Yen Leong Chua

GM-CSF–Licensed CD11b+ Lung Dendritic Cells Orchestrate Th2 Immunity to Blomia tropicalis

The Blomia tropicalis dust mite is prevalent in tropical and subtropical regions of the world. Although it is a leading cause of asthma, little is known how it induces allergy. Using a novel murine asthma model induced by intranasal exposure to B. tropicalis, we observed that a single intranasal sensitization to B. tropicalis extract induces strong Th2 priming in the lung draining lymph node. Resident CD11b+ dendritic cells (DCs) preferentially transport Ag from the lung to the draining lymph node and are crucial for the initiation of Th2 CD4+ T cell responses. As a consequence, mice selectively deficient in CD11b+ DCs exhibited attenuated Th2 responses and more importantly did not develop any allergic inflammation. Conversely, mice deficient in CD103+ DCs and CCR2-dependent monocyte-derived DCs exhibited similar allergic inflammation compared with their wild-type counterparts. We also show that CD11b+ DCs constitutively express higher levels of GM-CSF receptor compared with CD103+ DCs and are thus selectively licensed by lung epithelial-derived GM-CSF to induce Th2 immunity. Taken together, our study identifies GM-CSF–licensed CD11b+ lung DCs as a key component for induction of Th2 responses and represents a potential target for therapeutic intervention in allergy.

CD44high Memory CD8 T Cells Synergize with CpG DNA to Activate Dendritic Cell IL-12p70 Production

Protective memory CD8 T cell responses are generally associated with the rapid and efficient acquisition of CTL function. However, the ability of memory CD8 T cells to modulate immune responses through interactions with dendritic cells (DCs) during the early states of secondary Ag exposure is poorly understood. In this study, we show that murine Ag-specific CD44high CD8 T cells, representing CD8 T cells of the memory phenotype, potently activate DCs to produce high levels of IL-12p70 in conjunction with stimulation of DCs with the TLR 9 ligand, unmethylated CpG DNA. IL-12p70 production was produced predominantly by CD8α+ DCs and plasmacytoid DCs, and mediated by CD8 T cell-derived cytokines IFN-γ, GM-CSF, TNF-α, and surface CD40L. We also find that CD44high memory phenotype CD8 T cells were better DC IL-12p70 stimulators than CD44low naive phenotype CD8 T cells, and this was attributed to higher levels of IFN-γ and GM-CSF produced by CD44high memory phenotype CD8 T cells during their Ag specific interaction with DCs. Our study identifies CpG DNA as the most effective TLR ligand that cooperates with CD8 T cells for DC IL-12p70 production, and suggests that effectiveness of memory CD8 T cells could be attributed to their ability to rapidly and effectively induce protective Th1 immunity during early stages of pathogen reinfection.

Gamma Interferon Regulates Contraction of the Influenza Virus-Specific CD8 T Cell Response and Limits the Size of the Memory Population

ABSTRACT The factors that regulate the contraction of the CD8 T cell response and the magnitude of the memory cell population against localized mucosal infections such as influenza are important for generation of efficient vaccines but are currently undefined. In this study, we used a mouse model of influenza to demonstrate that the absence of gamma interferon (IFN-γ) or IFN-γ receptor 1 (IFN-γR1) leads to aberrant contraction of antigen-specific CD8 T cell responses. The increased accumulation of the effector CD8 T cell population was independent of viral load. Reduced contraction was associated with an increased fraction of CD8 T cells expressing the interleukin-7 receptor (IL-7R) at the peak of the response, resulting in enhanced numbers of memory/memory precursor cells in IFN-γ−/− and IFN-γR−/− compared to wild-type (WT) mice. Blockade of IL-7 within the lungs of IFN-γ−/− mice restored the contraction of influenza virus-specific CD8 T cells, indicating that IL-7R is important for survival and is not simply a consequence of the lack of IFN-γ signaling. Finally, enhanced CD8 T cell recall responses and accelerated viral clearance were observed in the IFN-γ−/− and IFN-γR−/− mice after rechallenge with a heterologous strain of influenza virus, confirming that higher frequencies of memory precursors are formed in the absence of IFN-γ signaling. In summary, we have identified IFN-γ as an important regulator of localized viral immunity that promotes the contraction of antigen-specific CD8 T cells and inhibits memory precursor formation, thereby limiting the size of the memory cell population after an influenza virus infection.

CD8 T Cells Regulate Allergic Contact Dermatitis by Modulating CCR2–Dependent TNF/iNOS–Expressing Ly6C+CD11b+ Monocytic Cells

Monocytes and their derived cells have critical roles in inflammation and immune defense. However, their function in skin diseases such as allergic contact dermatitis remains poorly defined. Using a model of contact hypersensitivity (CHS) toward 2,4-dinitrochlorobenzene, we show that Ly6C+ CD11b+ monocytic cells participate in the pathophysiology of CHS and their accumulation is regulated by effector CD8 T cells. These Ly6C+ CD11b+ monocytic cells are the primary contributors of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) and derive from Ly6C(hi)CCR2+ monocytes, as they were absent in non-inflamed skin and accumulate as a consequence of inflammation in a C-C chemokine receptor type 2 (CCR2)-dependent manner. Importantly, CCR2(-/-) mice, or wild-type mice depleted of monocytes via clodronate liposomes, display a marked decrease in TNF-α and iNOS expression accompanied by attenuated skin inflammation. Using transgenic mice and antibody depletion, we show that effector CD8 T cells regulate the accumulation of Ly6C+ CD11b+ monocytic cells through IL-17 and activate them for TNF-α and iNOS through IFN-γ. CD8 T cell-derived IFN-γ was also critical for the accumulation of the major histocompatibility complex II-expressing Ly6C+ CD11b+ subset, which expressed intermediate levels of CD11c and costimulatory molecules. Taken together, our findings provide further insight into the pathophysiology of allergic contact dermatitis by showing that CD8 T cells regulate the inflammatory cascade through TNF/iNOS-expressing Ly6C+ CD11b+ monocytic cells.

Priming with high and low respiratory allergen dose induces differential CD4+ T helper type 2 cells and IgE/IgG1 antibody responses in mice

Sensitization of allergic patients normally takes place over several years and is the result of repeated exposure to low levels of allergen. Most mouse asthma models use a high dose of allergen administered over a short period. We have investigated the role of dose in the immune response to an inhaled respiratory allergen (Blomia tropicalis). We observed the effect of priming dose on the allergic response in mice intranasally immunized with low (0·5 μg) and high (50 μg) doses of B. tropicalis extract and killed 1 day after the last challenge. For both doses of allergen, T helper type 2 (Th2) cells and Th2 cytokines were evident as well as eosinophilic inflammation accompanied by mucus hyper‐secretion. By contrast, IgE and IgG1 antibody responses were normally only detected at high‐dose priming. To investigate the mechanism for these effects, we found group 2 innate lymphoid cells (ILC2s) were increased 48 hr after challenge in the low‐dose‐treated but not the high‐dose‐treated mice. Furthermore, we determined whether repeated low‐dose exposure with different priming protocols could induce an antibody response. Repeated low‐dose exposure to 0·5 μg three times weekly for 4 weeks (cumulative 6 μg) had the same effect as a shorter high‐dose exposure (cumulative 80 μg) and increasing cumulative dose induced antibody responses. These data indicate that low doses of allergen are sufficient to prime Th2 cells and ILC2s, but insufficient to induce antibody responses. Cumulative exposure to small amounts of allergen induces both Th2 and antibody responses and may better reflect natural sensitization.

CD40L Expression Allows CD8+ T Cells to Promote Their Own Expansion and Differentiation through Dendritic Cells

CD8+ T cells play an important role in providing protective immunity against a wide range of pathogens, and a number of different factors control their activation. Although CD40L-mediated CD40 licensing of dendritic cells (DCs) by CD4+ T cells is known to be necessary for the generation of a robust CD8+ T cell response, the contribution of CD8+ T cell-expressed CD40L on DC licensing is less clear. We have previously shown that CD8+ T cells are able to induce the production of IL-12 p70 by DCs in a CD40L-dependent manner, providing some evidence that CD8+ T cell-mediated activation of DCs is possible. To better understand the role of CD40L on CD8+ T cell responses, we generated and characterized CD40L-expressing CD8+ T cells both in vitro and in vivo. We found that CD40L was expressed on 30–50% of effector CD8+ T cells when stimulated and that this expression was transient. The expression of CD40L on CD8+ T cells promoted the proliferation and differentiation of both the CD40L-expressing CD8+ T cells and the bystander effector CD8+ T cells. This process occurred via a cell-extrinsic manner and was mediated by DCs. These data demonstrate the existence of a mechanism where CD8+ T cells and DCs cooperate to maximize CD8+ T cell responses.

Blomia tropicalis –Specific TCR Transgenic Th2 Cells Induce Inducible BALT and Severe Asthma in Mice by an IL-4/IL-13–Dependent Mechanism

Previous studies have highlighted the importance of lung-draining lymph nodes in the respiratory allergic immune response, whereas the lung parenchymal immune system has been largely neglected. We describe a new in vivo model of respiratory sensitization to Blomia tropicalis, the principal asthma allergen in the tropics, in which the immune response is focused on the lung parenchyma by transfer of Th2 cells from a novel TCR transgenic mouse, specific for the major B. tropicalis allergen Blo t 5, that targets the lung rather than the draining lymph nodes. Transfer of highly polarized transgenic CD4 effector Th2 cells, termed BT-II, followed by repeated inhalation of Blo t 5 expands these cells in the lung >100-fold, and subsequent Blo t 5 challenge induced decreased body temperature, reduction in movement, and a fall in specific lung compliance unseen in conventional mouse asthma models following a physiological allergen challenge. These mice exhibit lung eosinophilia; smooth muscle cell, collagen, and goblet cell hyperplasia; hyper IgE syndrome; mucus plugging; and extensive inducible BALT. In addition, there is a fall in total lung volume and forced expiratory volume at 100 ms. These pathophysiological changes were substantially reduced and, in some cases, completely abolished by administration of neutralizing mAbs specific for IL-4 and IL-13 on weeks 1, 2, and 3. This IL-4/IL-13–dependent inducible BALT model will be useful for investigating the pathophysiological mechanisms that underlie asthma and the development of more effective drugs for treating severe asthma.

Molecular engineering of a therapeutic antibody for Blo t 5–induced allergic asthma

To the Editor: Epidemiological studies have established a causal link between sensitization to house dust mite (HDM) allergens and the development of allergic asthma. Despite the coexistence of the 2 major dust mite species in the tropics and dual sensitization profiles in atopic individuals, IgE responses targeting Blomia tropicalis have limited cross-reactivity with Dermatophagoides pteronyssinus. Among all known allergens of B tropicalis, Blo t 5 is the principal sensitizing allergen in tropical countries. Blo t 5 has a high allergenic potency with more than 70% of IgE reactivity in subjects with asthma and 96.7% in children with asthma. As such, Blo t 5 is a potential target for therapeutic approaches in B tropicalis–mediated allergic asthma. Previous attempts at neutralizing allergens have been made using allergen-specific polyclonal IgG antibodies. In this study, we sought to explore the potential of using engineered allergenspecific mAbs as a novel treatment modality for HDM-driven allergic asthma. To facilitate this, we first isolated an mAb with high affinity for Blo t 5 (see Fig E1, B, in this article’s Online Repository at www. jacionline.org). Epitope mapping studies with western and immuno-dot blot revealed that this mAb binds a linear epitope on an exposed surface of the Blo t 5 protein (Fig E1, C-E). Interestingly, our antibody targets a region of Blo t 5 previously implicated as a conformational IgE-binding epitope. Moreover, a dose-dependent inhibition of Blo t 5–specific IgE binding from allergic patients’ sera was observed when coincubated with increasing concentrations of anti–Blo t 5 mAb (Fig 1, A). This implies that our mAb may be binding the Blo t 5 antigen in a way that occludes the binding site of Blo t 5–specific IgE in patients’ serum by steric hindrance or distorting the Blo t 5 protein conformation such that Blo t 5–specific IgE in patients’ sera can no longer bind. We then engendered chimeric variants in which the murine constant region (Fc) of the antibody was replaced with the constant domains of human IgG1, IgG1-LALA, and IgG4-S228P (Fig 1, B). Integrities of the expressed antibodies were verified by SDS-PAGE under reducing and nonreducing conditions. The chimeric antibodies were assembled with a heavy and light chain (Fig 1, C) and were correctly folded into full-length IgGs as depicted by bands at 150 kDa and larger on a nonreducing gel (Fig 1, D). Binding specificities for Blo t 5 were retained despite themolecular manipulations, with comparable affinities in the nM range as the murine IgG1 antibody (Fig 1, E). The chimeric anti–Blo t 5 mAbs were tested for their ability to inhibit allergen-induced human basophil activation. Expression of CD63 was upregulated on stimulation with Blo t extract and anti-IgE (Fig 1, F) and close to 60% of this response was inhibited in the presence of the chimeric antibodies. The extent of inhibition was the strongest and weakest for the chimeric

Blomia tropicalis allergen 5 (Blo t 5) T-cell epitopes and their ability to suppress the allergic immune response

Blomia tropicalis is the major asthma allergen in the tropics comparable to Dermatophagoides pteronyssinus. However, little is known about the B. tropicalis epitopes recognized by T cells. Our aim was to identify the T‐cell epitopes in the major B. tropicalis allergen, Blo t 5, and investigate the potential of the corresponding peptides to inhibit the allergic inflammatory lung response. C57BL/6 mice were immunized with plasmid DNA encoding Blo t 5 and T‐cell epitopes identified using the interferon‐γ ELISPOT assay with 15‐mer overlapping peptides. C57BL/6 mice were sensitized with bone‐marrow‐derived dendritic cells (BMDC) pulsed with Blo t 5 allergen followed by intranasal Blo t 5 challenge. Two H‐2b restricted epitopes (Bt576–90 and Bt5106–115) were recognized by CD4 T cells specific for Blo t 5, but no CD8 epitopes were identified. In mice sensitized with Blo t 5‐pulsed BMDC and challenged with intranasal Blo t 5 Bt576–90 and Bt5106–115, peptide‐specific CD4 T cells were found to secrete the T helper type 2 cytokines interleukin‐5 and interleukin‐13. Intradermal administration of synthetic peptides encoding the identified T‐cell epitopes suppressed allergic airway inflammation to further allergen challenges. Hence, we have identified novel CD4 T‐cell epitopes specific for Blo t 5 and demonstrated that these peptides could be employed therapeutically to suppress the T‐cell response in a murine model of allergic airway inflammation.