Diversity of group 1 innate lymphoid cells in human tissues.

Group 1 innate lymphoid cells (ILC1s) are cytotoxic and interferon gamma-producing lymphocytes lacking antigen-specific receptors, which include ILC1s and natural killer (NK) cells. In mice, ILC1s differ from NK cells, as they develop independently of the NK-specifying transcription factor EOMES, while requiring the repressor ZFP683 (ZNF683 in humans) for tissue residency. Here we identify highly variable ILC1 subtypes across tissues through investigation of human ILC1 diversity by single-cell RNA sequencing and flow cytometry. The intestinal epithelium contained abundant mature EOMES- ILC1s expressing PRDM1 rather than ZNF683, alongside a few immature TCF7+PRDM1- ILC1s. Other tissues harbored NK cells expressing ZNF683 and EOMES transcripts; however, EOMES protein content was variable. These ZNF683+ NK cells are tissue-imprinted NK cells phenotypically resembling ILC1s. The tissue ILC1-NK spectrum also encompassed conventional NK cells and NK cells distinguished by PTGDS expression. These findings establish a foundation for evaluating phenotypic and functional changes within the NK-ILC1 spectrum in diseases.

TSLP: from allergy to cancer.

The cytokine TSLP has been shown to be a key factor in maintaining immune homeostasis and regulating inflammatory responses at mucosal barriers. While the role of TSLP in type 2 immune responses has been investigated extensively, recent studies have found an expanding role for TSLP in inflammatory diseases and cancer. In this Review, we will highlight major recent advances in TSLP biology, along with results from emerging clinical trials of anti-TSLP agents used for the treatment of a variety of inflammatory conditions.

A tumor-myeloid cell axis, mediated via the cytokines IL-1α and TSLP, promotes the progression of breast cancer.

Tumors actively manipulate the immune response through the production of factors that attract immune cells and subsequently alter their ability to recognize and effectively remove the tumor. While this mechanism for evading the immune system is an important aspect of tumor survival, the factors that serve as primary growth factors for the tumor are less understood. Here we demonstrate a previously unknown mechanism by which breast-cancer cells manipulate tumor-infiltrating myeloid cells to maintain their survival. Tumor-derived interleukin 1α (IL-1α), acting on infiltrating myeloid cells, induced the expression of a critical tumor survival factor, the cytokine TSLP. TSLP promoted the survival of the tumor cells through induction of the expression of the anti-apoptotic molecule Bcl-2. TSLP signaling was also required for metastasis to the lungs. These studies define a novel IL-1α-TSLP-mediated crosstalk between tumor-infiltrating myeloid cells and tumor cells in the control of metastatic breast cancer.

Publisher Correction: A tumor-myeloid cell axis, mediated via the cytokines IL-1α and TSLP, promotes the progression of breast cancer.

In the version of this article initially published, the far right label along the horizontal axis of the right plot of Fig. 1e (4T1-Tslp-/- 4-6), the middle and right labels along the horizontal axis of the far right plot of Fig. 3d (4T1-Tslpr-/- 4-6 and 4T1-Tslpr-/- 2-3, respectively), and the far right label along the horizontal axis of Fig. 6h (4T1-Tslpr-/- 2-3) were incorrect. The correct labels are as follows: Fig. 1e, 4T1-Tslpr-/- 4-6; Fig. 3d, 4T1-Tslp-/- 2-3 and 4T1-Tslpr-/- 4-6, respectively; and Fig. 6h, 4T1-Tslpr-/- 4-6. Also, Fig. 4e was incorrectly a duplicate of an adjacent panel. The errors have been corrected in the HTML and PDF version of the article.

Direct control of regulatory T cells by keratinocytes.

Environmental challenges to epithelial cells trigger gene expression changes that elicit context-appropriate immune responses. We found that the chromatin remodeler Mi-2ß controls epidermal homeostasis by regulating the genes involved in keratinocyte and immune-cell activation to maintain an inactive state. Mi-2ß depletion resulted in rapid deployment of both a pro-inflammatory and an immunosuppressive response in the skin. A key target of Mi-2ß in keratinocytes is the pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP). Loss of TSLP receptor (TSLPR) signaling specifically in regulatory T (Treg) cells prevented their activation and permitted rapid progression from a skin pro-inflammatory response to a lethal systemic condition. Thus, in addition to their well-characterized role in pro-inflammatory responses, keratinocytes also directly support immune-suppressive responses that are critical for re-establishing organismal homeostasis.

Conditioning of naive CD4(+) T cells for enhanced peripheral Foxp3 induction by nonspecific bystander inflammation.

Inflammation induced during infection can both promote and suppress immunity. This contradiction suggests that inflammatory cytokines affect the immune system in a context-dependent manner. Here we show that nonspecific bystander inflammation conditions naive CD4(+) T cells for enhanced peripheral Foxp3 induction and reduced effector differentiation. This results in inhibition of immune responses in vivo via a Foxp3-dependent effect on antigen-specific naive CD4(+) T cell precursors. Such conditioning may have evolved to allow immunity to infection while limiting subsequent autoimmunity caused by release of self-antigens in the wake of infection. Furthermore, this phenomenon suggests a mechanistic explanation for the idea that early tuning of the immune system by infection affects the long-term quality of immune regulation.

A regulatory role for TGF-ß signaling in the establishment and function of the thymic medulla.

Medullary thymic epithelial cells (mTECs) are critical in establishing and maintaining the appropriate microenvironment for negative selection and maturation of immunocompetent T cells with a self-tolerant T cell antigen receptor repertoire. Cues that direct proliferation and maturation of mTECs are provided by members of the tumor necrosis factor (TNF) superfamily expressed on developing thymocytes. Here we demonstrate a negative role of the morphogen TGF-ß in tempering these signals under physiological conditions, limiting both growth and function of the thymic medulla. Eliminating TGF-ß signaling specifically in TECs or by pharmacological means increased the size of the mTEC compartment, enhanced negative selection and functional maturation of medullary thymocytes as well as the production of regulatory T cells, thus reducing the autoreactive potential of peripheral T cells.

SKI Regulates Medullary Thymic Epithelial Cell Differentiation to Control Peripheral T Cell Responses in Mice.

The thymus is an important site for the establishment of an appropriate immune response through positive and negative selection of developing T cells. During selection, developing T cells interact with cortical and medullary thymic epithelial cells (TECs), termed cTECs and mTECs, respectively. Using a Foxn1Cre+/-SKIfl/fl mouse model, we found that TEC-specific deletion of SKI reduced the mTEC compartment in the thymus and that tissue-restricted Ag expression in mTECs was altered. This decrease in the medullary area led to a decrease in CD4 thymocyte cellularity; however, mature CD4 cellularity in the spleen remained normal. Interestingly, naive CD4 T cells purified from SKI-deleted mice showed a defect in proliferation in vitro after global TCR stimulation, and these mice were significantly protected from developing experimental autoimmune encephalomyelitis compared with the control mice. Overall, our findings suggest that SKI signaling in the thymus regulates mTEC differentiation and function as well as downstream peripheral T cell responses and provide evidence for targeting SKI in T cell-driven autoimmune diseases such as multiple sclerosis.

Oligodendrocyte-derived IL-33 functions as a microglial survival factor during neuroinvasive flavivirus infection.

In order to recover from infection, organisms must balance robust immune responses to pathogens with the tolerance of immune-mediated pathology. This balance is particularly critical within the central nervous system, whose complex architecture, essential function, and limited capacity for self-renewal render it susceptible to both pathogen- and immune-mediated pathology. Here, we identify the alarmin IL-33 and its receptor ST2 as critical for host survival to neuroinvasive flavivirus infection. We identify oligodendrocytes as the critical source of IL-33, and microglia as the key cellular responders. Notably, we find that the IL-33/ST2 axis does not impact viral control or adaptive immune responses; rather, it is required to promote the activation and survival of microglia. In the absence of intact IL-33/ST2 signaling in the brain, neuroinvasive flavivirus infection triggered aberrant recruitment of monocyte-derived peripheral immune cells, increased neuronal stress, and neuronal cell death, effects that compromised organismal survival. These findings identify IL-33 as a critical mediator of CNS tolerance to pathogen-initiated immunity and inflammation.

The transcription factor STAT5 is critical in dendritic cells for the development of TH2 but not TH1 responses.

Dendritic cells (DCs) are critical in immune responses, linking innate and adaptive immunity. We found here that DC-specific deletion of the transcription factor STAT5 was not critical for development but was required for T helper type 2 (TH2), but not TH1, allergic responses in both the skin and lungs. Loss of STAT5 in DCs led to the inability to respond to thymic stromal lymphopoietin (TSLP). STAT5 was required for TSLP-dependent DC activation, including upregulation of the expression of costimulatory molecules and chemokine production. Furthermore, TH2 responses in mice with DC-specific loss of STAT5 resembled those seen in mice deficient in the receptor for TSLP. Our results show that the TSLP-STAT5 axis in DCs is a critical component for the promotion of type 2 immunity at barrier surfaces.

Inhaled Fine Particles Induce Alveolar Macrophage Death and Interleukin-1α Release to Promote Inducible Bronchus-Associated Lymphoid Tissue Formation.

Particulate pollution is thought to function as an adjuvant that can induce allergic responses. However, the exact cell types and immunological factors that initiate the lung-specific immune responses are unclear. We found that upon intratracheal instillation, particulates such as aluminum salts and silica killed alveolar macrophages (AMs), which then released interleukin-1α (IL-1α) and caused inducible bronchus-associated lymphoid tissue (iBALT) formation in the lung. IL-1α release continued for up to 2 weeks after particulate exposure, and type-2 allergic immune responses were induced by the inhalation of antigen during IL-1α release and iBALT formation, even long after particulate instillation. Recombinant IL-1α was sufficient to induce iBALTs, which coincided with subsequent immunoglobulin E responses, and IL-1-receptor-deficient mice failed to induce iBALT formation. Therefore, the AM-IL-1α-iBALT axis might be a therapeutic target for particulate-induced allergic inflammation.

Investigating Thymic Epithelial Cell Diversity Using Systems Biology.

The thymus is an intricate organ consisting of a diverse population of thymic epithelial cells (TECs). Cortical and medullary TECs and their subpopulations have distinct roles in coordinating the development and selection of functionally competent and self-tolerant T cells. Recent advances made in technologies such as single-cell RNA sequencing have made it possible to investigate and resolve the heterogeneity in TECs. These findings have provided further understanding of the molecular mechanisms regulating TEC function and expression of tissue-restricted Ags. In this brief review, we focus on the newly characterized subsets of TECs and their diversity in relation to their functions in supporting T cell development. We also discuss recent discoveries in expression of self-antigens in the context of TEC development as well as the cellular and molecular changes occurring during embryonic development to thymic involution.

Early-life pulmonary viral infection leads to long-term functional and lower airway structural changes in the lungs.

Early-life respiratory virus infections have been correlated with enhanced development of childhood asthma. In particular, significant numbers of respiratory syncytial virus (RSV)-hospitalized infants go on to develop lung disease. It has been suggested that early-life viral infections may lead to altered lung development or repair that negatively impacts lung function later in life. Our data demonstrate that early-life RSV infection modifies lung structure, leading to decreased lung function. At 5 wk postneonatal RSV infection, significant defects are observed in baseline pulmonary function test (PFT) parameters consistent with decreased lung function as well as enlarged alveolar spaces. Lung function changes in the early-life RSV-infected group continue at 3 mo of age. The altered PFT and structural changes induced by early-life RSV were mitigated in TSLPR-/- mice that have previously been shown to have reduced immune cell accumulation associated with a persistent Th2 environment. Importantly, long-term effects were demonstrated using a secondary RSV infection 3 mo following the initial early-life RSV infection and led to significant additional defects in lung function, with severe mucus deposition within the airways, and consolidation of the alveolar spaces. These studies suggest that early-life respiratory viral infection leads to alterations in lung structure/repair that predispose to diminished lung function later in life.NEW & NOTEWORTHY These studies outline a novel finding that early-life respiratory virus infection can alter lung structure and function long-term. Importantly, the data also indicate that there are critical links between inflammatory responses and subsequent events that produce a more severe pathogenic response later in life. The findings provide additional data to support that early-life infections during lung development can alter the trajectory of airway function.

CBLB Deficiency in Human CD4+ T Cells Results in Resistance to T Regulatory Suppression through Multiple Mechanisms.

Cbl-b is a negative regulator of T cell activation, and in murine models, a lack of Cblb results in resistance of T effector (Teff) cells to T regulatory (Treg) cells, a feature of T cells in many autoimmune diseases. Here, we used trackable gene editing approaches to knock out CBLB in primary human CD4+ T cells. We found that CBLB-knockout (CBLB-KO) CD4+ T cells were hyperproliferative and produced excessive amounts of IL-2. CBLB-KO CD4+ T cells were resistant to Treg suppression in vitro, which was partially reversed by blockade of IL-2. RNA-sequencing and puromycin incorporation assays demonstrated that CBLB-KO CD4+ T cells can overcome Treg suppression on the transcriptional and translational levels, resulting in the overproduction of cytokines to drive the proliferation and activation of Teff cells. These findings highlight a potential mechanism of Teff resistance in human autoimmune disease and the power of gene editing primary T cells to explore disease mechanisms.

Distinct Epithelial-Innate Immune Cell Transcriptional Circuits Underlie Airway Hyperresponsiveness in Asthma.

Rationale: Indirect airway hyperresponsiveness (AHR) is a highly specific feature of asthma, but the underlying mechanisms responsible for driving indirect AHR remain incompletely understood. Objectives: To identify differences in gene expression in epithelial brushings obtained from individuals with asthma who were characterized for indirect AHR in the form of exercise-induced bronchoconstriction (EIB). Methods: RNA-sequencing analysis was performed on epithelial brushings obtained from individuals with asthma with EIB (n = 11) and without EIB (n = 9). Differentially expressed genes (DEGs) between the groups were correlated with measures of airway physiology, sputum inflammatory markers, and airway wall immunopathology. On the basis of these relationships, we examined the effects of primary airway epithelial cells (AECs) and specific epithelial cell-derived cytokines on both mast cells (MCs) and eosinophils (EOS). Measurements and Main Results: We identified 120 DEGs in individuals with and without EIB. Network analyses suggested critical roles for IL-33-, IL-18-, and IFN-γ-related signaling among these DEGs. IL1RL1 expression was positively correlated with the density of MCs in the epithelial compartment, and IL1RL1, IL18R1, and IFNG were positively correlated with the density of intraepithelial EOS. Subsequent ex vivo modeling demonstrated that AECs promote sustained type 2 (T2) inflammation in MCs and enhance IL-33-induced T2 gene expression. Furthermore, EOS increase the expression of IFNG and IL13 in response to both IL-18 and IL-33 as well as exposure to AECs. Conclusions: Circuits involving epithelial interactions with MCs and EOS are closely associated with indirect AHR. Ex vivo modeling indicates that epithelial-dependent regulation of these innate cells may be critical in indirect AHR and modulating T2 and non-T2 inflammation in asthma.

Rhinovirus infection of the airway epithelium enhances mast cell immune responses via epithelial-derived interferons.

BACKGROUND: Mast cells (MCs) within the airway epithelium in asthma are closely related to airway dysfunction, but cross talk between airway epithelial cells (AECs) and MCs in asthma remains incompletely understood. Human rhinovirus (RV) infections are key triggers for asthma progression, and AECs from individuals with asthma may have dysregulated antiviral responses. OBJECTIVE: We utilized primary AECs in an ex vivo coculture model system to examine cross talk between AECs and MCs after epithelial rhinovirus infection. METHODS: Primary AECs were obtained from 11 children with asthma and 10 healthy children, differentiated at air-liquid interface, and cultured in the presence of laboratory of allergic diseases 2 (LAD2) MCs. AECs were infected with rhinovirus serogroup A 16 (RV16) for 48 hours. RNA isolated from both AECs and MCs underwent RNA sequencing. Direct effects of epithelial-derived interferons on LAD2 MCs were examined by real-time quantitative PCR. RESULTS: MCs increased expression of proinflammatory and antiviral genes in AECs. AECs demonstrated a robust antiviral response after RV16 infection that resulted in significant changes in MC gene expression, including upregulation of genes involved in antiviral responses, leukocyte activation, and type 2 inflammation. Subsequent ex vivo modeling demonstrated that IFN-ß induces MC type 2 gene expression. The effects of AEC donor phenotype were small relative to the effects of viral infection and the presence of MCs. CONCLUSIONS: There is significant cross talk between AECs and MCs, which are present in the epithelium in asthma. Epithelial-derived interferons not only play a role in viral suppression but also further alter MC immune responses including specific type 2 genes.

Chitin activates parallel immune modules that direct distinct inflammatory responses via innate lymphoid type 2 and γδ T cells.

Chitin, a polysaccharide constituent of many allergens and parasites, initiates innate type 2 lung inflammation through incompletely defined pathways. We show that inhaled chitin induced expression of three epithelial cytokines, interleukin-25 (IL-25), IL-33, and thymic stromal lymphopoietin (TSLP), which nonredundantly activated resident innate lymphoid type 2 cells (ILC2s) to express IL-5 and IL-13 necessary for accumulation of eosinophils and alternatively activated macrophages (AAMs). In the absence of all three epithelial cytokines, ILC2s normally populated the lung but failed to increase IL-5 and IL-13. Although eosinophils and AAMs were attenuated, neutrophil influx remained normal without these epithelial cytokines. Genetic ablation of ILC2s, however, enhanced IL-1ß, TNFα, and IL-23 expression, increased activation of IL-17A-producing γδ T cells, and prolonged neutrophil influx. Thus, chitin elicited patterns of innate cytokines that targeted distinct populations of resident lymphoid cells, revealing divergent but interacting pathways underlying the tissue accumulation of specific types of inflammatory myeloid cells.

Conserved IFN Signature between Adult and Pediatric Eosinophilic Esophagitis.

Eosinophilic esophagitis (EoE) is an allergic inflammatory disease of the esophagus that occurs in both children and adults. Previous studies of affected tissue from pediatric cohorts have identified prominent signatures of eosinophilia and type 2 inflammation. However, the details of the immune response in adults with EoE are still being elucidated. To determine whether EoE in adults shares inflammatory profiles with those observed in children, we performed RNA sequencing of paired human esophageal biopsies and blood samples from adults with EoE or gastroesophageal reflux disease. Unbiased analysis of differentially expressed genes in tissue revealed a strong IFN signature that was significantly enriched in EoE patients as compared with patients with gastroesophageal reflux disease. Both type I and type II IFN-responsive genes were upregulated in adult biopsies, but not in blood. A similar increase in expression of IFN gene sets was observed in pediatric EoE biopsies as compared with non-EoE samples, and in public pediatric and adult RNA-sequencing data. Finally, we found that human peripheral CD4+ T cells from children with EoE produce IFN-γ upon activation with EoE-causal allergens. Together, this work identifies a conserved IFN signature in pediatric and adult EoE, highlighting a role for non-type 2 inflammatory networks in the disease process in humans.

Context-Dependent miR-21 Regulation of TLR7-Mediated Autoimmune and Foreign Antigen-Driven Antibody-Forming Cell and Germinal Center Responses.

MicroRNAs (miRNAs) are involved in healthy B cell responses and the loss of tolerance in systemic lupus erythematosus (SLE), although the role of many miRNAs remains poorly understood. Dampening miR-21 activity was previously shown to reduce splenomegaly and blood urea nitrogen levels in SLE-prone mice, but the detailed cellular responses and mechanism of action remains unexplored. In this study, using the TLR7 agonist, imiquimod-induced SLE model, we observed that loss of miR-21 in Sle1b mice prevented the formation of plasma cells and autoantibody-producing Ab-forming cells (AFCs) without a significant effect on the magnitude of the germinal center (GC) response. We further observed reduced dendritic cell and monocyte numbers in the spleens of miR-21-deficient Sle1b mice that were associated with reduced IFN, proinflammatory cytokines, and effector CD4+ T cell responses. RNA sequencing analysis on B cells from miR-21-deficient Sle1b mice revealed reduced activation and response to IFN, and cytokine and target array analysis revealed modulation of numerous miR-21 target genes in response to TLR7 activation and type I IFN stimulation. Our findings in the B6.Sle1bYaa (Sle1b Yaa) spontaneous model recapitulated the miR-21 role in TLR7-induced responses with an additional role in autoimmune GC and T follicular helper responses. Finally, immunization with T-dependent Ag revealed a role for miR-21 in foreign Ag-driven GC and Ab, but not AFC, responses. Our data suggest a potential multifaceted, context-dependent role for miR-21 in autoimmune and foreign Ag-driven AFC and GC responses. Further study is warranted to delineate the cell-intrinsic requirements and mechanisms of miR-21 during infection and SLE development.

TSLP-Driven Chromatin Remodeling and Trained Systemic Immunity after Neonatal Respiratory Viral Infection.

Our studies have previously shown a role for persistent TSLP production in the lungs of mice after early-life respiratory syncytial virus (RSV) infection that leads to an altered immune phenotype, including accumulation of "inflammatory" dendritic cells (DC). This study investigates the role of TSLP driving systemic trained immunity in DC in early-life RSV-infected mice. Bone marrow-derived DCs (BMDC) from early-life RSV-infected mice at 4 wk postinfection showed enhanced expression of costimulatory molecules and cytokines, including Tslp, that regulate immune cell function. The adoptive transfer of BMDC grown from early-life RSV-infected mice was sufficient to exacerbate allergic disease development. The addition of recombinant TSLP during differentiation of BMDC from naive mice induced a similar altered phenotype as BMDC grown from early-life RSV-infected mice, suggesting a role for TSLP in the phenotypic changes. To assess the role of TSLP in these changes, global transcriptomic characterization of TSLPR-/- BMDC infected with RSV was performed and showed a higher upregulation of type 1 IFN genes and concomitant downregulation of inflammatory genes. Assay for transposase-accessible chromatin using sequencing analysis demonstrated that TSLPR-/- BMDC had a parallel gain in physical chromatin accessibility near type 1 genes and loss in accessibility near genes related to RSV pathology, with IFN regulatory factor 4 (IRF4) and STAT3 predicted as top transcription factors binding within differentially accessible regions in wild-type. Importantly, these studies show that in the absence of TSLP signaling, BMDC are able to mount an appropriate type 1 IFN-associated antiviral response to RSV. In summary, RSV-induced TSLP alters chromatin structure in DC to drive trained innate immunity and activates pathogenic gene programs in mice.