A mouse DRG genetic toolkit reveals morphological and physiological diversity of somatosensory neuron subtypes.

Dorsal root ganglia (DRG) somatosensory neurons detect mechanical, thermal, and chemical stimuli acting on the body. Achieving a holistic view of how different DRG neuron subtypes relay neural signals from the periphery to the CNS has been challenging with existing tools. Here, we develop and curate a mouse genetic toolkit that allows for interrogating the properties and functions of distinct cutaneous targeting DRG neuron subtypes. These tools have enabled a broad morphological analysis, which revealed distinct cutaneous axon arborization areas and branching patterns of the transcriptionally distinct DRG neuron subtypes. Moreover, in vivo physiological analysis revealed that each subtype has a distinct threshold and range of responses to mechanical and/or thermal stimuli. These findings support a model in which morphologically and physiologically distinct cutaneous DRG sensory neuron subtypes tile mechanical and thermal stimulus space to collectively encode a wide range of natural stimuli.

A DRG genetic toolkit reveals molecular, morphological, and functional diversity of somatosensory neuron subtypes.

Mechanical and thermal stimuli acting on the skin are detected by morphologically and physiologically distinct sensory neurons of the dorsal root ganglia (DRG). Achieving a holistic view of how this diverse neuronal population relays sensory information from the skin to the central nervous system (CNS) has been challenging with existing tools. Here, we used transcriptomic datasets of the mouse DRG to guide development and curation of a genetic toolkit to interrogate transcriptionally defined DRG neuron subtypes. Morphological analysis revealed unique cutaneous axon arborization areas and branching patterns of each subtype. Physiological analysis showed that subtypes exhibit distinct thresholds and ranges of responses to mechanical and/or thermal stimuli. The somatosensory neuron toolbox thus enables comprehensive phenotyping of most principal sensory neuron subtypes. Moreover, our findings support a population coding scheme in which the activation thresholds of morphologically and physiologically distinct cutaneous DRG neuron subtypes tile multiple dimensions of stimulus space.

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.

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.

Single-cell profiling of CNS border compartment leukocytes reveals that B cells and their progenitors reside in non-diseased meninges.

The CNS is ensheathed by the meninges and cerebrospinal fluid, and recent findings suggest that these CNS-associated border tissues have complex immunological functions. Unlike myeloid lineage cells, lymphocytes in border compartments have yet to be thoroughly characterized. Based on single-cell transcriptomics, we here identified a highly location-specific composition and expression profile of tissue-resident leukocytes in CNS parenchyma, pia-enriched subdural meninges, dura mater, choroid plexus and cerebrospinal fluid. The dura layer of the meninges contained a large population of B cells under homeostatic conditions in mice and rats. Murine dura B cells exhibited slow turnover and long-term tissue residency, and they matured in experimental neuroinflammation. The dura also contained B lineage progenitors at the pro-B cell stage typically not found outside of bone marrow, without direct influx from the periphery or the skull bone marrow. This identified the dura as an unexpected site of B cell residence and potentially of development in both homeostasis and neuroinflammation.

Single-cell transcriptome analysis defines heterogeneity of the murine pancreatic ductal tree.

To study disease development, an inventory of an organ's cell types and understanding of physiologic function is paramount. Here, we performed single-cell RNA-sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify osteopontin as a regulator of this fate decision as well as human duct cell dedifferentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis.

Cytokines and transcription factors in the differentiation of CD4+ T helper cell subsets and induction of tissue inflammation and autoimmunity.

CD4+T helper (Th) cells are critical in homeostasis and host defense but are also central to the development of various autoimmune diseases if they become dysregulated. Specifically, pathogenic Th1 and Th17 cells contribute to autoimmune inflammation whereas Treg and Tr1 cells are important for maintaining immune tolerance and resolution of inflammation, respectively. Cytokines trigger signaling pathways in naive T cells that induce lineage-defining transcription factors that direct their differentiation into the distinct T helper cell subsets. It has become clear that the differentiation of T helper cells is not only influenced by the cytokine milieu but also by their metabolic state, cues from the microbiota and the tissue they reside in. A comprehensive understanding how these various stimuli contribute to T helper cell differentiation and phenotype could potentially provide novel ways for therapeutic intervention in autoimmunity and tissue inflammation.

Checkpoint Blockade Immunotherapy Induces Dynamic Changes in PD-1-CD8+ Tumor-Infiltrating T Cells.

An improved understanding of the anti-tumor CD8+ T cell response after checkpoint blockade would enable more informed and effective therapeutic strategies. Here we examined the dynamics of the effector response of CD8+ tumor-infiltrating lymphocytes (TILs) after checkpoint blockade therapy. Bulk and single-cell RNA profiles of CD8+ TILs after combined Tim-3+PD-1 blockade in preclinical models revealed significant changes in the transcriptional profile of PD-1- TILs. These cells could be divided into subsets bearing characterstics of naive-, effector-, and memory-precursor-like cells. Effector- and memory-precursor-like TILs contained tumor-antigen-specific cells, exhibited proliferative and effector capacity, and expanded in response to different checkpoint blockade therapies across different tumor models. The memory-precursor-like subset shared features with CD8+ T cells associated with response to checkpoint blockade in patients and was compromised in the absence of Tcf7. Expression of Tcf7/Tcf1 was requisite for the efficacy of diverse immunotherapies, highlighting the importance of this transcriptional regulator in the development of effective CD8+ T cell responses upon immunotherapy.

Schwann cells promote post-traumatic nerve inflammation and neuropathic pain through MHC class II.

The activation of T helper cells requires antigens to be exposed on the surface of antigen presenting cells (APCs) via MHC class II (MHC-II) molecules. Expression of MHC-II is generally limited to professional APCs, but other cell types can express MHC-II under inflammatory conditions. However, the importance of these conditional APCs is unknown. We and others have previously shown that Schwann cells are potentially conditional APCs, but the functional relevance of MHC-II expression by Schwann cells has not been studied in vivo. Here, we conditionally deleted the MHC-II ß-chain from myelinating Schwann cells in mice and investigated how this influenced post-traumatic intraneural inflammation and neuropathic pain using the chronic constriction injury (CCI) model. We demonstrate that deletion of MHC-II in myelinating Schwann cells reduces thermal hyperalgesia and, to a lesser extent, also diminishes mechanical allodynia in CCI in female mice. This was accompanied by a reduction of intraneural CD4+ T cells and greater preservation of preferentially large-caliber axons. Activation of T helper cells by MHC-II on Schwann cells thus promotes post-traumatic axonal loss and neuropathic pain. Hence, we provide experimental evidence that Schwann cells gain antigen-presenting function in vivo and modulate local immune responses and diseases in the peripheral nerves.

RBPJ Controls Development of Pathogenic Th17 Cells by Regulating IL-23 Receptor Expression.

Interleukin-17 (IL-17)-producing helper T cells (Th17 cells) play an important role in autoimmune diseases. However, not all Th17 cells induce tissue inflammation or autoimmunity. Th17 cells require IL-23 receptor (IL-23R) signaling to become pathogenic. The transcriptional mechanisms controlling the pathogenicity of Th17 cells and IL-23R expression are unknown. Here, we demonstrate that the canonical Notch signaling mediator RBPJ is a key driver of IL-23R expression. In the absence of RBPJ, Th17 cells fail to upregulate IL-23R, lack stability, and do not induce autoimmune tissue inflammation in vivo, whereas overexpression of IL-23R rescues this defect and promotes pathogenicity of RBPJ-deficient Th17 cells. RBPJ binds and trans-activates the Il23r promoter and induces IL-23R expression and represses anti-inflammatory IL-10 production in Th17 cells. We thus find that Notch signaling influences the development of pathogenic and non-pathogenic Th17 cells by reciprocally regulating IL-23R and IL-10 expression.

CD5L/AIM Regulates Lipid Biosynthesis and Restrains Th17 Cell Pathogenicity.

Th17 cells play a critical role in host defense against extracellular pathogens and tissue homeostasis but can induce autoimmunity. The mechanisms implicated in balancing "pathogenic" and "non-pathogenic" Th17 cell states remain largely unknown. We used single-cell RNA-seq to identify CD5L/AIM as a regulator expressed in non-pathogenic, but not in pathogenic Th17 cells. Although CD5L does not affect Th17 differentiation, it is a functional switch that regulates the pathogenicity of Th17 cells. Loss of CD5L converts non-pathogenic Th17 cells into pathogenic cells that induce autoimmunity. CD5L mediates this effect by modulating the intracellular lipidome, altering fatty acid composition and restricting cholesterol biosynthesis and, thus, ligand availability for Rorγt, the master transcription factor of Th17 cells. Our study identifies CD5L as a critical regulator of the Th17 cell functional state and highlights the importance of lipid metabolism in balancing immune protection and disease induced by T cells.

Deletion of the de novo DNA methyltransferase Dnmt3a promotes lung tumor progression.

Alterations in DNA methylation have been associated with genome-wide hypomethylation and regional de novo methylation in numerous cancers. De novo methylation is mediated by the de novo methyltransferases Dnmt3a and 3b, but only Dnmt3b has been implicated in promoting cancer by silencing of tumor-suppressor genes. In this study, we have analyzed the role of Dnmt3a in lung cancer by using a conditional mouse tumor model. We show that Dnmt3a deficiency significantly promotes tumor growth and progression but not initiation. Changes in gene expression show that Dnmt3a deficiency affects key steps in cancer progression, such as angiogenesis, cell adhesion, and cell motion, consistent with accelerated and more malignant growth. Our results suggest that Dnmt3a may act like a tumor-suppressor gene in lung tumor progression and may be a critical determinant of lung cancer malignancy.

De novo DNA methylation by Dnmt3a and Dnmt3b is dispensable for nuclear reprogramming of somatic cells to a pluripotent state.

Induced pluripotent stem cells (iPSCs) are generated from somatic cells by the transduction of defined transcription factors, and this process involves dynamic changes in DNA methylation. While the reprogramming of somatic cells is accompanied by demethylation of pluripotency genes, the functional importance of de novo DNA methylation has not been clarified. Here, using loss-of-function studies, we generated iPSCs from fibroblasts that were deficient in de novo DNA methylation mediated by Dnmt3a and Dnmt3b. These iPSCs reactivated pluripotency genes, underwent self-renewal, and showed restricted developmental potential that was rescued upon reintroduction of Dnmt3a and Dnmt3b. We conclude that de novo DNA methylation by Dnmt3a and Dnmt3b is dispensable for nuclear reprogramming of somatic cells.