Bridging the gap between scientific discoveries and clinical application at the Utrecht Science Park.

Associate Professor Victor Peperzak from the University Medical Center (UMC) Utrecht in the Netherlands discusses the opportunities and challenges of building a research group in the Netherlands. Victor highlights the scientific strengths of UMC Utrecht in juxtaposition with other universities and centers around the Netherlands, and highlights the collaborative nature of the Dutch research scene.

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.

Immunology in the Copenhagen area: a renewed focus on diseases of the skin.

The Maersk Tower in the heart of Copenhagen, Denmark, is home to the Leo Foundation Skin Immunology Research Centre (SIC) and the broader Department of Microbiology and Immunology (ISIM) of the University of Copenhagen. In this article, ICB Deputy Editor Associate Professor Jonathan Coquet interviews Professor Liv Eidsmo and discusses her research career, interest in skin diseases and what inspired her move to Copenhagen.

A conversation on allergy: recognizing the past and looking to the future.

Allergy is an ever-evolving group of disorders, which includes asthma, atopic dermatitis, rhinitis and food allergies and that currently affects over 1 billion people worldwide. This group of disorders has exploded in incidence since around the start of the 20th century, implying that genetics is not solely responsible for its development but that environmental factors have an important role. Here, Fabio Luciani and Jonathan Coquet, in their role as editors at Immunology & Cell Biology, asked nine prominent researchers in the field of allergy to define the term 'allergy', discuss the role of genetics and the environment, nominate the most important discoveries of the past decade and describe the best strategies to combat allergy at the population level going forward.

Recombinant multimeric dog allergen prevents airway hyperresponsiveness in a model of asthma marked by vigorous TH 2 and TH 17 cell responses.

BACKGROUND: Allergy to dogs affects around 10% of the population in developed countries. Immune therapy of allergic patients with dog allergen extracts has shown limited therapeutic benefit. METHODS: We established a mouse model of dog allergy by repeatedly administering dog dander and epithelium extracts via the intranasal route. We also assessed the efficacy of a recombinant multimeric protein containing Can f 1, f 2, f 4 and f 6 in preventing inflammatory responses to dog extracts. RESULTS: Repeated inhalation of dog extracts induced infiltration of the airways by TH 2 cells, eosinophils and goblet cells, reminiscent of the house dust mite (HDM) model of asthma. Dog extracts also induced robust airway hyperresponsiveness and promoted TH 17 cell responses, which was associated with a high neutrophilic infiltration of the airways. scRNA-Seq analysis of T helper cells in the airways pinpointed a unique gene signature for TH 17 cells. Analysis of T-cell receptors depicted a high frequency of clones that were shared between TH 17, TH 2 and suppressive Treg cells, indicative of a common differentiation trajectory for these subsets. Importantly, sublingual administration of multimeric Can f 1-2-4-6 protein prior to sensitization reduced airway hyperresponsiveness and type 2-mediated inflammation in this model. CONCLUSION: Dog allergen extracts induce robust TH 2 and TH 17 cell-mediated responses in mice. Recombinant Can f 1-2-4-6 can induce tolerance to complex dog allergen extracts.

The CD27 and CD70 costimulatory pathway inhibits effector function of T helper 17 cells and attenuates associated autoimmunity.

T helper 17 (Th17) cells protect against infection but also promote inflammation and autoimmunity. Therefore, the factors that govern Th17 cell differentiation are of special interest. The CD27 and CD70 costimulatory pathway impeded Th17 effector cell differentiation and associated autoimmunity in a mouse model of multiple sclerosis. CD27 or CD70 deficiency exacerbated disease, whereas constitutive CD27 signaling strongly reduced disease incidence and severity. CD27 signaling did not impact master regulators of T helper cell lineage commitment but selectively repressed transcription of the key effector molecules interleukin-17 (IL-17) and the chemokine receptor CCR6 in differentiating Th17 cells. CD27 mediated this repression at least in part via the c-Jun N-terminal kinase (JNK) pathway that restrained IL-17 and CCR6 expression in differentiating Th17 cells. CD27 signaling also resulted in epigenetic silencing of the Il17a gene. Thus, CD27 costimulation via JNK signaling, transcriptional, and epigenetic effects suppresses Th17 effector cell function and associated pathological consequences.

GPR43 regulates marginal zone B-cell responses to foreign and endogenous antigens.

Marginal zone (MZ) B cells are innate-like B cells that produce polyreactive antibodies with an affinity for microbial molecular patterns and carbohydrate ligands. MZ B cells have been shown to be important in mediating immunity to various bacteria including Streptococcus pneumoniae and are also implicated in inflammatory syndromes including lupus erythematosus. The intestinal microbiota is responsible for producing short-chain fatty acids, which can regulate immune cell function by several mechanisms including ligation of the G-protein-coupled receptor (GPR)43. Herein, we show that MZ B cells express Gpr43 messenger RNA and that the absence of this receptor impacts on MZ B-cell surface marker expression and antibody production. In T-cell-independent responses to the hapten 4-hydroxy-3-nitrophenylacetic acid (NP), mice deficient in GPR43 displayed higher serum titers of NP-specific antibodies. Moreover, in response to a pneumococcal polysaccharide vaccine, GPR43-deficient mice developed robust serum antibody responses and had markedly increased numbers of splenic antibody-secreting cells, compared with control mice. Finally, serum immunoglobulin M autoantibodies to double-stranded DNA and phosphatidylcholine were increased in resting 10-15-week-old mice lacking GPR43. Taken together, mice lacking GPR43 have heightened antibody responses to T-cell-independent antigens, which may be a result of impaired regulation of MZ B cells.

The Role of PPAR-γ in Allergic Disease.

PURPOSE OF REVIEW: The incidence of allergic diseases such as asthma, rhinitis and atopic dermatitis has risen at an alarming rate over the last century. Thus, there is a clear need to understand the critical factors that drive such pathologic immune responses. Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a nuclear receptor that has emerged as an important regulator of multiple cell types involved in the inflammatory response to allergens; from airway epithelial cells to T Helper (TH) cells. RECENT FINDINGS: Initial studies suggested that agonists of PPAR-γ could be employed to temper allergic inflammation, suppressing pro-inflammatory gene expression programs in epithelial cells. Several lines of work now suggest that PPAR-γ plays an essential in promoting 'type 2' immune responses that are typically associated with allergic disease. PPAR-γ has been found to promote the functions of TH2 cells, type 2 innate lymphoid cells, M2 macrophages and dendritic cells, regulating lipid metabolism and directly inducing effector gene expression. Moreover, preclinical models of allergy in gene-targeted mice have increasingly implicated PPAR-γ in driving allergic inflammation. Herein, we highlight the contrasting roles of PPAR-γ in allergic inflammation and hypothesize that the availability of environmental ligands for PPAR-γ may be at the heart of the rise in allergic diseases worldwide.

Atrophy of skin-draining lymph nodes predisposes for impaired immune responses to secondary infection in mice with chronic intestinal nematode infection.

Intestinal nematodes suppress immune responses in the context of allergy, gut inflammation, secondary infection and vaccination. Several mechanisms have been proposed for this suppression including alterations in Th2 cell differentiation and increased Treg cell suppressive function. In this study, we show that chronic nematode infection leads to reduced peripheral responses to vaccination because of a generalized reduction in the available responsive lymphocyte pool. We found that superficial skin-draining lymph nodes (LNs) in mice that are chronically infected with the intestinal nematode Heligmosomides polygyrus, do not reach the same cellularity as worm-free mice upon subsequent BCG infection in the skin. B cells and T cells, all declined in skin-draining LN of H. polygyrus-infected mice, resulting in LNs atrophy and altered lymphocyte composition. Importantly, anti-helminthic treatment improved lymphocyte numbers in skin-draining LN, indicating that time after de-worming is critical to regain full-scale LN cellularity. De-worming, and time for the skin LN to recover cellularity, also mended responses to Bacille Calmette-Guerin (BCG) in the LN draining the footpad injection site. Thus, our findings show that chronic nematode infection leads to a paucity of lymphocytes in peripheral lymph nodes, which acts to reduce the efficacy of immune responses at these sites.

Multi-faceted inhibition of dendritic cell function by CD4+Foxp3+ regulatory T cells.

CTLA-4 is required for CD4+Foxp3+ regulatory T (Treg) cell function, but its mode of action remains incompletely defined. Herein we generated Ctla-4ex2fl/flFoxp3-Cre mice with Treg cells exclusively expressing a naturally occurring, ligand-independent isoform of CTLA-4 (liCTLA-4) that cannot interact with the costimulatory molecules CD80 and CD86. The mice did not exhibit any signs of effector T cell activation early in life, however, at 6 months of age they exhibited excessive T cell activation and inflammation in lungs. In contrast, mice with Treg cells completely lacking CTLA-4 developed lymphoproliferative disease characterized by multi-organ inflammation early in life. In vitro, Treg cells exclusively expressing liCTLA-4 inhibited CD80 and CD86 expression on dendritic cells (DC). Conversely, Treg cells required the extra-cellular part of CTLA-4 to up-regulate expression of the co-inhibitory molecule PD-L2 on DCs. Transcriptomic analysis of suppressed DCs revealed that Treg cells induced a specific immunosuppressive program in DCs.

Acute Loss of Apolipoprotein E Triggers an Autoimmune Response That Accelerates Atherosclerosis.

Objective- Dyslipidemia is a component of the metabolic syndrome, an established risk factor for atherosclerotic cardiovascular disease, and is also observed in various autoimmune and chronic inflammatory conditions. However, there are limited opportunities to study the impact of acquired dyslipidemia on cardiovascular and immune pathology. Approach and Results- We designed a model system that allows for the conversion to a state of acute hyperlipidemia in adult life, so that the consequences of such a transition could be observed, through conditionally deleting APOE (apolipoprotein E) in the adult mouse. The transition to hypercholesterolemia was accompanied by adaptive immune responses, including the expansion of T lymphocyte helper cell 1, T follicular helper cell, and T regulatory subsets and the formation of germinal centers. Unlike steady-state Apoe-/- mice, abrupt loss of APOE induced rapid production of antibodies recognizing rheumatoid disease autoantigens. Genetic ablation of the germinal center reduced both autoimmunity and atherosclerosis, indicating that the immune response that follows loss of APOE is independent of atherosclerosis but nevertheless promotes plaque development. Conclusions- Our findings suggest that immune activation in response to hyperlipidemia could contribute to a wide range of inflammatory autoimmune diseases, including atherosclerosis.

The importance of co-stimulation in the orchestration of T helper cell differentiation.

Upon their activation, CD4 T cells can differentiate into distinct T helper cell subsets with specialised functions. Different T helper cell subsets produce specific cytokines that mediate beneficial and sometimes detrimental effects, depending on the infection or disease setting. CD4 T-cell priming relies on signals delivered by the T-cell antigen receptor, co-stimulatory receptors and cytokine receptors on the CD4 T-cell surface. Cytokine receptors are well known to deliver instructive signals that direct T helper cell differentiation. However, it is less appreciated that co-stimulatory receptors also exert potent modulatory effects on this process. In this review, we outline the contribution of co-stimulatory and co-inhibitory receptors to the process of T helper cell differentiation, focusing on those pathways for which the underlying mechanisms are best known. Herein, we depict the physiological context of T-cell priming and emphasise the impact of cell-cell communication on directing T helper cell differentiation.

Maintenance of caecal homeostasis by diverse adaptive immune cells in the rhesus macaque.

Objectives: The caecum bridges the small and large intestine and plays a front-line role in discriminating gastrointestinal antigens. Although dysregulated in acute and chronic conditions, the tissue is often overlooked immunologically. Methods: To address this issue, we applied single-cell transcriptomic-V(D)J sequencing to FACS-isolated CD45+ caecal patch/lamina propria leukocytes from a healthy (5-year-old) female rhesus macaque ex vivo and coupled these data to VDJ deep sequencing reads from haematopoietic tissues. Results: We found caecal NK cells and ILC3s to co-exist with a spectrum of effector T cells partially derived from SOX4 + recent thymic emigrants. Tolerogenic Vγ8Vδ1-T cells, plastic CD4+ T helper cells and GZMK + EOMES + and TMIGD2 + tissue-resident memory CD8+ T cells were present and differed metabolically. An IL13 + GATA3 + Th2 subset expressing eicosanoid pathway enzymes was accompanied by IL1RL1 + GATA3 + regulatory T cells and a minor proportion of IgE+ plasma cells (PCs), illustrating tightly regulated type 2 immunity devoid of ILC2s. In terms of B lymphocyte lineages, caecal patch antigen-presenting memory B cells sat alongside germinal centre cells undergoing somatic hypermutation and differentiation into IGF1 + PCs. Prototypic gene expression signatures decreased across PC clusters, and notably, expanded IgA clonotypes could be traced in VDJ deep sequencing reads from additional compartments, including the bone marrow, supporting that these cells contribute a steady stream of systemic antibodies. Conclusions: The data advance our understanding of caecal immunological function, revealing processes involved in barrier maintenance and molecular networks relevant to disease.

Human CD127 negative ILC2s show immunological memory.

ILC2s are key players in type 2 immunity and contribute to maintaining homeostasis. ILC2s are also implicated in the development of type 2 inflammation-mediated chronic disorders like asthma. While memory ILC2s have been identified in mouse, it is unknown whether human ILC2s can acquire immunological memory. Here, we demonstrate the persistence of CD45RO, a marker previously linked to inflammatory ILC2s, in resting ILC2s that have undergone prior activation. A high proportion of these cells concurrently reduce the expression of the canonical ILC marker CD127 in a tissue-specific manner. Upon isolation and in vitro stimulation of CD127-CD45RO+ ILC2s, we observed an augmented ability to proliferate and produce cytokines. CD127-CD45RO+ ILC2s are found in both healthy and inflamed tissues and display a gene signature of cell activation. Similarly, mouse memory ILC2s show reduced expression of CD127. Our findings suggest that human ILC2s can acquire innate immune memory and warrant a revision of the current strategies to identify human ILC2s.

Laboratory mice with a wild microbiota generate strong allergic immune responses.

Allergic disorders are caused by a combination of hereditary and environmental factors. The hygiene hypothesis postulates that early-life microbial exposures impede the development of subsequent allergic disease. Recently developed "wildling" mice are genetically identical to standard laboratory specific pathogen-free (SPF) mice but are housed under seminatural conditions and have rich microbial exposures from birth. Thus, by comparing conventional SPF mice with wildlings, we can uncouple the impact of lifelong microbial exposures from genetic factors on the allergic immune response. We found that wildlings developed larger populations of antigen-experienced T cells than conventional SPF mice, which included interleukin-10-producing CD4 T cells specific for commensal Lactobacilli strains and allergy-promoting T helper 2 (TH2) cells. In models of airway exposure to house dust mite (HDM), recombinant interleukin-33, or Alternaria alternata, wildlings developed strong allergic inflammation, characterized by eosinophil recruitment, goblet cell metaplasia, and antigen-specific immunoglobulin G1 (IgG1) and IgE responses. Wildlings developed robust de novo TH2 cell responses to incoming allergens, whereas preexisting TH2 cells could also be recruited into the allergic immune response in a cytokine-driven and TCR-independent fashion. Thus, wildling mice, which experience diverse and lifelong microbial exposures, were not protected from developing pathological allergic immune responses. Instead, wildlings mounted robust allergic responses to incoming allergens, shedding new light on the hygiene hypothesis.

Human resident liver myeloid cells protect against metabolic stress in obesity.

Although multiple populations of macrophages have been described in the human liver, their function and turnover in patients with obesity at high risk of developing non-alcoholic fatty liver disease (NAFLD) and cirrhosis are currently unknown. Herein, we identify a specific human population of resident liver myeloid cells that protects against the metabolic impairment associated with obesity. By studying the turnover of liver myeloid cells in individuals undergoing liver transplantation, we find that liver myeloid cell turnover differs between humans and mice. Using single-cell techniques and flow cytometry, we determine that the proportion of the protective resident liver myeloid cells, denoted liver myeloid cells 2 (LM2), decreases during obesity. Functional validation approaches using human 2D and 3D cultures reveal that the presence of LM2 ameliorates the oxidative stress associated with obese conditions. Our study indicates that resident myeloid cells could be a therapeutic target to decrease the oxidative stress associated with NAFLD.

Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralizing SARS-CoV-2 nanobodies.

Conventional approaches to isolate and characterize nanobodies are laborious. We combine phage display, multivariate enrichment, next-generation sequencing, and a streamlined screening strategy to identify numerous anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nanobodies. We characterize their potency and specificity using neutralization assays and hydrogen/deuterium exchange mass spectrometry (HDX-MS). The most potent nanobodies bind to the receptor binding motif of the receptor binding domain (RBD), and we identify two exceptionally potent members of this category (with monomeric half-maximal inhibitory concentrations around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the Beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 µg/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.

Probabilistic classification of anti-SARS-CoV-2 antibody responses improves seroprevalence estimates.

Objectives: Population-level measures of seropositivity are critical for understanding the epidemiology of an emerging pathogen, yet most antibody tests apply a strict cutoff for seropositivity that is not learnt in a data-driven manner, leading to uncertainty when classifying low-titer responses. To improve upon this, we evaluated cutoff-independent methods for their ability to assign likelihood of SARS-CoV-2 seropositivity to individual samples. Methods: Using robust ELISAs based on SARS-CoV-2 spike (S) and the receptor-binding domain (RBD), we profiled antibody responses in a group of SARS-CoV-2 PCR+ individuals (n = 138). Using these data, we trained probabilistic learners to assign likelihood of seropositivity to test samples of unknown serostatus (n = 5100), identifying a support vector machines-linear discriminant analysis learner (SVM-LDA) suited for this purpose. Results: In the training data from confirmed ancestral SARS-CoV-2 infections, 99% of participants had detectable anti-S and -RBD IgG in the circulation, with titers differing > 1000-fold between persons. In data of otherwise healthy individuals, 7.2% (n = 367) of samples were of uncertain serostatus, with values in the range of 3-6SD from the mean of pre-pandemic negative controls (n = 595). In contrast, SVM-LDA classified 6.4% (n = 328) of test samples as having a high likelihood (> 99% chance) of past infection, 4.5% (n = 230) to have a 50-99% likelihood, and 4.0% (n = 203) to have a 10-49% likelihood. As different probabilistic approaches were more consistent with each other than conventional SD-based methods, such tools allow for more statistically-sound seropositivity estimates in large cohorts. Conclusion: Probabilistic antibody testing frameworks can improve seropositivity estimates in populations with large titer variability.