Single cell transcriptomics of cerebrospinal fluid cells from patients with recent-onset narcolepsy.

Narcolepsy is a rare cause of hypersomnolence and may be associated or not with cataplexy, i.e. sudden muscle weakness. These forms are designated narcolepsy-type 1 (NT1) and -type 2 (NT2), respectively. Notable characteristics of narcolepsy are that most patients carry the HLA-DQB1*06:02 allele and NT1-patients have strongly decreased levels of hypocretin-1 (synonym orexin-A) in the cerebrospinal fluid (CSF). The pathogenesis of narcolepsy is still not completely understood but the strong HLA-bias and increased frequencies of CD4+ T cells reactive to hypocretin in the peripheral blood suggest autoimmune processes in the hypothalamus. Here we analyzed the transcriptomes of CSF-cells from twelve NT1 and two NT2 patients by single cell RNAseq (scRNAseq). As controls, we used CSF cells from patients with multiple sclerosis, radiologically isolated syndrome, and idiopathic intracranial hypertension. From 27,255 CSF cells, we identified 20 clusters of different cell types and found significant differences in three CD4+ T cell and one monocyte clusters between narcolepsy and multiple sclerosis patients. Over 1000 genes were differentially regulated between patients with NT1 and other diseases. Surprisingly, the most strongly upregulated genes in narcolepsy patients as compared to controls were coding for the genome-encoded MTRNR2L12 and MTRNR2L8 peptides, which are homologous to the mitochondria-encoded HUMANIN peptide that is known playing a role in other neurological diseases including Alzheimer's disease.

The immunopathogenesis of narcolepsy type 1.

Narcolepsy type 1 (NT1) is a chronic sleep disorder resulting from the loss of a small population of hypothalamic neurons that produce wake-promoting hypocretin (HCRT; also known as orexin) peptides. An immune-mediated pathology for NT1 has long been suspected given its exceptionally tight association with the MHC class II allele HLA-DQB1*06:02, as well as recent genetic evidence showing associations with polymorphisms of T cell receptor genes and other immune-relevant loci and the increased incidence of NT1 that has been observed after vaccination with the influenza vaccine Pandemrix. The search for both self-antigens and foreign antigens recognized by the pathogenic T cell response in NT1 is ongoing. Increased T cell reactivity against HCRT has been consistently reported in patients with NT1, but data demonstrating a primary role for T cells in neuronal destruction are currently lacking. Animal models are providing clues regarding the roles of autoreactive CD4+ and CD8+ T cells in the disease. Elucidation of the pathogenesis of NT1 will allow for the development of targeted immunotherapies at disease onset and could serve as a model for other immune-mediated neurological diseases.

Immunohistologic Features of Cerebral Venous Thrombosis Due to Vaccine-Induced Immune Thrombotic Thrombocytopenia.

OBJECTIVES: Vaccine-induced immune thrombotic thrombocytopenia (VITT), a recently described entity characterized by thrombosis at unusual locations such as cerebral venous sinus and splanchnic vein, has been rarely described after adenoviral-encoded COVID-19 vaccines. In this study, we report the immunohistological correlates in 3 fatal cases of cerebral venous thrombosis related to VITT analyzed at an academic medical center. METHODS: Detailed neuropathologic studies were performed in 3 cases of cerebral venous thrombosis related to VITT after adenoviral COVID-19 vaccination. RESULTS: Autopsy revealed extensive cerebral vein thrombosis in all 3 cases. Polarized thrombi were observed with a high density of neutrophils in the core and a low density in the tail. Endothelial cells adjacent to the thrombus were largely destroyed. Markers of neutrophil extracellular trap and complement activation were present at the border and within the cerebral vein thrombi. SARS-CoV-2 spike protein was detected within the thrombus and in the adjacent vessel wall. DISCUSSION: Data indicate that neutrophils and complement activation associated with antispike immunity triggered by the vaccine is probably involved in the disease process.

Effects of a Small-Molecule Perforin Inhibitor in a Mouse Model of CD8 T Cell-Mediated Neuroinflammation.

BACKGROUND AND OBJECTIVES: Alteration of the blood-brain barrier (BBB) at the interface between blood and CNS parenchyma is prominent in most neuroinflammatory diseases. In several neurologic diseases, including cerebral malaria and Susac syndrome, a CD8 T cell-mediated targeting of endothelial cells of the BBB (BBB-ECs) has been implicated in pathogenesis. METHODS: In this study, we used an experimental mouse model to evaluate the ability of a small-molecule perforin inhibitor to prevent neuroinflammation resulting from cytotoxic CD8 T cell-mediated damage of BBB-ECs. RESULTS: Using an in vitro coculture system, we first identified perforin as an essential molecule for killing of BBB-ECs by CD8 T cells. We then found that short-term pharmacologic inhibition of perforin commencing after disease onset restored motor function and inhibited the neuropathology. Perforin inhibition resulted in preserved BBB-EC viability, maintenance of the BBB, and reduced CD8 T-cell accumulation in the brain and retina. DISCUSSION: Therefore, perforin-dependent cytotoxicity plays a key role in the death of BBB-ECs inflicted by autoreactive CD8 T cells in a preclinical model and potentially represents a therapeutic target for CD8 T cell-mediated neuroinflammatory diseases, such as cerebral malaria and Susac syndrome.

Tissue-resident CD8 T cells in central nervous system inflammatory diseases: present at the crime scene and guilty.

Tissue-resident memory T cells (TRM) represent a subset of antigen-experienced T cells that are constantly retained in a given tissue with limited trafficking through the circulation. These cells are characterized by expression of molecules enabling their tissue anchoring and downregulation of molecules promoting tissue egress. They reside at sites of previous antigen encounter and their number increases with age. TRM have been shown to provide rapid and efficient protection against tissue reinfection and TRM density correlates with efficient antitumor responses. Intriguingly, the density of CD8 TRM is increased in the central nervous system (CNS) of patients with neuroinflammatory diseases such as multiple sclerosis, or suffering from neurodegenerative diseases. In this review, we discuss current knowledge regarding the diversity of CNS-resident CD8 T cells and their role in CNS autoimmunity. Given their likely contribution to the protracted course of several inflammatory diseases of the CNS, their therapeutic targeting becomes an important challenge.

Immune cells impede repair of old neurons.

Interfering with age-related neuroimmune interactions promotes nerve regeneration.

Influenza vaccination induces autoimmunity against orexinergic neurons in a mouse model for narcolepsy.

Narcolepsy with cataplexy or narcolepsy type 1 is a disabling chronic sleep disorder resulting from the destruction of orexinergic neurons in the hypothalamus. The tight association of narcolepsy with HLA-DQB1*06:02 strongly suggest an autoimmune origin to this disease. Furthermore, converging epidemiological studies have identified an increased incidence for narcolepsy in Europe following Pandemrix® vaccination against the 2009-2010 pandemic 'influenza' virus strain. The potential immunological link between the Pandemrix® vaccination and narcolepsy remains, however, unknown. Deciphering these mechanisms may reveal pathways potentially at play in most cases of narcolepsy. Here, we developed a mouse model allowing to track and study the T-cell response against 'influenza' virus haemagglutinin, which was selectively expressed in the orexinergic neurons as a new self-antigen. Pandemrix® vaccination in this mouse model resulted in hypothalamic inflammation and selective destruction of orexin-producing neurons. Further investigations on the relative contribution of T-cell subsets in this process revealed that haemagglutinin-specific CD4 T cells were necessary for the development of hypothalamic inflammation, but insufficient for killing orexinergic neurons. Conversely, haemagglutinin-specific CD8 T cells could not initiate inflammation but were the effectors of the destruction of orexinergic neurons. Additional studies revealed pathways potentially involved in the disease process. Notably, the interferon-γ pathway was proven essential, as interferon-γ-deficient CD8 T cells were unable to elicit the loss of orexinergic neurons. Our work demonstrates that an immunopathological process mimicking narcolepsy can be elicited by immune cross-reactivity between a vaccine antigen and a neuronal self-antigen. This process relies on a synergy between autoreactive CD4 and CD8 T cells for disease development. This work furthers our understanding of the mechanisms and pathways potentially involved in the development of a neurological side effect due to a vaccine and, likely, to narcolepsy in general.

Tissue-resident CD8+ T cells drive compartmentalized and chronic autoimmune damage against CNS neurons.

The mechanisms underlying the chronicity of autoimmune diseases of the central nervous system (CNS) are largely unknown. In particular, it is unclear whether tissue-resident memory T cells (TRM) contribute to lesion pathogenesis during chronic CNS autoimmunity. Here, we observed that a high frequency of brain-infiltrating CD8+ T cells exhibit a TRM-like phenotype in human autoimmune encephalitis. Using mouse models of neuronal autoimmunity and a combination of T single-cell transcriptomics, high-dimensional flow cytometry, and histopathology, we found that pathogenic CD8+ T cells behind the blood-brain barrier adopt a characteristic TRM differentiation program, and we revealed their phenotypic and functional heterogeneity. In the diseased CNS, autoreactive tissue-resident CD8+ T cells sustained focal neuroinflammation and progressive loss of neurons, independently of recirculating CD8+ T cells. Consistently, a large fraction of autoreactive tissue-resident CD8+ T cells exhibited proliferative potential as well as proinflammatory and cytotoxic properties. Persistence of tissue-resident CD8+ T cells in the CNS and their functional output, but not their initial differentiation, were crucially dependent on CD4+ T cells. Collectively, our results point to tissue-resident CD8+ T cells as essential drivers of chronic CNS autoimmunity and suggest that therapies targeting this compartmentalized autoreactive T cell subset might be effective for treating CNS autoimmune diseases.

Toward identification of personalized immunological profiles in multiple sclerosis.

The diversity of four previously unidentified autoantigens found in multiple sclerosis mirrors its notorious clinical variability.

Narcolepsy - clinical spectrum, aetiopathophysiology, diagnosis and treatment.

Narcolepsy is a rare brain disorder that reflects a selective loss or dysfunction of orexin (also known as hypocretin) neurons of the lateral hypothalamus. Narcolepsy type 1 (NT1) is characterized by excessive daytime sleepiness and cataplexy, accompanied by sleep-wake symptoms, such as hallucinations, sleep paralysis and disturbed sleep. Diagnosis is based on these clinical features and supported by biomarkers: evidence of rapid eye movement sleep periods soon after sleep onset; cerebrospinal fluid orexin deficiency; and positivity for HLA-DQB1*06:02. Symptomatic treatment with stimulant and anticataplectic drugs is usually efficacious. This Review focuses on our current understanding of how genetic, environmental and immune-related factors contribute to a prominent (but not isolated) orexin signalling deficiency in patients with NT1. Data supporting the view of NT1 as a hypothalamic disorder affecting not only sleep-wake but also motor, psychiatric, emotional, cognitive, metabolic and autonomic functions are presented, along with uncertainties concerning the 'narcoleptic borderland', including narcolepsy type 2 (NT2). The limitations of current diagnostic criteria for narcolepsy are discussed, and a possible new classification system incorporating the borderland conditions is presented. Finally, advances and obstacles in the symptomatic and causal treatment of narcolepsy are reviewed.

Robust Control of a Brain-Persisting Parasite through MHC I Presentation by Infected Neurons.

Control of CNS pathogens by CD8 T cells is key to avoid fatal neuroinflammation. Yet, the modalities of MHC I presentation in the brain are poorly understood. Here, we analyze the antigen presentation mechanisms underlying CD8 T cell-mediated control of the Toxoplasma gondii parasite in the CNS. We show that MHC I presentation of an efficiently processed model antigen (GRA6-OVA), even when not expressed in the bradyzoite stage, reduces cyst burden and dampens encephalitis in C57BL/6 mice. Antigen presentation assays with infected primary neurons reveal a correlation between lower MHC I presentation of tachyzoite antigens by neurons and poor parasite control in vivo. Using conditional MHC I-deficient mice, we find that neuronal MHC I presentation is required for robust restriction of T. gondii in the CNS during chronic phase, showing the importance of MHC I presentation by CNS neurons in the control of a prevalent brain pathogen.

Shared T cell receptor chains in blood memory CD4+ T cells of narcolepsy type 1 patients.

Convergent evidence points to the involvement of T cells in the pathogenesis of narcolepsy type 1 (NT1). Here, we hypothesized that expanded disease-specific T cell clones could be detected in the blood of NT1 patients. We compared the TCR repertoire of circulating antigen-experienced CD4+ and CD8+ T cells from 13 recently diagnosed NT1 patients and 11 age-, sex-, and HLA-DQB1*06:02-matched healthy controls. We detected a bias in the usage of TRAV3 and TRAV8 families, with public CDR3α motifs only present in CD4+ T cells from patients with NT1. These findings may offer a unique tool to identify disease-relevant antigens.

Hepatic T Cell Tolerance Induction in An Inflammatory Environment.

For the development of autoimmune hepatitis (AIH), genetic predisposition and environmental triggers are of major importance. Although experimental AIH can be induced in genetically susceptible mice, the low precursor frequency of autoreactive T cells hampers a deeper analysis of liver-specific T cells. Here, we established a system where the model antigen hemagglutinin (HA) is expressed exclusively in hepatocytes of Rosa26-HA mice following administration of a replication deficient adenovirus expressing Cre recombinase (Ad-Cre). Under these conditions, hepatocytes mimic the generation of altered-self neoantigens. To follow autoreactive T cells during AIH, we adoptively transferred HA--specific Cl4-TCR and 6.5-TCR T cells into Ad-Cre infected -Rosa26-HA mice. Alternatively, Rosa26-HA mice have been crossed with TCR transgenic mice that were infected with Ad-Cre to break hepatic tolerance and induce the expression of the HA antigen as a hepatic self-antigen. Surprisingly, neither adoptive transfer nor a very high precursor frequency of autoreactive T cells was able to break tolerance in the context of adenoviral infection. The low proliferation of the antigen experienced autoreactive T cells despite the presence of the autoantigen and inflammation points to anergy as a potential tolerance mechanism. This model underscores the crucial importance of genetic susceptibility to break tolerance against hepatic autoantigens.

Inflammatory CNS disease caused by immune checkpoint inhibitors: status and perspectives.

Cancer treatment strategies based on immune stimulation have recently entered the clinical arena, with unprecedented success. Immune checkpoint inhibitors (ICIs) work by indiscriminately promoting immune responses, which target tumour-associated antigens or tumour-specific mutations. However, the augmented immune response, most notably the T cell response, can cause either direct neurotoxicity or, more commonly, indirect neurotoxic effects through systemic or local inflammatory mechanisms or autoimmune mechanisms. Consequently, patients treated with ICIs are susceptible to CNS disease, including paraneoplastic neurological syndromes, encephalitis, multiple sclerosis and hypophysitis. In this Opinion article, we introduce the mechanisms of action of ICIs and review their adverse effects on the CNS. We highlight the importance of early detection of these neurotoxic effects, which should be distinguished from brain metastasis, and the need for early detection of neurotoxicity. It is crucial that physicians are well informed of these neurological adverse effects, given the anticipated increase in the use of immunotherapies to treat cancer.

Endothelial cells and lymphatics at the interface between the immune and central nervous systems: implications for multiple sclerosis.

PURPOSE OF REVIEW: The central nervous system (CNS) has a unique relationship with the immune system. This review highlights the distinct roles of lymphatic vessels and endothelial cells in the interface between CNS and immune cells and invites to revisit the concept of CNS immune privilege. RECENT FINDINGS: T cells can follow several routes to penetrate the CNS parenchyma but may also benefit, together with antigen-loaded presenting cells, from the newly described lymphatic network to exit the CNS. CNS endothelial cells (EC) critically positioned at the interface between circulating immune cells and the CNS regulate the multistep cascade for immune cell trafficking into the CNS. They can also be considered as semiprofessional antigen-presenting cells through their ability to present antigens to T cells and to regulate their activation through co-stimulatory and inhibitory molecules. SUMMARY: The lymphatic network linking the CNS to draining lymph nodes may contribute to the inflammatory reaction occurring in multiple sclerosis (MS). The abundance and strategic positioning of endothelial cells at the blood-brain barrier level most likely endow them with an important role in controlling local adaptive immune responses, rendering them potential therapeutic targets in neuro-inflammatory such as MS.

High-dimensional single-cell analysis reveals the immune signature of narcolepsy.

Narcolepsy type 1 is a devastating neurological sleep disorder resulting from the destruction of orexin-producing neurons in the central nervous system (CNS). Despite its striking association with the HLA-DQB1*06:02 allele, the autoimmune etiology of narcolepsy has remained largely hypothetical. Here, we compared peripheral mononucleated cells from narcolepsy patients with HLA-DQB1*06:02-matched healthy controls using high-dimensional mass cytometry in combination with algorithm-guided data analysis. Narcolepsy patients displayed multifaceted immune activation in CD4+ and CD8+ T cells dominated by elevated levels of B cell-supporting cytokines. Additionally, T cells from narcolepsy patients showed increased production of the proinflammatory cytokines IL-2 and TNF. Although it remains to be established whether these changes are primary to an autoimmune process in narcolepsy or secondary to orexin deficiency, these findings are indicative of inflammatory processes in the pathogenesis of this enigmatic disease.

Foxo3 Transcription Factor Drives Pathogenic T Helper 1 Differentiation by Inducing the Expression of Eomes.

The transcription factor Foxo3 plays a crucial role in myeloid cell function but its role in lymphoid cells remains poorly defined. Here, we have shown that Foxo3 expression was increased after T cell receptor engagement and played a specific role in the polarization of CD4+ T cells toward pathogenic T helper 1 (Th1) cells producing interferon-γ (IFN-γ) and granulocyte monocyte colony stimulating factor (GM-CSF). Consequently, Foxo3-deficient mice exhibited reduced susceptibility to experimental autoimmune encephalomyelitis. At the molecular level, we identified Eomes as a direct target gene for Foxo3 in CD4+ T cells and we have shown that lentiviral-based overexpression of Eomes in Foxo3-deficient CD4+ T cells restored both IFN-γ and GM-CSF production. Thus, the Foxo3-Eomes pathway is central to achieve the complete specialized gene program required for pathogenic Th1 cell differentiation and development of neuroinflammation.

CD8 T cell-mediated killing of orexinergic neurons induces a narcolepsy-like phenotype in mice.

Narcolepsy with cataplexy is a rare and severe sleep disorder caused by the destruction of orexinergic neurons in the lateral hypothalamus. The genetic and environmental factors associated with narcolepsy, together with serologic data, collectively point to an autoimmune origin. The current animal models of narcolepsy, based on either disruption of the orexinergic neurotransmission or neurons, do not allow study of the potential autoimmune etiology. Here, we sought to generate a mouse model that allows deciphering of the immune mechanisms leading to orexin(+) neuron loss and narcolepsy development. We generated mice expressing the hemagglutinin (HA) as a "neo-self-antigen" specifically in hypothalamic orexin(+) neurons (called Orex-HA), which were transferred with effector neo-self-antigen-specific T cells to assess whether an autoimmune process could be at play in narcolepsy. Given the tight association of narcolepsy with the human leukocyte antigen (HLA) HLA-DQB1*06:02 allele, we first tested the pathogenic contribution of CD4 Th1 cells. Although these T cells readily infiltrated the hypothalamus and triggered local inflammation, they did not elicit the loss of orexin(+) neurons or clinical manifestations of narcolepsy. In contrast, the transfer of cytotoxic CD8 T cells (CTLs) led to both T-cell infiltration and specific destruction of orexin(+) neurons. This phenotype was further aggravated upon repeated injections of CTLs. In situ, CTLs interacted directly with MHC class I-expressing orexin(+) neurons, resulting in cytolytic granule polarization toward neurons. Finally, drastic neuronal loss caused manifestations mimicking human narcolepsy, such as cataplexy and sleep attacks. This work demonstrates the potential role of CTLs as final effectors of the immunopathological process in narcolepsy.