A T cell redirection platform for co-targeting dual antigens on solid tumors.

In order to direct T cells to specific features of solid cancer cells, we engineered a bispecific antibody format, named Dual Antigen T cell Engager (DATE), by fusing a single-chain variable fragment targeting CD3 to a tumor-targeting antigen-binding fragment. In this format, multiple novel paratopes against different tumor antigens were able to recruit T-cell cytotoxicity to tumor cells in vitro and in an in vivo pancreatic ductal adenocarcinoma xenograft model. Since unique surface antigens in solid tumors are limited, in order to enhance selectivity, we further engineered "double-DATEs" targeting two tumor antigens simultaneously. The double-DATE contains an additional autonomous variable heavy-chain domain, which binds a second tumor antigen without itself eliciting a cytotoxic response. This novel modality provides a strategy to enhance the selectivity of immune redirection through binary targeting of native tumor antigens. The modularity and use of a common, stable human framework for all components enables a pipeline approach to rapidly develop a broad repertoire of tailored DATEs and double-DATEs with favorable biophysical properties and high potencies and selectivities.

Pathogenic TH17 inflammation is sustained in the lungs by conventional dendritic cells and Toll-like receptor 4 signaling.

BACKGROUND: Mechanisms that elicit mucosal TH17 cell responses have been described, yet how these cells are sustained in chronically inflamed tissues remains unclear. OBJECTIVE: We sought to understand whether maintenance of lung TH17 inflammation requires environmental agents in addition to antigen and to identify the lung antigen-presenting cell (APC) types that sustain the self-renewal of TH17 cells. METHODS: Animals were exposed repeatedly to aspiration of ovalbumin alone or together with environmental adjuvants, including common house dust extract (HDE), to test their role in maintaining lung inflammation. Alternatively, antigen-specific effector/memory TH17 cells, generated in culture with CD4+ T cells from Il17a fate-mapping mice, were adoptively transferred to assess their persistence in genetically modified animals lacking distinct lung APC subsets or cell-specific Toll-like receptor (TLR) 4 signaling. TH17 cells were also cocultured with lung APC subsets to determine which of these could revive their expansion and activation. RESULTS: TH17 cells and the consequent neutrophilic inflammation were poorly sustained by inhaled antigen alone but were augmented by inhalation of antigen together with HDE. This was associated with weight loss and changes in lung physiology consistent with interstitial lung disease. The effect of HDE required TLR4 signaling predominantly in lung hematopoietic cells, including CD11c+ cells. CD103+ and CD11b+ conventional dendritic cells interacted directly with TH17 cells in situ and revived the clonal expansion of TH17 cells both ex vivo and in vivo, whereas lung macrophages and B cells could not. CONCLUSION: TH17-dependent inflammation in the lungs can be sustained by persistent TLR4-mediated activation of lung conventional dendritic cells.

TNF is required for TLR ligand-mediated but not protease-mediated allergic airway inflammation.

Asthma is associated with exposure to a wide variety of allergens and adjuvants. The extent to which overlap exists between the cellular and molecular mechanisms triggered by these various agents is poorly understood, but it might explain the differential responsiveness of patients to specific therapies. In particular, it is unclear why some, but not all, patients benefit from blockade of TNF. Here, we characterized signaling pathways triggered by distinct types of adjuvants during allergic sensitization. Mice sensitized to an innocuous protein using TLR ligands or house dust extracts as adjuvants developed mixed eosinophilic and neutrophilic airway inflammation and airway hyperresponsiveness (AHR) following allergen challenge, whereas mice sensitized using proteases as adjuvants developed predominantly eosinophilic inflammation and AHR. TLR ligands, but not proteases, induced TNF during allergic sensitization. TNF signaled through airway epithelial cells to reprogram them and promote Th2, but not Th17, development in lymph nodes. TNF was also required during the allergen challenge phase for neutrophilic and eosinophilic inflammation. In contrast, TNF was dispensable for allergic airway disease in a protease-mediated model of asthma. These findings might help to explain why TNF blockade improves lung function in only some patients with asthma.

The TLR4-TRIF pathway can protect against the development of experimental allergic asthma.

The Toll-like receptor (TLR) adaptor proteins myeloid differentiating factor 88 (MyD88) and Toll, interleukin-1 receptor and resistance protein (TIR) domain-containing adaptor inducing interferon-ß (TRIF) comprise the two principal limbs of the TLR signalling network. We studied the role of these adaptors in the TLR4-dependent inhibition of allergic airway disease and induction of CD4+ ICOS+ T cells by nasal application of Protollin™, a mucosal adjuvant composed of TLR2 and TLR4 agonists. Wild-type (WT), Trif-/- or Myd88-/- mice were sensitized to birch pollen extract (BPEx), then received intranasal Protollin followed by consecutive BPEx challenges. Protollin's protection against allergic airway disease was TRIF-dependent and MyD88-independent. TRIF deficiency diminished the CD4+ ICOS+ T-cell subsets in the lymph nodes draining the nasal mucosa, as well as their recruitment to the lungs. Overall, TRIF deficiency reduced the proportion of cervical lymph node and lung CD4+ ICOS+ Foxp3- cells, in particular. Adoptive transfer of cervical lymph node cells supported a role for Protollin-induced CD4+ ICOS+ cells in the TRIF-dependent inhibition of airway hyper-responsiveness. Hence, our data demonstrate that stimulation of the TLR4-TRIF pathway can protect against the development of allergic airway disease and that a TRIF-dependent adjuvant effect on CD4+ ICOS+ T-cell responses may be a contributing mechanism.

Mechanical consequences of allergic induced remodeling on mice airway resistance and compressibility.

The effect of remodeling on airway function is uncertain. It may affect airway compressibility during forced expirations differently than airflow resistance, providing a tool for its assessment. The aim of the current study was to compare the effects of acute and chronic antigen challenge on methacholine-induced bronchoconstriction assessed from resistance and maximal tidal expiratory flow. Balb/C mice were sensitized with ovalbumin (OVA) and challenged either daily for three days with intra-nasal OVA or daily for 5 days and three times a week for 5 subsequent weeks. Acute and chronic allergen challenge induced airway hyperresponsiveness (AHR) to methacholine. However the relationship between maximal tidal expiratory flow and resistance during methacholine challenge was different between the two conditions, suggesting that the determinants of AHR are not identical following acute and chronic allergen exposure. We conclude that the contrast of changes in maximal tidal expiratory flow and respiratory resistance during methacholine-induced bronchoconstriction may allow the detection of the mechanical consequences of airway remodeling.

Concomitant exposure to ovalbumin and endotoxin augments airway inflammation but not airway hyperresponsiveness in a murine model of asthma.

Varying concentrations of lipopolysaccharide (LPS) in ovalbumin (OVA) may influence the airway response to allergic sensitization and challenge. We assessed the contribution of LPS to allergic airway inflammatory responses following challenge with LPS-rich and LPS-free commercial OVA. BALB/c mice were sensitized with LPS-rich OVA and alum and then underwent challenge with the same OVA (10 µg intranasally) or an LPS-free OVA. Following challenge, bronchoalveolar lavage (BAL), airway responsiveness to methacholine and the lung regulatory T cell population (Treg) were assessed. Both OVA preparations induced BAL eosinophilia but LPS-rich OVA also evoked BAL neutrophilia. LPS-free OVA increased interleukin (IL)-2, IL-4 and IL-5 whereas LPS-rich OVA additionally increased IL-1ß, IL-12, IFN-γ, TNF-α and KC. Both OVA-challenged groups developed airway hyperresponsiveness. TLR4-deficient mice challenged with either OVA preparation showed eosinophilia but not neutrophilia and had increased IL-5. Only LPS-rich OVA challenged mice had increased lung Tregs and LPS-rich OVA also induced in vitro Treg differentiation. LPS-rich OVA also induced a Th1 cytokine response in human peripheral blood mononuclear cells.We conclude that LPS-rich OVA evokes mixed Th1, Th2 and innate immune responses through the TLR-4 pathway, whereas LPS-free OVA evokes only a Th2 response. Contaminating LPS is not required for induction of airway hyperresponsiveness but amplifies the Th2 inflammatory response and is a critical mediator of the neutrophil, Th1 and T regulatory cell responses to OVA.

Inhaled birch pollen extract induces airway hyperresponsiveness via oxidative stress but independently of pollen-intrinsic NADPH oxidase activity, or the TLR4-TRIF pathway.

Oxidative stress in allergic asthma may result from oxidase activity or proinflammatory molecules in pollens. Signaling via TLR4 and its adaptor Toll-IL-1R domain-containing adapter inducing IFN-ß (TRIF) has been implicated in reactive oxygen species-mediated acute lung injury and in Th2 immune responses. We investigated the contributions of oxidative stress and TLR4/TRIF signaling to experimental asthma induced by birch pollen exposure exclusively via the airways. Mice were exposed to native or heat-inactivated white birch pollen extract (BPEx) intratracheally and injected with the antioxidants, N-acetyl-L-cysteine or dimethylthiourea, prior to sensitization, challenge, or all allergen exposures, to assess the role of oxidative stress and pollen-intrinsic NADPH oxidase activity in allergic sensitization, inflammation, and airway hyperresponsiveness (AHR). Additionally, TLR4 signaling was antagonized concomitantly with allergen exposure, or the development of allergic airway disease was evaluated in TLR4 or TRIF knockout mice. N-acetyl-L-cysteine inhibited BPEx-induced eosinophilic airway inflammation and AHR except when given exclusively during sensitization, whereas dimethylthiourea was inhibitory even when administered with the sensitization alone. Heat inactivation of BPEx had no effect on the development of allergic airway disease. Oxidative stress-mediated AHR was also TLR4 and TRIF independent; however, TLR4 deficiency decreased, whereas TRIF deficiency increased BPEx-induced airway inflammation. In conclusion, oxidative stress plays a significant role in allergic sensitization to pollen via the airway mucosa, but the pollen-intrinsic NADPH oxidase activity and TLR4 or TRIF signaling are unnecessary for the induction of allergic airway disease and AHR. Pollen extract does, however, activate TLR4, thereby enhancing airway inflammation, which is restrained by the TRIF-dependent pathway.

ICOS-expressing CD4 T cells induced via TLR4 in the nasal mucosa are capable of inhibiting experimental allergic asthma.

Modulation of adaptive immune responses via the innate immune pattern recognition receptors, such as the TLRs, is an emerging strategy for vaccine development. We investigated whether nasal rather than intrapulmonary application of Protollin, a mucosal adjuvant composed of TLR2 and TLR4 ligands, is sufficient to elicit protection against murine allergic lower airway disease. Wild-type, Tlr2(-/-), or Tlr4(-/-) BALB/c mice were sensitized to a birch pollen allergen extract (BPEx), then received either intranasal or intrapulmonary administrations of Protollin or Protollin admixed with BPEx, followed by consecutive daily BPEx challenges. Nasal application of Protollin or Protollin admixed with BPEx was sufficient to inhibit allergic lower airway disease with minimal collateral lung inflammation. Inhibition was dependent on TLR4 and was associated with the induction of ICOS in cells of the nasal mucosa and on both CD4+Foxp3+ and CD4+Foxp3- T cells of the draining lymph nodes (LNs), as well as their recruitment to the lungs. Adoptive transfer of cervical LN CD4+ICOS+, but not CD4+ICOS-, cells inhibited BPEx-induced airway hyperresponsiveness and bronchoalveolar lavage eosinophilia. Thus, our data indicate that expansion of resident ICOS-expressing CD4+ T cells of the cervical LNs by nasal mucosal TLR4 stimulation may inhibit the development of allergic lower airway disease in mice.

Intravenous immunoglobulin attenuates airway inflammation through induction of forkhead box protein 3-positive regulatory T cells.

BACKGROUND: Intravenous immunoglobulin (IVIG) is a frequently used disease-modifying therapy for a large spectrum of autoimmune and inflammatory conditions, yet its mechanisms of action are incompletely understood. Using a robust murine model of antigen-driven allergic airways disease, we have demonstrated that IVIG markedly improves ovalbumin (OVA)-induced airway hyperresponsiveness characterized by 4- to 6-fold enhancement in regulatory T (Treg) cells in pulmonary and associated lymphoid tissues. OBJECTIVE: We sought to determine whether IVIG induces antigen-specific Treg cells and to address cellular interactions that lead to induction of Treg cells by IVIG. METHODS: C57Bl/6 mice were sensitized and challenged by means of intranasal OVA exposure. IVIG or albumin control was administered 24 hours before challenge. Treg cells were tracked by using green fluorescent protein (GFP)-forkhead box protein 3 (Foxp3) knock-in reporter mice (Foxp3(GFP)), and Treg cell and dendritic cell (DC) phenotypes and activities were elucidated by using coculture and flow cytometry. RESULTS: IVIG therapy of OVA-sensitized and OVA-challenged mice induced antigen-specific forkhead box protein 3 (Foxp3)-positive Treg cells from non-Treg cell precursors. The induced Treg cells home specifically to the lungs and draining lymph nodes and have greatly potentiated suppressive activity compared with that seen in Treg cells purified from control mice. Induction of Treg cells is mediated by tolerogenic DCs generated after IVIG exposure. Compared with albumin-treated, OVA-exposed mice, IVIG-primed DCs express altered Notch ligands, including increased Delta-4 and reduced Jagged-1 levels, reflecting decreased T(H)2 polarization. Furthermore, IVIG-primed DCs can stimulate Treg cell differentiation from uncommitted Foxp3(-)CD4(+) T cells ex vivo, and adoptive transfer of IVIG-primed DCs abrogates airway hyperresponsiveness and induces Treg cells. CONCLUSION: The anti-inflammatory effects of IVIG therapy can be mediated by the immunomodulation of DCs, creating a bridge that induces antigen-specific, highly suppressive Treg cells.

Sites of allergic airway smooth muscle remodeling and hyperresponsiveness are not associated in the rat.

The cause-and-effect relationship between airway smooth muscle (ASM) remodeling and airway hyperresponsiveness (AHR) following allergen challenge is not well established. Using a rat model of allergen-induced ASM remodeling we explored the relationship between the site of ASM remodeling and AHR. Brown Norway rats, sensitized and challenged (3 times at 5-day intervals) with ovalbumin, were intranasally administered 0.1 mg/kg budesonide 24 and 1 h before challenge. Airway responses to aerosolized methacholine were assessed 48 h or 1 wk after three challenges. Airways were stained and analyzed for total airway wall area, area of smooth muscle-specific α-actin, and goblet cell hyperplasia, and the constant-phase model was used to resolve the changes in respiratory system mechanics into large airway and peripheral lung responses. After three ovalbumin challenges, there was a significant increase in ASM area and in the total wall area in all sized airways as well as an increase in goblet cells in the central airways. Budesonide inhibited ASM growth and central airway goblet cell hyperplasia following ovalbumin challenges. Budesonide also inhibited small but not large airway total wall area. AHR was attributable to excessive responses of the small airways, whereas responsiveness of the large airways was unchanged. Budesonide did not inhibit AHR after repeated challenge. We conclude that ASM remodeling induced by repeated allergen challenges involves the entire bronchial tree, whereas AHR reflects alterations in the lung periphery. Prevention of ASM remodeling by corticosteroid does not abrogate AHR.

Combined forced oscillation and forced expiration measurements in mice for the assessment of airway hyperresponsiveness.

BACKGROUND: Pulmonary function has been reported in mice using negative pressure-driven forced expiratory manoeuvres (NPFE) and the forced oscillation technique (FOT). However, both techniques have always been studied using separate cohorts of animals or systems. The objective of this study was to obtain NPFE and FOT measurements at baseline and following bronchoconstriction from a single cohort of mice using a combined system in order to assess both techniques through a refined approach. METHODS: Groups of allergen- or sham-challenged ovalbumin-sensitized mice that were either vehicle (saline) or drug (dexamethasone 1 mg/kg ip)-treated were studied. Surgically prepared animals were connected to an extended flexiVent system (SCIREQ Inc., Montreal, Canada) permitting NPFE and FOT measurements. Lung function was assessed concomitantly by both techniques at baseline and following doubling concentrations of aerosolized methacholine (MCh; 31.25 - 250 mg/ml). The effect of the NPFE manoeuvre on respiratory mechanics was also studied. RESULTS: The expected exaggerated MCh airway response of allergic mice and its inhibition by dexamethasone were detected by both techniques. We observed significant changes in FOT parameters at either the highest (Ers, H) or the two highest (Rrs, RN, G) MCh concentrations. The flow-volume (F-V) curves obtained following NPFE manoeuvres demonstrated similar MCh concentration-dependent changes. A dexamethasone-sensitive decrease in the area under the flow-volume curve at the highest MCh concentration was observed in the allergic mice. Two of the four NPFE parameters calculated from the F-V curves, FEV0.1 and FEF50, also captured the expected changes but only at the highest MCh concentration. Normalization to baseline improved the sensitivity of NPFE parameters at detecting the exaggerated MCh airway response of allergic mice but had minimal impact on FOT responses. Finally, the combination with FOT allowed us to demonstrate that NPFE induced persistent airway closure that was reversible by deep lung inflation. CONCLUSIONS: We conclude that FOT and NPFE can be concurrently assessed in the same cohort of animals to determine airway mechanics and expiratory flow limitation during methacholine responses, and that the combination of the two techniques offers a refined control and an improved reproducibility of the NPFE.

Overview of asthma; the place of the T cell.

Asthma is an inflammatory disease which is associated with activated T cells in the airway wall. The contribution of the T lymphocyte to inflammation in asthma has been extensively studied through descriptions of T cell subsets in the airway wall of asthmatic patients and from animal and cellular models. Allergy-driven airway disease is mediated primarily by the T helper (Th)2 cell subset. Other subsets, such as Th1, Th17, invariant natural killer T and CD8+ T cells likely contribute to the development, and possibly the progression of established disease. Resolution of inflammation is controlled in part by regulatory T cells. Therapies directed at T cells and their cytokines have been disappointing in asthma despite, in some instances, promising results on allergen challenge. This suggests that the induction of asthma may be T-cell-mediated and allergen-triggered, whereas disease may be sustained and exacerbated by other mechanisms.