Sweet splicing.

Glucose is the main source of energy for cells. In this issue of Cell, a study now shows that glucose has additional non-energetic functions, acting as a biomolecular cue that regulates alternative splicing during epidermal differentiation. As keratinocytes differentiate, glucose associates with RNA-binding protein DDX21 and modulates its interaction properties, which modifies splicing decisions.

Adaptive sequence divergence forged new neurodevelopmental enhancers in humans.

Searches for the genetic underpinnings of uniquely human traits have focused on human-specific divergence in conserved genomic regions, which reflects adaptive modifications of existing functional elements. However, the study of conserved regions excludes functional elements that descended from previously neutral regions. Here, we demonstrate that the fastest-evolved regions of the human genome, which we term "human ancestor quickly evolved regions" (HAQERs), rapidly diverged in an episodic burst of directional positive selection prior to the human-Neanderthal split, before transitioning to constraint within hominins. HAQERs are enriched for bivalent chromatin states, particularly in gastrointestinal and neurodevelopmental tissues, and genetic variants linked to neurodevelopmental disease. We developed a multiplex, single-cell in vivo enhancer assay to discover that rapid sequence divergence in HAQERs generated hominin-unique enhancers in the developing cerebral cortex. We propose that a lack of pleiotropic constraints and elevated mutation rates poised HAQERs for rapid adaptation and subsequent susceptibility to disease.

IKAROS and AIOLOS directly regulate AP-1 transcriptional complexes and are essential for NK cell development.

Ikaros transcription factors are essential for adaptive lymphocyte function, yet their role in innate lymphopoiesis is unknown. Using conditional genetic inactivation, we show that Ikzf1/Ikaros is essential for normal natural killer (NK) cell lymphopoiesis and IKZF1 directly represses Cish, a negative regulator of interleukin-15 receptor resulting in impaired interleukin-15 receptor signaling. Both Bcl2l11 and BIM levels, and intrinsic apoptosis were increased in Ikzf1-null NK cells, which in part accounts for NK lymphopenia as both were restored to normal levels when Ikzf1 and Bcl2l11 were co-deleted. Ikzf1-null NK cells presented extensive transcriptional alterations with reduced AP-1 transcriptional complex expression and increased expression of Ikzf2/Helios and Ikzf3/Aiolos. IKZF1 and IKZF3 directly bound AP-1 family members and deletion of both Ikzf1 and Ikzf3 in NK cells resulted in further reductions in Jun/Fos expression and complete loss of peripheral NK cells. Collectively, we show that Ikaros family members are important regulators of apoptosis, cytokine responsiveness and AP-1 transcriptional activity.

IL-23 stabilizes an effector Treg cell program in the tumor microenvironment.

Interleukin-23 (IL-23) is a proinflammatory cytokine mainly produced by myeloid cells that promotes tumor growth in various preclinical cancer models and correlates with adverse outcomes. However, as to how IL-23 fuels tumor growth is unclear. Here, we found tumor-associated macrophages to be the main source of IL-23 in mouse and human tumor microenvironments. Among IL-23-sensing cells, we identified a subset of tumor-infiltrating regulatory T (Treg) cells that display a highly suppressive phenotype across mouse and human tumors. The use of three preclinical models of solid cancer in combination with genetic ablation of Il23r in Treg cells revealed that they are responsible for the tumor-promoting effect of IL-23. Mechanistically, we found that IL-23 sensing represents a crucial signal driving the maintenance and stabilization of effector Treg cells involving the transcription factor Foxp3. Our data support that targeting the IL-23/IL-23R axis in cancer may represent a means of eliciting antitumor immunity.

Endocytosis Inhibition in Humans to Improve Responses to ADCC-Mediating Antibodies.

A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies.

Runx3 drives a CD8+ T cell tissue residency program that is absent in CD4+ T cells.

Tissue-resident memory T cells (TRM cells) provide rapid and superior control of localized infections. While the transcription factor Runx3 is a critical regulator of CD8+ T cell tissue residency, its expression is repressed in CD4+ T cells. Here, we show that, as a direct consequence of this Runx3-deficiency, CD4+ TRM cells lacked the transforming growth factor (TGF)-ß-responsive transcriptional network that underpins the tissue residency of epithelial CD8+ TRM cells. While CD4+ TRM cell formation required Runx1, this, along with the modest expression of Runx3 in CD4+ TRM cells, was insufficient to engage the TGF-ß-driven residency program. Ectopic expression of Runx3 in CD4+ T cells incited this TGF-ß-transcriptional network to promote prolonged survival, decreased tissue egress, a microanatomical redistribution towards epithelial layers and enhanced effector functionality. Thus, our results reveal distinct programming of tissue residency in CD8+ and CD4+ TRM cell subsets that is attributable to divergent Runx3 activity.

Functional Ontogeny of Hypothalamic Agrp Neurons in Neonatal Mouse Behaviors.

Hypothalamic Agrp neurons regulate food ingestion in adult mice. Whether these neurons are functional before animals start to ingest food is unknown. Here, we studied the functional ontogeny of Agrp neurons during breastfeeding using postnatal day 10 mice. In contrast to adult mice, we show that isolation from the nursing nest, not milk deprivation or ingestion, activated Agrp neurons. Non-nutritive suckling and warm temperatures blunted this effect. Using in vivo fiber photometry, neonatal Agrp neurons showed a rapid increase in activity upon isolation from the nest, an effect rapidly diminished following reunion with littermates. Neonates unable to release GABA from Agrp neurons expressed blunted emission of isolation-induced ultrasonic vocalizations. Chemogenetic overactivation of these neurons further increased emission of these ultrasonic vocalizations, but not milk ingestion. We uncovered important functional properties of hypothalamic Agrp neurons during mouse development, suggesting these neurons facilitate offspring-to-caregiver bonding.

A Pan-cancer Transcriptome Analysis Reveals Pervasive Regulation through Alternative Promoters.

Most human protein-coding genes are regulated by multiple, distinct promoters, suggesting that the choice of promoter is as important as its level of transcriptional activity. However, while a global change in transcription is recognized as a defining feature of cancer, the contribution of alternative promoters still remains largely unexplored. Here, we infer active promoters using RNA-seq data from 18,468 cancer and normal samples, demonstrating that alternative promoters are a major contributor to context-specific regulation of transcription. We find that promoters are deregulated across tissues, cancer types, and patients, affecting known cancer genes and novel candidates. For genes with independently regulated promoters, we demonstrate that promoter activity provides a more accurate predictor of patient survival than gene expression. Our study suggests that a dynamic landscape of active promoters shapes the cancer transcriptome, opening new diagnostic avenues and opportunities to further explore the interplay of regulatory mechanisms with transcriptional aberrations in cancer.

Discrete tissue microenvironments instruct diversity in resident memory T cell function and plasticity.

Tissue-resident memory T (TRM) cells are non-recirculating cells that exist throughout the body. Although TRM cells in various organs rely on common transcriptional networks to establish tissue residency, location-specific factors adapt these cells to their tissue of lodgment. Here we analyze TRM cell heterogeneity between organs and find that the different environments in which these cells differentiate dictate TRM cell function, durability and malleability. We find that unequal responsiveness to TGFß is a major driver of this diversity. Notably, dampened TGFß signaling results in CD103- TRM cells with increased proliferative potential, enhanced function and reduced longevity compared with their TGFß-responsive CD103+ TRM counterparts. Furthermore, whereas CD103- TRM cells readily modified their phenotype upon relocation, CD103+ TRM cells were comparatively resistant to transdifferentiation. Thus, despite common requirements for TRM cell development, tissue adaptation of these cells confers discrete functional properties such that TRM cells exist along a spectrum of differentiation potential that is governed by their local tissue microenvironment.

Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

Distinct epigenomic landscapes underlie tissue-specific memory T cell differentiation.

The memory CD8+ T cell pool contains phenotypically and transcriptionally heterogeneous subsets with specialized functions and recirculation patterns. Here, we examined the epigenetic landscape of CD8+ T cells isolated from seven non-lymphoid organs across four distinct infection models, alongside their circulating T cell counterparts. Using single-cell transposase-accessible chromatin sequencing (scATAC-seq), we found that tissue-resident memory T (TRM) cells and circulating memory T (TCIRC) cells develop along distinct epigenetic trajectories. We identified organ-specific transcriptional regulators of TRM cell development, including FOSB, FOS, FOSL1, and BACH2, and defined an epigenetic signature common to TRM cells across organs. Finally, we found that although terminal TEX cells share accessible regulatory elements with TRM cells, they are defined by TEX-specific epigenetic features absent from TRM cells. Together, this comprehensive data resource shows that TRM cell development is accompanied by dynamic transcriptome alterations and chromatin accessibility changes that direct tissue-adapted and functionally distinct T cell states.

Analysis of Genetically Diverse Macrophages Reveals Local and Domain-wide Mechanisms that Control Transcription Factor Binding and Function.

Non-coding genetic variation is a major driver of phenotypic diversity and allows the investigation of mechanisms that control gene expression. Here, we systematically investigated the effects of >50 million variations from five strains of mice on mRNA, nascent transcription, transcription start sites, and transcription factor binding in resting and activated macrophages. We observed substantial differences associated with distinct molecular pathways. Evaluating genetic variation provided evidence for roles of ∼100 TFs in shaping lineage-determining factor binding. Unexpectedly, a substantial fraction of strain-specific factor binding could not be explained by local mutations. Integration of genomic features with chromatin interaction data provided evidence for hundreds of connected cis-regulatory domains associated with differences in transcription factor binding and gene expression. This system and the >250 datasets establish a substantial new resource for investigation of how genetic variation affects cellular phenotypes.

Transcriptomic and epigenetic mechanisms underlying myeloid diversity in the lung.

The lung is inhabited by resident alveolar and interstitial macrophages as well as monocytic cells that survey lung tissues. Each cell type plays distinct functional roles under homeostatic and inflammatory conditions, but mechanisms establishing their molecular identities and functional potential remain poorly understood. In the present study, systematic evaluation of transcriptomes and open chromatin of alveolar macrophages (AMs), interstitial macrophages (IMs) and lung monocytes from two mouse strains enabled inference of common and cell-specific transcriptional regulators. We provide evidence that these factors drive selection of regulatory landscapes that specify distinct phenotypes of AMs and IMs and entrain qualitatively different responses to toll-like receptor 4 signaling in vivo. These studies reveal a striking divergence in a fundamental innate immune response pathway in AMs and establish a framework for further understanding macrophage diversity in the lung.

Developmental plasticity allows outside-in immune responses by resident memory T cells.

Central memory T (TCM) cells patrol lymph nodes and perform conventional memory responses on restimulation: proliferation, migration and differentiation into diverse T cell subsets while also self-renewing. Resident memory T (TRM) cells are parked within single organs, share properties with terminal effectors and contribute to rapid host protection. We observed that reactivated TRM cells rejoined the circulating pool. Epigenetic analyses revealed that TRM cells align closely with conventional memory T cell populations, bearing little resemblance to recently activated effectors. Fully differentiated TRM cells isolated from small intestine epithelium exhibited the potential to differentiate into TCM cells, effector memory T cells and TRM cells on recall. Ex-TRM cells, former intestinal TRM cells that rejoined the circulating pool, heritably maintained a predilection for homing back to their tissue of origin on subsequent reactivation and a heightened capacity to redifferentiate into TRM cells. Thus, TRM cells can rejoin the circulation but are advantaged to re-form local TRM when called on.

Mouse transcriptome reveals potential signatures of protection and pathogenesis in human tuberculosis.

Although mouse infection models have been extensively used to study the host response to Mycobacterium tuberculosis, their validity in revealing determinants of human tuberculosis (TB) resistance and disease progression has been heavily debated. Here, we show that the modular transcriptional signature in the blood of susceptible mice infected with a clinical isolate of M. tuberculosis resembles that of active human TB disease, with dominance of a type I interferon response and neutrophil activation and recruitment, together with a loss in B lymphocyte, natural killer and T cell effector responses. In addition, resistant but not susceptible strains of mice show increased lung B cell, natural killer and T cell effector responses in the lung upon infection. Notably, the blood signature of active disease shared by mice and humans is also evident in latent TB progressors before diagnosis, suggesting that these responses both predict and contribute to the pathogenesis of progressive M. tuberculosis infection.

Single-cell protein expression profiling resolves circulating and resident memory T cell diversity across tissues and infection contexts.

Memory CD8+ T cells can be broadly divided into circulating (TCIRCM) and tissue-resident memory T (TRM) populations. Despite well-defined migratory and transcriptional differences, the phenotypic and functional delineation of TCIRCM and TRM cells, particularly across tissues, remains elusive. Here, we utilized an antibody screening platform and machine learning prediction pipeline (InfinityFlow) to profile >200 proteins in TCIRCM and TRM cells in solid organs and barrier locations. High-dimensional analyses revealed unappreciated heterogeneity within TCIRCM and TRM cell lineages across nine different organs after either local or systemic murine infection models. Additionally, we demonstrated the relative effectiveness of strategies allowing for the selective ablation of TCIRCM or TRM populations across organs and identified CD55, KLRG1, CXCR6, and CD38 as stable markers for characterizing memory T cell function during inflammation. Together, these data and analytical framework provide an in-depth resource for memory T cell classification in both steady-state and inflammatory conditions.

COQ4 is required for the oxidative decarboxylation of the C1 carbon of coenzyme Q in eukaryotic cells.

Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum. Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role but also via the oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis and shed light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.

Intravital mucosal imaging of CD8+ resident memory T cells shows tissue-autonomous recall responses that amplify secondary memory.

CD8+ T cell immunosurveillance dynamics influence the outcome of intracellular infections and cancer. Here we used two-photon intravital microscopy to visualize the responses of CD8+ resident memory T cells (TRM cells) within the reproductive tracts of live female mice. We found that mucosal TRM cells were highly motile, but paused and underwent in situ division after local antigen challenge. TRM cell reactivation triggered the recruitment of recirculating memory T cells that underwent antigen-independent TRM cell differentiation in situ. However, the proliferation of pre-existing TRM cells dominated the local mucosal recall response and contributed most substantially to the boosted secondary TRM cell population. We observed similar results in skin. Thus, TRM cells can autonomously regulate the expansion of local immunosurveillance independently of central memory or proliferation in lymphoid tissue.

Environmental enteric dysfunction induces regulatory T cells that inhibit local CD4+ T cell responses and impair oral vaccine efficacy.

Environmental enteric dysfunction (EED) is a gastrointestinal inflammatory disease caused by malnutrition and chronic infection. EED is associated with stunting in children and reduced efficacy of oral vaccines. To study the mechanisms of oral vaccine failure during EED, we developed a microbiota- and diet-dependent mouse EED model. Analysis of E. coli-labile toxin vaccine-specific CD4+ T cells in these mice revealed impaired CD4+ T cell responses in the small intestine and but not the lymph nodes. EED mice exhibited increased frequencies of small intestine-resident RORγT+FOXP3+ regulatory T (Treg) cells. Targeted deletion of RORγT from Treg cells restored small intestinal vaccine-specific CD4 T cell responses and vaccine-mediated protection upon challenge. However, ablation of RORγT+FOXP3+ Treg cells made mice more susceptible to EED-induced stunting. Our findings provide insight into the poor efficacy of oral vaccines in EED and highlight how RORγT+FOXP3+ Treg cells can regulate intestinal immunity while leaving systemic responses intact.