Ocular antigen does not cause disease unless presented in the context of inflammation.

Ocular antigens are sequestered behind the blood-retina barrier and the ocular environment protects ocular tissues from autoimmune attack. The signals required to activate autoreactive T cells and allow them to cause disease in the eye remain in part unclear. In particular, the consequences of peripheral presentation of ocular antigens are not fully understood. We examined peripheral expression and presentation of ocular neo-self-antigen in transgenic mice expressing hen egg lysozyme (HEL) under a retina-specific promoter. High levels of HEL were expressed in the eye compared to low expression throughout the lymphoid system. Adoptively transferred naïve HEL-specific CD4+ T cells proliferated in the eye draining lymph nodes, but did not induce uveitis. By contrast, systemic infection with a murine cytomegalovirus (MCMV) engineered to express HEL induced extensive proliferation of transferred naïve CD4+ T cells, and significant uveoretinitis. In this model, wild-type MCMV, lacking HEL, did not induce overt uveitis, suggesting that disease is mediated by antigen-specific peripherally activated CD4+ T cells that infiltrate the retina. Our results demonstrate that retinal antigen is presented to T cells in the periphery under physiological conditions. However, when the same antigen is presented during viral infection, antigen-specific T cells access the retina and autoimmune uveitis ensues.

Gene therapy with recombinant adeno-associated vectors for neovascular age-related macular degeneration: 1 year follow-up of a phase 1 randomised clinical trial.

BACKGROUND: Neovascular, or wet, age-related macular degeneration causes central vision loss and represents a major health problem in elderly people, and is currently treated with frequent intraocular injections of anti-VEGF protein. Gene therapy might enable long-term anti-VEGF therapy from a single treatment. We tested the safety of rAAV.sFLT-1 in treatment of wet age-related macular degeneration with a single subretinal injection. METHODS: In this single-centre, phase 1, randomised controlled trial, we enrolled patients with wet age-related macular degeneration at the Lions Eye Institute and the Sir Charles Gairdner Hospital (Nedlands, WA, Australia). Eligible patients had to be aged 65 years or older, have age-related macular degeneration secondary to active subfoveal choroidal neovascularisation, with best corrected visual acuity (BCVA) of 3/60-6/24 and 6/60 or better in the other eye. Patients were randomly assigned (3:1) to receive either 1 × 10(10) vector genomes (vg; low-dose rAAV.sFLT-1 group) or 1 × 10(11) vg (high-dose rAAV.sFLT-1 group), or no gene-therapy treatment (control group). Randomisation was done by sequential group assignment. All patients and investigators were unmasked. Staff doing the assessments were masked to the study group at study visits. All patients received ranibizumab at baseline and week 4, and rescue treatment during follow-up based on prespecified criteria including BCVA measured on the Early Treatment Diabetic Retinopathy Study (EDTRS) scale, optical coherence tomography, and fluorescein angiography. The primary endpoint was ocular and systemic safety. This trial is registered with ClinicalTrials.gov, number NCT01494805. FINDINGS: From Dec 16, 2011, to April 5, 2012, we enrolled nine patients of whom eight were randomly assigned to receive either intervention (three patients in the low-dose rAAV.sFLT-1 group and three patients in the high-dose rAAV.sFLT-1 group) or no treatment (two patients in the control group). Subretinal injection of rAAV.sFLT-1 was highly reproducible. No drug-related adverse events were noted; procedure-related adverse events (subconjunctival or subretinal haemorrhage and mild cell debris in the anterior vitreous) were generally mild and self-resolving. There was no evidence of chorioretinal atrophy. Clinical laboratory assessments generally remained unchanged from baseline. Four (67%) of six patients in the treatment group required zero rescue injections, and the other two (33%) required only one rescue injection each. INTERPRETATION: rAAV.sFLT-1 was safe and well tolerated. These results support ocular gene therapy as a potential long-term treatment option for wet age-related macular degeneration. FUNDING: National Health and Medical Research Council of Australia, Richard Pearce Bequest, Lions Save Sight Foundation, Brian King Fellowship, and Avalanche Biotechnologies, Inc.

Kupffer cell-monocyte communication is essential for initiating murine liver progenitor cell-mediated liver regeneration.

UNLABELLED: Liver progenitor cells (LPCs) are necessary for repair in chronic liver disease because the remaining hepatocytes cannot replicate. However, LPC numbers also correlate with disease severity and hepatocellular carcinoma risk. Thus, the progenitor cell response in diseased liver may be regulated to optimize liver regeneration and minimize the likelihood of tumorigenesis. How this is achieved is currently unknown. Human and mouse diseased liver contain two subpopulations of macrophages with different ontogenetic origins: prenatal yolk sac-derived Kupffer cells and peripheral blood monocyte-derived macrophages. We examined the individual role(s) of Kupffer cells and monocyte-derived macrophages in the induction of LPC proliferation using clodronate liposome deletion of Kupffer cells and adoptive transfer of monocytes, respectively, in the choline-deficient, ethionine-supplemented diet model of liver injury and regeneration. Clodronate liposome treatment reduced initial liver monocyte numbers together with the induction of injury and LPC proliferation. Adoptive transfer of monocytes increased the induction of liver injury, LPC proliferation, and tumor necrosis factor-α production. CONCLUSION: Kupffer cells control the initial accumulation of monocyte-derived macrophages. These infiltrating monocytes are in turn responsible for the induction of liver injury, the increase in tumor necrosis factor-α, and the subsequent proliferation of LPCs.

Acute GVHD results in a severe DC defect that prevents T-cell priming and leads to fulminant cytomegalovirus disease in mice.

Viral infection is a common, life-threatening complication after allogeneic bone marrow transplantation (BMT), particularly in the presence of graft-versus-host disease (GVHD). Using cytomegalovirus (CMV) as the prototypic pathogen, we have delineated the mechanisms responsible for the inability to mount protective antiviral responses in this setting. Although CMV infection was self-limiting after syngeneic BMT, in the presence of GVHD after allogeneic BMT, CMV induced a striking cytopathy resulting in universal mortality in conjunction with a fulminant necrotizing hepatitis. Critically, GVHD induced a profound dendritic cell (DC) defect that led to a failure in the generation of CMV-specific CD8(+) T-cell responses. This was accompanied by a defect in antiviral CD8(+) T cells. In combination, these defects dramatically limited antiviral T-cell responses. The transfer of virus-specific cells circumvented the DC defects and provided protective immunity, despite concurrent GVHD. These data demonstrate the importance of avoiding GVHD when reconstructing antiviral immunity after BMT, and highlight the mechanisms by which the adoptive transfer of virus-specific T cells overcome the endogenous defects in priming invoked by GVHD.

A natural genetic variant of granzyme B confers lethality to a common viral infection.

Many immune response genes are highly polymorphic, consistent with the selective pressure imposed by pathogens over evolutionary time, and the need to balance infection control with the risk of auto-immunity. Epidemiological and genomic studies have identified many genetic variants that confer susceptibility or resistance to pathogenic micro-organisms. While extensive polymorphism has been reported for the granzyme B (GzmB) gene, its relevance to pathogen immunity is unexplored. Here, we describe the biochemical and cytotoxic functions of a common allele of GzmB (GzmBW) common in wild mouse. While retaining 'Asp-ase' activity, GzmBW has substrate preferences that differ considerably from GzmBP, which is common to all inbred strains. In vitro, GzmBW preferentially cleaves recombinant Bid, whereas GzmBP activates pro-caspases directly. Recombinant GzmBW and GzmBP induced equivalent apoptosis of uninfected targets cells when delivered with perforin in vitro. Nonetheless, mice homozygous for GzmBW were unable to control murine cytomegalovirus (MCMV) infection, and succumbed as a result of excessive liver damage. Although similar numbers of anti-viral CD8 T cells were generated in both mouse strains, GzmBW-expressing CD8 T cells isolated from infected mice were unable to kill MCMV-infected targets in vitro. Our results suggest that known virally-encoded inhibitors of the intrinsic (mitochondrial) apoptotic pathway account for the increased susceptibility of GzmBW mice to MCMV. We conclude that different natural variants of GzmB have a profound impact on the immune response to a common and authentic viral pathogen.

TRAIL+ NK cells control CD4+ T cell responses during chronic viral infection to limit autoimmunity.

Natural killer (NK) cells have been reported to control adaptive immune responses that occur in lymphoid organs at the early stages of immune challenge. The physiological purpose of such regulatory activity remains unclear, because it generally does not confer a survival advantage. We found that NK cells specifically eliminated activated CD4(+) T cells in the salivary gland during chronic murine cytomegalovirus (MCMV) infection. This was dependent on TNF-related apoptosis inducing ligand (TRAIL) expression by NK cells. Although NK cell-mediated deletion of CD4(+) T cells prolonged the chronicity of infection, it also constrained viral-induced autoimmunity. In the absence of this activity, chronic infection was associated with a Sjogren's-like syndrome characterized by focal lymphocytic infiltration into the glands, production of autoantibodies, and reduced saliva and tear secretion. Thus, NK cells are an important homeostatic control that balances the efficacy of adaptive immune responses with the risk of developing autoimmunity.

The early monocytic response to cytomegalovirus infection is MyD88 dependent but occurs independently of common inflammatory cytokine signals.

Cytomegalovirus latently infects myeloid cells; however, the acute effects of the virus on this cell subset are poorly characterised. We demonstrate that systemic cytomegalovirus infection induced rapid activation of monocytes in the bone marrow, characterised by upregulation of CD69, CD11c, Ly6C and M-CSF receptor. Activated bone marrow monocytes were more sensitive to M-CSF and less sensitive to granulocyte-monocyte colony stimulating factor in vitro, resulting in the generation of more macrophages and fewer dendritic cells, respectively. Monocyte activation was also observed in the periphery and resulted in significant accumulation of monocytes in the spleen. MyD88 expression was required within the haematopoietic compartment to initiate monocyte activation and recruitment. However, monocytes lacking MyD88 were activated and recruited in the presence of MyD88-sufficient cells in mixed bone marrow chimeras, indicating that once initiated, the process was MyD88 independent. Interestingly, we found that monocyte activation occurred in the absence of the common inflammatory cytokines, namely type I interferons (IFNs), IL-6, TNF-α and IL-1 as well as the NLRP3 inflammasome adaptor protein, ASC. We also excluded a role for the chemokine-like protein MCK-2 (m131/129) expressed by murine CMV. Taken together, these results challenge the notion that a single inflammatory cytokine mediates activation and recruitment of monocytes in response to infection.

A chemokine-like viral protein enhances alpha interferon production by plasmacytoid dendritic cells but delays CD8+ T cell activation and impairs viral clearance.

Murine cytomegalovirus encodes numerous proteins that act on a variety of pathways to modulate the innate and adaptive immune responses. Here, we demonstrate that a chemokine-like protein encoded by murine cytomegalovirus activates the early innate immune response and delays adaptive immunity, thereby impairing viral clearance. The protein, m131/129 (also known as MCK-2), is not required to establish infection in the spleen; however, a mutant virus lacking m131/129 was cleared more rapidly from this organ. In the absence of m131/129 expression, there was enhanced activation of dendritic cells (DC), and virus-specific CD8(+) T cells were recruited into the immune response earlier. Viral mutants lacking m131/129 elicited weaker production of alpha interferon (IFN-α) at 40 h postinfection, indicating that this protein exerts its effects during early rounds of viral replication in the spleen. Furthermore, while wild-type and mutant viruses activated plasmacytoid dendritic cells (pDC) equally at this time, as measured by the upregulation of costimulatory molecules, the presence of m131/129 stimulated more pDC to secrete IFN-α, accounting for the stronger IFN-α response than from the wild-type virus. These data provide evidence for a novel immunomodulatory function of a viral chemokine and expose the multifunctionality of immune evasion proteins. In addition, these results broaden our understanding of the interplay between innate and adaptive immunity.

CpG pretreatment enhances antiviral T-cell immunity against cytomegalovirus.

Major histocompatibility complex class I-restricted T-cell immunity is essential to control infection with cytomegalovirus (CMV), a clinically important virus that causes significant disease in immunocompromised individuals. Cross-presentation is considered the primary mode of antigen presentation to generate protective antiviral CD8⁺ T-cell immunity. Herpesviruses, including CMV, encode numerous proteins that interfere with direct antigen presentation, leading to the paradigm that T-cell immunity to these pathogens necessitates cross-presentation. However, the antigen presentation requirements needed to generate a protective T-cell response to CMV remain unknown. Here, we show that a fully functional antiviral CD8⁺ T-cell response can be generated in a system where cross-presentation is shut down by pretreatment with CpG. Notably, in this setting, CD8⁺ T cells demonstrate accelerated control of infection, and organ pathology is limited. These data indicate that protective antiviral T-cell immunity to CMV is generated by direct presentation and can be enhanced by pretreatment with CpG.

Cell-based therapies for ocular inflammation.

Since the plasticity and the potential for re-programming cells has become widely accepted, there has been great interest in cell-based therapies. These are being applied to a range of diseases, not least ocular diseases, where it is assumed that there is a reduced risk of immune rejection although this may be more perceived than real. There are two broad classes of cell-based therapies: those aimed at restoring structure and function of specific tissues and cells; and those directed towards restoring immunological homeostasis by controlling the damaging effects of inflammatory disease. Stem cells of all types represent the first group and prototypically have been used with the aim of regenerating failing cells. In contrast, immune cells have been suggested as potential modulators of inflammation. However, there is functional overlap in these two applications, with some types of stem cells, such as mesenchymal stem cells, demonstrating a potent immunomodulatory effect. This review summarises recent information on cell based therapies for ocular disease, with special emphasis on ocular inflammatory disease, and explores current uses, potential and limitations.

Interferon γ-dependent migration of microglial cells in the retina after systemic cytomegalovirus infection.

Microglial cells are the resident macrophages of the central nervous system and participate in both innate and adaptive immune responses but can also lead to exacerbation of neurodegenerative pathologies after viral infections. Microglia in the outer layers of the retina and the subretinal space are thought to be involved in retinal diseases where low-grade chronic inflammation and oxidative stress play a role. This study investigated the effect of systemic infection with murine cytomegalovirus on the distribution and dynamics of retinal microglia cells. Systemic infection with murine cytomegalovirus elicited a significant increase in the number of microglia in the subretinal space and an accumulation of iris macrophages, along with morphological signs of activation. Interferon γ (IFN-γ)-deficient mice failed to induce changes in microglia distribution. Bone marrow chimera experiments confirmed that microglial cells in the subretinal space were not recruited from the circulating monocyte pool, but rather represented an accumulation of resident microglial cells from within the retina. Our results demonstrate that a systemic viral infection can lead to IFN-γ-mediated accumulation of microglia into the outer retinal layers and offer proof of concept that systemic viral infections alter the ocular microenvironment and therefore, may influence the course of diseases such as macular degeneration, diabetic retinopathy, or autoimmune uveitis, where low-grade inflammation is implicated.

Flt3 inhibitor AC220 is a potent therapy in a mouse model of myeloproliferative disease driven by enhanced wild-type Flt3 signaling.

High levels of expression of wild-type Flt3 characterize many hematopoietic proliferative diseases and neoplasms, providing a potential therapeutic target. Using the c-Cbl RING finger mutant mouse as a model of a myeloproliferative disease (MPD) driven by wild-type Flt3, in the present study, we show that treatment with the Flt3 kinase inhibitor AC220 blocks MPD development by targeting Flt3(+) multipotent progenitors (MPPs). We found that daily administration of AC220 caused a marked reduction in Flt3 expression, induction of quiescence, and a significant loss of MPPs within 4 days. Unexpectedly, a robust Flt3 ligand-associated proliferative recovery response soon followed, preventing further loss of MPPs. However, continued AC220 treatment limited MPP recovery and maintained reduced, steady-state levels of cycling MPPs that express low levels of Flt3. Therefore, a finely tuned balance between the opposing forces of AC220 and Flt3 ligand production was established; whereas the Flt3 ligand blunted the inhibitory effects of AC220, the disease was held in remission for as long as therapy was continued. The net effect is a potent therapy indicating that patients with c-Cbl mutations, or those with similarly enhanced Flt3 signaling, may respond well to AC220 even after the induction of high levels of Flt3 ligand.

Kinetics of ocular and systemic antigen-specific T-cell responses elicited during murine cytomegalovirus retinitis.

Cytomegalovirus (CMV) reactivation in the retina of immunocompromized patients is a cause of significant morbidity as it can lead to blindness. The adaptive immune response is critical in controlling murine CMV (MCMV) infection in MCMV-susceptible mouse strains. CD8(+) T cells limit systemic viral replication in the acute phase of infection and are essential to contain latent virus. In this study, we provide the first evaluation of the kinetics of anti-viral T-cell responses after subretinal infection with MCMV. The acute response was characterized by a rapid expansion phase, with infiltration of CD8(+) T cells into the infected retina, followed by a contraction phase. MCMV-specific T cells displayed biphasic kinetics with a first peak at day 12 and contraction by day 18 followed by sustained recruitment of these cells into the retina at later time points post-infection. MCMV-specific CD8(+) T cells were also observed in the draining cervical lymph nodes and the spleen. Presentation of viral epitopes and activation of CD8(+) T cells was widespread and could be detected in the spleen and the draining lymph nodes, but not in the retina or iris. Moreover, after intraocular infection, antigen-specific cytotoxic activity was detectable and exhibited kinetics equivalent to those observed after intraperitoneal infection with the same viral dose. These data provide novel insights of how and where immune responses are initiated when viral antigen is present in the subretinal space.

Restricted aeroallergen access to airway mucosal dendritic cells in vivo limits allergen-specific CD4+ T cell proliferation during the induction of inhalation tolerance.

Chronic innocuous aeroallergen exposure attenuates CD4(+) T cell-mediated airways hyperresponsiveness in mice; however, the mechanism(s) remain unclear. We examined the role of airway mucosal dendritic cell (AMDC) subsets in this process using a multi-OVA aerosol-induced tolerance model in sensitized BALB/c mice. Aeroallergen capture by both CD11b(lo) and CD11b(hi) AMDC and the delivery of OVA to airway draining lymph nodes by CD8α(-) migratory dendritic cells (DC) were decreased in vivo (but not in vitro) when compared with sensitized but nontolerant mice. This was functionally significant, because in vivo proliferation of OVA-specific CD4(+) T cells was suppressed in airway draining lymph nodes of tolerized mice and could be restored by intranasal transfer of OVA-pulsed and activated exogenous DC, indicating a deficiency in Ag presentation by endogenous DC arriving from the airway mucosa. Bone marrow-derived DC Ag-presenting function was suppressed in multi-OVA tolerized mice, and allergen availability to airway APC populations was limited after multi-OVA exposure, as indicated by reduced OVA and dextran uptake by airway interstitial macrophages, with diffusion rather than localization of OVA across the airway mucosal surface. These data indicate that inhalation tolerance limits aeroallergen capture by AMDC subsets through a mechanism of bone marrow suppression of DC precursor function coupled with reduced Ag availability in vivo at the airway mucosa, resulting in limited Ag delivery to lymph nodes and hypoproliferation of allergen-specific CD4(+) T cells.

Monocytes from children with clinically stable cystic fibrosis show enhanced expression of Toll-like receptor 4.

SUMMARY: Lung disease in patients with cystic fibrosis (CF) is characterized by recurrent bacterial respiratory infections and intense airway inflammation. Pattern recognition receptors such as Toll-like receptor 2 (TLR2) and TLR4 identify bacterial pathogens and activate the innate immune response. We therefore hypothesized that increased expression of these receptors would be found on circulating immune cells from children with CF. A cohort of 66 young children (median age 3 years) with CF was studied and compared to both healthy controls (n = 14) and children without CF who were being investigated for recurrent respiratory infections (non-CF disease controls; n = 17) of a similar age. Surface expression of TLR2 and TLR4 on peripheral blood monocytes was analyzed using flow cytometry. TLR4 expression was significantly higher in patients with CF compared to healthy controls (P = 0.017) and non-CF disease controls (P = 0.025) but did not vary according to the presence or absence of pulmonary infection with Gram-negative or Gram-positive bacteria (P = 0.387) in the CF group. In contrast, TLR2 expression was similar across all three study groups (P = 0.930). The increased surface expression of TLR4 seen in young children with CF appears to be related to having CF per se and not related to current pulmonary infection.

Innate immunity defines the capacity of antiviral T cells to limit persistent infection.

Effective immunity requires the coordinated activation of innate and adaptive immune responses. Natural killer (NK) cells are central innate immune effectors, but can also affect the generation of acquired immune responses to viruses and malignancies. How NK cells influence the efficacy of adaptive immunity, however, is poorly understood. Here, we show that NK cells negatively regulate the duration and effectiveness of virus-specific CD4+ and CD8+ T cell responses by limiting exposure of T cells to infected antigen-presenting cells. This impacts the quality of T cell responses and the ability to limit viral persistence. Our studies provide unexpected insights into novel interplays between innate and adaptive immune effectors, and define the critical requirements for efficient control of viral persistence.

Lung homing T-cell generation is dependent on strength and timing of antigen delivery to lymph nodes.

Inhaled allergens are known for their immediate and ongoing effects in the respiratory tract (RT). In this report, we track inhaled antigen in normal mice for 7 days and find that while it is cleared from the airways, inhaled antigen persists in peripheral lung tissue and the draining lymph nodes (DLNs). The persistence of antigen led to ongoing presentation in the lymph nodes, but not the lungs, that decreased with time in direct proportion with the frequency of antigen-bearing RT dendritic cells (DCs). There was evidence of functional changes among the antigen-bearing DCs in the lymph nodes, as the expression of CD40, CD80 and CD86 were modulated over the course of 7 days. At the same time, there was a decrease in both CD4(+) T-cell proliferation in lymph nodes and the generation of recirculating CD4(+) T cells. However, early presentation of lower doses of inhaled antigen also resulted in a decrease in CD4(+) T-cell proliferation and recirculation. Thus, T-cell recirculation depends on the strength of stimulus in the DLNs and is produced by a combination of the dose of antigen delivered to the RT, DC migration and co-stimulatory molecule expression. These results provide an important insight into the fate of inhaled antigen in vivo and the influence of persistent antigen presentation on T-cell activation in the lymph nodes.

Interactions between innate antiviral and atopic immunoinflammatory pathways precipitate and sustain asthma exacerbations in children.

Severe asthma exacerbations in children requiring hospitalization are typically associated with viral infection and occur almost exclusively among atopics, but the significance of these comorbidities is unknown. We hypothesized that underlying interactions between immunoinflammatory pathways related to responses to aeroallergen and virus are involved, and that evidence of these interactions is detectable in circulating cells during exacerbations. To address this hypothesis we used a genomics-based approach involving profiling of PBMC subpopulations collected during exacerbation vs convalescence by microarray and flow cytometry. We demonstrate that circulating T cells manifest the postactivated "exhausted" phenotype during exacerbations, whereas monocyte/dendritic cell populations display up-regulated CCR2 expression accompanied by phenotypic changes that have strong potential for enhancing local inflammation after their recruitment to the atopic lung. Notably, up-regulation of FcepsilonR1, which is known to markedly amplify capacity for allergen uptake/presentation to Th2 effector cells via IgE-mediated allergen capture, and secondarily programming of IL-4/IL-13-dependent IL-13R(+) alternatively activated macrophages that have been demonstrated in experimental settings to be a potent source of autocrine IL-13 production. We additionally show that this disease-associated activation profile can be reproduced in vitro by cytokine exposure of atopic monocytes, and furthermore that IFN-alpha can exert both positive and negative roles in the process. Our findings suggest that respiratory viral infection in atopic children may initiate an atopy-dependent cascade that amplifies and sustains airway inflammation initiated by innate antiviral immunity via harnessing underlying atopy-associated mechanisms. These interactions may account for the unique susceptibility of atopics to severe viral-induced asthma exacerbations.

Airway hyperresponsiveness is associated with activated CD4+ T cells in the airways.

It is widely accepted that atopic asthma depends on an allergic response in the airway, yet the immune mechanisms that underlie the development of airway hyperresponsiveness (AHR) are poorly understood. Mouse models of asthma have been developed to study the pathobiology of this disease, but there is considerable strain variation in the induction of allergic disease and AHR. The aim of this study was to compare the development of AHR in BALB/c, 129/Sv, and C57BL/6 mice after sensitization and challenge with ovalbumin (OVA). AHR to methacholine was measured using a modification of the forced oscillation technique in anesthetized, tracheostomized mice to distinguish between airway and parenchymal responses. Whereas all strains showed signs of allergic sensitization, BALB/c was the only strain to develop AHR, which was associated with the highest number of activated (CD69(+)) CD4(+) T cells in the airway wall and the highest levels of circulating OVA-specific IgG(1). AHR did not correlate with total or antigen-specific IgE. We assessed the relative contribution of CD4(+) T cells and specific IgG(1) to the development of AHR in BALB/c mice using adoptive transfer of OVA-specific CD4(+) T cells from DO11.10 mice. AHR developed in these mice in a progressive fashion following multiple OVA challenges. There was no evidence that antigen-specific antibody had a synergistic effect in this model, and we concluded that the number of antigen-specific T cells activated and recruited to the airway wall was crucial for development of AHR.

Allergen-enhanced thrombomodulin (blood dendritic cell antigen 3, CD141) expression on dendritic cells is associated with a TH2-skewed immune response.

BACKGROUND: Dendritic cells (DCs) are important in allergic diseases such as asthma, although little is known regarding the mechanisms by which DCs induce T(H)2-polarized responses in atopic individuals. It has been suggested that intrinsic properties of allergens can directly stimulate T(H)2 polarizing functions of DCs, but little is known of the underlying mechanisms. OBJECTIVE: To identify novel genes expressed by house dust mite (HDM) allergen-exposed DCs. METHODS: We screened for allergen-induced gene expression by microarray, and validated differentially expressed genes at the mRNA and protein levels. RESULTS: Thrombomodulin (CD141, blood dendritic cell antigen 3) expression by microarray was higher on HDM-stimulated DCs from atopic (relative to nonatopic) individuals. These findings were confirmed at both the mRNA and protein levels in an independent group. Purified thrombomodulin(+) DCs induced a strongly T(H)2-polarized cytokine response by allergen-specific T cells compared with DCs lacking thrombomodulin. In vivo, thrombomodulin(+) circulating DCs were significantly more frequent in subjects with HDM allergy and asthma, compared with control subjects. Furthermore, thrombomodulin expression in blood leukocytes was higher in children with acute asthma than at convalescence 6 weeks later. CONCLUSION: Thrombomodulin expression on DCs may be involved in the pathogenesis of atopy and asthma.