Development and dysfunction of structural cells in eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a disorder characterized by dysfunction and chronic local inflammation of the esophagus. The incidence and prevalence of EoE are increasing worldwide. The mechanisms responsible are poorly understood, and effective treatment options are limited. From the lumen outward, the esophagus comprises stratified squamous epithelium, lamina propria, and muscle. The tissue-specific nature of EoE strongly suggests that structural cells in the esophagus are involved in the EoE diathesis. Epithelial basal cell hyperplasia and dilated intercellular spaces are cardinal features of EoE. Some patients with EoE develop lamina propria fibrosis, strictures, or esophageal muscle dysmotility. Clinical symptoms of EoE are only weakly correlated with peak eosinophil count, implying that other cell types contribute to EoE pathogenesis. Epithelial, endothelial, muscle, and fibroblast cells can each initiate inflammation and repair, regulate tissue resident immune cells, recruit peripheral leukocytes, and tailor adaptive immune cell responses. A better understanding of how structural cells maintain tissue homeostasis, respond to cell-intrinsic and cell-extrinsic stressors, and exacerbate and/or resolve inflammatory responses in the esophagus is needed. This knowledge will facilitate the development of more efficacious treatment strategies for EoE that can restore homeostasis of both hematopoietic and structural elements in the esophagus.

A randomized double-blinded trial to assess recurrence of systemic allergic reactions following COVID-19 mRNA vaccination.

BACKGROUND: Systemic allergic reactions (sARs) following coronavirus disease 2019 (COVID-19) mRNA vaccines were initially reported at a higher rate than after traditional vaccines. OBJECTIVE: We aimed to evaluate the safety of revaccination in these individuals and to interrogate mechanisms underlying these reactions. METHODS: In this randomized, double-blinded, phase 2 trial, participants aged 16 to 69 years who previously reported a convincing sAR to their first dose of COVID-19 mRNA vaccine were randomly assigned to receive a second dose of BNT162b2 (Comirnaty) vaccine and placebo on consecutive days in a blinded, 1:1 crossover fashion at the National Institutes of Health. An open-label BNT162b2 booster was offered 5 months later if the second dose did not result in severe sAR. None of the participants received the mRNA-1273 (Spikevax) vaccine during the study. The primary end point was recurrence of sAR following second dose and booster vaccination; exploratory end points included biomarker measurements. RESULTS: Of 111 screened participants, 18 were randomly assigned to receive study interventions. Eight received BNT162b2 second dose followed by placebo; 8 received placebo followed by BNT162b2 second dose; 2 withdrew before receiving any study intervention. All 16 participants received the booster dose. Following second dose and booster vaccination, sARs recurred in 2 participants (12.5%; 95% CI, 1.6 to 38.3). No sAR occurred after placebo. An anaphylaxis mimic, immunization stress-related response (ISRR), occurred more commonly than sARs following both vaccine and placebo and was associated with higher predose anxiety scores, paresthesias, and distinct vital sign and biomarker changes. CONCLUSIONS: Our findings support revaccination of individuals who report sARs to COVID-19 mRNA vaccines. Distinct clinical and laboratory features may distinguish sARs from ISRRs.

Notch signaling regulates antigen sensitivity of naive CD4+ T cells by tuning co-stimulation.

Adaptive immune responses begin when naive CD4(+) T cells engage peptide+major histocompatibility complex class II and co-stimulatory molecules on antigen-presenting cells (APCs). Notch signaling can influence effector functions in differentiated CD4(+) T helper and T regulatory cells. Whether and how ligand-induced Notch signaling influences the initial priming of CD4(+) T cells has not been addressed. We have found that Delta Like Ligand 4 (DLL4)-induced Notch signaling potentiates phosphatidylinositol 3-OH kinase (PI3K)-dependent signaling downstream of the T cell receptor+CD28, allowing naive CD4(+) T cells to respond to lower doses of antigen. In vitro, DLL4-deficient APCs were less efficient stimulators of CD4(+) T cell activation, metabolism, proliferation, and cytokine secretion. With deletion of DLL4 from CD11c(+) APCs in vivo, these deficits translated to an impaired ability to mount an effective CD4(+)-dependent anti-tumor response. These data implicate Notch signaling as an important regulator of adaptive immune responses.

Dendritic cells Trigger IFN-γ secretion by NK cells independent of IL-12 and IL-18.

It is commonly believed that IL-12 produced by DCs in response to pathogens is the first signal that stimulates the production of IFN-γ by NK cells. However, IL-12 production by DCs in response to bacterial LPS depends on either engagement of CD40 by CD40L on activated T cells or IFN-γ from NK cells. This suggests that during the primary immune response, NK cells produce IFN-γ before IL-12 production by DCs. Here, using single-cell measurements, cell sorting and mouse lines deficient in IL-12, IL-23, type I IFN receptor and the IL-18 receptor, we show that a subset of BM-derived DCs characterized by low expression of MHC class II (MHCIIlow ) stimulates IFN-γ production by NK cells. The expression of Toll-like Receptor (TLR) 4 on DCs but not NK cells was required for such NK-derived IFN-γ. In addition, soluble factor(s) produced by LPS-activated MHCIIlow DCs were sufficient to induce IFN-γ production by NK cells independent of IL-12, IL-23, and IL-18. This response was enhanced in the presence of a low dose of IL-2. These results delineate a previously unknown pathway of DC-mediated IFN-γ production by NK cells, which is independent of commonly known cytokines.

Critical Adverse Impact of IL-6 in Acute Pneumovirus Infection.

Severe respiratory virus infections feature robust local host responses that contribute to disease severity. Immunomodulatory strategies that limit virus-induced inflammation may be of critical importance, notably in the absence of antiviral vaccines. In this study, we examined the role of the pleiotropic cytokine IL-6 in acute infection with pneumonia virus of mice (PVM), a natural rodent pathogen that is related to respiratory syncytial virus and that generates local inflammation as a feature of severe infection. In contrast to Influenza A, PVM is substantially less lethal in IL-6 -/- mice than it is in wild-type, a finding associated with diminished neutrophil recruitment and reduced fluid accumulation in lung tissue. Ly6Chi proinflammatory monocytes are recruited in response to PVM via a CCR2-dependent mechanism, but they are not a major source of IL-6 nor do they contribute to lethal sequelae of infection. By contrast, alveolar macrophages are readily infected with PVM in vivo; ablation of alveolar macrophages results in prolonged survival in association with a reduction in virus-induced IL-6. Finally, as shown previously, administration of immunobiotic Lactobacillus plantarum to the respiratory tracts of PVM-infected mice promoted survival in association with diminished levels of IL-6. We demonstrated in this study that IL-6 suppression is a critical feature of the protective mechanism; PVM-infected IL-6 -/- mice responded to low doses of L. plantarum, and administration of IL-6 overcame L. plantarum-mediated protection in PVM-infected wild-type mice. Taken together, these results connect the actions of IL-6 to PVM pathogenesis and suggest cytokine blockade as a potential therapeutic modality in severe infection.

Cutting Edge: Quantitative Determination of CD40L Threshold for IL-12 and IL-23 Production from Dendritic Cells.

Early secretion of IL-12 by mouse dendritic cells (DCs) instructs T cells to make IFN-γ. However, only activated, but not naive T cells are able to license DCs for IL-12 production. We hypothesized that it might be due to different levels of CD40L expression on the surface of these cells, as CD40 signals are required for IL-12 production. Using quantitative cell-free systems incorporating CD40L in lipid bilayers combined with total internal reflection fluorescence microscopy and flow cytometry, we show that as low as ∼200 CD40L molecules/µm2 in combination with IL-4 is sufficient to induce IL-12 production by DCs. Remarkably, CD40L alone is adequate to induce IL-23 secretion by DCs. Thus, although activated T cells have somewhat higher levels of CD40L, it is the combination of CD40L and the cytokines they secrete that licenses DCs and influences the effector class of the immune response.

TGFß prevents IgE-mediated allergic disease by restraining T follicular helper 2 differentiation.

Allergic diseases are common, affecting more than 20% of the population. Genetic variants in the TGFß pathway are strongly associated with atopy. To interrogate the mechanisms underlying this association, we examined patients and mice with Loeys-Dietz syndrome (LDS) who harbor missense mutations in the kinase domain of TGFΒR1/2. We demonstrate that LDS mutations lead to reduced TGFß signaling and elevated total and allergen-specific IgE, despite the presence of wild-type T regulatory cells in a chimera model. Germinal center activity was enhanced in LDS and characterized by a selective increase in type 2 follicular helper T cells (TFH2). Expression of Pik3cg was increased in LDS TFH cells and associated with reduced levels of the transcriptional repressor SnoN. PI3Kγ/mTOR signaling in LDS naïve CD4+ T cells was elevated after T cell receptor cross-linking, and pharmacologic inhibition of PI3Kγ or mTOR prevented exaggerated TFH2 and antigen-specific IgE responses after oral antigen exposure in an adoptive transfer model. Naïve CD4+ T cells from nonsyndromic allergic individuals also displayed decreased TGFß signaling, suggesting that our mechanistic discoveries may be broadly relevant to allergic patients in general. Thus, TGFß plays a conserved, T cell-intrinsic, and nonredundant role in restraining TFH2 development via the PI3Kγ/mTOR pathway and thereby protects against allergic disease.

Presenilins regulate alphabeta T cell development by modulating TCR signaling.

TCRalphabeta signaling is crucial for the maturation of CD4 and CD8 T cells, but the role of the Notch signaling pathway in this process is poorly understood. Genes encoding Presenilin (PS) 1/2 were deleted to prevent activation of the multiple Notch receptors expressed by developing thymocytes. PS1/2 knockout thymocyte precursors inefficiently generate CD4 T cells, a phenotype that is most pronounced when thymocytes bear a single major histocompatibility complex (MHC) class II-restricted T cell receptor (TCR). Diminished T cell production correlated with evidence of impaired TCR signaling, and could be rescued by manipulations that enhance MHC recognition. Although Notch appears to directly regulate binary fate decisions in many systems, these findings suggest a model in which PS-dependent Notch signaling influences positive selection and the development of alphabeta T cells by modifying TCR signal transduction.

A genome catalog of the early-life human skin microbiome.

Metagenome-assembled genomes have greatly expanded the reference genomes for skin microbiome. However, the current reference genomes are largely based on samples from adults in North America and lack representation from infants and individuals from other continents. Here we used ultra-deep shotgun metagenomic sequencing to profile the skin microbiota of 215 infants at age 2-3 months and 12 months who were part of the VITALITY trial in Australia as well as 67 maternally-matched samples. Based on the infant samples, we present the Early-Life Skin Genomes (ELSG) catalog, comprising 9,194 bacterial genomes from 1,029 species, 206 fungal genomes from 13 species, and 39 eukaryotic viral sequences. This genome catalog substantially expands the diversity of species previously known to comprise human skin microbiome and improves the classification rate of sequenced data by 25%. The protein catalog derived from these genomes provides insights into the functional elements such as defense mechanisms that distinguish early-life skin microbiome. We also found evidence for vertical transmission at the microbial community, individual skin bacterial species and strain levels between mothers and infants. Overall, the ELSG catalog uncovers the skin microbiome of a previously underrepresented age group and population and provides a comprehensive view of human skin microbiome diversity, function, and transmission in early life.

Epithelial-intrinsic defects in TGFßR signaling drive local allergic inflammation manifesting as eosinophilic esophagitis.

Allergic diseases are a global health challenge. Individuals harboring loss-of-function variants in transforming growth factor-ß receptor (TGFßR) genes have an increased prevalence of allergic disorders, including eosinophilic esophagitis. Allergic diseases typically localize to mucosal barriers, implicating epithelial dysfunction as a cardinal feature of allergic disease. Here, we describe an essential role for TGFß in the control of tissue-specific immune homeostasis that provides mechanistic insight into these clinical associations. Mice expressing a TGFßR1 loss-of-function variant identified in atopic patients spontaneously develop disease that clinically, immunologically, histologically, and transcriptionally recapitulates eosinophilic esophagitis. In vivo and in vitro, TGFßR1 variant-expressing epithelial cells are hyperproliferative, fail to differentiate properly, and overexpress innate proinflammatory mediators, which persist in the absence of lymphocytes or external allergens. Together, our results support the concept that TGFß plays a fundamental, nonredundant, epithelial cell-intrinsic role in controlling tissue-specific allergic inflammation that is independent of its role in adaptive immunity.

A genome catalog of the early-life human skin microbiome.

BACKGROUND: Metagenome-assembled genomes have greatly expanded the reference genomes for skin microbiome. However, the current reference genomes are largely based on samples from adults in North America and lack representation from infants and individuals from other continents. RESULTS: Here we use deep shotgun metagenomic sequencing to profile the skin microbiota of 215 infants at age 2-3 months and 12 months who are part of the VITALITY trial in Australia as well as 67 maternally matched samples. Based on the infant samples, we present the Early-Life Skin Genomes (ELSG) catalog, comprising 9483 prokaryotic genomes from 1056 species, 206 fungal genomes from 13 species, and 39 eukaryotic viral sequences. This genome catalog substantially expands the diversity of species previously known to comprise human skin microbiome and improves the classification rate of sequenced data by 21%. The protein catalog derived from these genomes provides insights into the functional elements such as defense mechanisms that distinguish early-life skin microbiome. We also find evidence for microbial sharing at the community, bacterial species, and strain levels between mothers and infants. CONCLUSIONS: Overall, the ELSG catalog uncovers the skin microbiome of a previously underrepresented age group and population and provides a comprehensive view of human skin microbiome diversity, function, and development in early life.

Unexpected role for the B cell-specific Src family kinase B lymphoid kinase in the development of IL-17-producing γδ T cells.

The Ag receptors on αß and γδ T cells differ not only in the nature of the ligands that they recognize but also in their signaling potential. We hypothesized that the differences in αß- and γδTCR signal transduction were due to differences in the intracellular signaling pathways coupled to these two TCRs. To investigate this, we used transcriptional profiling to identify genes encoding signaling molecules that are differentially expressed in mature αß and γδ T cell populations. Unexpectedly, we found that B lymphoid kinase (Blk), a Src family kinase expressed primarily in B cells, is expressed in γδ T cells but not in αß T cells. Analysis of Blk-deficient mice revealed that Blk is required for the development of IL-17-producing γδ T cells. Furthermore, Blk is expressed in lymphoid precursors and, in this capacity, plays a role in regulating thymus cellularity during ontogeny.

Notch signaling in CD4 and CD8 T cell development.

Because Notch often acts in concert with other signaling pathways, it is able to regulate a diverse set of biological processes in a cell-context dependent manner. In lymphocytes, Notch is essential for specifying the T cell fate and for promoting early stages of T cell differentiation. At later stages of development, Notch signaling is proposed to direct CD4 versus CD8 T lineage commitment. This hypothesis has been challenged by recent studies of conditional Presenilin-deficient mice showing that Notch promotes the selection and maturation of CD4 and CD8 T cells by potentiating TCR signal transduction in immature thymocytes. While similar conclusions have not been reported with conditional mutation of other downstream mediators of Notch activation, it appears that functional inhibition may not have been achieved at a comparable stage of development and/or analogous issues have not been addressed. The differences also question whether in thymocytes Notch signals only through the canonical pathway. Further study of conditional mutants, signaling intermediates, and transcriptional regulators are needed to elucidate how Notch facilitates TCR signaling in generating mature T cells.

Activation-induced modification in the CD3 complex of the gammadelta T cell receptor.

The T cell antigen receptor complexes expressed on alphabeta and gammadelta T cells differ not only in their respective clonotypic heterodimers but also in the subunit composition of their CD3 complexes. The gammadelta T cell receptors (TCRs) expressed on ex vivo gammadelta T cells lack CD3delta, whereas alphabeta TCRs contain CD3delta. While this result correlates with the phenotype of CD3delta(-/-) mice, in which gammadelta T cell development is unaffected, it is inconsistent with the results of previous studies reporting that CD3delta is a component of the gammadelta TCR. Since earlier studies examined the subunit composition of gammadelta TCRs expressed on activated and expanded peripheral gammadelta T cells or gammadelta TCR(+) intestinal intraepithelial lymphocytes, we hypothesized that activation and expansion may lead to changes in the CD3 subunit composition of the gammadelta TCR. Here, we report that activation and expansion do in fact result in the inclusion of a protein, comparable in mass and mobility to CD3delta, in the gammadelta TCR. Further analyses revealed that this protein is not CD3delta, but instead is a differentially glycosylated form of CD3gamma. These results provide further evidence for a major difference in the subunit composition of alphabeta- and gammadelta TCR complexes and raise the possibility that modification of CD3gamma may have important functional consequences in activated gammadelta T cells.

Amyloid precursor family proteins are expressed by thymic and lymph node stromal cells but are not required for lymphocyte development.

Pharmacological inhibitors that block amyloid precursor protein (APP) cleavage and the formation of senile plaques are under development for the treatment of familial Alzheimer's disease. Unfortunately, many inhibitors that block gamma-secretase-mediated cleavage of APP also have immunosuppressive side effects. In addition to APP, numerous other proteins undergo gamma-secretase-mediated cleavage. In order to develop safer inhibitors, it is necessary to determine which of the gamma-secretase substrates contribute to the immunosuppressive effects. Because APP family members are widely expressed and are reported to influence calcium flux, transcription and apoptosis, they could be important for normal lymphocyte maturation. We find that APP and amyloid precursor-like protein 2 are expressed by stromal cells of thymus and lymph nodes, but not by lymphocytes. Although signals provided by thymic stromal cells are critical for normal T cell differentiation, lymphocyte development proceeds unperturbed in mice deficient for these APP family members.

Bone Marrow-Derived Dendritic Cell Cultures from RAG-/- Mice Include IFN-γ-Producing NK Cells.

Dendritic cells (DCs) play a key role in the initiation of an immune response and are known as "professional" APCs because of their ability to activate naive T cells. A widely used method to generate DCs in vitro is to culture bone marrow (BM) cells or blood monocytes in the presence of GM-CSF and IL-4. In this study, we show that a small population of NK cells residing in the BM of RAG-/-, but not RAG-/- γc chain-/- mice, remain in the DC culture and is the source of IFN-γ produced after stimulation with LPS. These cells, which may represent early promoters of LPS-induced responses, have to be taken into account when interpreting experiments using BM-derived DCs.

Impact of eosinophil-peroxidase (EPX) deficiency on eosinophil structure and function in mouse airways.

Eosinophil peroxidase (EPX) is a major constituent of the large cytoplasmic granules of both human and mouse eosinophilic leukocytes. Human EPX deficiency is a rare, autosomal-recessive disorder limited to the eosinophil lineage. Our intent was to explore the impact of EPX gene deletion on eosinophil content, structure, and function. In response to repetitive intranasal challenge with a filtrate of the allergen, Alternaria alternata, we found significantly fewer eosinophils peripherally and in the respiratory tracts of EPX-/- mice compared to wild-type controls; furthermore, both the major population (Gr1-/lo ) and the smaller population of Gr1hi eosinophils from EPX-/- mice displayed lower median fluorescence intensities (MFIs) for Siglec F. Quantitative evaluation of transmission electron micrographs of lung eosinophils confirmed the relative reduction in granule outer matrix volume in cells from the EPX-/- mice, a finding analogous to that observed in human EPX deficiency. Despite the reduced size of the granule matrix, the cytokine content of eosinophils isolated from allergen-challenged EPX-/- and wild-type mice were largely comparable to one another, although the EPX-/- eosinophils contained reduced concentrations of IL-3. Other distinguishing features of lung eosinophils from allergen-challenged EPX-/- mice included a reduced fraction of surface TLR4+ cells and reduced MFI for NOD1. Interestingly, the EPX gene deletion had no impact on eosinophil-mediated clearance of gram-negative Haemophilus influenzae from the airways. As such, although no clinical findings have been associated with human EPX deficiency, our findings suggest that further evaluation for alterations in eosinophil structure and function may be warranted.

Receptor signals and nuclear events in CD4 and CD8 T cell lineage commitment.

MHC specificity in positive selection is a major determinant in the CD4/CD8 T cell lineage decision. Previous studies support the view that quantitative differences in T cell receptor (TCR) signaling in immature CD4+CD8+ double positive thymocytes leads to an instructive bias in CD4/CD8 T cell lineage commitment that must be re-inforced in subsequent selection steps to ensure that MHC-restricted antigen recognition is linked to appropriate effector functions in mature T cells. Recent work has further defined the TCR signaling pathways involved in this process, but a major effort has been made to identify transcription factors and other regulators of CD4 and CD8 T cell lineage commitment. Methods and screens for detecting changes in gene expression, associated with TCR signaling in positive selection and lineage determination, are starting to provide a better understanding of these complex developmental processes.

Hematopoietic reconstitution of neonatal immunocompetent mice to study conditions with a perinatal window of susceptibility.

Efficient hematopoietic reconstitution of wild type mice requires preconditioning. Established experimental protocols exist to transplant hematopoietic stem cells into lethally irradiated or chemically myeloablated adult mice or unirradiated immunodeficient mice. We sought to develop a protocol to reconstitute immuno-replete neonatal mice. We describe irradiation and injection procedures for two-day old mice that lead to efficient long-term reconstitution of primary and secondary lymphoid organs. We demonstrate that the frequencies of lymphoid and myeloid cells in primary and secondary lymphoid organs are indistinguishable from unirradiated uninjected sex- and age-matched control animals by 5 weeks post-reconstitution. Thus, this system will facilitate studies aimed at understanding the developmental and environmental mechanisms that contribute to conditions that have a window of susceptibility during the perinatal period.

In Vivo Depletion of T Lymphocytes.

In vivo depletion of T lymphocytes is a means of studying the role of specific T cell populations during defined phases of in vivo immune responses. In this unit, a protocol is provided for injecting monoclonal antibodies (mAbs) into wild-type adult mice. Depletion of the appropriate subset of cells is verified by flow cytometry analysis of lymph node and spleen cell suspensions in pilot experiments. Once conditions have been established, depleted mice can be used to study the impact of T cell subsets on a variety of in vivo immune responses. The depleted condition may be maintained by repeated injections of the monoclonal antibody, or reversed by normal thymopoiesis following discontinuation of antibody administration.