Vaccine-elicited B- and T-cell immunity to SARS-CoV-2 is impaired in chronic lung disease patients.

Background: While vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) provides significant protection from coronavirus disease 2019, the protection afforded to individuals with chronic lung disease is less well established. This study seeks to understand how chronic lung disease impacts SARS-CoV-2 vaccine-elicited immunity. Methods: Deep immune phenotyping of humoral and cell-mediated responses to the SARS-CoV-2 vaccine was performed in patients with asthma, COPD and interstitial lung disease (ILD) compared to healthy controls. Results: 48% of vaccinated patients with chronic lung diseases had reduced antibody titres to the SARS-CoV-2 vaccine antigen relative to healthy controls. Vaccine antibody titres were significantly reduced among asthma (p<0.035), COPD (p<0.022) and a subset of ILD patients as early as 3-4 months after vaccination, correlating with decreased vaccine-specific memory B-cells in circulation. Vaccine-specific memory T-cells were significantly reduced in patients with asthma (CD8+ p<0.004; CD4+ p<0.023) and COPD (CD8+ p<0.008) compared to healthy controls. Impaired T-cell responsiveness was also observed in a subset of ILD patients (CD8+ 21.4%; CD4+ 42.9%). Additional heterogeneity between healthy and disease cohorts was observed among bulk and vaccine-specific follicular T-helper cells. Conclusions: Deep immune phenotyping of the SARS-CoV-2 vaccine response revealed the complex nature of vaccine-elicited immunity and highlights the need for more personalised vaccination schemes in patients with underlying lung conditions.

Vaccine-elicited B and T cell immunity to SARS-CoV-2 is impaired in chronic lung disease patients.

The protection afforded by vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to individuals with chronic lung disease is not well established. To understand how chronic lung disease impacts SARS-CoV-2 vaccine-elicited immunity we performed deep immunophenotyping of the humoral and cell mediated SARS-CoV-2 vaccine response in an investigative cohort of vaccinated patients with diverse pulmonary conditions including asthma, chronic obstructive pulmonary disease (COPD), and interstitial lung disease (ILD). Compared to healthy controls, 48% of vaccinated patients with chronic lung diseases had reduced antibody titers to the SARS-CoV-2 vaccine antigen as early as 3-4 months after vaccination, correlating with decreased vaccine-specific memory B cells. Vaccine-specific CD4 and CD8 T cells were also significantly reduced in patients with asthma, COPD, and a subset of ILD patients compared to healthy controls. These findings reveal the complex nature of vaccine-elicited immunity in high-risk patients with chronic lung disease.

Single cell analysis of host response to helminth infection reveals the clonal breadth, heterogeneity, and tissue-specific programming of the responding CD4+ T cell repertoire.

The CD4+ T cell response is critical to host protection against helminth infection. How this response varies across different hosts and tissues remains an important gap in our understanding. Using IL-4-reporter mice to identify responding CD4+ T cells to Nippostrongylus brasiliensis infection, T cell receptor sequencing paired with novel clustering algorithms revealed a broadly reactive and clonally diverse CD4+ T cell response. While the most prevalent clones and clonotypes exhibited some tissue selectivity, most were observed to reside in both the lung and lung-draining lymph nodes. Antigen-reactivity of the broader repertoires was predicted to be shared across both tissues and individual mice. Transcriptome, trajectory, and chromatin accessibility analysis of lung and lymph-node repertoires revealed three unique but related populations of responding IL-4+ CD4+ T cells consistent with T follicular helper, T helper 2, and a transitional population sharing similarity with both populations. The shared antigen reactivity of lymph node and lung repertoires combined with the adoption of tissue-specific gene programs allows for the pairing of cellular and humoral responses critical to the orchestration of anti-helminth immunity.

Local induction of bladder Th1 responses to combat urinary tract infections.

Given the high frequency of urinary tract infections (UTIs) and their recurrence, there is keen interest in developing effective UTI vaccines. Currently, most vaccine studies, including those in humans, involve parenteral vaccination aimed at evoking and sustaining elevated levels of systemic antibody directed at the uropathogens. In view of recent reports of aberrant Th2-biased bladder immune responses to infection, we hypothesized that immunizing mice intravesically with antigens from uropathogenic Escherichia coli (UPEC) combined with a Th1-skewing adjuvant could correct this defect and promote protection against UTIs. Here we report that compared with mice immunized subcutaneously with this vaccine combination, intravesically immunized mice were markedly more protected from UTIs because of their distinctive ability to recruit Th1 cells into the bladder. This mode of vaccination was effective even in mice that experienced multiple UTIs and displayed pronounced aberrant bladder immune responses. Thus, intravesical vaccination with one or more UPEC antigens to induce bladder Th1 responses represents a superior strategy to combat UTIs, especially in UTI-prone subjects.

The Heterogeneity, Origins, and Impact of Migratory iILC2 Cells in Anti-helminth Immunity.

Soil-transmitted helminths represent a major global health burden with infections and infection-related comorbidities causing significant reductions in the quality of life for individuals living in endemic areas. Repeated infections and chronic colonization by these large extracellular worms in mammals led to the evolution of type-2 immunity characterized by the production of the type-2 cytokines interleukin (IL)-4, IL-5, and IL-13. Although a number of adaptive and innate immune cells produce type-2 cytokines, a key cellular source in the context of helminth infection is group 2 innate lymphoid cells (ILC2s). ILC2s promote mucosal barrier homeostasis, integrity, and repair by rapidly responding to epithelial cues in mucosal tissues. Though tissue-resident ILC2s (nILC2s) have been studied in detail over the last decade, considerably less is known with regard to a subset of inflammatory ILC2s (iILC2s) that migrate to the lungs of mice early after Nippostrongylus brasiliensis infection and are potent early producers of type-2 cytokines. This review will discuss the relationship and differences between nILC2s and iILC2s that establish their unique roles in anti-helminth immunity. We have placed particular emphasis on studies investigating iILC2 origin, function, and their potential long-term contribution to tissue-resident ILC2 reservoirs in settings of helminth infection.

BATF acts as an essential regulator of IL-25-responsive migratory ILC2 cell fate and function.

A transitory, interleukin-25 (IL-25)-responsive, group 2 innate lymphoid cell (ILC2) subset induced during type 2 inflammation was recently identified as iILC2s. This study focuses on understanding the significance of this population in relation to tissue-resident nILC2s in the lung and intestine. RNA-sequencing and pathway analysis revealed the AP-1 superfamily transcription factor BATF (basic leucine zipper transcription factor, activating transcription factor-like) as a potential modulator of ILC2 cell fate. Infection of BATF-deficient mice with Nippostrongylus brasiliensis showed a selective defect in IL-25-mediated helminth clearance and a corresponding loss of iILC2s in the lung characterized as IL-17RBhigh, KLRG1high, BATFhigh, and Arg1low BATF deficiency selectively impaired iILC2s because it had no impact on tissue-resident nILC2 frequency or function. Pulmonary-associated iILC2s migrated to the lung after infection, where they represented an early source of IL-4 and IL-13. Although the composition of ILC2s in the small intestine was distinct from those in the lung, their frequency and IL-13 expression remained dependent on BATF, which was also required for optimal goblet and tuft cell hyperplasia. Findings support IL-25-responsive ILC2s as early sentinels of mucosal barrier integrity.

Sox4 Promotes Atoh1-Independent Intestinal Secretory Differentiation Toward Tuft and Enteroendocrine Fates.

BACKGROUND & AIMS: The intestinal epithelium is maintained by intestinal stem cells (ISCs), which produce postmitotic absorptive and secretory epithelial cells. Initial fate specification toward enteroendocrine, goblet, and Paneth cell lineages requires the transcription factor Atoh1, which regulates differentiation of the secretory cell lineage. However, less is known about the origin of tuft cells, which participate in type II immune responses to parasite infections and appear to differentiate independently of Atoh1. We investigated the role of Sox4 in ISC differentiation. METHODS: We performed experiments in mice with intestinal epithelial-specific disruption of Sox4 (Sox4fl/fl:vilCre; SOX4 conditional knockout [cKO]) and mice without disruption of Sox4 (control mice). Crypt- and single-cell-derived organoids were used in assays to measure proliferation and ISC potency. Lineage allocation and gene expression changes were studied by immunofluorescence, real-time quantitative polymerase chain reaction, and RNA-seq analyses. Intestinal organoids were incubated with the type 2 cytokine interleukin 13 and gene expression was analyzed. Mice were infected with the helminth Nippostrongylus brasiliensis and intestinal tissues were collected 7 days later for analysis. Intestinal tissues collected from mice that express green fluorescent protein regulated by the Atoh1 promoter (Atoh1GFP mice) and single-cell RNA-seq analysis were used to identify cells that coexpress Sox4 and Atoh1. We generated SOX4-inducible intestinal organoids derived from Atoh1fl/fl:vilCreER (ATOH1 inducible knockout) mice and assessed differentiation. RESULTS: Sox4cKO mice had impaired ISC function and secretory differentiation, resulting in decreased numbers of tuft and enteroendocrine cells. In control mice, numbers of SOX4+ cells increased significantly after helminth infection, coincident with tuft cell hyperplasia. Sox4 was activated by interleukin 13 in control organoids; SOX4cKO mice had impaired tuft cell hyperplasia and parasite clearance after infection with helminths. In single-cell RNA-seq analysis, Sox4+/Atoh1- cells were enriched for ISC, progenitor, and tuft cell genes; 12.5% of Sox4-expressing cells coexpressed Atoh1 and were enriched for enteroendocrine genes. In organoids, overexpression of Sox4 was sufficient to induce differentiation of tuft and enteroendocrine cells-even in the absence of Atoh1. CONCLUSIONS: We found Sox4 promoted tuft and enteroendocrine cell lineage allocation independently of Atoh1. These results challenge the longstanding model in which Atoh1 is the sole regulator of secretory differentiation in the intestine and are relevant for understanding epithelial responses to parasitic infection.

Using Cytokine Reporter Mice to Visualize Type-2 Immunity In Vivo.

Type-2 cytokine production plays a critical role in the context of type 2 immunity and allergic inflammation. Interleukin-4 (IL-4) and IL-13 are key modulators of the cell-mediated and humoral immune hallmarks most commonly associated with type-2 immune responses. However, production of these cytokines by lymphocytes and their tissue localization has been difficult to detect in vivo. As such, the field has relied heavily on ex vivo restimulation and in vitro differentiation assays to understand type-2 cytokine biology. Although these studies have greatly informed our understanding of type-2 cytokine regulation, it is becoming increasingly clear that the data does not always provide a true accounting of the complexity of type-2 immune cell biology in vivo. Described below is a protocol used to detect IL-4-competent and protein-producing cells in the lung and lymph nodes of mice after infection with a helminth. Importantly, this protocol has also been used to successfully identify reporter expression and cell function in vivo using various other cytokine-reporter systems.

Live Imaging of IL-4-Expressing T Follicular Helper Cells in Explanted Lymph Nodes.

The generation of class-switched, high-affinity, antibody-producing B cells plays a critical role in the establishment of type 2 immunity to intestinal helminths as well as in the pathogenesis of allergy and asthma. The generation of these high-affinity, antibody-producing B cells occurs in germinal centers (GC) and relies on interactions with follicular dendritic cells (FDCs) and T follicular helper (Tfh) cells. One critical mediator produced by Tfh cells in GCs is interleukin-4 (IL-4). Tfh-derived IL-4 drives class switching to type 2 antibody isotypes IgE and IgG1 and is required for high-affinity IgG1 production. In vivo detection of IL-4-expressing Tfh cells is required to better understand the role of these cells during the GC response. Detection of IL-4-expressing cells has been greatly improved by the generation of the IL-44get reporter mice, which read out IL-4 expression as green fluorescent protein (GFP). Much has been learned from these mice with regard to type 2 immunity using flow cytometry and immunohistochemistry. However, these methods do not allow the study of cellular behavior and interactions in real time. In contrast, multi-photon microscopy allows for deep tissue imaging and tracking of multiple cell types in intact tissues over time. Here, we describe a protocol for in vivo detection of IL-4-expressing Tfh cells in an explanted popliteal lymph node by multi-photon microscopy. The dynamics of Tfh cell motility and their interactions with FDC networks in the GCs were analyzed.

Library Preparation for ATAC-Sequencing of Mouse CD4+ T Cells Isolated from the Lung and Lymph Nodes After Helminth Infection.

Although conventional methods such as MNase-seq, DNase-seq, and ChIP-seq have been used effectively to assess chromatin and locus accessibility at the genome level, these techniques generally require large numbers of input cells. As such, much of what we understand in terms of epigenetic regulation and locus accessibility in CD4+ T cell subsets comes from in vitro culture systems, which allow for the production of large numbers of polarized T cells. However, obtaining such numbers directly ex vivo from tissues of individual mice is difficult. Here we describe a method combining cytokine reporter mice and Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) to identify genome wide locus accessibility in a small number of cytokine-expressing CD4+ T cells. This method takes you from cell isolation to library generation and quality control to query. Because the Il4 and Ifng loci are reciprocally regulated in polarized CD4+ T cell subsets (Th1 vs. Th2), we investigated the ability of this approach to identify transposase integration in both IL-4- and IFN-γ-expressing CD4+ T cells isolated directly from the lung and lymph nodes after helminth infection.

Notch signaling represents an important checkpoint between follicular T-helper and canonical T-helper 2 cell fate.

Type-2 immunity is regulated by two distinct CD4+ T-cell subsets. T follicular helper (Tfh) cells are required for humoral hallmarks of type-2 inflammation. T-helper type-2 (Th2) cells orchestrate type-2 inflammation in peripheral tissues, such as the lung and intestine. Given the importance of Notch signaling in the establishment of other CD4+ T-helper cell subsets, we investigated whether canonical Notch activation could differentially impact Tfh and Th2 cell fate during the induction of type-2 immunity. These studies show that Tfh cell, but not Th2 cell, generation and function is reliant on Notch signaling. While early Tfh cell specification is influenced by functional Notch ligands on classical dendritic cells, functional Notch ligands on cells other than dendritic cells, T cells, B cells, and follicular dendritic cells are sufficient to achieve full Tfh cell commitment. These findings identify Notch signaling as an early lineage-determining factor between Tfh and Th2 cell fate.

γδ T Cells and B Cells.

γδ T cells constitute the third arm of a tripartite adaptive immune system in jawed vertebrates, besides αß T cells and B cells. Like the other two lymphocyte-types, they express diverse antigen receptors, capable of specific ligand recognition. Functionally, γδ T cells represent a system of differentiated subsets, sometimes engaged in cross-regulation, which ultimately determines their effect on other components of the immune system, including B cells and antibodies. γδ T cells are capable of providing help to B cells in antibody production. More recently it became clear that γδ T cells influence B cell differentiation during the peripheral stages of B cell development, control levels of circulating immunoglobulin (all subclasses), and affect production of autoantibodies. Because of this relationship between γδ T cells and B cells, the extensive variation of γδ T cells among human individuals might be expected to modulate their humoral responsiveness.

BATF Modulates the Th2 Locus Control Region and Regulates CD4+ T Cell Fate during Antihelminth Immunity.

The AP-1 factor basic leucine zipper transcription factor, ATF-like (BATF) is important for CD4+ Th17, Th9, and follicular Th cell development. However, its precise role in Th2 differentiation and function remains unclear, and the requirement for BATF in nonallergic settings of type-2 immunity has not been explored. In this article, we show that, in response to parasitic helminths, Batf-/- mice are unable to generate follicular Th and Th2 cells. As a consequence, they fail to establish productive type-2 immunity during primary and secondary infection. Batf-/- CD4+ T cells do not achieve type-2 cytokine competency, which implies that BATF plays a key role in the regulation of IL-4 and IL-13. In contrast to Th17 and Th9 cell subsets in which BATF binds directly to promoter and enhancer regions to regulate cytokine expression, our results show that BATF is significantly enriched at Rad50 hypersensitivity site (RHS)6 and RHS7 of the locus control region relative to AP-1 sites surrounding type-2 cytokine loci in Th2 cells. Indeed, Batf-/- CD4+ T cells do not obtain permissive epigenetic modifications within the Th2 locus, which were linked to RHS6 and RHS7 function. In sum, these findings reveal BATF as a central modulator of peripheral and humoral hallmarks of type-2 immunity and begin to elucidate a novel mechanism by which it regulates type-2 cytokine production through its modification of the Th2 locus control region.

The differential expression of IL-4 and IL-13 and its impact on type-2 immunity.

Allergic disease represents a significant global health burden, and disease incidence continues to rise in urban areas of the world. As such, a better understanding of the basic immune mechanisms underlying disease pathology are key to developing therapeutic interventions to both prevent disease onset as well as to ameliorate disease morbidity in those individuals already suffering from a disorder linked to type-2 inflammation. Two factors central to type-2 immunity are interleukin (IL)-4 and IL-13, which have been linked to virtually all major hallmarks associated with type-2 inflammation. Therefore, IL-4 and IL-13 and their regulatory pathways represent ideal targets to suppress disease. Despite sharing many common regulatory pathways and receptors, these cytokines perform very distinct functions during a type-2 immune response. This review summarizes the literature surrounding the function and expression of IL-4 and IL-13 in CD4+ T cells and innate immune cells. It highlights recent findings in vivo regarding the differential expression and non-canonical regulation of IL-4 and IL-13 in various immune cells, which likely play important and underappreciated roles in type-2 immunity.

A novel model for IFN-γ-mediated autoinflammatory syndromes.

Autoinflammatory disease and hyperinflammatory syndromes represent a growing number of diseases associated with inappropriately controlled inflammation in multiple organs. Systemic inflammation commonly results from dysregulated activation of innate immune cells, and therapeutic targeting of the IL-1ß pathway has been used to ameliorate some of these diseases. Some hyperinflammatory syndromes, however, such as hemophagocytic lymphohistiocytosis and the newly classified proteasome disability syndromes, are refractory to such treatments, suggesting that other factors or environmental stressors may be contributing. In comparing two cytokine reporter mouse strains, we identify IFN-γ as a mediator of systemic autoinflammatory disease. Chronically elevated levels of IFN-γ resulted in progressive multiorgan inflammation and two copies of the mutant allele resulted in increased mortality accompanied by myeloproliferative disease. Disease was alleviated by genetic deletion of T-bet. These studies raise the possibility that therapeutics targeting the IFN-γ pathway might be effective in hyperinflammatory conditions refractory to IL-1ß-targeted therapies.

Age-dependent hepatic lymphoid organization directs successful immunity to hepatitis B.

Hepatitis B virus (HBV) is a major human pathogen that causes immune-mediated hepatitis. Successful immunity to HBV is age dependent: viral clearance occurs in most adults, whereas neonates and young children usually develop chronic infection. Using a mouse model of HBV infection, we sought mechanisms underpinning the age-dependent outcome of HBV and demonstrated that hepatic macrophages facilitate lymphoid organization and immune priming within the adult liver and promote successful immunity. In contrast, lymphoid organization and immune priming was greatly diminished in the livers of young mice, and of macrophage-depleted adult mice, leading to abrogated HBV immunity. Furthermore, we found that CXCL13, which is involved in B lymphocyte trafficking and lymphoid architecture and development, is expressed in an age-dependent manner in both adult mouse and human hepatic macrophages and plays an integral role in facilitating an effective immune response against HBV. Taken together, these results identify some of the immunological mechanisms necessary for effective control of HBV.

Marking and quantifying IL-17A-producing cells in vivo.

Interleukin (IL)-17A plays an important role in host defense against a variety of pathogens and may also contribute to the pathogenesis of autoimmune diseases. However, precise identification and quantification of the cells that produce this cytokine in vivo have not been performed. We generated novel IL-17A reporter mice to investigate expression of IL-17A during Klebsiella pneumoniae infection and during experimental autoimmune encephalomyelitis, conditions previously demonstrated to potently induce IL-17A production. In both settings, the majority of IL-17A was produced by non-CD4(+) T cells, particularly γδ T cells, but also invariant NKT cells and other CD4(-)CD3ε(+) cells. As measured in dual-reporter mice, IFN-γ-producing Th1 cells greatly outnumbered IL-17A-producing Th17 cells throughout both challenges. Production of IL-17A by cells from unchallenged mice or by non-T cells under any condition was not evident. Administration of IL-1ß and/or IL-23 elicited rapid production of IL-17A by γδ T cells, invariant NKT cells and other CD4(-)CD3ε(+) cells in vivo, demonstrating that these cells are poised for rapid cytokine production and likely comprise the major sources of this cytokine during acute immunologic challenges.

Divergent expression patterns of IL-4 and IL-13 define unique functions in allergic immunity.

Interleukin 4 (IL-4) and IL-13 are critical for responses to parasitic helminthes. We used genetically engineered reporter mice to assess the temporal and spatial production of these cytokines in vivo. In lymph nodes, IL-4, but not IL-13, was made by follicular helper T cells (T(FH) cells). In contrast, tissue type 2 helper T cells (T(H)2 cells) produced both cytokines. There was also divergent production of IL-4 and IL-13 among cells of the innate immune system, whereby basophils produced IL-4, whereas innate helper type 2 cells (Ih2 cells) produced IL-13. IL-13 production by T(H)2 and Ih2 cells was dependent on the transcription factor GATA-3, which was present in large amounts in these cells, and in contrast to the small amount of GATA-3 in T(FH) cells and basophils. The distinct localization and cellular expression of IL-4 and IL-13 explains their unique roles during allergic immunity.

Systemically dispersed innate IL-13-expressing cells in type 2 immunity.

Type 2 immunity is a stereotyped host response to allergens and parasitic helminths that is sustained in large part by the cytokines IL-4 and IL-13. Recent advances have called attention to the contributions by innate cells in initiating adaptive immunity, including a novel lineage-negative population of cells that secretes IL-13 and IL-5 in response to the epithelial cytokines IL-25 and IL-33. Here, we use IL-4 and IL-13 reporter mice to track lineage-negative innate cells that arise during type 2 immunity or in response to IL-25 and IL-33 in vivo. Unexpectedly, lineage-negative IL-25 (and IL-33) responsive cells are widely distributed in tissues of the mouse and are particularly prevalent in mesenteric lymph nodes, spleen, and liver. These cells expand robustly in response to exogenous IL-25 or IL-33 and after infection with the helminth Nippostrongylus brasiliensis, and they are the major innate IL-13-expressing cells under these conditions. Activation of these cells using IL-25 is sufficient for worm clearance, even in the absence of adaptive immunity. Widely dispersed innate type 2 helper cells, which we designate Ih2 cells, play an integral role in type 2 immune responses.