Polo-like kinase 1 (PLK1) is a novel CARD14-binding protein in keratinocytes.

Caspase recruitment domain (CARD)-containing protein 14 (CARD14) is an intracellular protein that mediates nuclear factor-kappa B (NF-ĸB) signaling and proinflammatory gene expression in skin keratinocytes. Several hyperactivating CARD14 mutations have been associated with psoriasis and other inflammatory skin diseases. CARD14-induced NF-ĸB signaling is dependent on the formation of a CARD14-BCL10-MALT1 (CBM) signaling complex, but upstream receptors and molecular mechanisms that activate and regulate CARD14 signaling are still largely unclear. Using unbiased affinity purification and mass spectrometry (AP-MS) screening, we discover polo-like kinase 1 (PLK1) as a novel CARD14-binding protein. CARD14-PLK1 binding is independent of the CARD14 CARD domain but involves a consensus phospho-dependent PLK1-binding motif in the CARD14 linker region (LR). Expression of the psoriasis-associated CARD14(E138A) variant in human keratinocytes induces the recruitment of PLK1 to CARD14-containing signalosomes in interphase cells, but does not affect the specific location of PLK1 in mitotic cells. Finally, disruption of the PLK1-binding motif in CARD14(E138A) increases CARD14-induced proinflammatory signaling and gene expression. Together, our data identify PLK1 as a novel CARD14-binding protein and indicate a negative regulatory role for PLK1 in CARD14 signaling.

Differential protease content of mast cells and the processing of IL-33 in Alternaria alternata induced allergic airway inflammation in mice.

Background: Recent in vitro studies strongly implicated mast cell-derived proteases as regulators of IL-33 activity by enzymatic cleavage in its central domain. A better understanding of the role of mast cell proteases on IL-33 activity in vivo is needed. We aimed to compare the expression of mast cell proteases in C57BL/6 and BALB/c mice, their role in the cleavage of IL-33 cytokine, and their contribution to allergic airway inflammation. Results: In vitro, full-length IL-33 protein was efficiently degraded by mast cell supernatants of BALB/c mice in contrast to the mast cell supernatants from C57BL/6 mice. RNAseq analysis indicated major differences in the gene expression profiles of bone marrow-derived mast cells from C57BL/6 and BALB/c mice. In Alternaria alternata (Alt) - treated C57BL/6 mice the full-length form of IL-33 was mainly present, while in BALB/c mice, the processed shorter form of IL-33 was more prominent. The observed cleavage pattern of IL-33 was associated with a nearly complete lack of mast cells and their proteases in the lungs of C57BL/6 mice. While most inflammatory cells were similarly increased in Alt-treated C57BL/6 and BALB/c mice, C57BL/6 mice had significantly more eosinophils in the bronchoalveolar lavage fluid and IL-5 protein levels in their lungs than BALB/c mice. Conclusion: Our study demonstrates that lung mast cells differ in number and protease content between the two tested mouse strains and could affect the processing of IL-33 and inflammatory outcome of Alt -induced airway inflammation. We suggest that mast cells and their proteases play a regulatory role in IL-33-induced lung inflammation by limiting its proinflammatory effect via the IL-33/ST2 signaling pathway.

Neutrophils Affect IL-33 Processing in Response to the Respiratory Allergen Alternaria alternata.

Future precision medicine requires further clarifying the mechanisms of inflammation in the severe endotypes of chronic airway diseases such as asthma and chronic rhinosinusitis (CRS). The presence of neutrophils in the airways is often associated with severe airway inflammation, while their precise contribution to the severe inflammation is largely unknown. We aimed to study the role of neutrophils in BALB/c and C57BL/6 mice exposed to Alternaria alternata (Alt). The mice were exposed to Alt extract for twelve hours or ten days to induce allergic airway inflammation. C57BL/6 mice exposed to Alt responded with eosinophilic infiltration and the characteristic IL-5 upregulation. In contrast, the inflammatory response to Alt extract in BALB/c mice was characterized by a neutrophilic response, high levels of G-CSF, and elastase in the lungs. The lack of neutrophils affected the processing of IL-33 in BALB/c mice, as was demonstrated by depletion of neutrophils through intraperitoneal injections of anti-Ly6G antibody. Our data identifies the key role of neutrophils in airway inflammation through IL-33 cleavage in the Alt-induced airway inflammation in mice, which could potentially underline the different endotypes in human disease.

Defining the combinatorial space of PKC::CARD-CC signal transduction nodes.

Signal transduction typically displays a so-called bow-tie topology: Multiple receptors lead to multiple cellular responses but the signals all pass through a narrow waist of central signaling nodes. One such signaling node for several inflammatory and oncogenic signaling pathways is the CARD-CC/BCL10/MALT1 (CBM) complexes, which get activated by protein kinase C (PKC)-mediated phosphorylation of the caspase activation and recruitment domain (CARD)-coiled-coil domain (CC) component. In humans, there are four CARD-CC family proteins (CARD9, CARD10, CARD11, and CARD14) and 9 true PKC isozymes (α to ι). At this moment, less than a handful of PKC::CARD-CC relationships are known. In order to explore the biologically relevant combinatorial space out of all 36 potential permutations in this two-component signaling event, we made use of CARD10-deficient human embryonic kidney 293T cells for subsequent pairwise cotransfections of all CARD-CC family members and all activated PKCs. Upon analysis of NF-κB-dependent reporter gene expression, we could define specific PKC::CARD-CC relationships. Surprisingly, as many as 21 PKC::CARD-CC functional combinations were identified. CARD10 was responsive to most PKCs, while CARD14 was mainly activated by PKCδ. The CARD11 activation profile was most similar to that of CARD9. We also discovered the existence of mixed protein complexes between different CARD-CC proteins, which was shown to influence their PKC response profile. Finally, multiple PKCs were found to use a common phosphorylation site to activate CARD9, while additional phosphorylation sites contribute to CARD14 activation. Together, these data reveal the combinatorial space of PKC::CARD-CC signal transduction nodes, which will be valuable for future studies on the regulation of CBM signaling.

Single-Chain Soluble Receptor Fusion Proteins as Versatile Cytokine Inhibitors.

Cytokines are small secreted proteins that among many functions also play key roles in the orchestration of inflammation in host defense and disease. Over the past years, a large number of biologics have been developed to target cytokines in disease, amongst which soluble receptor fusion proteins have shown some promise in pre-clinical studies. We have previously shown proof-of-concept for the therapeutic targeting of interleukin (IL)-33 in airway inflammation using a newly developed biologic, termed IL-33trap, comprising the ectodomains of the cognate receptor ST2 and the co-receptor IL-1RAcP fused into a single-chain recombinant fusion protein. Here we extend the biophysical and biological characterization of IL-33trap variants, and show that IL-33trap is a stable protein with a monomeric profile both at physiological temperatures and during liquid storage at 4°C. Reducing the N-glycan heterogeneity and complexity of IL-33trap via GlycoDelete engineering neither affects its stability nor its inhibitory activity against IL-33. We also report that IL-33trap specifically targets biologically active IL-33 splice variants. Finally, we document the generation and antagonistic activity of a single-chain IL-4/13trap, which inhibits both IL-4 and IL-13 signaling. Collectively, these results illustrate that single-chain soluble receptor fusion proteins against IL-4, IL-13, and IL-33 are novel biologics that might not only be of interest for research purposes and further interrogation of the role of their target cytokines in physiology and disease, but may also complement monoclonal antibodies for the treatment of allergic and other inflammatory diseases.

ST2 as checkpoint target for colorectal cancer immunotherapy.

Immune checkpoint blockade immunotherapy delivers promising clinical results in colorectal cancer (CRC). However, only a fraction of cancer patients develop durable responses. The tumor microenvironment (TME) negatively impacts tumor immunity and subsequently clinical outcomes. Therefore, there is a need to identify other checkpoint targets associated with the TME. Early-onset factors secreted by stromal cells as well as tumor cells often help recruit immune cells to the TME, among which are alarmins such as IL-33. The only known receptor for IL-33 is stimulation 2 (ST2). Here we demonstrated that high ST2 expression is associated with poor survival and is correlated with low CD8+ T cell cytotoxicity in CRC patients. ST2 is particularly expressed in tumor-associated macrophages (TAMs). In preclinical models of CRC, we demonstrated that ST2-expressing TAMs (ST2+ TAMs) were recruited into the tumor via CXCR3 expression and exacerbated the immunosuppressive TME; and that combination of ST2 depletion using ST2-KO mice with anti-programmed death 1 treatment resulted in profound growth inhibition of CRC. Finally, using the IL-33trap fusion protein, we suppressed CRC tumor growth and decreased tumor-infiltrating ST2+ TAMs. Together, our findings suggest that ST2 could serve as a potential checkpoint target for CRC immunotherapy.

Stabilization of the TAK1 adaptor proteins TAB2 and TAB3 is critical for optimal NF-κB activation.

TAB2 and TAB3 bind to K63-linked polyubiquitin chains and recruit the critical kinase MAP3K7 (TAK1). The polyubiquitin-recruited TAK1/TAB2/TAB3 complex comes in close proximity with the IKK (IKKα/IKKß/IKKγ) complex, which is recruited to M1-linked polyubiquitin chains via the IKKγ (NEMO) component. Together, the two complexes activate the NF-κB family of transcription factors. NF-κB transcription factors are critical mediators of pro-inflammatory signals and must be tightly regulated at multiple levels. Recently, it was discovered that one such point of regulation occurs at the level of TAB2 and TAB3 protein stability by the deubiquitinase USP15. Comment on: https://doi.org/10.1111/febs.15202.

IL-33trap is a novel IL-33-neutralizing biologic that inhibits allergic airway inflammation.

BACKGROUND: The emergence of IL-33 as a key molecular player in the development and propagation of widespread inflammatory diseases, including asthma and atopic dermatitis, has established the need for effective IL-33-neutralizing biologics. OBJECTIVE: Here we describe the development and validation of a new antagonist of IL-33, termed IL-33trap, which combines the extracellular domains of the IL-33 receptor (ST2) and its coreceptor, IL-1 receptor accessory protein, into a single fusion protein. METHODS: We produced and purified recombinant IL-33trap from human cells and analyzed its IL-33-binding affinity and IL-33 antagonistic activity in cultured cells and mice. IL-33trap activity was also benchmarked with a recombinant soluble ST2 corresponding to the naturally occurring IL-33 decoy receptor. Finally, we studied the effect of IL-33trap in the Alternaria alternata mouse model of allergic airway inflammation. RESULTS: In vitro IL-33trap binds IL-33 and inhibits IL-33 activity to a much stronger degree than soluble ST2. Furthermore, IL-33trap inhibits eosinophil infiltration, splenomegaly, and production of signature cytokines in splenic lymphocytes and lung tissue on IL-33 injection. Finally, administration of IL-33trap at the time of allergen challenge inhibits inflammatory responses in a preclinical mouse model of acute allergic airway inflammation. CONCLUSIONS: IL-33trap is a novel IL-33 antagonist that outperforms the natural IL-33 decoy receptor and shows anti-inflammatory activities in a preclinical mouse model of acute allergic airway inflammation when administered at the time of allergen challenge.

Novel carboxylate-based glycolipids: TLR4 antagonism, MD-2 binding and self-assembly properties.

New monosaccharide-based lipid A analogues were rationally designed through MD-2 docking studies. A panel of compounds with two carboxylate groups as phosphates bioisosteres, was synthesized with the same glucosamine-bis-succinyl core linked to different unsaturated and saturated fatty acid chains. The binding of the synthetic compounds to purified, functional recombinant human MD-2 was studied by four independent methods. All compounds bound to MD-2 with similar affinities and inhibited in a concentration-dependent manner the LPS-stimulated TLR4 signaling in human and murine cells, while being inactive as TLR4 agonists when provided alone. A compound of the panel was tested in vivo and was not able to inhibit the production of proinflammatory cytokines in animals. This lack of activity is probably due to strong binding to serum albumin, as suggested by cell experiments in the presence of the serum. The interesting self-assembly property in solution of this type of compounds was investigated by computational methods and microscopy, and formation of large vesicles was observed by cryo-TEM microscopy.

Molecular mechanisms of IL-33-mediated stromal interactions in cancer metastasis.

Molecular mechanisms underlying the cancer stroma in metastasis need further exploration. Here, we discovered that cancer-associated fibroblasts (CAFs) produced high levels of IL-33 that acted on tumor-associated macrophages (TAMs), causing them to undergo the M1 to M2 transition. Genomic profiling of metastasis-related genes in the IL-33-stimulated TAMs showed a >200-fold increase of MMP9. Signaling analysis demonstrated the IL-33-ST2-NF-κB-MMP9-laminin pathway that governed tumor stroma-mediated metastasis. In mouse and human fibroblast-rich pancreatic cancers, genetic deletion of IL-33, ST2, or MMP9 markedly blocked metastasis. Pharmacological inhibition of NF-κB and MMP9 also blocked cancer metastasis. Deletion of IL-33, ST2, or MMP9 restored laminin, a key basement membrane component associated with tumor microvessels. Together, our data provide mechanistic insights on the IL-33-NF-κB-MMP9-laminin axis that mediates the CAF-TAM-committed cancer metastasis. Thus, targeting the CAF-TAM-vessel axis provides an outstanding therapeutic opportunity for cancer treatment.

IL-33 signalling contributes to pollutant-induced allergic airway inflammation.

BACKGROUND: Clinical and experimental studies have identified a crucial role for IL-33 and its receptor ST2 in allergic asthma. Inhalation of traffic-related pollutants, such as diesel exhaust particles (DEP), facilitates the development of asthma and can cause exacerbations of asthma. However, it is unknown whether IL-33/ST2 signalling contributes to the enhancing effects of air pollutants on allergic airway responses. OBJECTIVE: We aim to investigate the functional role of IL-33/ST2 signalling in DEP-enhanced allergic airway responses, using an established murine model. METHODS: C57BL/6J mice were exposed to saline, DEP alone, house dust mite (HDM) alone or combined DEP+HDM. To inhibit IL-33 signalling, recombinant soluble ST2 (r-sST2) was given prophylactically (ie, during the whole experimental protocol) or therapeutically (ie, at the end of the experimental protocol). Airway hyperresponsiveness and the airway inflammatory responses were assessed in bronchoalveolar lavage fluid (BALF) and lung. RESULTS: Combined exposure to DEP+HDM increased IL-33 and ST2 expression in lung, elevated inflammatory responses and bronchial hyperresponsiveness compared to saline, sole DEP or sole HDM exposure. Prophylactic interference with the IL-33/ST2 signalling pathway impaired the DEP-enhanced allergic airway inflammation in the BALF, whereas effects on lung inflammation and airway hyperresponsiveness were minimal. Treatment with r-sST2 at the end of the experimental protocol did not modulate the DEP-enhanced allergic airway responses. CONCLUSION: Our data suggest that the IL-33/ST2 pathway contributes to the onset of DEP-enhanced allergic airway inflammation.

Staphylococcus aureus Induces a Mucosal Type 2 Immune Response via Epithelial Cell-derived Cytokines.

RATIONALE: Chronic rhinosinusitis with nasal polyps is characterized by a T-helper cell type 2-skewed upper airway inflammation. Mucosal Staphylococcus aureus colonization is found in the majority of patients with nasal polyps. S. aureus is known to induce type 2 cytokine release via enterotoxins. OBJECTIVES: To investigate the impact of non-enterotoxin-producing S. aureus on type 2 cytokine release. METHODS: TSLP (thymic stromal lymphopoietin), IL-33, and type 2 cytokines were assessed in a human mucosal tissue model upon S. aureus infection. MEASUREMENTS AND MAIN RESULTS: S. aureus exposure increased the expression of IL-33, TSLP, IL-5, and IL-13 in nasal polyp tissue, accompanied by elevated expression levels of TSLP and IL-33 receptors, predominantly on CD3+ T cells. S. aureus infection led to the release of TSLP, but not IL-33, IL-5, or IL-13, from healthy inferior turbinate tissue. In contrast, S. epidermidis did not induce any epithelial cell-derived cytokines in nasal polyp or healthy tissue. S. aureus infection also increased the release of IL-33 and TSLP in BEAS-2B epithelial cells, accompanied by activation of NF-κB (nuclear factor κB) pathways. Incubation with CU-CPT22, a specific Toll-like receptor 2 antagonist, significantly reduced the S. aureus-induced release of TSLP and IL-33, and the activity of the NF-κB signal in BEAS-2B cells. CONCLUSIONS: This study demonstrates for the first time that S. aureus can directly induce epithelial cell-derived cytokine release via binding to Toll-like receptor 2, and may thereby propagate type 2 cytokine expression in nasal polyp tissue.

Structure-Activity Relationship in Monosaccharide-Based Toll-Like Receptor 4 (TLR4) Antagonists.

The structure-activity relationship was investigated in a series of synthetic TLR4 antagonists formed by a glucosamine core linked to two phosphate esters and two linear carbon chains. Molecular modeling showed that the compounds with 10, 12, and 14 carbons chains are associated with higher stabilization of the MD-2/TLR4 antagonist conformation than in the case of the C16 variant. Binding experiments with human MD-2 showed that the C12 and C14 variants have higher affinity than C10, while the C16 variant did not interact with the protein. The molecules, with the exception of the C16 variant, inhibited the LPS-stimulated TLR4 signal in human and murine cells, and the antagonist potency mirrored the MD-2 affinity calculated from in vitro binding experiments. Fourier-transform infrared, nuclear magnetic resonance, and small angle X-ray scattering measurements suggested that the aggregation state in aqueous solution depends on fatty acid chain lengths and that this property can influence TLR4 activity in this series of compounds.

Dichotomous function of IL-33 in health and disease: From biology to clinical implications.

Interleukin (IL)-33 is a cytokine that is released from epithelial and endothelial cells at barrier surfaces upon tissue stress or damage to operate as an alarmin. IL-33 has been primarily implicated in the induction of T helper (Th) 2 type immune responses. Therefore, IL-33 has attracted a lot of interest as a potential therapeutic target in asthma and other allergic diseases. Over the years, it has become clear that IL-33 has a much broader activity and also contributes to Th1 immunity, expanding the possibilities for therapeutic modulation of IL-33 activity to multiple inflammatory diseases. However, more recently IL-33 has also been shown to mediate immunosuppression and tissue repair by activating regulatory T cells (Treg) and promoting M2 macrophage polarization. These pleiotropic activities of IL-33 illustrate the need for a tight molecular regulation of IL-33 activity, and have to be taken into account when IL-33 or its receptor is targeted for therapeutic modulation. Here we review the multiple molecular mechanisms that regulate IL-33 activity and describe how IL-33 can shape innate and adaptive immune responses by promoting Th1, Th2 and Treg function. Finally, we will discuss the possibilities for therapeutic modulation of IL-33 signaling as well as possible safety issues.

The IL-33/ST2 axis is crucial in type 2 airway responses induced by Staphylococcus aureus-derived serine protease-like protein D.

BACKGROUND: Chronic airway inflammatory diseases, such as chronic rhinosinusitis with nasal polyps and asthma, show increased nasal Staphylococcus aureus colonization. Staphylococcus aureus-derived serine protease-like protein (Spl) D and other closely related proteases secreted by S aureus have recently been identified as inducers of allergic asthma in human subjects and mice, but their mechanism of action is largely unknown. OBJECTIVE: We investigated the role of recombinant SplD in driving TH2-biased responses and IgE formation in a murine model of allergic asthma. METHODS: Allergic asthma was induced in C57BL/6 J wild-type mice, Toll-like receptor (TLR) 4 knockout (Tlr4-/-) mice, and recombination-activating gene (Rag2) knockout (Rag2-/-) mice by means of repeated intratracheal applications of SplD. Inflammatory parameters in the airways were assessed by means of flow cytometry, ELISA, Luminex, and immunohistochemistry. Serum SplD-specific IgE levels were analyzed by using ELISA. RESULTS: We observed that repeated intratracheal exposure to SplD led to IL-33 and eotaxin production, eosinophilia, bronchial hyperreactivity, and goblet cell hyperplasia in the airways. Blocking IL-33 activity with a soluble ST2 receptor significantly decreased the numbers of eosinophils, IL-13+ type 2 innate lymphoid cells and IL-13+CD4+ T cells and IL-5 and IL-13 production by lymph node cells but had no effect on IgE production. SplD-induced airway inflammation and IgE production were largely dependent on the presence of the functional adaptive immune system and independent of TLR4 signaling. CONCLUSION: The S aureus-derived protein SplD is a potent allergen of S aureus and induces a TH2-biased inflammatory response in the airways in an IL-33-dependent but TRL4-independent manner. The soluble ST2 receptor could be an efficient strategy to interfere with SplD-induced TH2 inflammation but does not prevent the allergic sensitization.

Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma.

The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor α-chain (IL-7Rα) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Rα extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency.

The PDGF-BB-SOX7 axis-modulated IL-33 in pericytes and stromal cells promotes metastasis through tumour-associated macrophages.

Signalling molecules and pathways that mediate crosstalk between various tumour cellular compartments in cancer metastasis remain largely unknown. We report a mechanism of the interaction between perivascular cells and tumour-associated macrophages (TAMs) in promoting metastasis through the IL-33-ST2-dependent pathway in xenograft mouse models of cancer. IL-33 is the highest upregulated gene through activation of SOX7 transcription factor in PDGF-BB-stimulated pericytes. Gain- and loss-of-function experiments validate that IL-33 promotes metastasis through recruitment of TAMs. Pharmacological inhibition of the IL-33-ST2 signalling by a soluble ST2 significantly inhibits TAMs and metastasis. Genetic deletion of host IL-33 in mice also blocks PDGF-BB-induced TAM recruitment and metastasis. These findings shed light on the role of tumour stroma in promoting metastasis and have therapeutic implications for cancer therapy.

NEMA image quality phantom measurements and attenuation correction in integrated PET/MR hybrid imaging.

BACKGROUND: In integrated PET/MR hybrid imaging the evaluation of PET performance characteristics according to the NEMA standard NU 2-2007 is challenging because of incomplete MR-based attenuation correction (AC) for phantom imaging. In this study, a strategy for CT-based AC of the NEMA image quality (IQ) phantom is assessed. The method is systematically evaluated in NEMA IQ phantom measurements on an integrated PET/MR system. METHODS: NEMA IQ measurements were performed on the integrated 3.0 Tesla PET/MR hybrid system (Biograph mMR, Siemens Healthcare). AC of the NEMA IQ phantom was realized by an MR-based and by a CT-based method. The suggested CT-based AC uses a template µ-map of the NEMA IQ phantom and a phantom holder for exact repositioning of the phantom on the systems patient table. The PET image quality parameters contrast recovery, background variability, and signal-to-noise ratio (SNR) were determined and compared for both phantom AC methods. Reconstruction parameters of an iterative 3D OP-OSEM reconstruction were optimized for highest lesion SNR in NEMA IQ phantom imaging. RESULTS: Using a CT-based NEMA IQ phantom µ-map on the PET/MR system is straightforward and allowed performing accurate NEMA IQ measurements on the hybrid system. MR-based AC was determined to be insufficient for PET quantification in the tested NEMA IQ phantom because only photon attenuation caused by the MR-visible phantom filling but not the phantom housing is considered. Using the suggested CT-based AC, the highest SNR in this phantom experiment for small lesions (<= 13 mm) was obtained with 3 iterations, 21 subsets and 4 mm Gaussian filtering. CONCLUSION: This study suggests CT-based AC for the NEMA IQ phantom when performing PET NEMA IQ measurements on an integrated PET/MR hybrid system. The superiority of CT-based AC for this phantom is demonstrated by comparison to measurements using MR-based AC. Furthermore, optimized PET image reconstruction parameters are provided for the highest lesion SNR in NEMA IQ phantom measurements.

Interleukin-21-Producing CD4(+) T Cells Promote Type 2 Immunity to House Dust Mites.

Asthma is a T helper 2 (Th2)-cell-mediated disease; however, recent findings implicate Th17 and innate lymphoid cells also in regulating airway inflammation. Herein, we have demonstrated profound interleukin-21 (IL-21) production after house dust mite (HDM)-driven asthma by using T cell receptor (TCR) transgenic mice reactive to Dermatophagoides pteronyssinus 1 and an IL-21GFP reporter mouse. IL-21-producing cells in the mediastinal lymph node (mLN) bore characteristics of T follicular helper (Tfh) cells, whereas IL-21(+) cells in the lung did not express CXCR5 (a chemokine receptor expressed by Tfh cells) and were distinct from effector Th2 or Th17 cells. Il21r(-/-) mice developed reduced type 2 responses and the IL-21 receptor (IL-21R) enhanced Th2 cell function in a cell-intrinsic manner. Finally, administration of recombinant IL-21 and IL-25 synergistically promoted airway eosinophilia primarily via effects on CD4(+) lymphocytes. This highlights an important Th2-cell-amplifying function of IL-21-producing CD4(+) T cells in allergic airway inflammation.