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.

Multiple environmental antigens may trigger autoimmunity in psoriasis through T-cell receptor polyspecificity.

Introduction: Psoriasis is a T-cell mediated autoimmune skin disease. HLA-C*06:02 is the main psoriasis-specific risk gene. Using a Vα3S1/Vß13S1 T-cell receptor (TCR) from a lesional psoriatic CD8+ T-cell clone we had discovered that, as an underlying pathomechanism, HLA-C*06:02 mediates an autoimmune response against melanocytes in psoriasis, and we had identified an epitope from ADAMTS-like protein 5 (ADAMTSL5) as a melanocyte autoantigen. The conditions activating the psoriatic autoimmune response in genetically predisposed individuals throughout life remain incompletely understood. Here, we aimed to identify environmental antigens that might trigger autoimmunity in psoriasis because of TCR polyspecificity. Methods: We screened databases with the peptide recognition motif of the Vα3S1/Vß13S1 TCR for environmental proteins containing peptides activating this TCR. We investigated the immunogenicity of these peptides for psoriasis patients and healthy controls by lymphocyte stimulation experiments and peptide-loaded HLA-C*06:02 tetramers. Results: We identified peptides from wheat, Saccharomyces cerevisiae, microbiota, tobacco, and pathogens that activated both the Vα3S1/Vß13S1 TCR and CD8+ T cells from psoriasis patients. Using fluorescent HLA-C*06:02 tetramers loaded with ADAMTSL5 or wheat peptides, we find that the same CD8+ T cells may recognize both autoantigen and environmental antigens. A wheat-free diet could alleviate psoriasis in several patients. Discussion: Our results show that due to TCR polyspecificity, several environmental antigens corresponding to previously suspected psoriasis risk conditions converge in the reactivity of a pathogenic psoriatic TCR and might thus be able to stimulate the psoriatic autoimmune response against melanocytes. Avoiding the corresponding environmental risk factors could contribute to the management of psoriasis.

Virus-reactive T cells expanded in aplastic anemia eliminate hematopoietic progenitor cells by molecular mimicry.

ABSTRACT: Acquired aplastic anemia is a bone marrow failure syndrome characterized by hypocellular bone marrow and peripheral blood pancytopenia. Frequent clinical responses to calcineurin inhibition and antithymocyte globulin strongly suggest critical roles for hematopoietic stem/progenitor cell-reactive T-cell clones in disease pathophysiology; however, their exact contribution and antigen specificities remain unclear. We determined differentiation states and targets of dominant T-cell clones along with their potential to eliminate hematopoietic progenitor cells in the bone marrow of 15 patients with acquired aplastic anemia. Single-cell sequencing and immunophenotyping revealed oligoclonal expansion and effector differentiation of CD8+ T-cell compartments. We reexpressed 28 dominant T-cell receptors (TCRs) of 9 patients in reporter cell lines to determine reactivity with (1) in vitro-expanded CD34+ bone marrow, (2) CD34- bone marrow, or (3) peptide pools covering immunodominant epitopes of highly prevalent viruses. Besides 5 cytomegalovirus-reactive TCRs, we identified 3 TCRs that recognized antigen presented on hematopoietic progenitor cells. T cells transduced with these TCRs eliminated hematopoietic progenitor cells of the respective patients in vitro. One progenitor cell-reactive TCR (11A5) also recognized an epitope of the Epstein-Barr virus-derived latent membrane protein 1 (LMP1) presented on HLA-A∗02:01. We identified 2 LMP1-related mimotopes within the human proteome as activating targets of TCR 11A5, providing proof of concept that molecular mimicry of viral and self-epitopes can drive T cell-mediated elimination of hematopoietic progenitor cells in aplastic anemia.

A genome-wide in vivo CRISPR screen identifies essential regulators of T cell migration to the CNS in a multiple sclerosis model.

Multiple sclerosis (MS) involves the infiltration of autoreactive T cells into the CNS, yet we lack a comprehensive understanding of the signaling pathways that regulate this process. Here, we conducted a genome-wide in vivo CRISPR screen in a rat MS model and identified 5 essential brakes and 18 essential facilitators of T cell migration to the CNS. While the transcription factor ETS1 limits entry to the CNS by controlling T cell responsiveness, three functional modules, centered around the adhesion molecule α4-integrin, the chemokine receptor CXCR3 and the GRK2 kinase, are required for CNS migration of autoreactive CD4+ T cells. Single-cell analysis of T cells from individuals with MS confirmed that the expression of these essential regulators correlates with the propensity of CD4+ T cells to reach the CNS. Our data thus reveal key regulators of the fundamental step in the induction of MS lesions.

Pairing of single-cell RNA analysis and T cell antigen receptor profiling indicates breakdown of T cell tolerance checkpoints in atherosclerosis.

Atherosclerotic plaques form in the inner layer of arteries triggering heart attacks and strokes. Although T cells have been detected in atherosclerosis, tolerance dysfunction as a disease driver remains unexplored. Here we examine tolerance checkpoints in atherosclerotic plaques, artery tertiary lymphoid organs and lymph nodes in mice burdened by advanced atherosclerosis, via single-cell RNA sequencing paired with T cell antigen receptor sequencing. Complex patterns of deteriorating peripheral T cell tolerance were observed being most pronounced in plaques followed by artery tertiary lymphoid organs, lymph nodes and blood. Affected checkpoints included clonal expansion of CD4+, CD8+ and regulatory T cells; aberrant tolerance-regulating transcripts of clonally expanded T cells; T cell exhaustion; Treg-TH17 T cell conversion; and dysfunctional antigen presentation. Moreover, single-cell RNA-sequencing profiles of human plaques revealed that the CD8+ T cell tolerance dysfunction observed in mouse plaques was shared in human coronary and carotid artery plaques. Thus, our data support the concept of atherosclerosis as a bona fide T cell autoimmune disease targeting the arterial wall.

Persistent virus-specific and clonally expanded antibody-secreting cells respond to induced self-antigen in the CNS.

B cells contribute to the pathogenesis of both cellular- and humoral-mediated central nervous system (CNS) inflammatory diseases through a variety of mechanisms. In such conditions, B cells may enter the CNS parenchyma and contribute to local tissue destruction. It remains unexplored, however, how infection and autoimmunity drive transcriptional phenotypes, repertoire features, and antibody functionality. Here, we profiled B cells from the CNS of murine models of intracranial (i.c.) viral infections and autoimmunity. We identified a population of clonally expanded, antibody-secreting cells (ASCs) that had undergone class-switch recombination and extensive somatic hypermutation following i.c. infection with attenuated lymphocytic choriomeningitis virus (rLCMV). Recombinant expression and characterisation of these antibodies revealed specificity to viral antigens (LCMV glycoprotein GP), correlating with ASC persistence in the brain weeks after resolved infection. Furthermore, these virus-specific ASCs upregulated proliferation and expansion programs in response to the conditional and transient induction of the LCMV GP as a neo-self antigen by astrocytes. This class-switched, clonally expanded, and mutated population persisted and was even more pronounced when peripheral B cells were depleted prior to autoantigen induction in the CNS. In contrast, the most expanded B cell clones in mice with persistent expression of LCMV GP in the CNS did not exhibit neo-self antigen specificity, potentially a consequence of local tolerance induction. Finally, a comparable population of clonally expanded, class-switched, and proliferating ASCs was detected in the cerebrospinal fluid of relapsing multiple sclerosis (RMS) patients. Taken together, our findings support the existence of B cells that populate the CNS and are capable of responding to locally encountered autoantigens.

Antigen-specific immune reactions by expanded CD8+ T cell clones from HLA-B*27-positive patients with spondyloarthritis.

Spondyloarthritis (SpA) is a chronic inflammatory disease that is tightly linked to HLA-B*27 but the pathophysiological basis of this link is still unknown. It is discussed whether either the instability of HLA-B*27 molecules triggers predominantly innate immune reactions or yet unknown antigenic peptides presented by HLA-B*27 induce adaptive autoimmune reactions by CD8+ T cells. To analyze the pathogenesis of SpA, we here investigated the T cell receptor (TCR) usage and whole transcriptomes of CD8+ single cells from synovial fluid of HLA-B*27-positive SpA patients and HLA-B*27-negative controls. In HLA-B*27-positive patients, we confirmed preferential expression of several TCR ß-chain families, found even more restricted usage of particular TCR α-chains, assigned matching TCR αß-chain pairs with homologous CDR3-sequences, and detected identical TCR-chains in different patients. Gene expression analyses by single cell mRNAseq revealed that genes specific for the tissue resident memory phenotype, exhaustion, and apoptosis were particularly highly expressed in expanded clonotypes from HLA-B*27-positive SpA patients. Together, several independent lines of evidence argue in favor of an (auto)antigenic peptide related pathogenesis.

Immune Phenotypes and Target Antigens of Clonally Expanded Bone Marrow T Cells in Treatment-Naïve Multiple Myeloma.

Multiple myeloma is a hematologic malignancy of monoclonal plasma cells that accumulate in the bone marrow. Despite their clinical and pathophysiologic relevance, the roles of bone marrow-infiltrating T cells in treatment-naïve patients are incompletely understood. We investigated whether clonally expanded T cells (i) were detectable in multiple myeloma bone marrow, (ii) showed characteristic immune phenotypes, and (iii) whether dominant clones recognized antigens selectively presented on multiple myeloma cells. Single-cell index sorting and T-cell receptor (TCR) αß sequencing of bone marrow T cells from 13 treatment-naïve patients showed dominant clonal expansion within CD8+ cytolytic effector compartments, and only a minority of expanded T-cell clones expressed the classic immune-checkpoint molecules PD-1, CTLA-4, or TIM-3. To identify their molecular targets, TCRs of 68 dominant bone marrow clones from five selected patients were reexpressed and incubated with multiple myeloma and non-multiple myeloma cells from corresponding patients. Only 1 of 68 TCRs recognized antigen presented on multiple myeloma cells. This TCR was HLA-C-restricted, self-peptide-specific and could be activated by multiple myeloma cells of multiple patients. The remaining dominant T-cell clones did not recognize multiple myeloma cells and were, in part, specific for antigens associated with chronic viral infections. In conclusion, we showed that dominant bone marrow T-cell clones in treatment-naïve patients rarely recognize antigens presented on multiple myeloma cells and exhibit low expression of classic immune-checkpoint molecules. Our data provide experimental context for experiences from clinical immune-checkpoint inhibition trials and will inform future T cell-dependent therapeutic strategies.

P2R Inhibitors Prevent Antibody-Mediated Complement Activation in an Animal Model of Neuromyelitis Optica : P2R Inhibitors Prevent Autoantibody Injury.

Purinergic 2 receptors (P2Rs) contribute to disease-related immune cell signaling and are upregulated in various pathological settings, including neuroinflammation. P2R inhibitors have been used to treat inflammatory diseases and can protect against complement-mediated cell injury. However, the mechanisms behind these anti-inflammatory properties of P2R inhibitors are not well understood, and their potential in CNS autoimmunity is underexplored. Here, we tested the effects of P2R inhibitors on glial toxicity in a mouse model of neuromyelitis optica spectrum disorder (NMOSD). NMOSD is a destructive CNS autoimmune disorder, in which autoantibodies against astrocytic surface antigen Aquaporin 4 (AQP4) mediate complement-dependent loss of astrocytes. Using two-photon microscopy in vivo, we found that various classes of P2R inhibitors prevented AQP4-IgG/complement-dependent astrocyte death. In vitro, these drugs inhibited the binding of AQP4-IgG or MOG-IgG to their antigen in a dose-dependent manner. Size-exclusion chromatography and circular dichroism spectroscopy revealed a partial unfolding of antibodies in the presence of various P2R inhibitors, suggesting a shared interference with IgG antibodies leading to their conformational change. Our study demonstrates that P2R inhibitors can disrupt complement activation by direct interaction with IgG. This mechanism is likely to influence the role of P2R inhibitors in autoimmune disease models and their therapeutic impact in human disease.

Rapid single-cell identification of Epstein-Barr virus-specific T-cell receptors for cellular therapy.

BACKGROUND AND AIMS: Epstein-Barr virus (EBV) is associated with solid and hematopoietic malignancies. After allogeneic stem cell transplantation, EBV infection or reactivation represents a potentially life-threatening condition with no specific treatment available in clinical routine. In vitro expansion of naturally occurring EBV-specific T cells for adoptive transfer is time-consuming and influenced by the donor's T-cell receptor (TCR) repertoire and requires a specific memory compartment that is non-existent in seronegative individuals. The authors present highly efficient identification of EBV-specific TCRs that can be expressed on human T cells and recognize EBV-infected cells. METHODS AND RESULTS: Mononuclear cells from six stem cell grafts were expanded in vitro with three HLA-B*35:01- or four HLA-A*02:01-presented peptides derived from six EBV proteins expressed during latent and lytic infection. Epitope-specific T cells expanded on average 42-fold and were single-cell-sorted and TCRαß-sequenced. To confirm specificity, 11 HLA-B*35:01- and six HLA-A*02:01-restricted dominant TCRs were expressed on reporter cell lines, and 16 of 17 TCRs recognized their presumed target peptides. To confirm recognition of virus-infected cells and assess their value for adoptive therapy, three selected HLA-B*35:01- and four HLA-A*02:01-restricted TCRs were expressed on human peripheral blood lymphocytes. All TCR-transduced cells recognized EBV-infected lymphoblastoid cell lines. CONCLUSIONS: The authors' approach provides sets of EBV epitope-specific TCRs in two different HLA contexts. Resulting cellular products do not require EBV-seropositive donors, can be adjusted to cell subsets of choice with exactly defined proportions of target-specific T cells, can be tracked in vivo and will help to overcome unmet clinical needs in the treatment and prophylaxis of EBV reactivation and associated malignancies.

GABAA Receptor Autoantibodies Decrease GABAergic Synaptic Transmission in the Hippocampal CA3 Network.

Autoimmune encephalitis associated with antibodies (Abs) against α1, ß3, and γ2 subunits of γ-aminobutyric acid receptor A (GABAAR) represents a severe form of encephalitis with refractory seizures and status epilepticus. Reduction in inhibitory GABAergic synaptic activity is linked to dysfunction of neuronal networks, hyperexcitability, and seizures. The aim in this study was to investigate the direct pathogenic effect of a recombinant GABAAR autoantibody (rAb-IP2), derived from the cerebrospinal fluid (CSF) of a patient with autoimmune GABAAR encephalitis, on hippocampal CA1 and CA3 networks. Acute brain slices from C57BL/6 mice were incubated with rAb-IP2. The spontaneous synaptic GABAergic transmission was measured using electrophysiological recordings in voltage-clamp mode. The GABAAR autoantibody rAb-IP2 reduced inhibitory postsynaptic signaling in the hippocampal CA1 pyramidal neurons with regard to the number of spontaneous inhibitory postsynaptic currents (sIPSCs) but did not affect their amplitude. In the hippocampal CA3 network, decreased number and amplitude of sIPSCs were detected, leading to decreased GABAergic synaptic transmission. Immunohistochemical staining confirmed the rAb-IP2 bound to hippocampal tissue. These findings suggest that GABAAR autoantibodies exert direct functional effects on both hippocampal CA1 and CA3 pyramidal neurons and play a crucial role in seizure generation in GABAAR autoimmune encephalitis.

ERAP1 Controls the Autoimmune Response against Melanocytes in Psoriasis by Generating the Melanocyte Autoantigen and Regulating Its Amount for HLA-C*06:02 Presentation.

Autoimmune diseases develop when autoantigens activate previously quiescent self-reactive lymphocytes. Gene-gene interaction between certain HLA class I risk alleles and variants of the endoplasmic reticulum aminopeptidase ERAP1 controls the risk for common immune-mediated diseases, including psoriasis, ankylosing spondylitis, and Behçet disease. The functional mechanisms underlying this statistical association are unknown. In psoriasis, HLA-C*06:02 mediates an autoimmune response against melanocytes by autoantigen presentation. Using various genetically modified cell lines together with an autoreactive psoriatic TCR in a TCR activation assay, we demonstrate in this study that in psoriasis, ERAP1 generates the causative melanocyte autoantigen through trimming N-terminal elongated peptide precursors to the appropriate length for presentation by HLA-C*06:02. An ERAP1 risk haplotype for psoriasis produced the autoantigen much more efficiently and increased HLA-C expression and stimulation of the psoriatic TCR by melanocytes significantly more than a protective haplotype. Compared with the overall HLA class I molecules, cell surface expression of HLA-C decreased significantly more upon ERAP1 knockout. The combined upregulation of ERAP1 and HLA-C on melanocytes in psoriasis lesions emphasizes the pathogenic relevance of their interaction in patients. We conclude that in psoriasis pathogenesis, the increased generation of an ERAP1-dependent autoantigen by an ERAP1 risk haplotype enhances the likelihood that autoantigen presentation by HLA-C*06:02 will exceed the threshold for activation of potentially autoreactive T cells, thereby triggering CD8+ T cell-mediated autoimmune disease. These data identify ERAP1 function as a central checkpoint and promising therapeutic target in psoriasis and possibly other HLA class I-associated diseases with a similar genetic predisposition.

Skin and gut imprinted helper T cell subsets exhibit distinct functional phenotypes in central nervous system autoimmunity.

Multidimensional single-cell analyses of T cells have fueled the debate about whether there is extensive plasticity or 'mixed' priming of helper T cell subsets in vivo. Here, we developed an experimental framework to probe the idea that the site of priming in the systemic immune compartment is a determinant of helper T cell-induced immunopathology in remote organs. By site-specific in vivo labeling of antigen-specific T cells in inguinal (i) or gut draining mesenteric (m) lymph nodes, we show that i-T cells and m-T cells isolated from the inflamed central nervous system (CNS) in a model of multiple sclerosis (MS) are distinct. i-T cells were Cxcr6+, and m-T cells expressed P2rx7. Notably, m-T cells infiltrated white matter, while i-T cells were also recruited to gray matter. Therefore, we propose that the definition of helper T cell subsets by their site of priming may guide an advanced understanding of helper T cell biology in health and disease.

Cross-reactivity of a pathogenic autoantibody to a tumor antigen in GABAA receptor encephalitis.

Encephalitis associated with antibodies against the neuronal gamma-aminobutyric acid A receptor (GABAA-R) is a rare form of autoimmune encephalitis. The pathogenesis is still unknown but autoimmune mechanisms were surmised. Here we identified a strongly expanded B cell clone in the cerebrospinal fluid of a patient with GABAA-R encephalitis. We expressed the antibody produced by it and showed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry that it recognizes the GABAA-R. Patch-clamp recordings revealed that it tones down inhibitory synaptic transmission and causes increased excitability of hippocampal CA1 pyramidal neurons. Thus, the antibody likely contributed to clinical disease symptoms. Hybridization to a protein array revealed the cross-reactive protein LIM-domain-only protein 5 (LMO5), which is related to cell-cycle regulation and tumor growth. We confirmed LMO5 recognition by immunoprecipitation and ELISA and showed that cerebrospinal fluid samples from two other patients with GABAA-R encephalitis also recognized LMO5. This suggests that cross-reactivity between GABAA-R and LMO5 is frequent in GABAA-R encephalitis and supports the hypothesis of a paraneoplastic etiology.

Archeological neuroimmunology: resurrection of a pathogenic immune response from a historical case sheds light on human autoimmune encephalomyelitis and multiple sclerosis.

Aim of our study was to identify the target auto-antigen in the central nervous system recognized by the immune system of a unique patient, who died more than 60 years ago from a disease with pathological changes closely resembling multiple sclerosis (MS), following a misguided immunization with lyophilized calf brain tissue. Total mRNA was isolated from formaldehyde fixed and paraffin embedded archival brain tissue containing chronic active inflammatory demyelinating lesions with inflammatory infiltrates rich in B-lymphocytes and plasma cells. Analysis of the transcriptome by next generation sequencing and reconstruction of the dominant antibody by bioinformatic tools revealed the presence of one strongly expanded B-cell clone, producing an autoantibody against a conformational epitope of myelin oligodendrocytes glycoprotein (MOG), similar to that recognized by the well characterized monoclonal anti-MOG antibody 8-18C5. The reconstructed antibody induced demyelination after systemic or intrathecal injection into animals with T-cell mediated encephalomyelitis. Our study suggests that immunization with bovine brain tissue in humans may-in a small subset of patients-induce a disease with an intermediate clinical and pathological presentation between MS and MOG-antibody associated inflammatory demyelinating disease (MOGAD).

An expanded parenchymal CD8+ T cell clone in GABAA receptor encephalitis.

The role of T cells in autoimmune encephalitis syndromes with autoantibodies against cell surface antigens is still enigmatic. Here we analyzed the T cell receptor repertoires of CD8+ and CD4+ T cells in a patient with "idiopathic" gamma-aminobutyric-acid-A receptor (GABAA -R) encephalitis by next-generation sequencing and single-cell analyses. We identified a CD8+ T cell clone that was strongly expanded in the cerebrospinal fluid and in the hippocampus but not in the operculo-insular cortex. By contrast, CD4+ T cells were polyclonal in these tissues. Such a strong clonal expansion suggests that CD8+ T cells may play a significant role in the pathogenesis.

CD8+ T cell-mediated endotheliopathy is a targetable mechanism of neuro-inflammation in Susac syndrome.

Neuroinflammation is often associated with blood-brain-barrier dysfunction, which contributes to neurological tissue damage. Here, we reveal the pathophysiology of Susac syndrome (SuS), an enigmatic neuroinflammatory disease with central nervous system (CNS) endotheliopathy. By investigating immune cells from the blood, cerebrospinal fluid, and CNS of SuS patients, we demonstrate oligoclonal expansion of terminally differentiated activated cytotoxic CD8+ T cells (CTLs). Neuropathological data derived from both SuS patients and a newly-developed transgenic mouse model recapitulating the disease indicate that CTLs adhere to CNS microvessels in distinct areas and polarize granzyme B, which most likely results in the observed endothelial cell injury and microhemorrhages. Blocking T-cell adhesion by anti-α4 integrin-intervention ameliorates the disease in the preclinical model. Similarly, disease severity decreases in four SuS patients treated with natalizumab along with other therapy. Our study identifies CD8+ T-cell-mediated endotheliopathy as a key disease mechanism in SuS and highlights therapeutic opportunities.

Early adaptive immune activation detected in monozygotic twins with prodromal multiple sclerosis.

Multiple sclerosis (MS) is a disabling disease of the CNS. Inflammatory features of MS include lymphocyte accumulations in the CNS and cerebrospinal fluid (CSF). The preclinical events leading to established MS are still enigmatic. Here we compared gene expression patterns of CSF cells from MS-discordant monozygotic twin pairs. Six "healthy" co-twins, who carry a maximal familial risk for developing MS, showed subclinical neuroinflammation (SCNI) with small MRI lesions. Four of these subjects had oligoclonal bands (OCBs). By single-cell RNA sequencing of 2752 CSF cells, we identified clonally expanded CD8+ T cells, plasmablasts, and, to a lesser extent, CD4+ T cells not only from MS patients but also from subjects with SCNI. In contrast to nonexpanded T cells, clonally expanded T cells showed characteristics of activated tissue-resident memory T (TRM) cells. The TRM-like phenotype was detectable already in cells from SCNI subjects but more pronounced in cells from patients with definite MS. Expanded plasmablast clones were detected only in MS and SCNI subjects with OCBs. Our data provide evidence for very early concomitant activation of 3 components of the adaptive immune system in MS, with a notable contribution of clonally expanded TRM-like CD8+ cells.