A chemogenetic screen reveals that Trpv1-expressing neurons control regulatory T cells in the gut.

Neuroimmune cross-talk participates in intestinal tissue homeostasis and host defense. However, the matrix of interactions between arrays of molecularly defined neuron subsets and of immunocyte lineages remains unclear. We used a chemogenetic approach to activate eight distinct neuronal subsets, assessing effects by deep immunophenotyping, microbiome profiling, and immunocyte transcriptomics in intestinal organs. Distinct immune perturbations followed neuronal activation: Nitrergic neurons regulated T helper 17 (TH17)-like cells, and cholinergic neurons regulated neutrophils. Nociceptor neurons, expressing Trpv1, elicited the broadest immunomodulation, inducing changes in innate lymphocytes, macrophages, and RORγ+ regulatory T (Treg) cells. Neuroanatomical, genetic, and pharmacological follow-up showed that Trpv1+ neurons in dorsal root ganglia decreased Treg cell numbers via the neuropeptide calcitonin gene-related peptide (CGRP). Given the role of these neurons in nociception, these data potentially link pain signaling with gut Treg cell function.

Expansion of mammary intraepithelial lymphocytes and intestinal inputs shape T cell dynamics in lactogenesis.

Pregnancy brings about profound changes to the mammary gland in preparation for lactation. Changes in immunocyte populations that accompany this rapid remodeling are incompletely understood. We comprehensively analyzed mammary T cells through all parous stages, revealing a marked increase in CD4+ and CD8+ T effector cells in late pregnancy and lactation. T cell expansion was partly dependent on microbial signals and included an increase in TCRαß+CD8αα+ cells with strong cytotoxic markers, located in the epithelium, that resemble intraepithelial lymphocytes of mucosal tissues. This relationship was substantiated by demonstrating T cell migration from gut to mammary gland in late pregnancy, by TCR clonotypes shared by intestine and mammary tissue in the same mouse, including intriguing gut TCR families. Putative counterparts of CD8αα+ IELs were found in human milk. Mammary T cells are thus poised to manage the transition from a non-mucosal tissue to a mucosal barrier during lactogenesis.

The meninges host a unique compartment of regulatory T cells that bulwarks adult hippocampal neurogenesis.

Our knowledge about the meningeal immune system has recently burgeoned, particularly our understanding of how innate and adaptive effector cells are mobilized to meet brain challenges. However, information on how meningeal immunocytes guard brain homeostasis in healthy individuals remains sparse. This study highlights the heterogeneous and polyfunctional regulatory-T (Treg) cell compartment in the meninges. A Treg subtype specialized in controlling Th1-cell responses and another known to control responses in B-cell follicles were substantial components of this compartment, foretelling that punctual Treg-cell ablation rapidly unleashed interferon-gamma production by meningeal lymphocytes, unlocked their access to the brain parenchyma, and altered meningeal B-cell profiles. Distally, the hippocampus assumed a reactive state, with morphological and transcriptional changes in multiple glial-cell types; within the dentate gyrus, neural stem cells showed exacerbated death and desisted from further differentiation, associated with inhibition of spatial-reference memory. Thus, meningeal Treg cells are a multifaceted bulwark to brain homeostasis at steady-state. One sentence summary: A distinct population of regulatory T cells in the murine meninges safeguards homeostasis by keeping local interferon-γ-producing lymphocytes in check, thereby preventing their invasion of the parenchyma, activation of hippocampal glial cells, death of neural stem cells, and memory decay.

Microheterogeneity in the Kinetics and Sex-Specific Response to Type I IFN.

The response to type I IFNs involves the rapid induction of prototypical IFN signature genes (ISGs). It is not known whether the tightly controlled ISG expression observed at the cell population level correctly represents the coherent responses of individual cells or whether it masks some heterogeneity in gene modules and/or responding cells. We performed a time-resolved single-cell analysis of the first 3 h after in vivo IFN stimulation in macrophages and CD4+ T and B lymphocytes from mice. All ISGs were generally induced in concert, with no clear cluster of faster- or slower-responding ISGs. Response kinetics differed between cell types: mostly homogeneous for macrophages, but with far more kinetic diversity among B and T lymphocytes, which included a distinct subset of nonresponsive cells. Velocity analysis confirmed the differences between macrophages in which the response progressed throughout the full 3 h, versus B and T lymphocytes in which it was rapidly curtailed by negative feedback and revealed differences in transcription rates between the lineages. In all cell types, female cells responded faster than their male counterparts. The ISG response thus seems to proceed as a homogeneous gene block, but with kinetics that vary between immune cell types and with sex differences that might underlie differential outcomes of viral infections.

Adipose-tissue Treg cells restrain differentiation of stromal adipocyte precursors to promote insulin sensitivity and metabolic homeostasis.

Regulatory T (Treg) cells in epidydimal visceral adipose tissue (eVAT) of lean mice and humans regulate metabolic homeostasis. We found that constitutive or punctual depletion of eVAT-Treg cells reined in the differentiation of stromal adipocyte precursors. Co-culture of these precursors with conditional medium from eVAT-Treg cells limited their differentiation in vitro, suggesting a direct effect. Transcriptional comparison of adipocyte precursors, matured in the presence or absence of the eVAT-Treg-conditioned medium, identified the oncostatin-M (OSM) signaling pathway as a key distinction. Addition of OSM to in vitro cultures blocked the differentiation of adipocyte precursors, while co-addition of anti-OSM antibodies reversed the ability of the eVAT-Treg-conditioned medium to inhibit in vitro adipogenesis. Genetic depletion of OSM (specifically in Treg) cells or of the OSM receptor (specifically on stromal cells) strongly impaired insulin sensitivity and related metabolic indices. Thus, Treg-cell-mediated control of local progenitor cells maintains adipose tissue and metabolic homeostasis, a regulatory axis seemingly conserved in humans.

AIRE relies on Z-DNA to flag gene targets for thymic T cell tolerization.

AIRE is an unconventional transcription factor that enhances the expression of thousands of genes in medullary thymic epithelial cells and promotes clonal deletion or phenotypic diversion of self-reactive T cells1-4. The biological logic of AIRE's target specificity remains largely unclear as, in contrast to many transcription factors, it does not bind to a particular DNA sequence motif. Here we implemented two orthogonal approaches to investigate AIRE's cis-regulatory mechanisms: construction of a convolutional neural network and leveraging natural genetic variation through analysis of F1 hybrid mice5. Both approaches nominated Z-DNA and NFE2-MAF as putative positive influences on AIRE's target choices. Genome-wide mapping studies revealed that Z-DNA-forming and NFE2L2-binding motifs were positively associated with the inherent ability of a gene's promoter to generate DNA double-stranded breaks, and promoters showing strong double-stranded break generation were more likely to enter a poised state with accessible chromatin and already-assembled transcriptional machinery. Consequently, AIRE preferentially targets genes with poised promoters. We propose a model in which Z-DNA anchors the AIRE-mediated transcriptional program by enhancing double-stranded break generation and promoter poising. Beyond resolving a long-standing mechanistic conundrum, these findings suggest routes for manipulating T cell tolerance.

Gut complement induced by the microbiota combats pathogens and spares commensals.

Canonically, the complement system is known for its rapid response to remove microbes in the bloodstream. However, relatively little is known about a functioning complement system on intestinal mucosal surfaces. Herein, we report the local synthesis of complement component 3 (C3) in the gut, primarily by stromal cells. C3 is expressed upon commensal colonization and is regulated by the composition of the microbiota in healthy humans and mice, leading to an individual host's specific luminal C3 levels. The absence of membrane attack complex (MAC) components in the gut ensures that C3 deposition does not result in the lysis of commensals. Pathogen infection triggers the immune system to recruit neutrophils to the infection site for pathogen clearance. Basal C3 levels directly correlate with protection against enteric infection. Our study reveals the gut complement system as an innate immune mechanism acting as a vigilant sentinel that combats pathogens and spares commensals.

A dynamic atlas of immunocyte migration from the gut.

Dysbiosis in the gut microbiota affects several systemic diseases, possibly by driving the migration of perturbed intestinal immunocytes to extraintestinal tissues. Combining Kaede photoconvertible mice and single-cell genomics, we generated a detailed map of migratory trajectories from the colon, at baseline, and in several models of intestinal and extraintestinal inflammation. All lineages emigrated from the colon in an S1P-dependent manner. B lymphocytes represented the largest contingent, with the unexpected circulation of nonexperienced follicular B cells, which carried a gut-imprinted transcriptomic signature. T cell emigration included distinct groups of RORγ+ and IEL-like CD160+ subsets. Gut inflammation curtailed emigration, except for dendritic cells disseminating to lymph nodes. Colon-emigrating cells distributed differentially to distinct sites of extraintestinal models of inflammation (psoriasis-like skin, arthritic synovium, and tumors). Thus, specific cellular trails originating in the gut and influenced by microbiota may shape peripheral immunity in varied ways.

Adipose-tissue regulatory T cells are a consortium of subtypes that evolves with age and diet.

Foxp3+CD4+ regulatory T (Treg) cells found within tissues regulate local immunity, inflammation, and homeostasis. Tregs in epididymal visceral adipose tissue (eVAT) are critical regulators of local and systemic inflammation and metabolism. During aging and under obesogenic conditions, eVAT Tregs undergo transcriptional and phenotypic changes and are important for containing inflammation and normalizing metabolic indices. We have employed single-cell RNA sequencing, single-cell Tra and Trb sequencing, adoptive transfers, photoconvertible mice, cellular interaction analyses, and in vitro cultures to dissect the evolving heterogeneity of eVAT Tregs with aging and obesity. Distinct Treg subtypes with distinguishable gene expression profiles and functional roles were enriched at differing ages and with differing diets. Like those in lean mice, eVAT Tregs in obese mice were not primarily recruited from the circulation but instead underwent local expansion and had a distinct and diversified T cell receptor repertoire. The different eVAT-Treg subtypes were specialized in different functions; for example, the subtypes enriched in lean, but not obese, mice suppressed adipogenesis. The existence of functionally divergent eVAT-Treg subtypes in response to obesogenic conditions presents possibilities for precision therapeutics in the context of obesity.

Homeostatic, repertoire and transcriptional relationships between colon T regulatory cell subsets.

Foxp3+ regulatory T cells (Tregs) in the colon are key to promoting peaceful coexistence with symbiotic microbes. Differentiated in either thymic or peripheral locations, and modulated by microbes and other cellular influencers, colonic Treg subsets have been identified through key transcription factors (TFs; Helios, Rorγ, Gata3, and cMaf), but their interrelationships are unclear. Applying a multimodal array of immunologic, genomic, and microbiological assays, we find more overlap than expected between populations. The key TFs (Rorγ, Helios, Gata3, and cMaf) play different roles, some essential for subset identity, others driving functional gene signatures. Functional divergence was clearest under challenge. Single-cell genomics revealed a spectrum of phenotypes between the Helios+ and Rorγ+ poles, different Treg-inducing bacteria inducing the same Treg phenotypes to varying degrees, not distinct populations. TCR repertoires in monocolonized mice revealed that Helios+ and Rorγ+ Tregs are related and cannot be uniquely equated to tTreg and pTreg. Comparison of spleen and colon repertoires revealed that 2 to 5% of clonotypes are shared between the locations. We propose that rather than the origin of their differentiation, tissue-specific cues dictate the spectrum of colonic Treg phenotypes.

Lymph node stromal cell responses to perinatal T cell waves, a temporal atlas.

The perinatal period is a critical time window in establishing T cell tolerance. Regulatory T cells (Tregs) made during the first 2 wk of life are key drivers of perinatal tolerance induction, but how these cells are generated and operate has not been established. To elucidate the unique environment murine perinatal Tregs encounter within the lymph nodes (LNs) as they first emerge from the thymus, and how it evolves over the succeeding days, we employed single-cell RNA sequencing to generate an atlas of the early LN niche. A highly dynamic picture emerged, the stromal cell compartment showing the most striking changes and putative interactions with other LN cell compartments. In particular, LN stromal cells showed increasing potential for lymphocyte interactions with age. Analogous studies on mice lacking α:ß T cells or enriched for autoreactive α:ß T cells revealed an acute stromal cell response to α:ß T cell dysfunction, largely reflecting dysregulation of Tregs. Punctual ablation of perinatal Tregs induced stromal cell activation that was dependent on both interferon-gamma signaling and activation of conventional CD4+ T cells. These findings elucidate some of the earliest cellular and molecular events in perinatal induction of T cell tolerance, providing a framework for future explorations.

A discrete 'early-responder' stromal-cell subtype orchestrates immunocyte recruitment to injured tissue.

Following acute injury, stromal cells promote tissue regeneration by a diversity of mechanisms. Time-resolved single-cell RNA sequencing of muscle mesenchymal stromal cells (MmSCs) responding to acute injury identified an 'early-responder' subtype that spiked on day 1 and expressed a notable array of transcripts encoding immunomodulators. IL-1ß, TNF-α and oncostatin M each strongly and rapidly induced MmSCs transcribing this immunomodulatory program. Macrophages amplified the program but were not strictly required for its induction. Transfer of the inflammatory MmSC subtype, tagged with a unique surface marker, into healthy hindlimb muscle induced inflammation primarily driven by neutrophils and macrophages. Among the abundant inflammatory transcripts produced by this subtype, Cxcl5 was stroma-specific and highly upregulated with injury. Depletion of this chemokine early after injury revealed a substantial impact on recruitment of neutrophils, a prolongation of inflammation to later times and an effect on tissue regeneration. Mesenchymal stromal cell subtypes expressing a comparable inflammatory program were found in a mouse model of muscular dystrophy and in several other tissues and pathologies in both mice and humans. These 'early-responder' mesenchymal stromal cells, already in place, permit rapid and coordinated mobilization and amplification of critical cell collaborators in response to injury.

Regulatory T cells shield muscle mitochondria from interferon-γ-mediated damage to promote the beneficial effects of exercise.

Exercise enhances physical performance and reduces the risk of many disorders such as cardiovascular disease, type 2 diabetes, dementia, and cancer. Exercise characteristically incites an inflammatory response, notably in skeletal muscles. Although some effector mechanisms have been identified, regulatory elements activated in response to exercise remain obscure. Here, we have addressed the roles of Foxp3+CD4+ regulatory T cells (Tregs) in the healthful activities of exercise via immunologic, transcriptomic, histologic, metabolic, and biochemical analyses of acute and chronic exercise models in mice. Exercise rapidly induced expansion of the muscle Treg compartment, thereby guarding against overexuberant production of interferon-γ and consequent metabolic disruptions, particularly mitochondrial aberrancies. The performance-enhancing effects of exercise training were dampened in the absence of Tregs. Thus, exercise is a natural Treg booster with therapeutic potential in disease and aging contexts.

FoxP3 recognizes microsatellites and bridges DNA through multimerization.

FoxP3 is a transcription factor (TF) essential for development of regulatory T cells (Tregs), a branch of T cells that suppress excessive inflammation and autoimmunity 1-5 . Molecular mechanisms of FoxP3, however, remain elusive. We here show that FoxP3 utilizes the Forkhead domain--a DNA binding domain (DBD) that is commonly thought to function as a monomer or dimer--to form a higher-order multimer upon binding to T n G repeat microsatellites. A cryo-electron microscopy structure of FoxP3 in complex with T 3 G repeats reveals a ladder-like architecture, where two double-stranded DNA molecules form the two "side rails" bridged by five pairs of FoxP3 molecules, with each pair forming a "rung". Each FoxP3 subunit occupies TGTTTGT within the repeats in the manner indistinguishable from that of FoxP3 bound to the Forkhead consensus motif (FKHM; TGTTTAC). Mutations in the "intra-rung" interface impair T n G repeat recognition, DNA bridging and cellular functions of FoxP3, all without affecting FKHM binding. FoxP3 can tolerate variable "inter-rung" spacings, explaining its broad specificity for T n G repeat-like sequences in vivo and in vitro . Both FoxP3 orthologs and paralogs show similar T n G repeat recognition and DNA bridging. These findings thus reveal a new mode of DNA recognition that involves TF homo-multimerization and DNA bridging, and further implicates microsatellites in transcriptional regulation and diseases.

FOXP3 recognizes microsatellites and bridges DNA through multimerization.

FOXP3 is a transcription factor that is essential for the development of regulatory T cells, a branch of T cells that suppress excessive inflammation and autoimmunity1-5. However, the molecular mechanisms of FOXP3 remain unclear. Here we here show that FOXP3 uses the forkhead domain-a DNA-binding domain that is commonly thought to function as a monomer or dimer-to form a higher-order multimer after binding to TnG repeat microsatellites. The cryo-electron microscopy structure of FOXP3 in a complex with T3G repeats reveals a ladder-like architecture, whereby two double-stranded DNA molecules form the two 'side rails' bridged by five pairs of FOXP3 molecules, with each pair forming a 'rung'. Each FOXP3 subunit occupies TGTTTGT within the repeats in a manner that is indistinguishable from that of FOXP3 bound to the forkhead consensus motif (TGTTTAC). Mutations in the intra-rung interface impair TnG repeat recognition, DNA bridging and the cellular functions of FOXP3, all without affecting binding to the forkhead consensus motif. FOXP3 can tolerate variable inter-rung spacings, explaining its broad specificity for TnG-repeat-like sequences in vivo and in vitro. Both FOXP3 orthologues and paralogues show similar TnG repeat recognition and DNA bridging. These findings therefore reveal a mode of DNA recognition that involves transcription factor homomultimerization and DNA bridging, and further implicates microsatellites in transcriptional regulation and diseases.

Mutations from patients with IPEX ported to mice reveal different patterns of FoxP3 and Treg dysfunction.

Mutations of the transcription factor FoxP3 in patients with "IPEX" (immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome) disrupt regulatory T cells (Treg), causing an array of multiorgan autoimmunity. To understand the functional impact of mutations across FoxP3 domains, without genetic and environmental confounders, six human FOXP3 missense mutations are engineered into mice. Two classes of mutations emerge from combined immunologic and genomic analyses. A mutation in the DNA-binding domain shows the same lymphoproliferation and multiorgan infiltration as complete FoxP3 knockouts but delayed by months. Tregs expressing this mutant FoxP3 are destabilized by normal Tregs in heterozygous females compared with hemizygous males. Mutations in other domains affect chromatin opening differently, involving different cofactors and provoking more specific autoimmune pathology (dermatitis, colitis, diabetes), unmasked by immunological challenges or incrossing NOD autoimmune-susceptibility alleles. This work establishes that IPEX disease heterogeneity results from the actual mutations, combined with genetic and environmental perturbations, explaining then the intra-familial variation in IPEX.

Hnf4 activates mimetic-cell enhancers to recapitulate gut and liver development within the thymus.

Mimetic cells are medullary thymic epithelial cells (mTECs) that mimic extra-thymic cell types to tolerize T cells to self-antigens. Here, we dissected the biology of entero-hepato mTECs, mimetic cells expressing gut- and liver-associated transcripts. Entero-hepato mTECs conserved their thymic identity yet accessed wide swaths of enterocyte chromatin and transcriptional programs via the transcription factors Hnf4α and Hnf4γ. Deletion of Hnf4α and Hnf4γ in TECs ablated entero-hepato mTECs and downregulated numerous gut- and liver-associated transcripts, with a primary contribution from Hnf4γ. Loss of Hnf4 impaired enhancer activation and CTCF redistribution in mTECs but did not impact Polycomb-mediated repression or promoter-proximal histone marks. By single-cell RNA sequencing, Hnf4 loss produced three distinct effects on mimetic cell state, fate, and accumulation. Serendipitously, a requirement for Hnf4 in microfold mTECs was discovered, which exposed a requirement for Hnf4γ in gut microfold cells and the IgA response. Study of Hnf4 in entero-hepato mTECs thus revealed mechanisms of gene control in the thymus and periphery alike.

Homeostatic, repertoire and transcriptional relationships between colon T regulatory cell subsets.

Foxp3 + regulatory T cells (Tregs) in the colon are key to promoting peaceful co-existence with symbiotic microbes. Differentiated in either thymic or peripheral locations, and modulated by microbes and other cellular influencers, colonic Treg subsets have been identified through key transcription factors (TF; Helios, Rorg, Gata3, cMaf), but their inter-relationships are unclear. Applying a multimodal array of immunologic, genomic, and microbiological assays, we find more overlap than expected between populations. The key TFs play different roles, some essential for subset identity, others driving functional gene signatures. Functional divergence was clearest under challenge. Single-cell genomics revealed a spectrum of phenotypes between the Helios+ and Rorγ+ poles, different Treg-inducing bacteria inducing the same Treg phenotypes to varying degrees, not distinct populations. TCR clonotypes in monocolonized mice revealed that Helios+ and Rorγ+ Tregs are related, and cannot be uniquely equated to tTreg and pTreg. We propose that rather than the origin of their differentiation, tissue-specific cues dictate the spectrum of colonic Treg phenotypes.

Regulatory T cells in the face of the intestinal microbiota.

Regulatory T cells (Treg cells) are key players in ensuring a peaceful coexistence with microorganisms and food antigens at intestinal borders. Startling new information has appeared in recent years on their diversity, the importance of the transcription factor FOXP3, how T cell receptors influence their fate and the unexpected and varied cellular partners that influence Treg cell homeostatic setpoints. We also revisit some tenets, maintained by the echo chambers of Reviews, that rest on uncertain foundations or are a subject of debate.

A variegated model of transcription factor function in the immune system.

Specific combinations of transcription factors (TFs) control the gene expression programs that underlie specialized immune responses. Previous models of TF function in immunocytes had restricted each TF to a single functional categorization [e.g., lineage-defining (LDTFs) vs. signal-dependent TFs (SDTFs)] within one cell type. Synthesizing recent results, we instead propose a variegated model of immunological TF function, whereby many TFs have flexible and different roles across distinct cell states, contributing to cell phenotypic diversity. We discuss evidence in support of this variegated model, describe contextual inputs that enable TF diversification, and look to the future to imagine warranted experimental and computational tools to build quantitative and predictive models of immunocyte gene regulatory networks.