Production and Function of Immunoglobulin A.

Among antibodies, IgA is unique because it has evolved to be secreted onto mucosal surfaces. The structure of IgA and the associated secretory component allow IgA to survive the highly proteolytic environment of mucosal surfaces but also substantially limit IgA's ability to activate effector functions on immune cells. Despite these characteristics, IgA is critical for both preventing enteric infections and shaping the local microbiome. IgA's function is determined by a distinct antigen-binding repertoire, composed of antibodies with a variety of specificities, from permissive polyspecificity to cross-reactivity to exquisite specificity to a single epitope, which act together to regulate intestinal bacteria. Development of the unique function and specificities of IgA is shaped by local cues provided by the gut-associated lymphoid tissue, driven by the constantly changing environment of the intestine and microbiota.

An epithelial cell-derived metabolite tunes immunoglobulin A secretion by gut-resident plasma cells.

Immunoglobulin A (IgA) secretion by plasma cells, terminally differentiated B cells residing in the intestinal lamina propria, assures microbiome homeostasis and protects the host against enteric infections. Exposure to diet-derived and commensal-derived signals provides immune cells with organizing cues that instruct their effector function and dynamically shape intestinal immune responses at the mucosal barrier. Recent data have described metabolic and microbial inputs controlling T cell and innate lymphoid cell activation in the gut; however, whether IgA-secreting lamina propria plasma cells are tuned by local stimuli is completely unknown. Although antibody secretion is considered to be imprinted during B cell differentiation and therefore largely unaffected by environmental changes, a rapid modulation of IgA levels in response to intestinal fluctuations might be beneficial to the host. In the present study, we showed that dietary cholesterol absorption and commensal recognition by duodenal intestinal epithelial cells lead to the production of oxysterols, evolutionarily conserved lipids with immunomodulatory functions. Using conditional cholesterol 25-hydroxylase deleter mouse line we demonstrated that 7α,25-dihydroxycholesterol from epithelial cells is critical to restrain IgA secretion against commensal- and pathogen-derived antigens in the gut. Intestinal plasma cells sense oxysterols via the chemoattractant receptor GPR183 and couple their tissue positioning with IgA secretion. Our findings revealed a new mechanism linking dietary cholesterol and humoral immune responses centered around plasma cell localization for efficient mucosal protection.

Antigen receptor signaling and cell death resistance controls intestinal humoral response zonation.

Immunoglobulin A (IgA) maintains commensal communities in the intestine while preventing dysbiosis. IgA generated against intestinal microbes assures the simultaneous binding to multiple, diverse commensal-derived antigens. However, the exact mechanisms by which B cells mount broadly reactive IgA to the gut microbiome remains elusive. Here, we have shown that IgA B cell receptor (BCR) is required for B cell fitness during the germinal center (GC) reaction in Peyer's patches (PPs) and for generation of gut-homing plasma cells (PCs). We demonstrate that IgA BCR drove heightened intracellular signaling in mouse and human B cells, and as a consequence, IgA+ B cells received stronger positive selection cues. Mechanistically, IgA BCR signaling offset Fas-mediated death, possibly rescuing low-affinity B cells to promote a broad humoral response to commensals. Our findings reveal an additional mechanism linking BCR signaling, B cell fate, and antibody production location, which have implications for how intestinal antigen recognition shapes humoral immunity.

Skin γδ T cell inflammatory responses are hardwired in the thymus by oxysterol sensing via GPR183 and calibrated by dietary cholesterol.

Dietary components and metabolites have a profound impact on immunity and inflammation. Here, we investigated how sensing of cholesterol metabolite oxysterols by γδ T cells impacts their tissue residency and function. We show that dermal IL-17-producing γδ T (Tγδ17) cells essential for skin-barrier homeostasis require oxysterols sensing through G protein receptor 183 (GPR183) for their development and inflammatory responses. Single-cell transcriptomics and murine reporter strains revealed that GPR183 on developing γδ thymocytes is needed for their maturation by sensing medullary thymic epithelial-cell-derived oxysterols. In the skin, basal keratinocytes expressing the oxysterol enzyme cholesterol 25-hydroxylase (CH25H) maintain dermal Tγδ17 cells. Diet-driven increases in oxysterols exacerbate Tγδ17-cell-mediated psoriatic inflammation, dependent on GPR183 on γδ T cells. Hence, cholesterol-derived oxysterols control spatially distinct but biologically linked processes of thymic education and peripheral function of dermal T cells, implicating diet as a focal parameter of dermal Tγδ17 cells.

The cholesterol metabolite 25-hydroxycholesterol restrains the transcriptional regulator SREBP2 and limits intestinal IgA plasma cell differentiation.

Diets high in cholesterol alter intestinal immunity. Here, we examined how the cholesterol metabolite 25-hydroxycholesterol (25-HC) impacts the intestinal B cell response. Mice lacking cholesterol 25-hydroxylase (CH25H), the enzyme generating 25-HC, had higher frequencies of immunoglobulin A (IgA)-secreting antigen-specific B cells upon immunization or infection. 25-HC did not affect class-switch recombination but rather restrained plasma cell (PC) differentiation. 25-HC was produced by follicular dendritic cells and increased in response to dietary cholesterol. Mechanistically, 25-HC restricted activation of the sterol-sensing transcription factor SREBP2, thereby regulating B cell cholesterol biosynthesis. Ectopic expression of SREBP2 in germinal center B cells induced rapid PC differentiation, whereas SREBP2 deficiency reduced PC output in vitro and in vivo. High-cholesterol diet impaired, whereas Ch25h deficiency enhanced, the IgA response against Salmonella and the resulting protection from systemic bacterial dissemination. Thus, a 25-HC-SREBP2 axis shapes the humoral response at the intestinal barrier, providing insight into the effect of high dietary cholesterol in intestinal immunity.

Dysbiosis exacerbates colitis by promoting ubiquitination and accumulation of the innate immune adaptor STING in myeloid cells.

Alterations in the cGAS-STING DNA-sensing pathway affect intestinal homeostasis. We sought to delineate the functional role of STING in intestinal inflammation. Increased STING expression was a feature of intestinal inflammation in mice with colitis and in humans afflicted with inflammatory bowel disease. Mice bearing an allele rendering STING constitutively active exhibited spontaneous colitis and dysbiosis, as well as progressive chronic intestinal inflammation and fibrosis. Bone marrow chimera experiments revealed STING accumulation in intestinal macrophages and monocytes as the initial driver of inflammation. Depletion of Gram-negative bacteria prevented STING accumulation in these cells and alleviated intestinal inflammation. STING accumulation occurred at the protein rather than transcript level, suggesting post-translational stabilization. We found that STING was ubiquitinated in myeloid cells, and this K63-linked ubiquitination could be elicited by bacterial products, including cyclic di-GMP. Our findings suggest a positive feedback loop wherein dysbiosis foments the accumulation of STING in intestinal myeloid cells, driving intestinal inflammation.

Dietary Cholesterol Metabolite Regulation of Tissue Immune Cell Development and Function.

Obesity is considered the primary environmental factor associated with morbidity and severity of wide-ranging inflammatory disorders. The molecular mechanism linking high-fat or cholesterol diet to imbalances in immune responses, beyond the increased production of generic inflammatory factors, is just beginning to emerge. Diet cholesterol by-products are now known to regulate function and migration of diverse immune cell subsets in tissues. The hydroxylated metabolites of cholesterol oxysterols as central regulators of immune cell positioning in lymphoid and mucocutaneous tissues is the focus of this review. Dedicated immunocyte cell surface receptors sense spatially distributed oxysterol tissue depots to tune cell metabolism and function, to achieve the "right place at the right time" axiom of efficient tissue immunity.

Hierarchy of signaling thresholds downstream of the T cell receptor and the Tec kinase ITK.

The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-associated proteins like IRF4. To regulate T cell activation programming, the TCR and the TCR proximal interleukin-2-inducible T cell kinase (ITK) simultaneously trigger many biochemically separate signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions, ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through the unequal activation of distinct signaling pathways, we examined Erk1/2 phosphorylation or nuclear factor of activated T cells (NFAT) and nuclear factor (NF)-κB translocation in naïve OT-I CD8+ cell nuclei. We observed the consistent digital activation of NFAT1 and Erk1/2, but NF-κB displayed dynamic, graded activation in response to variation in TCR signal strength, tunable by treatment with an ITK inhibitor. Inhibitor-treated cells showed the dampened induction of AP-1 factors Fos and Fosb, NF-κB response gene transcripts, and survival factor Il2 transcripts. ATAC sequencing analysis also revealed that genomic regions most sensitive to ITK inhibition were enriched for NF-κB and AP-1 motifs. Specific inhibition of NF-κB during peptide stimulation tuned the expression of early gene products like c-Fos. Together, these data indicate a key role for ITK in orchestrating the optimal activation of separate TCR downstream pathways, specifically aiding NF-κB activation. More broadly, we revealed a mechanism by which variations in TCR signal strength can produce patterns of graded gene expression in activated T cells.

Persistent antigen and germinal center B cells sustain T follicular helper cell responses and phenotype.

T follicular helper (Tfh) cells provide help to B cells and are crucial for establishment of germinal center (GC) reactions, including production of high-affinity antibodies and generation of memory B cells and long-lived plasma cells. Here we report that the magnitude of the Tfh cell response was dictated by the amount of antigen and directly correlated with the magnitude of the GC B cell response. In addition, maintenance of the Tfh cell phenotype required sustained antigenic stimulation by GC B cells. In lymphopenic conditions, a strong and prolonged Tfh cell response led to bystander B cell activation, hypergammaglobulinemia, and production of poly- and self-reactive antibodies. These data demonstrate that antigen dose determines the size and duration of the Tfh cell response and GC reaction, highlight the transient nature of the Tfh cell phenotype, and suggest a link between overstimulation of Tfh cells and the development of dysregulated humoral immune responses.

C-C chemokine receptor 6-regulated entry of TH-17 cells into the CNS through the choroid plexus is required for the initiation of EAE.

Interleukin 17-producing T helper cells (T(H)-17 cells) are important in experimental autoimmune encephalomyelitis, but their route of entry into the central nervous system (CNS) and their contribution relative to that of other effector T cells remain to be determined. Here we found that mice lacking CCR6, a chemokine receptor characteristic of T(H)-17 cells, developed T(H)-17 responses but were highly resistant to the induction of experimental autoimmune encephalomyelitis. Disease susceptibility was reconstituted by transfer of wild-type T cells that entered into the CNS before disease onset and triggered massive CCR6-independent recruitment of effector T cells across activated parenchymal vessels. The CCR6 ligand CCL20 was constitutively expressed in epithelial cells of choroid plexus in mice and humans. Our results identify distinct molecular requirements and ports of lymphocyte entry into uninflamed versus inflamed CNS and suggest that the CCR6-CCL20 axis in the choroid plexus controls immune surveillance of the CNS.

Peyer's patches: organizing B-cell responses at the intestinal frontier.

Secondary lymphoid tissues share the important function of bringing together antigens and rare antigen-specific lymphocytes to foster induction of adaptive immune responses. Peyer's patches (PPs) are unique compared to other secondary lymphoid tissues in their continual exposure to an enormous diversity of microbiome- and food-derived antigens and in the types of pathogens they encounter. Antigens are delivered to PPs by specialized microfold (M) epithelial cells and they may be captured and presented by resident dendritic cells (DCs). In accord with their state of chronic microbial antigen exposure, PPs exhibit continual germinal center (GC) activity. These GCs not only contribute to the generation of B cells and plasma cells producing somatically mutated gut antigen-specific IgA antibodies but have also been suggested to support non-specific antigen diversification of the B-cell repertoire. Here, we review current understanding of how PPs foster B-cell encounters with antigen, how they favor isotype switching to the secretory IgA isotype, and how their GC responses may uniquely contribute to mucosal immunity.

Clathrin light chains' role in selective endocytosis influences antibody isotype switching.

Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor ß receptor 2 (TGFßR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the ß2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin's role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules.

Microbiota encoded fatty-acid metabolism expands tuft cells to protect tissues homeostasis during Clostridioides difficile infection in the large intestine.

Metabolic byproducts of the intestinal microbiota are crucial in maintaining host immune tone and shaping inter-species ecological dynamics. Among these metabolites, succinate is a driver of tuft cell (TC) differentiation and consequent type 2 immunity-dependent protection against invading parasites in the small intestine. Succinate is also a growth enhancer of the nosocomial pathogen Clostridioides difficile in the large intestine. To date, no research has shown the role of succinate in modulating TC dynamics in the large intestine, or the relevance of this immune pathway to C. difficile pathophysiology. Here we reveal the existence of a three-way circuit between commensal microbes, C. difficile and host epithelial cells which centers around succinate. Through selective microbiota depletion experiments we demonstrate higher levels of type 2 cytokines leading to expansion of TCs in the colon. We then demonstrate the causal role of the microbiome in modulating colonic TC abundance and subsequent type 2 cytokine induction using rational supplementation experiments with fecal transplants and microbial consortia of succinate-producing bacteria. We show that administration of a succinate-deficient Bacteroides thetaiotaomicron knockout (Δfrd) significantly reduces the enhanced type 2 immunity in mono-colonized mice. Finally, we demonstrate that mice prophylactically administered with the consortium of succinate-producing bacteria show reduced C. difficile-induced morbidity and mortality compared to mice administered with heat-killed bacteria or the vehicle. This effect is reduced in a partial tuft cell knockout mouse, Pou2f3+/-, and nullified in the tuft cell knockout mouse, Pou2f3-/-, confirming that the observed protection occurs via the TC pathway. Succinate is an intermediary metabolite of the production of short-chain fatty acids, and its concentration often increases during dysbiosis. The first barrier to enteric pathogens alike is the intestinal epithelial barrier, and host maintenance and strengthening of barrier integrity is vital to homeostasis. Considering our data, we propose that activation of TC by the microbiota-produced succinate in the colon is a mechanism evolved by the host to counterbalance microbiome-derived cues that facilitate invasion by intestinal pathogens.

Distinct components of nucleoside-modified messenger RNA vaccines cooperate to instruct efficient germinal center responses.

Nucleoside-modified mRNA vaccines elicit protective antibodies through their ability to promote T follicular helper (Tfh) cells. The lipid nanoparticle (LNP) component of mRNA vaccines possesses inherent adjuvant activity. However, to what extent the nucleoside-modified mRNA can be sensed and contribute to Tfh cell responses remains largely undefined. Herein, we deconvoluted the signals induced by LNP and mRNA that instruct dendritic cells (DCs) to promote Tfh cell differentiation. We demonstrated that the nucleoside-modified mRNA drives the production of type I interferons that act on DCs to induce their maturation and the induction of Th1-biased Tfh responses. Conversely, LNP favors the acquisition of a Tfh cell-inducing program in DCs, a stronger Th2 polarization in Tfh cells, and allows for rapid mRNA translation by DCs within the draining lymph node. Our work unravels distinct adjuvant features of mRNA and LNP necessary for the induction of Tfh cells, with implications for vaccine design.

Gut-associated lymphoid tissue attrition associates with response to anti-α4ß7 therapy in ulcerative colitis.

Vedolizumab (VDZ) is a first-line treatment in ulcerative colitis (UC) that targets the α4ß7- mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) axis. To determine the mechanisms of action of VDZ, we examined five distinct cohorts of patients with UC. A decrease in naïve B and T cells in the intestines and gut-homing (ß7+) plasmablasts in circulation of VDZ-treated patients suggested that VDZ targets gut-associated lymphoid tissue (GALT). Anti-α4ß7 blockade in wild-type and photoconvertible (KikGR) mice confirmed a loss of GALT size and cellularity because of impaired cellular entry. In VDZ-treated patients with UC, treatment responders demonstrated reduced intestinal lymphoid aggregate size and follicle organization and a reduction of ß7+IgG+ plasmablasts in circulation, as well as IgG+ plasma cells and FcγR-dependent signaling in the intestine. GALT targeting represents a previously unappreciated mechanism of action of α4ß7-targeted therapies, with major implications for this therapeutic paradigm in UC.

Embryonic type 3 innate lymphoid cells sense maternal dietary cholesterol to control local Peyer's patch development.

Lymphoid tissue inducer (LTi) cells develop during intrauterine life and rely on developmental programs to initiate the organogenesis of secondary lymphoid organs (SLOs). This evolutionary conserved process endows the fetus with the ability to orchestrate the immune response after birth and to react to the triggers present in the environment. While it is established that LTi function can be shaped by maternal-derived cues and is critical to prepare the neonate with a functional scaffold to mount immune response, the cellular mechanisms that control anatomically distinct SLO organogenesis remain unclear. We discovered that LTi cells forming Peyer's patches, gut-specific SLOs, require the coordinated action of two migratory G protein coupled receptors (GPCR) GPR183 and CCR6. These two GPCRs are uniformly expressed on LTi cells across SLOs, but their deficiency specifically impacts Peyer's patch formation, even when restricted to fetal window. The unique CCR6 ligand is CCL20, while the ligand for GPR183 is the cholesterol metabolite 7α,25-Dihydroxycholesterol (7α,25-HC), whose production is controlled by the enzyme cholesterol 25-hydroxylase (CH25H). We identified a fetal stromal cell subset that expresses CH25H and attracts LTi cells in the nascent Peyer's patch anlagen. GPR183 ligand concentration can be modulated by the cholesterol content in the maternal diet and impacts LTi cell maturation in vitro and in vivo, highlighting a link between maternal nutrients and intestinal SLO organogenesis. Our findings revealed that in the fetal intestine, cholesterol metabolite sensing by GPR183 in LTi cells for Peyer's patch formation is dominant in the duodenum, the site of cholesterol absorption in the adult. This anatomic requirement suggests that embryonic, long-lived non-hematopoietic cells might exploit adult metabolic functions to ensure highly specialized SLO development in utero.

Gut-associated lymphoid tissue attrition associates with response to anti-α4ß7 therapy in ulcerative colitis.

Targeting the α4ß7-MAdCAM-1 axis with vedolizumab (VDZ) is a front-line therapeutic paradigm in ulcerative colitis (UC). However, mechanism(s) of action (MOA) of VDZ remain relatively undefined. Here, we examined three distinct cohorts of patients with UC (n=83, n=60, and n=21), to determine the effect of VDZ on the mucosal and peripheral immune system. Transcriptomic studies with protein level validation were used to study drug MOA using conventional and transgenic murine models. We found a significant decrease in colonic and ileal naïve B and T cells and circulating gut-homing plasmablasts (ß7+) in VDZ-treated patients, pointing to gut-associated lymphoid tissue (GALT) targeting by VDZ. Murine Peyer's patches (PP) demonstrated a significant loss cellularity associated with reduction in follicular B cells, including a unique population of epithelium-associated B cells, following anti-α4ß7 antibody (mAb) administration. Photoconvertible (KikGR) mice unequivocally demonstrated impaired cellular entry into PPs in anti-α4ß7 mAb treated mice. In VDZ-treated, but not anti-tumor necrosis factor-treated UC patients, lymphoid aggregate size was significantly reduced in treatment responders compared to non-responders, with an independent validation cohort further confirming these data. GALT targeting represents a novel MOA of α4ß7-targeted therapies, with major implications for this therapeutic paradigm in UC, and for the development of new therapeutic strategies.

Bruton's Tyrosine Kinase Supports Gut Mucosal Immunity and Commensal Microbiome Recognition in Autoimmune Arthritis.

Bruton's tyrosine kinase (Btk) deficiency preferentially eliminates autoreactive B cells while sparing normal humoral responses, but has not been studied in mucosal immunity. Commensal microbes and intact BTK signaling have been independently shown to be essential for arthritis development in K/BxN mice. Here, we examine how BTK-mediated signaling interfaces with the gut microbiome. Btk-deficient K/BxN mice were found to have small Peyer's Patches with reduced germinal center and IgA class-switched B cells. IgA-switched plasma cells in small intestines were reduced, especially in villi of Btk-deficient mice. IgH CDR3 sequencing showed similar V gene diversity and somatic hypermutation frequency despite Btk deficiency but showed reduced CDR3 amino acid polarity, suggesting potential qualitative differences in the gut plasma cell repertoire. Small intestinal IgA was low and IgA coating of commensal bacteria was reduced. IgA-seq showed a shift in small intestinal microbes that are normally IgA-coated into the uncoated fraction in Btk-deficient mice. Overall, this study shows that BTK supports normal intestinal IgA development in response to commensals. This manuscript was previously published as a preprint at: https://www.biorxiv.org/content/10.1101/2021.03.10.434762v2.

Epithelial HNF4A shapes the intraepithelial lymphocyte compartment via direct regulation of immune signaling molecules.

Hepatocyte nuclear factor 4 α (HNF4A) is a highly conserved nuclear receptor that has been associated with ulcerative colitis. In mice, HNF4A is indispensable for the maintenance of intestinal homeostasis, yet the underlying mechanisms are poorly characterized. Here, we demonstrate that the expression of HNF4A in intestinal epithelial cells (IECs) is required for the proper development and composition of the intraepithelial lymphocyte (IEL) compartment. HNF4A directly regulates expression of immune signaling molecules including butyrophilin-like (Btnl) 1, Btnl6, H2-T3, and Clec2e that control IEC-IEL crosstalk. HNF4A selectively enhances the expansion of natural IELs that are TCRγδ+ or TCRαß+CD8αα+ to shape the composition of IEL compartment. In the small intestine, HNF4A cooperates with its paralog HNF4G, to drive expression of immune signaling molecules. Moreover, the HNF4A-BTNL regulatory axis is conserved in human IECs. Collectively, these findings underscore the importance of HNF4A as a conserved transcription factor controlling IEC-IEL crosstalk and suggest that HNF4A maintains intestinal homeostasis through regulation of the IEL compartment.

CD8+ T Cells Require ITK-Mediated TCR Signaling for Migration to the Intestine.

The Tec kinase IL-2-inducible T cell kinase (ITK) regulates the expression of TCR-induced genes. Itk-/- T cell responses are impaired but not absent. ITK inhibition prevented colitis disease progression and impaired T cell migration to the colon in mice. To examine the function of ITK in T cell migration to the intestine, we examined the number of gut T cells in Itk-/- mice and then evaluated their expression of gut-homing receptors. Combined with in vitro murine T cell stimulation and in vivo migration assay using congenic B6 mice, we demonstrated an essential role for ITK in T cell migration to the intestine in mice. Reconstitution of Itk-/- mouse CD8+ T cells with IFN regulatory factor 4 restored gut-homing properties, providing mechanistic insight into the function of ITK-mediated signaling in CD8+ T cell migration to the intestinal mucosa in mice.