic treatment with anti-OX40 antibody on CD4+ T cells in the lung and BALF. Cells in the lung after exposure to HDM for 5 weeks along with continuous administration of antiOX40 antibody were analyzed by multicolor flow cytometry. Total numbers of (A, B) CD4+ T cells, (C, D) activated CD4+ T cells (CD4+ CD44hi CD25+ CD62Llo ), and (E, F) effector memory like CD4+ T cells (CD4+ CD44hi CD25− CD62Llo ) in the (A, C, E) lung and (B, D, F) BALF. Data are presented as mean + SEM and are from one experiment, with n = 8 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control.
injected at week 6 (from day 35) and continued twice weekly for a further 3 weeks during concomitant HDM exposure. Lung and BALF samples were then collected and analyzed by multicolor flow cytometry after the 8th week of HDM exposure. As shown in Figure 6, therapeutic treatment of the mice with 100 mg/kg anti-OX40 antibody resulted in a significant reduction in the number of eosinophils and neutrophils in BALF. Moreover, the total number of CD4+ T cells in the lung (Fig. 7A) and BALF (Fig. 7B) was reduced in mice treated with 100 mg/kg anti-OX40 antibody, and to a lesser extent with 30 mg/kg (90 and 34% reduction (relative to vehicle control) in the lung, respectively). In concurrence with our prophylactic data, the CD4+ T-cell subsets most susceptible to inhibition by OX40 blockade were acti-
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
vated CD4+ T cells and effector memory like CD4+ T cells with a respective reduction in the lung of 90 and 91% (relative to vehicle control) with 100 mg/kg anti-OX40 antibody (Fig. 7C and E). This effect was mirrored in the airways, albeit to a lesser extent (Fig. 7D and F). OX40 blockade also resulted in a significant decrease of Th2 and Th17 cell numbers in the lung (Fig. 8A and B; 50 and 40%, respectively, at 100 mg/kg, relative to vehicle control) and in the BALF (Fig. 8C and D; 55 and 57% for Th2and IL-17-producing cells, respectively, at 100 mg/kg relative to vehicle control). A reduction in IL-10-positive cells was also noted and is shown in Supporting Information Figure 7. Altogether these data show that blockade of OX40 attenuates established inflammation of the lung and airways by reducing
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Figure 3. Effect of prophylactic treatment with anti-OX40 antibody on Th2 and Th17 cells in the lung and BALF. Cytokine production by CD4+ T cells in the lung after exposure to HDM for 5 weeks along with continuous administration of anti-OX40 antibody was analyzed by intracellular cytokine staining by multicolor flow cytometry. The number of (A, B) Th2 cells (CD4+ T1ST2+ ), (C, D) IL-13+ CD4+ T cells, and (E, F) Th17 cells in the (A, C, E) lung and (B, D, F) BALF. Data are presented as mean ± SEM and are from one experiment, with n = 8 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control.
the number of activated and effector memory CD4+ T cells, thus implicating the interaction between OX40 and OX40L in the perpetuation of allergic airways disease.
Blocking OX40 reduces Th2 cytokine production by PBMCs from HDM allergic donors Allergens such as those derived from HDM can induce proliferative responses and Th2 cytokine production in sensitized individuals. PMBCs from HDM allergic donors, when stimulated in vitro with HDM extract, produce high levels of the Th2 cytokines IL-5 and IL-13 compared to nonallergic donors (Fig. 9B and C). This is in contrast to the levels of Th1 cytokines, which are comparable between allergic and nonallergic donors (Fig. 9A). To understand whether the efficacy observed in murine models would translate to atopy in man, we used HDM allergic donors to investigate the effect of OX40 blockade on Th2 cytokine production following activation of PBMCs with HDM extract. PBMCs were stimulated with C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
HDM extract for 6 days in the presence of 50 μg/mL anti-OX40 or control antibody. Since large variations in response were found across donors and experiments, the data were pooled and represented as percent inhibition. (Data from an individual representative donor are shown in Supporting Information Fig. 1.) Treatment with the OX40 antibody significantly inhibited IL-5 and IL-13 production by 53 and 71%, respectively, compared to PBMCs cultured without antibody (Fig. 10). The control antibody did not inhibit Th2 cytokine production indicating that anti-OX40 treatment may be an effective treatment for allergic Th2-driven asthma.
Blocking OX40 inhibits both primary and secondary CD4+ T-cell responses to HDM antigens in vitro OX40 plays a central role in the generation of memory T-cell responses and in the promotion of effector T-cell survival after www.eji-journal.eu
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Figure 4. Effect of prophylactic treatment with anti-OX40 antibody on respiratory dynamics induced by methacholine, airway remodeling, and goblet cell hyperplasia. (A) Airway response of vehicle control or anti-OX40-treated mice to methacholine (24 h after exposure to HDM). Data presented as mean Penh + SEM and are from one experiment, with n = 7–8 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control. Representative photomicrographs of airway sections stained with (B, C) Masson’s trichrome or (D, E) AB/PAS from mice exposed to HDM and treated with (B, D) vehicle or (C, E) anti-OX40 antibody. Scale bar: 100 μm. Images are representative of one experiment.
antigen priming. We therefore hypothesized that treatment of human CD4+ T cells with an anti-OX40 antibody would not only inhibit a primary antigen response in vitro through costimulation blockade, but may also inhibit a secondary response to the same antigen due to a reduction in survival and effector function of antigen-specific memory T cells. To investigate this, CD4+ T cells from HDM allergic donors were stimulated with autologous APCs pulsed with HDM extract in the presence of 10 μg/mL anti-OX40 or control antibody for 7 days. At day 7, cytokine production was analyzed to evaluate the primary in vitro T-cell response. Since large vari-
ations in response were found across donors and experiments, the data were pooled and represented as percent inhibition. (Data from an individual representative donor are shown in Supporting Information Fig. 2.) Following the primary stimulation, the anti-OX40 antibody inhibited IL-5 production by 48% compared to cells cultured with no antibody (Fig. 11A). The CD4+ T cells were then washed and rested for 3 days without stimulation before being cultured with fresh autologous APCs pulsed with HDM for 3 days. No further antibodies were added during the secondary in vitro stimulation. After 3 days, the magnitude of the secondary immune response was measured by
Figure 5. Effect of prophylactic treatment with anti-OX40 antibody on levels of HDMspecific IgG1 and IgE. Levels of (A) HDMspecific IgG1 and (B) IgE were measured by ELISA in plasma taken from mice treated with vehicle or anti-OX40 antibody in a chronic HDM model of allergic airway inflammation. Data presented as mean ± SEM and are from one experiment, with n = 7–8 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control.
Figure 6. Effect of therapeutic treatment with anti-OX40 antibody on granulocytes in the BALF. Cells in the lung after exposure to HDM for 8 weeks with administration of anti-OX40 antibody from week 6 were analyzed by multicolor flow cytometry. Graphs show the numbers of (A) eosinophils and (B) neutrophils in the BALF. Data presented as mean + SEM and are from one experiment, with n = 9–10 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 7. Effect of anti-OX40 antibody therapeutic treatment on CD4+ cells in the lung and BALF. Mice were exposed to HDM for 8 weeks and dosed with anti-OX40 antibody from week 6. Cells in the lung were analyzed by multicolor flow cytometry. Graphs show the number of (A, B) total CD4+ T cells, (C, D) activated CD4+ T cells (CD4+ CD25+ CD44hi CD62Llo ), and (E, F) effector memory like CD4+ T cells (CD4+ CD25− CD44hi CD62Llo ) in the (A, C, E) lung and (B, D, F) BALF after therapeutic treatment with anti-OX40. Data presented as mean + SEM and are from one experiment, with n = 10 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control.
cytokine analysis and the number of activated CD4+ T cells was assessed by flow cytometry. Cells cultured with the anti-OX40 antibody during the primary stimulation produced less IL-5 following secondary stimulation compared to those treated with control antibody (Fig. 11A; 75% reduction compared to untreated cells). Flow cytometry analysis of the T cells treated with the antiOX40 antibody showed a decrease in the proportion of activated CD4+ CD25+ T cells after the secondary stimulation compared to cells treated with control antibody (15 and 38%, respectively; Fig. 11B). Since there was no antibody present in the culture during the secondary stimulation, these data suggest that an anti-OX40 antibody may be able to modulate antigen-specific human T cells and alter their potential to respond to the same antigen by reducing activation and effector function. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Discussion In this study, we have examined the role of the OX40/OX40L pathway in allergic asthma by utilizing a blocking OX40 antibody in a chronic HDM model of allergic lung inflammation in conjunction with human in vitro systems using cells isolated from allergic individuals. We have sought to understand the potential mechanism of action of OX40 blockade through detailed analysis of different T-cell populations that accumulate in the lung during the ongoing disease process. First, our data demonstrate that OX40 blockade inhibits both eosinophil and neutrophil infiltration into the airways and importantly into the lung tissue. This finding is relevant since it is clear that there is a group of patients with persistent eosinophilic inflammation who fail to respond to current therapies such as inhaled corticosteroids. Moreover, it is postulated
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Immunomodulation
Figure 8. Effect of anti-OX40 antibody therapeutic treatment on Th2 and Th17 cells in the lung and BALF. Mice were exposed to HDM for 8 weeks and dosed with anti-OX40 antibody from week 6. Cytokine production by CD4+ T cells in the lung and BALF was analyzed by intracellular cytokine staining by flow cytometry. Graphs show the number of (A, C) Th2 (CD4+ T1ST2+ ) and (B, D) Th17 cells in the (A, B) lung and (C, D) BALF. Data presented as mean + SEM and are from one experiment, with n = 10 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The significance of the data was evaluated by one-way ANOVA with Bonferroni’s multiple comparison test compared to vehicle control.
Figure 10. Effect of an anti-OX40 antibody on Th2 cytokine production from human PBMCs. PBMCs from HDM allergic donors were isolated and stimulated with HDM in the presence of anti-OX40 or control antibody for 6 days. Levels of Th2 cytokines were measured using MSD. Data are shown as mean + SEM (n = 3 replicates) and are pooled from four donors. Percent inhibition was calculated relative to cells cultured with no antibody. *p
