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.

FEAST: A flow cytometry-based toolkit for interrogating microglial engulfment of synaptic and myelin proteins.

Although engulfment is a hallmark of microglia function, fully validated platforms that facilitate high-throughput quantification of this process are lacking. Here, we present FEAST (Flow cytometric Engulfment Assay for Specific Target proteins), which enables interrogation of in vivo engulfment of synaptic material by brain resident macrophages at single-cell resolution. We optimize FEAST for two different analyses: quantification of fluorescent material inside live cells and of engulfed endogenous proteins within fixed cells. To overcome false-positive engulfment signals, we introduce an approach suitable for interrogating engulfment in microglia from perfusion-fixed tissue. As a proof-of-concept for the specificity and versatility of FEAST, we examine the engulfment of synaptic proteins after optic nerve crush and of myelin in two mouse models of demyelination (treatment with cuprizone and injections of lysolecithin). We find that microglia, but not brain-border associated macrophages, engulf in these contexts. Our work underscores how FEAST can be utilized to gain critical insight into functional neuro-immune interactions that shape development, homeostasis, and disease.

Bacteria hijack a meningeal neuroimmune axis to facilitate brain invasion.

The meninges are densely innervated by nociceptive sensory neurons that mediate pain and headache1,2. Bacterial meningitis causes life-threatening infections of the meninges and central nervous system, affecting more than 2.5 million people a year3-5. How pain and neuroimmune interactions impact meningeal antibacterial host defences are unclear. Here we show that Nav1.8+ nociceptors signal to immune cells in the meninges through the neuropeptide calcitonin gene-related peptide (CGRP) during infection. This neuroimmune axis inhibits host defences and exacerbates bacterial meningitis. Nociceptor neuron ablation reduced meningeal and brain invasion by two bacterial pathogens: Streptococcus pneumoniae and Streptococcus agalactiae. S. pneumoniae activated nociceptors through its pore-forming toxin pneumolysin to release CGRP from nerve terminals. CGRP acted through receptor activity modifying protein 1 (RAMP1) on meningeal macrophages to polarize their transcriptional responses, suppressing macrophage chemokine expression, neutrophil recruitment and dural antimicrobial defences. Macrophage-specific RAMP1 deficiency or pharmacological blockade of RAMP1 enhanced immune responses and bacterial clearance in the meninges and brain. Therefore, bacteria hijack CGRP-RAMP1 signalling in meningeal macrophages to facilitate brain invasion. Targeting this neuroimmune axis in the meninges can enhance host defences and potentially produce treatments for bacterial meningitis.

Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain.

A key aspect of nearly all single-cell sequencing experiments is dissociation of intact tissues into single-cell suspensions. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression. Here, we demonstrate that use of enzymatic dissociation on brain tissue induces an aberrant ex vivo gene expression signature, most prominently in microglia, which is prevalent in published literature and can substantially confound downstream analyses. To address this issue, we present a rigorously validated protocol that preserves both in vivo transcriptional profiles and cell-type diversity and yield across tissue types and species. We also identify a similar signature in postmortem human brain single-nucleus RNA-sequencing datasets, and show that this signature is induced in freshly isolated human tissue by exposure to elevated temperatures ex vivo. Together, our results provide a methodological solution for preventing artifactual gene expression changes during fresh tissue digestion and a reference for future deeper analysis of the potential confounding states present in postmortem human samples.

A RIPK1-regulated inflammatory microglial state in amyotrophic lateral sclerosis.

Microglial-derived inflammation has been linked to a broad range of neurodegenerative and neuropsychiatric conditions, including amyotrophic lateral sclerosis (ALS). Using single-cell RNA sequencing, a class of Disease-Associated Microglia (DAMs) have been characterized in neurodegeneration. However, the DAM phenotype alone is insufficient to explain the functional complexity of microglia, particularly with regard to regulating inflammation that is a hallmark of many neurodegenerative diseases. Here, we identify a subclass of microglia in mouse models of ALS which we term RIPK1-Regulated Inflammatory Microglia (RRIMs). RRIMs show significant up-regulation of classical proinflammatory pathways, including increased levels of Tnf and Il1b RNA and protein. We find that RRIMs are highly regulated by TNFα signaling and that the prevalence of these microglia can be suppressed by inhibiting receptor-interacting protein kinase 1 (RIPK1) activity downstream of the TNF receptor 1. These findings help to elucidate a mechanism by which RIPK1 kinase inhibition has been shown to provide therapeutic benefit in mouse models of ALS and may provide an additional biomarker for analysis in ongoing phase 2 clinical trials of RIPK1 inhibitors in ALS.

Weight(s) of complicity.

International non-governmental organisations (INGO) face a dilemma when deciding whether to intervene in crisis situations where their efforts can be exploited or co-opted by others: intervene and risk becoming complicit with wrongdoing or sit on the sidelines and consign vulnerable people to the ravages of neglect or oppression. In "'He who helps the guilty, shares the crime'? INGOs, moral narcissism and complicity in wrongdoing," Buth et al argue that concerns about complicity often stifle ethical debate and encourage moral narcissism. We argue that neglecting concerns about complicity can foster a different form of moral narcissism and that where worries of complicity are present, aid efforts face three types of risk: risks to others created by contributing to wrongful acts or bad outcomes; risks to the moral integrity of the INGO and its personnel; and risks to social trust in the INGO. In the end, we challenge the assumption that there is a unique, ethically best way to reconcile these values. We suggest that the causes of justice and humanity might be better served by a diverse community of INGOs who each gives different weight to these concerns, than if each INGO adopts the same framework for reconciling these competing demands.

Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes.

Microglia, the resident immune cells of the brain, rapidly change states in response to their environment, but we lack molecular and functional signatures of different microglial populations. Here, we analyzed the RNA expression patterns of more than 76,000 individual microglia in mice during development, in old age, and after brain injury. Our analysis uncovered at least nine transcriptionally distinct microglial states, which expressed unique sets of genes and were localized in the brain using specific markers. The greatest microglial heterogeneity was found at young ages; however, several states-including chemokine-enriched inflammatory microglia-persisted throughout the lifespan or increased in the aged brain. Multiple reactive microglial subtypes were also found following demyelinating injury in mice, at least one of which was also found in human multiple sclerosis lesions. These distinct microglia signatures can be used to better understand microglia function and to identify and manipulate specific subpopulations in health and disease.

CD47 Protects Synapses from Excess Microglia-Mediated Pruning during Development.

Microglia regulate synaptic circuit remodeling and phagocytose synaptic material in the healthy brain; however, the mechanisms directing microglia to engulf specific synapses and avoid others remain unknown. Here, we demonstrate that an innate immune signaling pathway protects synapses from inappropriate removal. The expression patterns of CD47 and its receptor, SIRPα, correlated with peak pruning in the developing retinogeniculate system, and mice lacking these proteins exhibited increased microglial engulfment of retinogeniculate inputs and reduced synapse numbers in the dorsal lateral geniculate nucleus. CD47-deficient mice also displayed increased functional pruning, as measured by electrophysiology. In addition, CD47 was found to be required for neuronal activity-mediated changes in engulfment, as microglia in CD47 knockout mice failed to display preferential engulfment of less active inputs. Taken together, these results demonstrate that CD47-SIRPα signaling prevents excess microglial phagocytosis and show that molecular brakes can be regulated by activity to protect specific inputs.

Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase.

We explored the utility of targeting anaplastic lymphoma kinase (ALK), a cell surface receptor overexpressed on pediatric solid tumors, using chimeric antigen receptor (CAR)-based immunotherapy. T cells expressing a CAR incorporating the single-chain variable fragment sequence of the ALK48 mAb linked to a 4-1BB-CD3ζ signaling domain lysed ALK-expressing tumor lines and produced interferon-gamma upon antigen stimulation but had limited anti-tumor efficacy in two xenograft models of human neuroblastoma. Further exploration demonstrated that cytokine production was highly dependent upon ALK target density and that target density of ALK on neuroblastoma cell lines was insufficient for maximal activation of CAR T cells. In addition, ALK CAR T cells demonstrated rapid and complete antigen-induced loss of receptor from the T cell surface via internalization. Using a model that simultaneously modulated antigen density and CAR expression, we demonstrated that CAR functionality is regulated by target antigen and CAR density and that low expression of either contributes to limited anti-tumor efficacy of the ALK CAR. These data suggest that stoichiometric relationships between CAR receptors and target antigens may significantly impact the anti-tumor efficacy of CAR T cells and that manipulation of these parameters could allow precise tuning of CAR T cell activity.

CCR7 deficient inflammatory Dendritic Cells are retained in the Central Nervous System.

Dendritic cells (DC) accumulate in the CNS during neuroinflammation, yet, how these cells contribute to CNS antigen drainage is still unknown. We have previously shown that after intracerebral injection, antigen-loaded bone marrow DC migrate to deep cervical lymph nodes where they prime antigen-specific T cells and exacerbate experimental autoimmune encephalomyelitis (EAE) in mice. Here, we report that DC migration from brain parenchyma is dependent upon the chemokine receptor CCR7. During EAE, both wild type and CCR7-/- CD11c-eYFP cells infiltrated into the CNS but cells that lacked CCR7 were retained in brain and spinal cord while wild type DC migrated to cervical lymph nodes. Retention of CCR7-deficient CD11c-eYFP cells in the CNS exacerbated EAE. These data are the first to show that CD11chigh DC use CCR7 for migration out of the CNS, and in the absence of this receptor they remain in the CNS in situ and exacerbate EAE.

Ethylatropine Bromide as a Peripherally Restricted Muscarinic Antagonist.

Quaternary ammonium analogues of atropine that are unable to cross the blood-brain barrier are used to alleviate peripheral muscarinic toxicity in animal models of epilepsy produced by systemic administration of pilocarpine or diisopropylfluorophosphate (DFP). Currently, methylatropine is the most popular and potent of these quaternary derivatives; however, it is expensive and produced in limited quantity. Here, we propose the use of ethylatropine bromide as an alternative to methylatropine. The synthesis of ethylatropine bromide is simple, inexpensive and has low environmental impact. We demonstrate the efficacy of ethylatropine bromide to antagonize the carbachol induced rise in intracellular calcium in a calcium mobilization assay, and its ability to prevent pilocarpine-induced total fluid secretions in mice without blocking pilocarpine-induced seizures. The ease of synthesis, cost effectiveness, and efficacy makes ethylatropine bromide a desirable alternative to methylatropine as a peripherally restricted acetylcholine receptor antagonist.

Reduction of MDSCs with All-trans Retinoic Acid Improves CAR Therapy Efficacy for Sarcomas.

Genetically engineered T cells expressing CD19-specific chimeric antigen receptors (CAR) have shown impressive activity against B-cell malignancies, and preliminary results suggest that T cells expressing a first-generation disialoganglioside (GD2)-specific CAR can also provide clinical benefit in patients with neuroblastoma. We sought to assess the potential of GD2-CAR therapies to treat pediatric sarcomas. We observed that 18 of 18 (100%) of osteosarcomas, 2 of 15 (13%) of rhabdomyosarcomas, and 7 of 35 (20%) of Ewing sarcomas expressed GD2. T cells engineered to express a third-generation GD2-CAR incorporating the 14g2a-scFv with the CD28, OX40, and CD3ζ signaling domains (14g2a.CD28.OX40.ζ) mediated efficient and comparable lysis of both GD2+ sarcoma and neuroblastoma cell lines in vitro However, in xenograft models, GD2-CAR T cells had no antitumor effect against GD2+ sarcoma, despite effectively controlling GD2+ neuroblastoma. We observed that pediatric sarcoma xenografts, but not neuroblastoma xenografts, induced large populations of monocytic and granulocytic murine myeloid-derived suppressor cells (MDSC) that inhibited human CAR T-cell responses in vitro Treatment of sarcoma-bearing mice with all-trans retinoic acid (ATRA) largely eradicated monocytic MDSCs and diminished the suppressive capacity of granulocytic MDSCs. Combined therapy using GD2-CAR T cells plus ATRA significantly improved antitumor efficacy against sarcoma xenografts. We conclude that retinoids provide a clinically accessible class of agents capable of diminishing the suppressive effects of MDSCs, and that co-administration of retinoids may enhance the efficacy of CAR therapies targeting solid tumors. Cancer Immunol Res; 4(10); 869-80. ©2016 AACR.

4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors.

Chimeric antigen receptors (CARs) targeting CD19 have mediated dramatic antitumor responses in hematologic malignancies, but tumor regression has rarely occurred using CARs targeting other antigens. It remains unknown whether the impressive effects of CD19 CARs relate to greater susceptibility of hematologic malignancies to CAR therapies, or superior functionality of the CD19 CAR itself. We show that tonic CAR CD3-ζ phosphorylation, triggered by antigen-independent clustering of CAR single-chain variable fragments, can induce early exhaustion of CAR T cells that limits antitumor efficacy. Such activation is present to varying degrees in all CARs studied, except the highly effective CD19 CAR. We further determine that CD28 costimulation augments, whereas 4-1BB costimulation reduces, exhaustion induced by persistent CAR signaling. Our results provide biological explanations for the antitumor effects of CD19 CARs and for the observations that CD19 CAR T cells incorporating the 4-1BB costimulatory domain are more persistent than those incorporating CD28 in clinical trials.

CCR2-dependent dendritic cell accumulation in the central nervous system during early effector experimental autoimmune encephalomyelitis is essential for effector T cell restimulation in situ and disease progression.

Dendritic cells (DCs)--although absent from the healthy CNS parenchyma--rapidly accumulate within brain and spinal cord tissue during neuroinflammation associated with experimental autoimmune encephalomyelitis (EAE; a mouse model of multiple sclerosis). Yet, although DCs have been appreciated for their role in initiating adaptive immune responses in peripheral lymphoid organ tissues, how DCs infiltrate the CNS and contribute to ongoing neuroinflammation in situ is poorly understood. In this study, we report the following: 1) CD11c(+) bone marrow-derived DCs and CNS-infiltrating DCs express chemokine receptor CCR2; 2) compared with CCR2(+/+) cells, adoptively transferred CCR2(-/-) bone marrow-derived DCs or DC precursors do not accumulate in the CNS during EAE, despite abundance in blood; 3) CCR2(-/-) DCs show less accumulation in the inflamed CNS in mixed bone marrow chimeras, when compared with CCR2(+/+) DCs; and 4) ablation of CCR2(+/+) DCs during EAE clinical onset delays progression and attenuates cytokine production by infiltrating T cells. Whereas the role of CCR2 in monocyte migration into the CNS has been implicated previously, the role of CCR2 in DC infiltration into the CNS has never been directly addressed. Our data suggest that CCR2-dependent DC recruitment to the CNS during ongoing neuroinflammation plays a crucial role in effector T cell cytokine production and disease progression, and signify that CNS-DCs and circulating DC precursors might be key therapeutic targets for suppressing ongoing neuroinflammation in CNS autoimmune diseases.

Mapping the accumulation of co-infiltrating CNS dendritic cells and encephalitogenic T cells during EAE.

Evidence from experimental autoimmune encephalomyelitis (EAE) suggests that CNS-infiltrating dendritic cells (DCs) are crucial for restimulation of coinfiltrating T cells. Here we systematically quantified and visualized the distribution and interaction of CNS DCs and T cells during EAE. We report marked periventricular accumulation of DCs and myelin-specific T cells during EAE disease onset prior to accumulation in the spinal cord, indicating that the choroid plexus-CSF axis is a CNS entry portal. Moreover, despite emphasis on spinal cord inflammation in EAE and in correspondence with MS pathology, inflammatory lesions containing interacting DCs and T cells are present in specific brain regions.

GRACE principles: recognizing high-quality observational studies of comparative effectiveness.

Nonrandomized comparative effectiveness studies contribute to clinical and biologic understanding of treatments by themselves, via subsequent confirmation in a more targeted randomized clinical trial, or through advances in basic science. Although methodological challenges and a lack of accepted principles to assess the quality of nonrandomized studies of comparative effectiveness have limited the practical use of these investigations, even imperfect studies can contribute useful information if they are thoughtfully designed, well conducted, carefully analyzed, and reported in a manner that addresses concerns from skeptical readers and reviewers. The GRACE (Good Research for Comparative Effectiveness) principles have been developed to help healthcare providers, researchers, journal readers, and editors evaluate the quality inherent in observational research studies of comparative effectiveness. The GRACE principles were developed by experienced academic and private sector researchers and were vetted over several years through presentation, critique, and consensus building among outcomes researchers, pharmacoepidemiologists, and other medical scientists and via formal review by the International Society of Pharmacoepidemiology. In contrast to other documents that guide systematic review and reporting, the GRACE principles are high-level concepts about good practice for nonrandomized comparative effectiveness research. The GRACE principles comprise a series of questions to guide evaluation. No scoring system is provided or encouraged, as interpretation of these observational studies requires weighing of all available evidence, tempered by judgment regarding the applicability of the studies to routine care.