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Epitope mapping using BMP heterodimers (BMP4/7, BMP2/6 and BMP2/7).

Bone morphogenetic proteins (BMP) have important but distinct roles in tissue homeostasis and disease, including carcinogenesis and tumor progression. A large number of BMP inhibitors are available to study BMP function; however, as most of these antagonists are promiscuous, evaluating specific effects of individual BMPs is not feasible. Because the oncogenic role of the different BMPs varies for each neoplasm, highly selective BMP inhibitors are required. Here, we describe the generation of three types of llama-derived heavy chain variable domains (VHH) that selectively bind to either BMP4, to BMP2 and 4, or to BMP2, 4, 5, and 6. These generated VHHs have high affinity to their targets and are able to inhibit BMP signaling. Epitope binning and docking modeling have shed light into the basis for their BMP specificity. As opposed to the wide structural reach of natural inhibitors, these small molecules target the grooves and pockets of BMPs involved in receptor binding. In organoid experiments, specific inhibition of BMP4 does not affect the activation of normal stem cells. Furthermore, in vitro inhibition of cancer-derived BMP4 noncanonical signals results in an increase of chemosensitivity in a colorectal cancer cell line. Therefore, because of their high specificity and low off-target effects, these VHHs could represent a therapeutic alternative for BMP4+ malignancies. Mol Cancer Ther; 14(11); 2527–40. ©2015 AACR.

Supplementary file containing Supplementary materials and methods, supplementary figure legends and supplementary references.

Summary of epitope binning experiments of the VHHs and controls

Overview lysis primary AML samples; Quality control during bTCE preparation; Supportive data on T-cell induced AML cell lysis and T-cell activation

Acute myeloid leukemia (AML) is a high-risk disease with a poor prognosis, particularly in elderly patients. Because current AML treatment relies primarily on untargeted therapies with severe side effects that limit patient eligibility, identification of novel therapeutic AML targets is highly desired. We recently described AT1413, an antibody produced by donor B cells of a patient with AML cured after allogeneic hematopoietic stem cell transplantation. AT1413 binds CD43s, a unique sialylated epitope on CD43, which is weakly expressed on normal myeloid cells and overexpressed on AML cells. Because of its selectivity for AML cells, we considered CD43s as a target for a bispecific T-cell–engaging antibody (bTCE) and generated a bTCE by coupling AT1413 to two T-cell–targeting fragments using chemo-enzymatic linkage. In vitro, AT1413 bTCE efficiently induced T-cell–mediated cytotoxicity toward different AML cell lines and patient-derived AML blasts, whereas endothelial cells with low binding capacity for AT1413 remained unaffected. In the presence of AML cells, AT1413 bTCE induced upregulation of T-cell activation markers, cytokine release, and T-cell proliferation. AT1413 bTCE was also effective in vivo. Mice either coinjected with human peripheral blood mononuclear cells or engrafted with human hematopoietic stem cells [human immune system (HIS) mice] were inoculated with an AML cell line or patient-derived primary AML blasts. AT1413 bTCE treatment strongly inhibited tumor growth and, in HIS mice, had minimal effects on normal human hematopoietic cells. Taken together, our results indicate that CD43s is a promising target for T-cell–engaging antibodies and that AT1413 holds therapeutic potential in a bTCE-format.Significance:These findings offer preclinical evidence for the therapeutic potential of a bTCE antibody that targets a sialylated epitope on CD43 in AML.

The family of innate lymphoid cells (ILCs) are composed of five canonical subsets, NK cells, ILC1, ILC2, ILC3 and Lymphoid tissue inducer cells. ILCs have important functions in early stages of immune response towards infectious agents. ILCs are highly plastic enabling rapid modification of their functions dependent on the type of microbe and tissue environment to optimally counter these microbes. Data that still accumulate in a rapid pace indicate that these cells are also involved in immunity against tumor cells. Paradoxically ILC subsets have been shown to have tumor suppressing and tumor promoting activities. In this brief review we provide a snapshot of our current knowledge of characteristics and functions of tumor infiltrating ILC subsets and speculate on how these cells can be harnessed to mediate anti-tumor immunity.

Food allergy is an increasingly prevalent disease driven by uncontrolled type 2 immune response. Currently, knowledge about the underlying mechanisms that initiate and promote the immune response to dietary allergens is limited. Patients with food allergy are commonly sensitized through the skin in their early life, later on developing allergy symptoms within the gastrointestinal tract. Food allergy results from a dysregulated type 2 response to food allergens, characterized by enhanced levels of IgE, IL-4, IL-5, and IL-13 with infiltration of mast cells, eosinophils, and basophils. Recent studies raised a possible role for the involvement of innate lymphoid cells (ILCs) in driving food allergy. Unlike lymphocytes, ILCs lack They represent a group of lymphocytes that lack specific antigen receptors. ILCs contribute to immune responses not only by releasing cytokines and other mediators but also by responding to cytokines produced by activated cells in their local microenvironment. Due to their localization at barrier surfaces of the airways, gut, and skin, ILCs form a link between the innate and adaptive immunity. This review summarizes recent evidence on how skin and gastrointestinal mucosal immune system contribute to both homeostasis and the development of food allergy, as well as the involvement of ILCs toward inflammatory processes and regulatory mechanisms.

Five subsets of ILCs are extensively described, Lymphoid Tissue inducer (LTi) cells, cytotoxic NK cells and non-cytotoxic helper ILC1s, ILC2s and ILC3s. So far, the main focus has been on the potent cytokine production by helper ILCs and their plastic nature that allows them to switch function and phenotype upon environmental changes. Recent advances in the field indicate the presence of cytotoxic helper ILCs that are distinct from conventional NK cells. In humans, these cytotoxic ILCs can develop from conventional helper ILCs whereas in mice this remains to be elucidated. In this review we discuss the identification, development and function of cytotoxic helper ILCs subsets in humans and mice.

Innate lymphoid cells (ILCs) are highly plastic and predominantly mucosal tissue-resident cells that contribute to both homeostasis and inflammation depending on the microenvironment. The discovery of naïve-like ILCs suggests an ILC differentiation process that is akin to naïve T cell differentiation. Delineating the mechanisms that underlie ILC differentiation in tissues is crucial for understanding ILC biology in health and disease. Here, we showed that tonsillar ILCs expressing CD45RA lacked proliferative activity, indicative of cellular quiescence. CD62L distinguished two subsets of CD45RA

Innate lymphoid cells (ILCs) are highly plastic and predominantly mucosal tissue-resident cells that contribute to both homeostasis and inflammation depending on the microenvironment. The discovery of naïve-like ILCs suggests an ILC differentiation process that is akin to naïve T cell differentiation. Delineating the mechanisms that underlie ILC differentiation in tissues is crucial for understanding ILC biology in health and disease. Here, we showed that tonsillar ILCs expressing CD45RA lacked proliferative activity, indicative of cellular quiescence. CD62L distinguished two subsets of CD45RA+ILCs. CD45RA+CD62L+ILCs (CD62L+ILCs) resembled circulating naïve ILCs because they lacked the transcriptional, metabolic, epigenetic, and cytokine production signatures of differentiated ILCs. CD45RA+CD62L−ILCs (CD62L−ILCs) were epigenetically similar to CD62L+ILCs but showed a transcriptional, metabolic, and cytokine production signature that was more akin to differentiated ILCs. CD62L+and CD62L−ILCs contained uni- and multipotent precursors of ILC1s/NK cells and ILC3s. Differentiation of CD62L+and CD62L−ILCs led to metabolic reprogramming including up-regulation of genes associated with glycolysis, which was needed for their effector functions after differentiation. CD62L−ILCs with preferential differentiation capacity toward IL-22–producing ILC3s accumulated in the inflamed mucosa of patients with inflammatory bowel disease. These data suggested distinct differentiation potential of CD62L+and CD62L−ILCs between tissue microenvironments and identified that manipulation of these cells is a possible approach to restore tissue-immune homeostasis.

For many high-risk haematologic malignancies, such as acute myeloid leukaemia, the success of therapy relies mainly on invoking a curative antitumour immune response. This can be achieved by inducing a graft-versus-leukaemia response following allogeneic haematopoietic cell transplantation. While the contribution of T cells and natural killer cells to graft-versus-leukaemia responses is established, the contribution of B cells and antibodies is relatively unexplored. This article reviews what is known about the contribution of B cells and tumour-specific antibody responses to a successful graft-versus-leukaemia response leading to eradication of the tumour.

Innate lymphoid cells (ILCs) are important for tissue homeostasis and for the initiation of immune responses. Based on their transcriptional regulation and cytokine profiles, ILCs can be categorized into five subsets with defined phenotypes and functional profiles, but they also have the ability to adapt to local environmental cues by changing these profiles. This plasticity raises the question of the extent to which the cytokine production profiles of ILCs are pre-programmed or are a reflection of the tissue microenvironment. Here, we review recent advances in research on ILCs, with a focus on the plasticity of these cells. We highlight the ability of ILCs to communicate with the surrounding microenvironment and discuss the possible consequences of ILC plasticity for our understanding of the biological roles of these cells. Finally, we discuss how we might use this knowledge of ILC plasticity to develop or improve options for the treatment of inflammatory diseases.

Antigen-specific memory B cells play a key role in the induction of immune tolerance to food allergens and clinical healing. Here, we characterized the role of allergen-specific B cells in immune tolerance induced by oral allergen-specific immunotherapy (OIT) and natural tolerance that developed in children who spontaneously outgrew cow’s milk allergy. Increased frequency of circulating milk allergen αS1-casein -specific B cells was observed after OIT and natural tolerance (NT). Milk desensitized subjects showed partial acquisition of tolerance phenotypic features induced tolerance, suggesting that desensitization is an earlier stage of tolerance. Immunoregulatory genes such as IL10RA and IGHG4 are significantly upregulated after OIT (desensitized and tolerance) versus NT. Secreted proteins from allergen-specific B cells revealed higher amounts of regulatory cytokines, IL-10 and TGF-β after OIT and NT. Taken together, allergen-specific B cells are essential elements in regulating food allergen tolerance in both OIT-received and naturally-resolved individuals.

Phenotypic definition of helper ILC1 and NK cells is problematic due to overlapping markers. Recently we showed the identification of cytotoxic ILC3s characterized by expression of CD94. Here we analyse CD127+ ILCs and NK cells in intestinal lamina propria from healthy donors and Crohn’s disease patients and identify two populations of CD127+CD94+ ILCs, designated population A and B, that can be distinguished on the expression of CD117, CD18 and cytotoxic molecules. Population B expresses granulysin, a cytotoxic molecule linked to bacterial lysis and/or chemotaxis of monocytes. Granulysin protein is secreted by population B cells upon stimulation with IL-15. Activation of population B in the presence of TGF-β strongly reduces the expression of cytotoxic effector molecules of population B. Strikingly, samples from individuals that suffer from active Crohn’s disease display enhanced frequencies of granulysin-expressing effector CD127+CD94+ ILCs in comparison to controls. Thus this study identifies group 1 ILC populations which accumulate in inflamed intestinal tissue of Crohn’s disease patients and may play a role in the pathology of the disease., Phenotypic markers that overlap between ILC1 and NK populations have impacted the robust and specific analysis of these immune cell populations. Employing scRNA sequencing here the authors identify CD127+ CD94+ innate lymphoid cells that express granulysin and perforin and are expanded in patients with Crohn’s disease.

Jan Egbert de Vries (Figure 1) is a cosmopolitan immunologist and an enthralling mentor with a large track record of innovative achievements in the fields of allergy and immunology. Jan was born in Strijen, a small town located in the Hollands Diep estuary in western Netherlands (NL). He spent his youth in the NL combining his studies with his passion for sports,becoming Dutch champion in decathlon. Shortly after his PhD, and like his fellow countryman Erasmus of Rotterdam, he started a long journey that brought him to France, California, Austria and Switzerland,although never settling in any of the cities he worked.Like Erasmus, he has been since an insatiable scholar ("Non est ulla studiorum satietas") and an inspiring mentor for a large number of students and collaborators.

Targeted cancer therapy with monoclonal antibodies has proven successful for different cancer types but is limited by the availability of suitable antibody targets. CD43s, a unique sialylated form of CD43 expressed by hematologic malignancies, is a recently identified target and antibodies interacting with CD43s may have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was derived from a high-risk AML patient who successfully cleared leukemia after allogeneic stem cell transplantation. Here we observed that AT1413 binds also to certain non-hematopoietic tumor cells, particularly melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody-dependent cellular cytotoxicity against short-term cultured patient-derived melanoma samples. However, AT1413 was unable to affect the growth of melanoma cells in vivo. To increase the efficacy of AT1413 as a therapeutic antibody, we generated two different formats of bispecific T-cell engaging antibodies (TCEs): one binding bivalently (bTCE) and the other monovalently (knob-in-hole; KiH) to both CD43s and CD3 epsilon. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with differences in potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth in these mice. These data indicate a broad therapeutic potential of AT1413 that includes AML and CD43s-expressing solid tumors that originate from CD43-negative tissues.

Background & Aims In order to design an effective vaccine against hepatitis C virus (HCV) infection, it is necessary to understand immune protection. A number of broadly reactive neutralizing antibodies have been isolated from B cells of HCV-infected patients. However, it remains unclear whether B cells producing such antibodies contribute to HCV clearance and long-term immune protection against HCV. Methods We analysed the B cell repertoire of 13 injecting drug users from the Amsterdam Cohort Study, who were followed up for a median of 17.5 years after primary infection. Individuals were classified into 2 groups based on the outcome of HCV infection: 5 who became chronically infected either after primary infection or after reinfection, and 8 who were HCV RNA negative following spontaneous clearance of ≥1 HCV infection(s). From each individual, 10,000 CD27+IgG+B cells, collected 0.75 year after HCV infection, were cultured to characterize the antibody repertoire. Results Using a multiplex flow cytometry-based assay to study the antibody binding to E1E2 from genotype 1 to 6, we found that a high frequency of cross-genotype antibodies was associated with spontaneous clearance of 1 or multiple infections (p = 0.03). Epitope specificity of these cross-genotype antibodies was determined by alanine mutant scanning in 4 individuals who were HCV RNA negative following spontaneous clearance of 1 or multiple infections. Interestingly, the cross-genotype antibodies were mainly antigenic region 3 (AR3)-specific and showed cross-neutralizing activity against HCV. In addition to AR3 antibodies, 3 individuals developed antibodies recognizing antigenic region 4, of which 1 monoclonal antibody showed cross-neutralizing capacity. Conclusions Together, these data suggest that a strong B cell response producing cross-genotype and neutralizing antibodies, especially targeting AR3, contributes to HCV clearance and long-term immune protection against HCV. Lay summary Although effective treatments against hepatitis C virus (HCV) are available, 500,000 people die from liver disease caused by HCV each year and approximately 1.75 million people are newly infected. This could be prevented by a vaccine. To design a vaccine against HCV, more insight into the role of antibodies in the protection against HCV infection is needed. In a cohort of injecting drug users, we found that antibodies interfering with virus cell entry, and recognizing multiple HCV genotypes, conferred long-term protection against chronic HCV infection.

Here we identify a group 2 innate lymphoid cell (ILC2) subpopulation that can convert into interleukin-17 (IL-17)-producing NKp44− ILC3-like cells. c-Kit and CCR6 define this ILC2 subpopulation that exhibits ILC3 features, including RORγt, enabling the conversion into IL-17-producing cells in response to IL-1β and IL-23. We also report a role for transforming growth factor-β in promoting the conversion of c-Kit− ILC2s into RORγt-expressing cells by inducing the upregulation of IL23R, CCR6 and KIT messenger RNA in these cells. This switch was dependent on RORγt and the downregulation of GATA-3. IL-4 was able to reverse this event, supporting a role for this cytokine in maintaining ILC2 identity. Notably, this plasticity has physiological relevance because a subset of RORγt+ ILC2s express the skin-homing receptor CCR10, and the frequencies of IL-17-producing ILC3s are increased at the expense of ILC2s within the lesional skin of patients with psoriasis.

International audience; Human immune system (HIS) mouse models provide a robust in vivo platform to study human immunity. Nevertheless, the signals that guide human lymphocyte differentiation in HIS mice remain poorly understood. Here, we have developed a novel Balb/c Rag2-/- Il2rg-/- SirpaNOD (BRGS) HIS mouse model expressing human HLA-A2 and -DR2 transgenes (BRGSA2DR2). When comparing BRGS and BRGSA2DR2 HIS mice engrafted with human CD34+ stem cells, a more rapid emergence of T cells in the circulation of hosts bearing human HLA was shown, which may reflect a more efficient human T-cell development in the mouse thymus. Development of CD4+ and CD8+ T cells was accelerated in BRGSA2DR2 HIS mice and generated more balanced B and T-cell compartments in peripheral lymphoid organs. Both B- and T-cell function appeared enhanced in the presence of human HLA transgenes with higher levels of class switched Ig, increased percentages of polyfunctional T cells and clear evidence for antigen-specific T-cell responses following immunization. Taken together, the presence of human HLA class I and II molecules can improve multiple aspects of human B- and T-cell homeostasis and function in the BRGS-based HIS mouse model.

Objective Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA). Aside from autoantibody production, the function of autoantigen-specific B cells remains poorly understood in the context of this disease. This study set out to elucidate autoantigen-specific B cell functions through the isolation and immortalization of unique citrullinated protein/peptide (CP)-reactive B cell clones from RA patients. Methods B cell clones from either the blood or synovial fluid of cyclic citrullinated peptide 2 (CCP2) antibody-positive RA patients were immortalized by genetic reprogramming with Bcl-6 and Bcl-xL. Enzyme-linked immunosorbent assay and flow cytometry were used to identify CCP2-reactive clones and to further characterize surface marker and cytokine expression as well as B cell receptor signaling competence. Global gene expression profiles were interrogated by RNA sequencing. Results Three unique CP-reactive memory B cell clones were generated from the blood or synovial fluid of 2 RA patients. CP-reactive memory B cells did not appear to be broadly cross-reactive, but rather had a fairly restricted epitope recognition profile. These clones were able to secrete both pro- and antiinflammatory cytokines and had a unique surface profile of costimulatory molecules and receptors, including CD40 and C5a receptor type 1, when compared to non-CP-reactive clones from the same patient. In addition, CP-reactive clones bound citrullinated protein, but not native protein, and could mobilize calcium in response to antigen binding. Conclusion CP-reactive memory B cells comprise a rare, seemingly oligoclonal population with restricted epitope specificity and distinct phenotypic and molecular characteristics suggestive of antigen-presenting cells. Cloning by genetic reprogramming opens new avenues to study the function of autoreactive memory B cells, especially in terms of antigen processing, presentation, and subsequent T cell polarization.

Historically, bispecific antibodies have been constructed through the genetic fusion of additional binding domains to the constant domains of the antibody heavy- or light chains. We present an alternative method for the introduction of additional functional domains to an antibody: site-specific chemo-enzymatic conjugation. This method relies on the combination of site-specific transpeptidases and bioorthogonal chemistry. Transpeptidases are used to site-specifically introduce chemical handles, which can then be used to couple new functional groups by means of a bioorthogonal chemical reaction. We demonstrate site-specific chemo-enzymatic linkage using the transpeptidase sortase (hereafter: sortase) and either a strain-promoted alkyne-azide cycloaddition (SPAAC) or an inverse-electron demand Diels-Alder reaction. Other transpeptidases and bioorthogonal reactions suitable for this purpose exist. Site-specific chemo-enzymatic linkage is a modular method. After introduction of a chemical handle in the antibody, any functional group of interest may then be attached. The modularity of this conjugation method allows for a ‘plug-and-play’ approach to prepare new antibody conjugates, thus bypassing the need for (potentially) laborious genetic fusions. Moreover, as sortase is used to specifically modify the exact C-termini of the antibody chains, the final product will be fused in a C-to-C orientation, which is impossible to achieve by genetic manipulations alone. Here we demonstrate the utility of site-specific chemo-enzymatic conjugation to prepare antibody heterodimers, bispecific T-cell engager antibodies, and immunocytokines, discussing purification methods and describing possible pitfalls.

International audience; Objective: Clinical trials of the anti-interleukin-17A (anti-IL-17A) antibody secukinumab have demonstrated a crucial role of the cytokine IL-17A in the pathogenesis of spondyloarthritis (SpA); however, its cellular source in this condition remains a matter of controversy. Group 3 innate lymphoid cells (ILC3s) have been recently identified as potent producers of proinflammatory cytokines, including IL-17A and IL-22, in a number of different tissues. This study was undertaken to characterize the presence and composition of ILCs, and investigate whether these cells are an important source of IL-17A, in the synovial tissue (ST) of patients with SpA.Methods: Matched ST, synovial fluid, and peripheral blood (PB) samples were obtained from SpA patients with actively inflamed knee joints. ILC subsets were characterized by flow cytometry. Gene expression analysis at the single-cell level was performed directly ex vivo and after in vitro activation. An IL-17A enzyme-linked immunospot assay was used to detect IL-17A-secreting cells.Results: ILCs, and particularly NKp44+ ILC3s, were expanded in inflamed arthritic joints. Single-cell expression analysis demonstrated that ST ILCs were clearly distinguishable from ST T cells and from their PB counterparts. Expression of the Th17 signature transcripts RORC, AHR, and IL23R was detected in a large proportion of ST ILC3s. These cells were capable of inducing expression of IL22 and CSF2, but not IL17A, in response to in vitro restimulation.Conclusion: Our findings demonstrate that absolute and relative numbers of ILC3s are enriched in the synovial joints of patients with SpA. However, these cells are not a significant source of IL-17A in this disease.

International audience; During T cell-dependent (TD) germinal center (GC) responses, naïve B cells are instructed to differentiate towards GC B cells (GCBC), high-affinity long-lived plasma cells (LLPC) or memory B cells (Bmem). Alterations in the B cell-fate choice could contribute to immune dysregulation leading to the loss of self-tolerance and the initiation of autoimmune disease. Here we show that mRNA levels of the transcription regulator BOB.1 are increased in the lymph node compartment of patients with rheumatoid arthritis (RA), a prototypical auto-immune disease caused by the loss of immunological tolerance. Investigating to what extent levels of BOB.1 impact B cells during TD immune responses we found that BOB.1 has a crucial role in determining the B cell-fate decision. High BOB.1 levels promote the generation of cells with phenotypic and functional characteristics of Bmem. Mechanistically, overexpression of BOB.1 drives ABF1 and suppresses BCL6, favouring Bmem over LLPC or recycling GCBC. Low levels of BOB.1 are sufficient for LLPC but not for Bmem differentiation. Our findings demonstrate a novel role for BOB.1 in B cells during TD GC responses and suggest that its dysregulation may contribute to the pathogenesis of RA by disturbing the B cell-fate determination.

The human CD117+ CRTH2− ILC population contains ILC precursors. Nagasawa et al. segregate this population by the mutually exclusive expression of KLRG1 and NKp46. KLRG1+ ILCs are biased toward the ILC2 lineage, whereas NKp46 clearly defines ILC3-lineage–biased cells., Recently, human ILCs that express CD117 and CD127 but lack CRTH2 and NKp44 have been shown to contain precursors of ILC1, ILC2, and ILC3. However, these ILCs have not been extensively characterized. We performed an unbiased hierarchical stochastic neighbor embedding (HSNE) analysis of the phenotype of peripheral blood CD117+ ILCs, which revealed the presence of three major subsets: the first expressed NKp46, the second expressed both NKp46 and CD56, and the third expressed KLRG1, but not NKp46 or CD56. Analysis of their cytokine production profiles and transcriptome revealed that NKp46+ ILCs predominantly develop into ILC3s; some of them can differentiate into ILC1/NK-like cells, but they are unable to develop into ILC2s. In contrast, KLRG1+ ILCs predominantly differentiate into ILC2s. Single-cell cultures demonstrate that KLRG1+ ILCs can also differentiate into other ILC subsets depending on the signals they receive. Epigenetic profiling of KLRG1+ ILCs is consistent with the broad differentiation potential of these cells.

Pneumonia represents a major health care burden and Gram‐negative bacteria provide an increasing therapeutic challenge at least in part through the emergence of multidrug‐resistant strains. IL‐33 is a multifunctional cytokine belonging to the IL‐1 family that can affect many different cell types. We sought here to determine the effect of recombinant IL‐33 on the host response during murine pneumonia caused by the common Gram‐negative pathogen Klebsiella pneumoniae. IL‐33 pretreatment prolonged survival for more than 1 day during lethal airway infection and decreased bacterial loads at the primary site of infection and distant organs. Postponed treatment with IL‐33 (3 h) also reduced bacterial growth and dissemination. IL‐33‐mediated protection was not observed in mice deficient for the IL‐33 receptor component IL‐1 receptor‐like 1. IL‐33 induced a brisk type 2 response, characterized by recruitment of type 2 innate lymphoid cells to the lungs and enhanced release of IL‐5 and IL‐13. However, neither absence of innate lymphoid cells or IL‐13, nor blocking of IL‐5 impacted on IL‐33 effects in mice infected with Klebsiella. Likewise, IL‐33 remained effective in reducing bacterial loads in mice lacking B, T, and natural killer T cells. Experiments using antibody‐mediated cell depletion indicated that neutrophils and inflammatory monocytes were of importance for antibacterial defense. The capacity of IL‐33 to restrict bacterial growth in the lungs was strongly reduced in mice depleted of both neutrophils and inflammatory monocytes, but not in mice selectively depleted of either one of these cell types. These results suggest that IL‐33 boosts host defense during bacterial pneumonia by a combined effect on neutrophils and inflammatory monocytes. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

The role of innate immune cells in allergen immunotherapy that confers immune tolerance to the sensitizing allergen is unclear. Here, we report a role of interleukin-10-producing type 2 innate lymphoid cells (IL-10+ ILC2s) in modulating grass-pollen allergy. We demonstrate that KLRG1+ but not KLRG1– ILC2 produced IL-10 upon activation with IL-33 and retinoic acid. These cells attenuated Th responses and maintained epithelial cell integrity. IL-10+ KLRG1+ ILC2s were lower in patients with grass-pollen allergy when compared to healthy subjects. In a prospective, double-blind, placebo-controlled trial, we demonstrated that the competence of ILC2 to produce IL-10 was restored in patients who received grass-pollen sublingual immunotherapy. The underpinning mechanisms were associated with the modification of retinol metabolic pathway, cytokine-cytokine receptor interaction, and JAK-STAT signaling pathways in the ILCs. Altogether, our findings underscore the contribution of IL-10+ ILC2s in the disease-modifying effect by allergen immunotherapy.