BACH2 regulates diversification of regulatory and proinflammatory chromatin states in TH17 cells.

Interleukin-17 (IL-17)-producing helper T (TH17) cells are heterogenous and consist of nonpathogenic TH17 (npTH17) cells that contribute to tissue homeostasis and pathogenic TH17 (pTH17) cells that mediate tissue inflammation. Here, we characterize regulatory pathways underlying TH17 heterogeneity and discover substantial differences in the chromatin landscape of npTH17 and pTH17 cells both in vitro and in vivo. Compared to other CD4+ T cell subsets, npTH17 cells share accessible chromatin configurations with regulatory T cells, whereas pTH17 cells exhibit features of both npTH17 cells and type 1 helper T (TH1) cells. Integrating single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq), we infer self-reinforcing and mutually exclusive regulatory networks controlling different cell states and predicted transcription factors regulating TH17 cell pathogenicity. We validate that BACH2 promotes immunomodulatory npTH17 programs and restrains proinflammatory TH1-like programs in TH17 cells in vitro and in vivo. Furthermore, human genetics implicate BACH2 in multiple sclerosis. Overall, our work identifies regulators of TH17 heterogeneity as potential targets to mitigate autoimmunity.

Stem-like intestinal Th17 cells give rise to pathogenic effector T cells during autoimmunity.

While intestinal Th17 cells are critical for maintaining tissue homeostasis, recent studies have implicated their roles in the development of extra-intestinal autoimmune diseases including multiple sclerosis. However, the mechanisms by which tissue Th17 cells mediate these dichotomous functions remain unknown. Here, we characterized the heterogeneity, plasticity, and migratory phenotypes of tissue Th17 cells in vivo by combined fate mapping with profiling of the transcriptomes and TCR clonotypes of over 84,000 Th17 cells at homeostasis and during CNS autoimmune inflammation. Inter- and intra-organ single-cell analyses revealed a homeostatic, stem-like TCF1+ IL-17+ SLAMF6+ population that traffics to the intestine where it is maintained by the microbiota, providing a ready reservoir for the IL-23-driven generation of encephalitogenic GM-CSF+ IFN-γ+ CXCR6+ T cells. Our study defines a direct in vivo relationship between IL-17+ non-pathogenic and GM-CSF+ and IFN-γ+ pathogenic Th17 populations and provides a mechanism by which homeostatic intestinal Th17 cells direct extra-intestinal autoimmune disease.

Metabolic modeling of single Th17 cells reveals regulators of autoimmunity.

Metabolism is a major regulator of immune cell function, but it remains difficult to study the metabolic status of individual cells. Here, we present Compass, an algorithm to characterize cellular metabolic states based on single-cell RNA sequencing and flux balance analysis. We applied Compass to associate metabolic states with T helper 17 (Th17) functional variability (pathogenic potential) and recovered a metabolic switch between glycolysis and fatty acid oxidation, akin to known Th17/regulatory T cell (Treg) differences, which we validated by metabolic assays. Compass also predicted that Th17 pathogenicity was associated with arginine and downstream polyamine metabolism. Indeed, polyamine-related enzyme expression was enhanced in pathogenic Th17 and suppressed in Treg cells. Chemical and genetic perturbation of polyamine metabolism inhibited Th17 cytokines, promoted Foxp3 expression, and remodeled the transcriptome and epigenome of Th17 cells toward a Treg-like state. In vivo perturbations of the polyamine pathway altered the phenotype of encephalitogenic T cells and attenuated tissue inflammation in CNS autoimmunity.

TH17 cell heterogeneity and its role in tissue inflammation.

Since their discovery almost two decades ago, interleukin-17-producing CD4+ T cells (TH17 cells) have been implicated in the pathogenesis of multiple autoimmune and inflammatory disorders. In addition, TH17 cells have been found to play an important role in tissue homeostasis, especially in the intestinal mucosa. Recently, the use of single-cell technologies, along with fate mapping and various mutant mouse models, has led to substantial progress in the understanding of TH17 cell heterogeneity in tissues and of TH17 cell plasticity leading to alternative T cell states and differing functions. In this Review, we discuss the heterogeneity of TH17 cells and the role of this heterogeneity in diverse functions of TH17 cells from homeostasis to tissue inflammation. In addition, we discuss TH17 cell plasticity and its incorporation into the current understanding of T cell subsets and alternative views on the role of TH17 cells in autoimmune and inflammatory diseases.

Targeting PGLYRP1 promotes antitumor immunity while inhibiting autoimmune neuroinflammation.

Co-inhibitory and checkpoint molecules suppress T cell function in the tumor microenvironment, thereby rendering T cells dysfunctional. Although immune checkpoint blockade is a successful treatment option for multiple human cancers, severe autoimmune-like adverse effects can limit its application. Here, we show that the gene encoding peptidoglycan recognition protein 1 (PGLYRP1) is highly coexpressed with genes encoding co-inhibitory molecules, indicating that it might be a promising target for cancer immunotherapy. Genetic deletion of Pglyrp1 in mice led to decreased tumor growth and an increased activation/effector phenotype in CD8+ T cells, suggesting an inhibitory function of PGLYRP1 in CD8+ T cells. Surprisingly, genetic deletion of Pglyrp1 protected against the development of experimental autoimmune encephalomyelitis, a model of autoimmune disease in the central nervous system. PGLYRP1-deficient myeloid cells had a defect in antigen presentation and T cell activation, indicating that PGLYRP1 might function as a proinflammatory molecule in myeloid cells during autoimmunity. These results highlight PGLYRP1 as a promising target for immunotherapy that, when targeted, elicits a potent antitumor immune response while protecting against some forms of tissue inflammation and autoimmunity.

Gut CD4+ T cell phenotypes are a continuum molded by microbes, not by TH archetypes.

CD4+ effector lymphocytes (Teff) are traditionally classified by the cytokines they produce. To determine the states that Teff cells actually adopt in frontline tissues in vivo, we applied single-cell transcriptome and chromatin analyses to colonic Teff cells in germ-free or conventional mice or in mice after challenge with a range of phenotypically biasing microbes. Unexpected subsets were marked by the expression of the interferon (IFN) signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic helper T cell (TH) subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as TH markers were distributed in a polarized continuum, which was functionally validated. Clones derived from single progenitors gave rise to both IFN-γ- and interleukin (IL)-17-producing cells. Most of the transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activities of activator protein (AP)-1 and IFN-regulatory factor (IRF) transcription factor (TF) families, not the canonical subset master regulators T-bet, GATA3 or RORγ.

Transcription factor TCF1 binds to RORγt and orchestrates a regulatory network that determines homeostatic Th17 cell state.

T helper (Th) 17 cells encompass a spectrum of cell states, including cells that maintain homeostatic tissue functions and pro-inflammatory cells that can drive autoimmune tissue damage. Identifying regulators that determine Th17 cell states can identify ways to control tissue inflammation and restore homeostasis. Here, we found that interleukin (IL)-23, a cytokine critical for inducing pro-inflammatory Th17 cells, decreased transcription factor T cell factor 1 (TCF1) expression. Conditional deletion of TCF1 in mature T cells increased the pro-inflammatory potential of Th17 cells, even in the absence of IL-23 receptor signaling, and conferred pro-inflammatory potential to homeostatic Th17 cells. Conversely, sustained TCF1 expression decreased pro-inflammatory Th17 potential. Mechanistically, TCF1 bound to RORγt, thereby interfering with its pro-inflammatory functions, and orchestrated a regulatory network that determined Th17 cell state. Our findings identify TCF1 as a major determinant of Th17 cell state and provide important insight for the development of therapies for Th17-driven inflammatory diseases.

T Helper Cell Cytokines Modulate Intestinal Stem Cell Renewal and Differentiation.

In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5+ ISCs. We show that MHCII+ Lgr5+ ISCs are non-conventional antigen-presenting cells in co-cultures with CD4+ T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5+ ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. In vivo genetic perturbation of Th cells or MHCII expression on Lgr5+ ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5+ ISCs, thus, orchestrate tissue-wide responses to external signals.

Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.

Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

Disruption of the IL-33-ST2-AKT signaling axis impairs neurodevelopment by inhibiting microglial metabolic adaptation and phagocytic function.

To accommodate the changing needs of the developing brain, microglia must undergo substantial morphological, phenotypic, and functional reprogramming. Here, we examined whether cellular metabolism regulates microglial function during neurodevelopment. Microglial mitochondria bioenergetics correlated with and were functionally coupled to phagocytic activity in the developing brain. Transcriptional profiling of microglia with diverse metabolic profiles revealed an activation signature wherein the interleukin (IL)-33 signaling axis is associated with phagocytic activity. Genetic perturbation of IL-33 or its receptor ST2 led to microglial dystrophy, impaired synaptic function, and behavioral abnormalities. Conditional deletion of Il33 from astrocytes or Il1rl1, encoding ST2, in microglia increased susceptibility to seizures. Mechanistically, IL-33 promoted mitochondrial activity and phagocytosis in an AKT-dependent manner. Mitochondrial metabolism and AKT activity were temporally regulated in vivo. Thus, a microglia-astrocyte circuit mediated by the IL-33-ST2-AKT signaling axis supports microglial metabolic adaptation and phagocytic function during early development, with implications for neurodevelopmental and neuropsychiatric disorders.

Induction of a colitogenic phenotype in Th1-like cells depends on interleukin-23 receptor signaling.

Interleukin-23 receptor plays a critical role in inducing inflammation and autoimmunity. Here, we report that Th1-like cells differentiated in vitro with IL-12 + IL-21 showed similar IL-23R expression to that of pathogenic Th17 cells using eGFP reporter mice. Fate mapping established that these cells did not transition through a Th17 cell state prior to becoming Th1-like cells, and we observed their emergence in vivo in the T cell adoptive transfer colitis model. Using IL-23R-deficient Th1-like cells, we demonstrated that IL-23R was required for the development of a highly colitogenic phenotype. Single-cell RNA sequencing analysis of intestinal T cells identified IL-23R-dependent genes in Th1-like cells that differed from those expressed in Th17 cells. The perturbation of one of these regulators (CD160) in Th1-like cells inhibited the induction of colitis. We thus uncouple IL-23R as a purely Th17 cell-specific factor and implicate IL-23R signaling as a pathogenic driver in Th1-like cells inducing tissue inflammation.

Deciphering cell states and genealogies of human haematopoiesis.

The human blood system is maintained through the differentiation and massive amplification of a limited number of long-lived haematopoietic stem cells (HSCs)1. Perturbations to this process underlie diverse diseases, but the clonal contributions to human haematopoiesis and how this changes with age remain incompletely understood. Although recent insights have emerged from barcoding studies in model systems2-5, simultaneous detection of cell states and phylogenies from natural barcodes in humans remains challenging. Here we introduce an improved, single-cell lineage-tracing system based on deep detection of naturally occurring mitochondrial DNA mutations with simultaneous readout of transcriptional states and chromatin accessibility. We use this system to define the clonal architecture of HSCs and map the physiological state and output of clones. We uncover functional heterogeneity in HSC clones, which is stable over months and manifests as both differences in total HSC output and biases towards the production of different mature cell types. We also find that the diversity of HSC clones decreases markedly with age, leading to an oligoclonal structure with multiple distinct clonal expansions. Our study thus provides a clonally resolved and cell-state-aware atlas of human haematopoiesis at single-cell resolution, showing an unappreciated functional diversity of human HSC clones and, more broadly, paving the way for refined studies of clonal dynamics across a range of tissues in human health and disease.

B-cell-specific checkpoint molecules that regulate anti-tumour immunity.

The role of B cells in anti-tumour immunity is still debated and, accordingly, immunotherapies have focused on targeting T and natural killer cells to inhibit tumour growth1,2. Here, using high-throughput flow cytometry as well as bulk and single-cell RNA-sequencing and B-cell-receptor-sequencing analysis of B cells temporally during B16F10 melanoma growth, we identified a subset of B cells that expands specifically in the draining lymph node over time in tumour-bearing mice. The expanding B cell subset expresses the cell surface molecule T cell immunoglobulin and mucin domain 1 (TIM-1, encoded by Havcr1) and a unique transcriptional signature, including multiple co-inhibitory molecules such as PD-1, TIM-3, TIGIT and LAG-3. Although conditional deletion of these co-inhibitory molecules on B cells had little or no effect on tumour burden, selective deletion of Havcr1 in B cells both substantially inhibited tumour growth and enhanced effector T cell responses. Loss of TIM-1 enhanced the type 1 interferon response in B cells, which augmented B cell activation and increased antigen presentation and co-stimulation, resulting in increased expansion of tumour-specific effector T cells. Our results demonstrate that manipulation of TIM-1-expressing B cells enables engagement of the second arm of adaptive immunity to promote anti-tumour immunity and inhibit tumour growth.

Calcitonin Gene-Related Peptide Negatively Regulates Alarmin-Driven Type 2 Innate Lymphoid Cell Responses.

Neuroimmune interactions have emerged as critical modulators of allergic inflammation, and type 2 innate lymphoid cells (ILC2s) are an important cell type for mediating these interactions. Here, we show that ILC2s expressed both the neuropeptide calcitonin gene-related peptide (CGRP) and its receptor. CGRP potently inhibited alarmin-driven type 2 cytokine production and proliferation by lung ILC2s both in vitro and in vivo. CGRP induced marked changes in ILC2 expression programs in vivo and in vitro, attenuating alarmin-driven proliferative and effector responses. A distinct subset of ILCs scored highly for a CGRP-specific gene signature after in vivo alarmin stimulation, suggesting CGRP regulated this response. Finally, we observed increased ILC2 proliferation and type 2 cytokine production as well as exaggerated responses to alarmins in mice lacking the CGRP receptor. Together, these data indicate that endogenous CGRP is a critical negative regulator of ILC2 responses in vivo.

Stem-like T cells in cancer and autoimmunity.

Stem-like T cells are characterized by their ability to self-renew, survive long-term, and give rise to a heterogeneous pool of effector and memory T cells. Recent advances in single-cell RNA-sequencing (scRNA-seq) and lineage tracing technologies revealed an important role for stem-like T cells in both autoimmunity and cancer. In cancer, stem-like T cells constitute an important arm of the anti-tumor immune response by giving rise to effector T cells that mediate tumor control. In contrast, in autoimmunity stem-like T cells perform an unfavorable role by forming a reservoir of long-lived autoreactive cells that replenish the pathogenic, effector T-cell pool and thereby driving disease pathology. This review provides background on the discovery of stem-like T cells and their function in cancer and autoimmunity. Moreover, the influence of the microbiota and metabolism on the stem-like T-cell pool is summarized. Lastly, the implications of our knowledge about stem-like T cells for clinical treatment strategies for cancer and autoimmunity will be discussed.

Fas Promotes T Helper 17 Cell Differentiation and Inhibits T Helper 1 Cell Development by Binding and Sequestering Transcription Factor STAT1.

The death receptor Fas removes activated lymphocytes through apoptosis. Previous transcriptional profiling predicted that Fas positively regulates interleukin-17 (IL-17)-producing T helper 17 (Th17) cells. Here, we demonstrate that Fas promoted the generation and stability of Th17 cells and prevented their differentiation into Th1 cells. Mice with T-cell- and Th17-cell-specific deletion of Fas were protected from induced autoimmunity, and Th17 cell differentiation and stability were impaired. Fas-deficient Th17 cells instead developed a Th1-cell-like transcriptional profile, which a new algorithm predicted to depend on STAT1. Experimentally, Fas indeed bound and sequestered STAT1, and Fas deficiency enhanced IL-6-induced STAT1 activation and nuclear translocation, whereas deficiency of STAT1 reversed the transcriptional changes induced by Fas deficiency. Thus, our computational and experimental approach identified Fas as a regulator of the Th17-to-Th1 cell balance by controlling the availability of opposing STAT1 and STAT3 to have a direct impact on autoimmunity.

Beyond the Tricks: The Science and Comparative Cognition of Magic.

Magic is an art form that has fascinated humans for centuries. Recently, the techniques used by magicians to make their audience experience the impossible have attracted the attention of psychologists, who, in just a couple of decades, have produced a large amount of research regarding how these effects operate, focusing on the blind spots in perception and roadblocks in cognition that magic techniques exploit. Most recently, this investigation has given a pathway to a new line of research that uses magic effects to explore the cognitive abilities of nonhuman animals. This new branch of the scientific study of magic has already yielded new evidence illustrating the power of magic effects as a psychological tool for nonhuman animals. This review aims to give a thorough overview of the research on both the human and nonhuman perception of magic effects by critically illustrating the most prominent works of both fields of inquiry.

To know or not to know? Curiosity and the value of prospective information in animals.

Humans and other animals often seek instrumental information to strategically improve their decisions in the present. Our curiosity also leads us to acquire non-instrumental information that is not immediately useful but can be encoded in memory and stored for use in the future by means of episodic recall. Despite its adaptive benefits and central role in human cognition, questions remain about the cognitive mechanisms and evolutionary origins that underpin curiosity. Here, we comparatively review recent empirical studies that some authors have suggested reflects curiosity in nonhuman animals. We focus on findings from laboratory tasks in which individuals can choose to gain advanced information about uncertain future outcomes, even though the information cannot be used to increase future rewards and is often costly. We explore the prevalence of preferences in these tasks across animals, discuss the theoretical advances that they have promoted, and outline some limitations in contemporary research. We also discuss several features of human curiosity that can guide future empirical research aimed at characterising and understanding curiosity in animals. Though the prevalence of curiosity in animals is actively debated, we surmise that investigating behavioural candidates for curiosity-motivated behaviour in a broader range of species and contexts, should help promote theoretical advances in our understanding of cognitive principles and evolutionary pressures that support curiosity-driven behaviour.

Exploring the perceptual inabilities of Eurasian jays (Garrulus glandarius) using magic effects.

In recent years, scientists have begun to use magic effects to investigate the blind spots in our attention and perception [G. Kuhn, Experiencing the Impossible: The Science of Magic (2019); S. Macknik, S. Martinez-Conde, S. Blakeslee, Sleights of Mind: What the Neuroscience of Magic Reveals about Our Everyday Deceptions (2010)]. Recently, we suggested that similar techniques could be transferred to nonhuman animal observers and that such an endeavor would provide insight into the inherent commonalities and discrepancies in attention and perception in human and nonhuman animals [E. Garcia-Pelegrin, A. K. Schnell, C. Wilkins, N. S. Clayton, Science 369, 1424-1426 (2020)]. Here, we performed three different magic effects (palming, French drop, and fast pass) to a sample of six Eurasian jays (Garrulus glandarius). These magic effects were specifically chosen as they utilize different cues and expectations that mislead the spectator into thinking one object has or has not been transferred from one hand to the other. Results from palming and French drop experiments suggest that Eurasian jays have different expectations from humans when observing some of these effects. Specifically, Eurasian jays were not deceived by effects that required them to expect an object to move between hands when observing human hand manipulations. However, similar to humans, Eurasian jays were misled by magic effects that utilize fast movements as a deceptive action. This study investigates how another taxon perceives the magician's techniques of deception that commonly deceive humans.