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Traumatic brain injury (TBI) and cerebrovascular injury are leading causes of disability and mortality worldwide. Systemic infections often accompany these disorders and can worsen outcomes. Recovery after brain injury depends on innate immunity, but the effect of infections on this process is not well understood. Here, we demonstrate that systemically introduced microorganisms and microbial products interfered with meningeal vascular repair after TBI in a type I interferon (IFN-I)-dependent manner, with sequential infections promoting chronic disrepair. Mechanistically, we discovered that MDA5-dependent detection of an arenavirus encountered after TBI disrupted pro-angiogenic myeloid cell programming via induction of IFN-I signaling. Systemic viral infection similarly blocked restorative angiogenesis in the brain parenchyma after intracranial hemorrhage, leading to chronic IFN-I signaling, blood–brain barrier leakage and a failure to restore cognitive–motor function. Our findings reveal a common immunological mechanism by which systemic infections deviate reparative programming after central nervous system injury and offer a new therapeutic target to improve recovery. Traumatic brain injury and stroke are commonly complicated by systemic infections, which impede recovery and lead to poor clinical outcomes. Using a mouse model, McGavern and colleagues show systemic microbial infections impair central nervous system revascularization and repair by a mechanism involving type I interferon signaling.

The blood–tumor barrier (BTB) limits the entry of effective chemotherapeutic agents into the brain for treatment of malignant tumors like glioblastoma. Poor drug entry across the BTB allows infiltrative glioma stem cells to evade therapy and develop treatment resistance. Regadenoson, an FDA-approved adenosine A2A receptor (A2AR) agonist, has been shown to increase drug delivery across the blood–brain barrier in non–tumor-bearing rodents without a defined mechanism of enhancing BTB permeability. Here, we characterize the time-dependent impact of regadenoson on brain endothelial cell interactions and paracellular transport, using mouse and rat brain endothelial cells and tumor models. In vitro, A2AR activation leads to disorganization of cytoskeletal actin filaments by 30 minutes, downregulation of junctional protein expression by 4 hours, and reestablishment of endothelial cell integrity by 8 hours. In rats bearing intracranial gliomas, regadenoson treatment results in increase of intratumoral temozolomide concentrations, yet no increased survival noted with combined temozolomide therapy. These findings demonstrate regadenoson's ability to induce brain endothelial structural changes among glioma to increase BTB permeability. The use of vasoactive mediators, like regadenoson, which transiently influences paracellular transport, should further be explored to evaluate their potential to enhance central nervous system treatment delivery to aggressive brain tumors. Implications: This study provides insight on the use of a vasoactive agent to increase exposure of the BTB to chemotherapy with intention to improve glioma treatment efficacy.

Cerebrovascular injuries can cause severe edema and inflammation that adversely affect human health. Here, we observed that recanalization after successful endovascular thrombectomy for acute large vessel occlusion was associated with cerebral edema and poor clinical outcomes in patients who experienced hemorrhagic transformation. To understand this process, we developed a cerebrovascular injury model using transcranial ultrasound that enabled spatiotemporal evaluation of resident and peripheral myeloid cells. We discovered that injurious and reparative responses diverged based on time and cellular origin. Resident microglia initially stabilized damaged vessels in a purinergic receptor-dependent manner, which was followed by an influx of myelomonocytic cells that caused severe edema. Prolonged blockade of myeloid cell recruitment with anti-adhesion molecule therapy prevented severe edema but also promoted neuronal destruction and fibrosis by interfering with vascular repair subsequently orchestrated by proinflammatory monocytes and proangiogenic repair-associated microglia (RAM). These data demonstrate how temporally distinct myeloid cell responses can contain, exacerbate and ultimately repair a cerebrovascular injury.

A role for microglia in neuropsychiatric diseases, including major depressive disorder (MDD), has been postulated. Regulation of microglial phenotype by immune receptors has become a central topic in many neurological conditions. We explored preclinical and clinical evidence for the role of the CD300f immune receptor in the fine regulation of microglial phenotype and its contribution to MDD. We found that a prevalent nonsynonymous single-nucleotide polymorphism (C/T, rs2034310) of the human CD300f receptor cytoplasmic tail inhibits the protein kinase C phosphorylation of a threonine and is associated with protection against MDD, mainly in women. Interestingly, CD300f −/− mice displayed several characteristic MDD traits such as augmented microglial numbers, increased interleukin 6 and interleukin 1 receptor antagonist messenger RNA, alterations in synaptic strength, and noradrenaline-dependent and persistent depressive-like and anhedonic behaviors in females. This behavioral phenotype could be potentiated inducing the lipopolysaccharide depression model. RNA sequencing and biochemical studies revealed an association with impaired microglial metabolic fitness. In conclusion, we report a clear association that links the function of the CD300f immune receptor with MDD in humans, depressive-like and anhedonic behaviors in female mice, and altered microglial metabolic reprogramming.

Chronic infection provokes alterations in inflammatory and suppressive pathways that potentially affect the function and integrity of multiple tissues, impacting both ongoing immune control and restorative immune therapies. Here we demonstrate that chronic lymphocytic choriomeningitis virus infection rapidly triggers severe thymic depletion, mediated by CD8 T cell-intrinsic type I interferon (IFN) and signal transducer and activator of transcription 2 (Stat2) signaling. Occurring temporal to T cell exhaustion, thymic cellularity reconstituted despite ongoing viral replication, with a rapid secondary thymic depletion following immune restoration by anti-programmed death-ligand 1 (PDL1) blockade. Therapeutic hematopoietic stem cell transplant (HSCT) during chronic infection generated new antiviral CD8 T cells, despite sustained virus replication in the thymus, indicating an impairment in negative selection. Consequently, low amounts of high-affinity self-reactive T cells also escaped the thymus following HSCT during chronic infection. Thus, by altering the stringency and partially impairing negative selection, the host generates new virus-specific T cells to replenish the fight against the chronic infection, but also has the potentially dangerous effect of enabling the escape of self-reactive T cells.

Central nervous system (CNS) infections are a major cause of human morbidity and mortality worldwide. Even patients that survive CNS infections can have lasting neurological dysfunction resulting from immune and pathogen induced pathology. Developing approaches to noninvasively track pathology and immunity in the infected CNS is crucial for patient management and development of new therapeutics. Here, we develop novel MRI-based approaches to monitor virus-specific CD8+ T cells and their relationship to cerebrovascular pathology in the living brain. We studied a relevant murine model in which a neurotropic virus (vesicular stomatitis virus) was introduced intranasally and then entered the brain via olfactory sensory neurons – a route exploited by many pathogens in humans. Using T2*-weighted high-resolution MRI, we identified small cerebral microbleeds as the earliest form of pathology associated with viral entry into the brain. Mechanistically, these microbleeds occurred in the absence of peripheral immune cells and were associated with infection of vascular endothelial cells. We monitored the adaptive response to this infection by developing methods to iron label and track individual virus specific CD8+ T cells by MRI. Transferred antiviral T cells were detected in the brain within a day of infection and were able to reduce cerebral microbleeds. These data demonstrate the utility of MRI in detecting the earliest pathological events in the virally infected CNS as well as the therapeutic potential of antiviral T cells in mitigating this pathology.

Repetitive mild traumatic brain injuries (mTBI) disrupt central nervous system (CNS) barriers, the erosion of which has been linked to long-term neurodegenerative and psychiatric conditions. Although much attention has been devoted to CNS vasculature following mTBI, little is known about the glia limitans superficialis - a barrier of surface-associated astrocytes that helps protect the CNS parenchyma and maintain homeostasis. Here, we identify the glia limitans superficialis as a crucial barrier surface whose breakdown after acute repeat mTBI facilitates increased cell death and recruitment of peripheral myelomonocytic cells. Using intravital microscopy, we show that brain resident microglia fortify this structure after a single mTBI yet fail to do so following secondary injury, which triggers massive recruitment of myelomonocytic cells from the periphery that contribute to further destruction of the glia limitans superficialis but not cortical cell death. We demonstrate instead that reactive oxygen species (ROS) generated in response to repeat head injury are largely responsible for enhanced cortical cell death, and therapeutic administration of the antioxidant, glutathione, markedly reduces this cell death, preserves the glia limitans, and prevents myelomonocytic cells from entering the brain parenchyma. Collectively, our findings underscore the importance of preserving the glia limitans superficialis after brain injury and offer a therapeutic means to protect this structure and the underlying cortex.

Summary CD8 T cells are essential for adaptive immunity against viral infections. Protease activated receptor 1 (PAR1) is expressed by CD8 T cells; however, its role in T cell effector function is not well defined. Here we show that in human CD8 T cells, PAR1 stimulation accelerates calcium mobilization. Furthermore, PAR1 is involved in cytotoxic T cell function by facilitating granule trafficking via actin polymerization and repositioning of the microtubule organizing center (MTOC) toward the immunological synapse. In vivo, PAR1−/− mice have reduced cytokine-producing T cells in response to a lymphocytic choriomeningitis virus (LCMV) infection and fail to efficiently control the virus. Specific deletion of PAR1 in LCMV GP33-specific CD8 T cells results in reduced expansion and diminished effector function. These data demonstrate that PAR1 plays a role in T cell activation and function, and this pathway could represent a new therapeutic strategy to modulate CD8 T cell effector function., Graphical abstract, Highlights • PAR1 signaling in human CD8 T cells accelerates TCR-induced calcium mobilization • PAR1 participates in the repositioning of the MTOC at the immunological synapse • PAR1 facilitates polarized secretion of cytotoxic granules at the immunological synapse • PAR1−/− Gp33-specific CD8 T cells show reduced expansion and effector function, Molecular biology; Immunology; Immune response

Multiple sclerosis (MS) lesions that do not resolve in the months after they form harbour ongoing demyelination and axon degeneration, and are identifiable in vivo by their paramagnetic rims on MRI scans1-3. Here, to define mechanisms underlying this disabling, progressive neurodegenerative state4-6 and foster development of new therapeutic agents, we used MRI-informed single-nucleus RNA sequencing to profile the edge of demyelinated white matter lesions at various stages of inflammation. We uncovered notable glial and immune cell diversity, especially at the chronically inflamed lesion edge. We define 'microglia inflamed in MS' (MIMS) and 'astrocytes inflamed in MS', glial phenotypes that demonstrate neurodegenerative programming. The MIMS transcriptional profile overlaps with that of microglia in other neurodegenerative diseases, suggesting that primary and secondary neurodegeneration share common mechanisms and could benefit from similar therapeutic approaches. We identify complement component 1q (C1q) as a critical mediator of MIMS activation, validated immunohistochemically in MS tissue, genetically by microglia-specific C1q ablation in mice with experimental autoimmune encephalomyelitis, and therapeutically by treating chronic experimental autoimmune encephalomyelitis with C1q blockade. C1q inhibition is a potential therapeutic avenue to address chronic white matter inflammation, which could be monitored by longitudinal assessment of its dynamic biomarker, paramagnetic rim lesions, using advanced MRI methods.

SUMMARY Most current tumor immunotherapy strategies leverage cytotoxic CD8+ T cells. Despite evidence for clinical potential of CD4+ tumor-infiltrating lymphocytes (TILs), their functional diversity limits our ability to harness their activity. Here, we use single-cell mRNA sequencing to analyze the response of tumor-specific CD4+ TILs and draining lymph node (dLN) T cells. Computational approaches to characterize subpopulations identify TIL transcriptomic patterns strikingly distinct from acute and chronic anti-viral responses and dominated by diversity among T-bet-expressing T helper type 1 (Th1)-like cells. In contrast, the dLN response includes T follicular helper (Tfh) cells but lacks Th1 cells. We identify a type I interferon-driven signature in Th1-like TILs and show that it is found in human cancers, in which it is negatively associated with response to checkpoint therapy. Our study provides a proof-of-concept methodology to characterize tumor-specific CD4+ T cell effector programs. Targeting these programs should help improve immunotherapy strategies., In Brief CD4+ T cells contribute to immune responses to tumors, but their functional diversity has hampered their utilization in clinical settings. Magen et al. use single-cell RNA sequencing to dissect the heterogeneity of CD4+ T cell responses to tumor antigens and reveal molecular divergences between anti-tumor and anti-viral responses., Graphical Abstract

Host response to viral infection is a highly regulated process involving engagement of various host factors, cytokines, chemokines, and stimulatory signals that pave the way for an antiviral immune response. The response is manifested in terms of viral sequestration, phagocytosis, and inhibition of genome replication, and, finally, if required, lymphocyte-mediated clearance of virally infected cells. During this process, cross-talk between viral and host factors can shape disease outcomes and immunopathology. Bone marrow stromal antigen 2 (BST-2), also know as tetherin, is induced by type I interferon produced in response to viral infections, as well as in certain cancers. BST-2 has been shown to be a host restriction factor of virus multiplication through its ability to physically tether budding virions and restrict viral spread. However, BST-2 has other roles in the host antiviral response. This review focuses on the diverse functions of BST-2 and its downstream signaling pathways in regulating host immune responses.

Purpose: Breast cancer diagnosed in young patients is often aggressive. Because primary breast tumors from young and older patients have similar mutational patterns, we hypothesized that the young host microenvironment promotes more aggressive metastatic disease. Experimental Design: Triple-negative or luminal B breast cancer cell lines were injected into young and older mice side-by-side to quantify lung, liver, and brain metastases. Young and older mouse brains, metastatic and naïve, were analyzed by flow cytometry. Immune populations were depleted using antibodies or a colony-stimulating factor-1 receptor (CSF-1R) inhibitor, and brain metastasis assays were conducted. Effects on myeloid populations, astrogliosis, and the neuroinflammatory response were determined. Results: Brain metastases were 2- to 4-fold higher in young as compared with older mouse hosts in four models of triple-negative or luminal B breast cancer; no age effect was observed on liver or lung metastases. Aged brains, naïve or metastatic, contained fewer resident CNS myeloid cells. Use of a CSF-1R inhibitor to deplete myeloid cells, including both microglia and infiltrating macrophages, preferentially reduced brain metastasis burden in young mice. Downstream effects of CSF-1R inhibition in young mice resembled that of an aged brain in terms of myeloid numbers, induction of astrogliosis, and Semaphorin 3A secretion within the neuroinflammatory response. Conclusions: Host microenvironmental factors contribute to the aggressiveness of triple-negative and luminal B breast cancer brain metastasis. CSF-1R inhibitors may hold promise for young brain metastasis patients.

Mutations in EPAS1, encoding hypoxia-inducible factor-2α (HIF-2α), were previously identified in a syndrome of multiple paragangliomas, somatostatinoma, and polycythemia. HIF-2α, when dimerized with HIF-1β, acts as an angiogenic transcription factor. Patients referred to the NIH for new, recurrent, and/or metastatic paraganglioma or pheochromocytoma were confirmed for EPAS1 gain-of-function mutation; imaging was evaluated for vascular malformations. We evaluated the Epas1A529V transgenic syndrome mouse model, corresponding to the mutation initially detected in the patients (EPAS1A530V), for vascular malformations via intravital 2-photon microscopy of meningeal vessels, terminal vascular perfusion with Microfil silicate polymer and subsequent intact ex vivo 14T MRI and micro-CT, and histologic sectioning and staining of the brain and identified pathologies. Further, we evaluated retinas from corresponding developmental time points (P7, P14, and P21) and the adult dura via immunofluorescent labeling of vessels and confocal imaging. We identified a spectrum of vascular malformations in all 9 syndromic patients and in all our tested mutant mice. Patient vessels had higher variant allele frequency than adjacent normal tissue. Veins of the murine retina and intracranial dura failed to regress normally at the expected developmental time points. These findings add vascular malformation as a new clinical feature of EPAS1 gain-of-function syndrome., We discovered vascular malformations due to failure of developmental vascular regression in patients with EPAS1 gain-of-function mutation syndrome and the corresponding transgenic mouse model.

Thymic stromal lymphopoietin (TSLP) is a cytokine that acts directly on CD4+ T cells and dendritic cells to promote progression of asthma, atopic dermatitis, and allergic inflammation. However, a direct role for TSLP in CD8+ T-cell primary responses remains controversial and its role in memory CD8+ T cell responses to secondary viral infection is unknown. Here, we investigate the role of TSLP in both primary and recall responses in mice using two different viral systems. Interestingly, TSLP limited the primary CD8+ T-cell response to influenza but did not affect T cell function nor significantly alter the number of memory CD8+ T cells generated after influenza infection. However, TSLP inhibited memory CD8+ T-cell responses to secondary viral infection with influenza or acute systemic LCMV infection. These data reveal a previously unappreciated role for TSLP on recall CD8+ T-cell responses in response to viral infection, findings with potential translational implications.

The neuroepithelium is a nasal barrier surface populated by olfactory sensory neurons that detect odorants in the airway and convey this information directly to the brain via axon fibers. This barrier surface is especially vulnerable to infection, yet respiratory infections rarely cause fatal encephalitis, suggesting a highly evolved immunological defense. Here, using a mouse model, we sought to understand the mechanism by which innate and adaptive immune cells thwart neuroinvasion by vesicular stomatitis virus (VSV), a potentially lethal virus that uses olfactory sensory neurons to enter the brain after nasal infection. Fate-mapping studies demonstrated that infected central nervous system (CNS) neurons were cleared noncytolytically, yet specific deletion of major histocompatibility complex class I (MHC I) from these neurons unexpectedly had no effect on viral control. Intravital imaging studies of calcium signaling in virus-specific CD8+ T cells revealed instead that brain-resident microglia were the relevant source of viral peptide-MHC I complexes. Microglia were not infected by the virus but were found to cross-present antigen after acquisition from adjacent neurons. Microglia depletion interfered with T cell calcium signaling and antiviral control in the brain after nasal infection. Collectively, these data demonstrate that microglia provide a front-line defense against a neuroinvasive nasal infection by cross-presenting antigen to antiviral T cells that noncytolytically cleanse neurons. Disruptions in this innate defense likely render the brain susceptible to neurotropic viruses like VSV that attempt to enter the CNS via the nose.

The central nervous system (CNS) is an immunologically specialized organ where restrictive barrier structures protect the parenchyma from inflammation and infection. This protection is important in preventing damage to non-renewable resident cell populations, such as neurons, responsible for functions ranging from executive to autonomic. Despite these barriers, the CNS can be infected through several entry portals, giving rise to meningitis and encephalitis. Following infection, resident cells recruit peripherally derived immune cells to sites of viral infection. In this review, we discuss recent advances in immune recruitment and entry at barrier structures as well as current immunotherapeutic strategies for the treatment of persistent viral infections.

There has been a growing interest in the use of manganese-enhanced MRI (MEMRI) for neuronal tract tracing in mammals, especially in rodents. For this MEMRI application, manganese solutions are usually directly injected into specific brain regions. Recently it was reported that manganese ions can diffuse through intact rat skull. Here the local manganese concentrations in the brain tissue after transcranial manganese application were quantified and the effectiveness of tracing from the area under the skull where delivery occurred was determined. It was established that transcranially applied manganese yields brain tissue enhancement dependent on the location of application on the skull and that manganese that enters the brain transcranially can trace to deeper brain areas.

BACKGROUNDCerebral malaria (CM) accounts for nearly 400,000 deaths annually in African children. Current dogma suggests that CM results from infected RBC (iRBC) sequestration in the brain microvasculature and resulting sequelae. Therapies targeting these events have been unsuccessful; findings in experimental models suggest that CD8+ T cells drive disease pathogenesis. However, these data have largely been ignored because corroborating evidence in humans is lacking. This work fills a critical gap in our understanding of CM pathogenesis that is impeding development of therapeutics.METHODSUsing multiplex immunohistochemistry, we characterized cerebrovascular immune cells in brain sections from 34 children who died from CM or other causes. Children were grouped by clinical diagnosis (CM+ or CM-), iRBC sequestration (Seqhi, Seqlo, Seq0) and HIV status (HIV+ or HIV-).RESULTSWe identified effector CD3+CD8+ T cells engaged on the cerebrovasculature in 69% of CM+ HIV- children. The number of intravascular CD3+CD8+ T cells was influenced by CM status (CM+ > CM-, P = 0.004) and sequestration level (Seqhi > Seqlo, P = 0.010). HIV coinfection significantly increased T cell numbers (P = 0.017) and shifted cells from an intravascular (P = 0.004) to perivascular (P < 0.0001) distribution.CONCLUSIONWithin the studied cohort, CM is associated with cerebrovascular engagement of CD3+CD8+ T cells, which is exacerbated by HIV coinfection. Thus, CD3+CD8+ T cells are highly promising targets for CM adjunctive therapy, opening new avenues for the treatment of this deadly disease.FUNDINGThis research was supported by the Intramural Research Program of the National Institutes of Health.

Summary Mammals evolved in the face of fluctuating food availability. How the immune system adapts to transient nutritional stress remains poorly understood. Here, we show that memory T cells collapsed in secondary lymphoid organs in the context of dietary restriction (DR) but dramatically accumulated within the bone marrow (BM), where they adopted a state associated with energy conservation. This response was coordinated by glucocorticoids and associated with a profound remodeling of the BM compartment, which included an increase in T cell homing factors, erythropoiesis, and adipogenesis. Adipocytes, as well as CXCR4-CXCL12 and S1P-S1P1R interactions, contributed to enhanced T cell accumulation in BM during DR. Memory T cell homing to BM during DR was associated with enhanced protection against infections and tumors. Together, this work uncovers a fundamental host strategy to sustain and optimize immunological memory during nutritional challenges that involved a temporal and spatial reorganization of the memory pool within “safe haven” compartments.

Barriers between circulation and the central nervous system (CNS) play a key role in the development and modulation of CNS immune responses. Structural variations in the vasculature traversing different anatomical regions within the CNS strongly influence where and how CNS immune responses first develop. Here, we provide an overview of cerebrovascular anatomy, focusing on the blood-CNS interface and how anatomical variations influence steady-state immunology in the compartment. We then discuss how CNS vasculature is affected by and influences the development of different pathophysiological states, such as CNS autoimmune disease, cerebrovascular injury, cerebral ischemia, and infection.

One of the major goals in the study of multiple sclerosis (MS) is to identify a beneficial therapeutic intervention that mimics the intrinsic reparative process and results in long- term clinical improvement. As yet, the therapeutic strategies tested in MS have failed to accomplish this task. However, one potential therapy that has shown some promise in rodent models of demyelination involves the administration of antibodies. Studies in var ious models of demyelination (virus-induced, autoimmune, and toxic) indicate that a sub set of autoantibodies with reactivity to CNS antigens promote remyelination. We have identified a prototypic germline IgMk monoclonal antibody, designated SCH 94.03, with reactivity to a surface antigen on oligodendrocytes that promotes CNS remyelination. This antibody has the phenotypic features of polyreactive physiological natural autoan tibodies. Additionally, treatment of MS patients with intravenous immunoglobulin, which contains these natural autoantibodies, may be efficacious in a subset of patients. We propose three mechanisms (direct stimulation of oligodendrocytes, immunomodulation, and opsonization of debris) by which polyreactive natural autoantibodies directed against CNS antigen may promote remyelination. Remyelination has the potential to not only improve conduction velocity but also may protect axons from injury and improve neuro logical function. NEUROSCIENTIST 5:19-28, 1999

Summary The generation of high-affinity neutralizing antibodies, the objective of most vaccine strategies, occurs in B cells within germinal centers (GCs) and requires rate-limiting “help” from follicular helper CD4+ T (Tfh) cells. Although Tfh differentiation is an attribute of MHC II-restricted CD4+ T cells, the transcription factors driving Tfh differentiation, notably Bcl6, are not restricted to CD4+ T cells. Here, we identified a requirement for the CD4+-specific transcription factor Thpok during Tfh cell differentiation, GC formation, and antibody maturation. Thpok promoted Bcl6 expression and bound to a Thpok-responsive region in the first intron of Bcl6. Thpok also promoted the expression of Bcl6-independent genes, including the transcription factor Maf, which cooperated with Bcl6 to mediate the effect of Thpok on Tfh cell differentiation. Our findings identify a transcriptional program that links the CD4+ lineage with Tfh differentiation, a limiting factor for efficient B cell responses, and suggest avenues to optimize vaccine generation.

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic T cell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immune cells, including T cells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by T cells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.

This protocol describes how to prepare mouse brain tissue for quantification of multiple inflammatory mediators using a multiplex bead-based immunoassay. It is important to have methods that allow quantification of multiple analytes from small amounts of tissue. Bio-Plex is a Luminex xMAP-based multiplex bead-based immunoassay technology that permits simultaneous analysis of up to 100 analytes from a single tissue sample. This assay has been used extensively to investigate analytes in plasma and serum samples as well as cultured and primary cells. Here, we describe a method for simultaneous analysis of 33 different inflammatory cytokines and chemokines from mouse brain tissue using the Bio-Plex Pro Mouse Chemokine Panel 33-Plex.

Most current tumor immunotherapy strategies leverage cytotoxic CD8+T cells. Despite evidence for clinical potential of CD4+tumor-infiltrating lymphocytes (TILs), their functional diversity has limited our ability to harness their activity. To address this issue, we have used single-cell mRNA sequencing to analyze the response of CD4+T cells specific for a defined recombinant tumor antigen, both in the tumor microenvironment and draining lymph nodes (dLN). Designing new computational approaches to characterize subpopulations, we identify TIL transcriptomic patterns strikingly distinct from those elicited by responses to infection, and dominated by diversity among T-bet-expressing T helper type 1 (Th1)-like cells. In contrast, the dLN response includes follicular helper (Tfh)-like cells but lacks Th1 cells. We identify a type I interferon-driven signature in Th1-like TILs, and show that it is found in human liver cancer and melanoma, in which it is negatively associated with response to checkpoint therapy. Our study unveils unsuspected differences between tumor and virus CD4+T cell responses, and provides a proof-of-concept methodology to characterize tumor specific CD4+T cell effector programs. Targeting these programs should help improve immunotherapy strategies.One Sentence SummarySingle-cell RNA sequencing reveals novel and highly diverse transcriptomic patterns characteristic of CD4+T cell responses to tumors.

Glioblastoma multiforme (GBM) is a universally lethal cancer of the central nervous system. Patients with GBM have a median survival of 14 months and a 5-year survival of less than 5%, a grim statistic that has remained unchanged over the last 50 years. GBM is intransigent for a variety of reasons. The immune system has a difficult time mounting a response against glioblastomas because they reside in the brain (an immunologically dampened compartment) and generate few neoantigens relative to other cancers. Glioblastomas inhabit the brain like sand in the grass and display a high degree of intra- and inter-tumoral heterogeneity, impeding efforts to therapeutically target a single pathway. Of all potential therapeutic strategies to date, virotherapy offers the greatest chance of counteracting each of the obstacles mounted by GBM. Virotherapy can xenogenize a tumor that is deft at behaving like “self”, triggering adaptive immune recognition in an otherwise immunologically quiet compartment. Viruses can also directly lyse tumor cells, creating damage and further stimulating secondary immune reactions that are detrimental to tumor growth. In this review, we summarize the basic immune mechanisms underpinning GBM immune evasion and the recent successes achieved using virotherapies.

Women diagnosed with breast cancer at a younger age (typically defined as < 40 years old) often have a poorer prognosis and an increased risk of brain metastasis compared to their older counterparts. Multivariate analyses accounting for differences in tumor characteristics have shown that age is an independent predictor of worse outcome. We therefore hypothesized that rather than intrinsic tumor properties, extrinsic microenvironmental factors contribute to age-related differences in aggressiveness. The effect of age was examined by injecting brain-selected breast cancer cells into young (2 – 6 months) and older (>12 months) mice. In four brain metastasis models examined, young mice developed 2- to 16-fold (p < 0.05) more brain metastases compared to older mice. The effect of age was not observed in mouse breast cancer models that metastasize to liver and lungs, suggesting that this is an organ-specific phenomenon. Flow cytometry-based immune-profiling of mouse brains showed that T-cells (CD4+, CD8+, and FOXP3+CD25+ regulatory T-cells), monocytes and neutrophils were elevated in brains with metastases, but the abundance of these populations did not vary dramatically with age. Furthermore, antibody-based depletion of T-cells, monocytes and neutrophils did not significantly alter brain metastasis development. Microglia, which are resident CNS myeloid cells, were 1.5-fold more abundant in young brains compared to older brains. Depletion of CNS myeloid cells using the colony stimulating factor-1-receptor inhibitor PLX3397 reduced brain metastatic tumor burden in young mice by 2.1-fold (p < 0.001). Importantly, loss of CNS myeloid cells/microglia, which are normally more activated in aged mice and thus may protect the older brain against metastasis, did not augment brain metastasis formation in older mice. These results suggest that the younger brain is more permissive for breast cancer metastasis and that targeting resident CNS myeloid cells may be an effective strategy to prevent brain metastasis development in younger patients.

T follicular helper (Tfh) cells are a specialized population of CD4+ T cells that provide help to B cells for the formation and maintenance germinal centers, and the production of high affinity class-switched antibodies, long-lived plasma cells, and memory B cells. As such, Tfh cells are essential for the generation of successful long-term humoral immunity and memory responses to vaccination and infection. Conversely, overproduction of Tfh cells has been associated with the generation of autoantibodies and autoimmunity. Data from gene-targeted mice, pharmacological inhibitors, as well as studies of human and mice expressing activating mutants have revealed that PI3Kδ is a key regulator of Tfh cell differentiation, acting downstream of ICOS to facilitate inactivation of FOXO1, repression of Klf2 and induction of Bcl6. Nonetheless, here we show that after acute LCMV infection, WT and activated-PI3Kδ mice (Pik3cdE1020K/+) show comparable ratios of Tfh:Th1 viral specific CD4+ T cells, despite higher polyclonal Tfh cells in Pik3cdE1020K/+ mice. Thus, the idea that PI3K activity primarily drives Tfh cell differentiation may be an oversimplification and PI3K-mediated pathways are likely to integrate multiple signals to promote distinct effector T cell lineages. The consequences of dysregulated Tfh cell generation will be discussed in the context of the human primary immunodeficiency “Activated PI3K-delta Syndrome” (APDS), also known as “p110 delta-activating mutation causing senescent T cells, lymphadenopathy and immunodeficiency” (PASLI). Overall, these data underscore a major role for PI3K signaling in the orchestration of T lymphocyte responses.

International audience; The transcription factor T-bet has been associated with increased susceptibility to systemic and organ-specific autoimmunity, but the mechanism by which T-bet expression promotes neuroinflammation remains unknown. In this study, we demonstrate a cardinal role of T-bet-dependent NKp46(+) innate lymphoid cells (ILCs) in the initiation of CD4(+) T(H)17-mediated neuroinflammation. Loss of T-bet specifically in NKp46(+) ILCs profoundly impaired the ability of myelin-reactive T(H)17 cells to invade central nervous system (CNS) tissue and protected the mice from autoimmunity. T-bet-dependent NKp46(+) ILCs localized in the meninges and acted as chief coordinators of meningeal inflammation by inducing the expression of proinflammatory cytokines, chemokines and matrix metalloproteinases, which together facilitated T cell entry into CNS parenchyma. Our findings uncover a detrimental role of T-bet-dependent NKp46(+) ILCs in the development of CNS autoimmune disease.

A continual dialogue exists between the central nervous system (CNS) and immune system that contributes to neural homeostasis as well as protection from microbes, repair following damage, autoimmune disease, and neurodegeneration. Characterization of resident and peripherally derived leukocyte populations within the central nervous system can provide valuable information regarding how these cells contribute to steady-state and inflammatory conditions. Flow cytometry provides a method to conduct detailed multi-parameter analyses of immune cells isolated from various tissues. This protocol provides a method to isolate leukocytes from brain, spinal cord, and meninges for flow cytometric analysis and provides a basic framework for phenotyping these cells. © 2018 by John Wiley & Sons, Inc.

The interferon inducible protein, BST-2 (or, tetherin), plays an important role in the innate antiviral defense system by inhibiting the release of many enveloped viruses. Consequently, viruses have evolved strategies to counteract the anti-viral activity of this protein. While the mechanisms by which BST-2 prevents viral dissemination have been defined, less is known about how this protein shapes the early viral distribution and immunological defense against pathogens during the establishment of persistence. Using the lymphocytic choriomeningitis virus (LCMV) model of infection, we sought insights into how the in vitro antiviral activity of this protein compared to the immunological defense mounted in vivo. We observed that BST-2 modestly reduced production of virion particles from cultured cells, which was associated with the ability of BST-2 to interfere with the virus budding process mediated by the LCMV Z protein. Moreover, LCMV does not encode a BST-2 antagonist, and viral propagation was not significantly restricted in cells that constitutively expressed BST-2. In contrast to this very modest effect in cultured cells, BST-2 played a crucial role in controlling LCMV in vivo. In BST-2 deficient mice, a persistent strain of LCMV was no longer confined to the splenic marginal zone at early times post-infection, which resulted in an altered distribution of LCMV-specific T cells, reduced T cell proliferation / function, delayed viral control in the serum, and persistence in the brain. These data demonstrate that BST-2 is important in shaping the anatomical distribution and adaptive immune response against a persistent viral infection in vivo., PLoS Pathogens, 14(7), art. no. e1007172; 2018

Tissue macrophages have an embryonic origin and can be replenished in some tissues under steady-state conditions by blood monocytes. However, little is known about the residency and properties of infiltrating monocytes after an inflammatory challenge. The meninges of the central nervous system (CNS) are populated by a dense network of macrophages that act as resident immune sentinels. Here we show that, following lymphocytic choriomeningitis virus infection, resident meningeal macrophages (MMs) acquired viral antigen and interacted directly with infiltrating cytotoxic T lymphocytes, which led to macrophage depletion. Concurrently, the meninges were infiltrated by inflammatory monocytes that engrafted the meningeal niche and remained in situ for months after viral clearance. This engraftment led to interferon-γ-dependent functional changes in the pool of MMs, including loss of bacterial and immunoregulatory sensors. Collectively, these data indicate that peripheral monocytes can engraft the meninges after an inflammatory challenge, imprinting the compartment with long-term defects in immune function.

The central nervous system (CNS) is an immunologically specialized tissue protected by a blood–brain barrier. The CNS parenchyma is enveloped by a series of overlapping membranes that are collectively referred to as the meninges. The meninges provide an additional CNS barrier, harbor a diverse array of resident immune cells, and serve as a crucial interface with the periphery. Recent studies have significantly advanced our understanding of meningeal immunity, demonstrating how a complex immune landscape influences CNS functions under steady-state and inflammatory conditions. The location and activation state of meningeal immune cells can profoundly influence CNS homeostasis and contribute to neurological disorders, but these cells are also well equipped to protect the CNS from pathogens. In this review, we discuss advances in our understanding of the meningeal immune repertoire and provide insights into how this CNS barrier operates immunologically under conditions ranging from neurocognition to inflammatory diseases.

Infiltrating monocyte-derived macrophages (MDMs) and resident microglia dominate central nervous system (CNS) injury sites. Differential roles for these cell populations after injury are beginning to be uncovered. Here, we show evidence that MDMs and microglia directly communicate with one another and differentially modulate each other’s functions. Importantly, microglia-mediated phagocytosis and inflammation are suppressed by infiltrating macrophages. In the context of spinal cord injury (SCI), preventing such communication increases microglial activation and worsens functional recovery. We suggest that macrophages entering the CNS provide a regulatory mechanism that controls acute and long-term microglia-mediated inflammation, which may drive damage in a variety of CNS conditions., Author summary The immune and the central nervous systems are now thought to be inextricably linked. In response to injury, the immune system shapes CNS recovery through a complex of molecular and cellular mediators. However, it is unclear how the kinetics, magnitude, and components of this response can be harnessed to improve CNS restoration. The two immune cells that dominate CNS lesions are resident microglia—already present before the injury—and infiltrating macrophages, which enter from the blood after injury. Both cells are thought to be critical to the outcome, yet it is unknown if, or how, they interact. To investigate this, we used mouse and human cells in microglia–macrophage coculture systems and an in vivo model of traumatic spinal cord injury. We show that infiltrating macrophages suppress key functions of microglia, such as removal of tissue debris and propagation of inflammation. Preventing macrophage–microglia communication increases microglial activation and worsens recovery. We suggest that infiltrating macrophages from the blood provide a natural control mechanism against detrimental acute and long-term microglial-mediated inflammation. Manipulation of the peripheral macrophages may provide a therapeutic treatment option to target microglial-mediated mechanisms that cause or exacerbate CNS injury and disease.

Innate and adaptive immune interactions within the central nervous system (CNS) and surrounding meninges contribute significantly to neural homeostasis as well as a variety of different neurological disorders. Two-photon laser scanning microscopy is a deep tissue imaging technique that provides a means to image immune cell dynamics and interactions in the living CNS with high spatial and temporal resolution. Optical access to the brain and meninges can be achieved through the creation of thinned skull windows, which can be made without inducing damage and inflammation in the underlying tissue. This protocol provides guidance on how to create a thinned skull window without causing CNS injury. We also describe a highly reproducible method to induce a mild traumatic brain injury using the thinned skull approach. © 2018 by John Wiley & Sons, Inc.

IL-7 is known to be vital for T cell homeostasis but has previously been presumed to be dispensable for TCR-induced activation. Here, we show that IL-7 is critical for the initial activation of CD4(+) T cells in that it provides some of the necessary early signaling components, such as activated STAT5 and Akt. Accordingly, short-term in vivo IL-7Rα blockade inhibited the activation and expansion of autoantigen-specific CD4(+) T cells and, when used to treat experimental autoimmune encephalomyelitis (EAE), prevented and ameliorated disease. Our studies demonstrate that IL-7 signaling is a prerequisite for optimal CD4(+) T cell activation and that IL-7R antagonism may be effective in treating CD4(+) T cell-mediated neuroinflammation and other autoimmune inflammatory conditions.

Significance IL-21 is a type I cytokine important for immune cell differentiation and function. We found that transcription factors STAT1 and STAT3 play partially opposing roles in IL-21 function in CD4 + T cells. Both STAT1 and STAT3 control IL-21–mediated gene regulation, with some genes, including Ifng , Tbx21 , and Il21 reciprocally regulated by these STATs. IFN-γ production was also differentially regulated by these STATs in vitro during CD4 + T-cell differentiation and in vivo during chronic lymphocytic choriomeningitis infection. Importantly, IL-21–induced IFNG and TBX21 expression was higher in CD4 + T cells from patients with autosomal dominant hyper-IgE syndrome or with STAT1 gain-of-function mutations, suggesting that dys-regulated IL-21–STAT signaling partially explains the clinical manifestations of these patients.

Cellular inhibitor of apoptosis proteins (c-IAP) 1 and 2 are widely expressed ubiquitin protein ligases that regulate a variety of cellular functions, including the sensitivity of T cells to costimulation. 4-1BB is a TNF receptor family member that signals via a complex that includes TRAF family members and the c-IAPs to upregulate NF-κB and ERK, and has been implicated in memory T-cell survival. Here, we show that effector and memory T cells from mice expressing a dominant negative E3-inactive c-IAP2 (c-IAP2(H570A)) have impaired signaling downstream of 4-1BB. When infected with lymphocytic choriomeningitis virus, unlike mice in which c-IAPs were acutely downregulated by c-IAP antagonists, the primary response of c-IAP2(H570A) mice was normal. However, the number of antigen-specific CD8(+) but not CD4(+) T cells declined more rapidly and to a greater extent in c-IAP2(H570A) mice than in WT controls. Studies with T-cell adoptive transfer demonstrated that the enhanced decay of memory cells was T-cell intrinsic. Thus, c-IAP E3 activity is required for 4-1BB coreceptor signaling and maintenance of CD8(+) T-cell memory.

Mild traumatic brain injury (mTBI) can cause meningeal vascular injury and cell death that spreads into the brain parenchyma and triggers local inflammation and recruitment of peripheral immune cells. The factors that dictate meningeal recovery after mTBI are unknown at present. Here we demonstrated that most patients who had experienced mTBI resolved meningeal vascular damage within 2–3 weeks, although injury persisted for months in a subset of patients. To understand the recovery process, we studied a mouse model of mTBI and found extensive meningeal remodeling that was temporally reliant on infiltrating myeloid cells with divergent functions. Inflammatory myelomonocytic cells scavenged dead cells in the lesion core, whereas wound-healing macrophages proliferated along the lesion perimeter and promoted angiogenesis through the clearance of fibrin and production of the matrix metalloproteinase MMP-2. Notably, a secondary injury experienced during the acute inflammatory phase aborted this repair program and enhanced inflammation, but a secondary injury experienced during the wound-healing phase did not. Our findings demonstrate that meningeal vasculature can undergo regeneration after mTBI that is dependent on distinct myeloid cell subsets.

Traumatic brain injury (TBI) is a leading cause of morbidity and disability, with a considerable socioeconomic burden. Heterogeneity of pathoanatomical subtypes and diversity in the pathogenesis and extent of injury contribute to differences in the course and outcome of TBI. Following the primary injury, extensive and lasting damage is sustained through a complex cascade of events referred to as “secondary injury”. Neuroinflammation is proposed as an important manipulable aspect of secondary injury in animal and human studies. Because neuroinflammation can be detrimental or beneficial, before developing immunomodulatory therapies, it is necessary to better understand the timing and complexity of the immune responses that follows TBI. With a rapidly increasing body of literature, there is a need for a clear summary of TBI neuroimmunology. This review presents our current understanding of the immune response to TBI in a chronological and compartment-based manner, highlighting early changes in gene expression and initial signaling pathways that lead to activation of innate and adaptive immunity. Based on recent advances in our understanding of innate immune cell activation, we propose a new paradigm to study innate immune cells following TBI that moves away from the existing M1/M2 classification of activation states towards a stimulus and disease-specific understanding of polarization state based on transcriptomic and proteomic profiling.

Proper development and function of the mammalian central nervous system (CNS) depend critically on the activity of parenchymal sentinels referred to as microglia. Although microglia were first described as ramified brain-resident phagocytes, research conducted over the past century has expanded considerably upon this narrow view and ascribed many functions to these dynamic CNS inhabitants. Microglia are now considered among the most versatile cells in the body, possessing the capacity to morphologically and functionally adapt to their ever-changing surroundings. Even in a resting state, the processes of microglia are highly dynamic and perpetually scan the CNS. Microglia are in fact vital participants in CNS homeostasis, and dysregulation of these sentinels can give rise to neurological disease. In this review, we discuss the exciting developments in our understanding of microglial biology, from their developmental origin to their participation in CNS homeostasis and pathophysiological states such as neuropsychiatric disorders, neurodegeneration, sterile injury responses, and infectious diseases. We also delve into the world of microglial dynamics recently uncovered using real-time imaging techniques.

Younger women (< 40 years old) diagnosed with breast cancer often have a poorer outcome and a higher risk of developing brain metastases compared to women diagnosed at an older age. Multi-variate analyses have shown that even after accounting for differences in primary tumor characteristics, young age is still an independent predictor of poorer outcome. We therefore hypothesize that rather than intrinsic tumor properties, age-related changes to microenvironmental factors can affect breast cancer metastasis. To test this hypothesis, human and mouse breast cancer cells were injected into young (< 6 month) and old (> 13 month) mice and metastatic tumor burden was quantified. Young mice injected with brain-seeking breast cancer cells (MDA-MB-231BR, 4T1-BR, and 99LN-BrM) developed significantly more brain metastases compared to their older counterparts. In contrast, age had no effect on lung metastatic tumor burden in five breast cancer models. The effect of age is organ-specific, and the young brain is more permissive for breast cancer metastasis. To gain mechanistic insight, the transcriptome of young and old mouse brains were analyzed by RNAseq, the metastatic microenvironment was analyzed by laser capture microdissection and mass spectrometry, immune populations have been identified by flow cytometry, and functional immune contributions analyzed by immunodepleting antibodies. Multiple brain immune subsets were altered with age. In vivo depletion experiments showed no significant contribution of CD4+ T-cells and GR1+ myeloid cells to baseline brain metastatic colonization. A subpopulation of microglia in aged metastatic brains had a high side-scatter profile, which is consistent with published reports that aged microglia are in a “pro-inflammatory” state. Depletion of microglia reduced baseline brain metastatic colonization by 50% and experiments are underway to determine their contribution to an age effect.

Traumatic brain injury (TBI) is increasingly appreciated to be highly prevalent and deleterious to neurological function 1, 2. At present no effective treatment options are available, and little is known about the complex cellular response to TBI during its acute phase. To gain novel insights into TBI pathogenesis, we developed a novel closed-skull brain injury model that mirrors some pathological features associated with mild TBI in humans and used long-term intravital microscopy to study the dynamics of the injury response from its inception. Here we demonstrate that acute brain injury induces vascular damage, meningeal cell death, and the generation of reactive oxygen species (ROS) that ultimately breach the glial limitans and promote spread of the injury into the parenchyma. In response, the brain elicits a neuroprotective, purinergic receptor dependent inflammatory response characterized by meningeal neutrophil swarming and microglial reconstitution of the damaged glial limitans. We additionally show that the skull bone is permeable to small molecular weight compounds and use this delivery route to modulate inflammation and therapeutically ameliorate brain injury through transcranial administration of the ROS scavenger, glutathione. Our results provide novel insights into the acute cellular response to TBI and a means to locally deliver therapeutic compounds to the site of injury.

Disseminated candidiasis primarily targets the kidneys and brain in mice and humans. Damage to these critical organs leads to the high mortality associated with such infections, and invasion across the blood-brain barrier can result in fungal meningoencephalitis. Candida albicans can penetrate a brain endothelial cell barrier in vitro through transcellular migration, but this mechanism has not been confirmed in vivo. MRI using the extracellular vascular contrast agent gadolinium diethylenetriaminepentaacetic acid demonstrated that integrity of the blood-brain barrier is lost during C. albicans invasion. Intravital two-photon laser scanning microscopy was used to provide the first real-time demonstration of C. albicans colonizing the living brain, where both yeast and filamentous forms of the pathogen were found. Furthermore, we adapted a previously described method utilizing MRI to monitor inflammatory cell recruitment into infected tissues in mice. Macrophages and other phagocytes were visualized in kidney and brain by the administration of ultrasmall iron oxide particles. In addition to obtaining new insights into the passage of C. albicans across the brain microvasculature, these imaging methods provide useful tools to study further the pathogenesis of C. albicans infections, to define the roles of Candida virulence genes in kidney versus brain infection and to assess new therapeutic measures for drug development.

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection that results in thousands of deaths each year, mostly in African children. The in vivo mechanisms underlying this fatal condition are not entirely understood. Using the animal model of experimental cerebral malaria (ECM), we sought mechanistic insights into the pathogenesis of CM. Fatal disease was associated with alterations in tight junction proteins, vascular breakdown in the meninges / parenchyma, edema, and ultimately neuronal cell death in the brainstem, which is consistent with cerebral herniation as a cause of death. At the peak of ECM, we revealed using intravital two-photon microscopy that myelomonocytic cells and parasite-specific CD8+ T cells associated primarily with the luminal surface of CNS blood vessels. Myelomonocytic cells participated in the removal of parasitized red blood cells (pRBCs) from cerebral blood vessels, but were not required for the disease. Interestingly, the majority of disease-inducing parasite-specific CD8+ T cells interacted with the lumen of brain vascular endothelial cells (ECs), where they were observed surveying, dividing, and arresting in a cognate peptide-MHC I dependent manner. These activities were critically dependent on IFN-γ, which was responsible for activating cerebrovascular ECs to upregulate adhesion and antigen-presenting molecules. Importantly, parasite-specific CD8+ T cell interactions with cerebral vessels were impaired in chimeric mice rendered unable to present EC antigens on MHC I, and these mice were in turn resistant to fatal brainstem pathology. Moreover, anti-adhesion molecule (LFA-1 / VLA-4) therapy prevented fatal disease by rapidly displacing luminal CD8+ T cells from cerebrovascular ECs without affecting extravascular T cells. These in vivo data demonstrate that parasite-specific CD8+ T cell-induced fatal vascular breakdown and subsequent neuronal death during ECM is associated with luminal, antigen-dependent interactions with cerebrovasculature., Author Summary Cerebral malaria (CM) is a severe and potentially fatal complication of malaria in humans that results in swelling and bleeding within the brain. The mechanisms that cause this fatal disease in humans are not completely understood. We studied an animal model known as experimental cerebral malaria to learn more about the factors that drive this disease process. Using a technique referred to as intravital microscopy, we captured movies of immune cells operating in the living brain as the disease developed. At the peak of disease, we observed evidence of immune cells interacting with and aggregating along blood vessels throughout the brain. These interactions were directly associated vascular leakage. This caused the brain to swell, which gave rise to an unsustainable pressure that ultimately killed neurons responsible for heart and lung function. The fatal swelling was induced by immune cells (referred to as T cells) interacting with bits of parasite presented by blood vessels in the brain. Removal of this parasite presentation protected the mice from fatal disease. We also evaluated a straightforward therapy that involved intravenous administration of antibodies that interfered with T cell sticking to blood vessels. Our movies revealed that this therapeutic approach rapidly displaced T cells from the blood vessels in the brain and prevented fatal disease.