HLA Associations of Intrathecal IgG Production against Specific Viruses in Multiple Sclerosis.

OBJECTIVE: Specific human leucocyte antigen (HLA) alleles are not only associated with higher risk to develop multiple sclerosis (MS) and other autoimmune diseases, but also with the severity of various viral and bacterial infections. Here, we analyzed the most specific biomarker for MS, that is, the polyspecific intrathecal IgG antibody production against measles, rubella, and varicella zoster virus (MRZ reaction), for possible HLA associations in MS. METHODS: We assessed MRZ reaction from 184 Swiss patients with MS and clinically isolated syndrome (CIS) and 89 Swiss non-MS/non-CIS control patients, and performed HLA sequence-based typing, to check for associations of positive MRZ reaction with the most prevalent HLA alleles. We used a cohort of 176 Swedish MS/CIS patients to replicate significant findings. RESULTS: Whereas positive MRZ reaction showed a prevalence of 38.0% in MS/CIS patients, it was highly specific (97.7%) for MS/CIS. We identified HLA-DRB1*15:01 and other tightly linked alleles of the HLA-DR15 haplotype as the strongest HLA-encoded risk factors for a positive MRZ reaction in Swiss MS/CIS (odds ratio [OR], 3.90, 95% confidence interval [CI] 2.05-7.46, padjusted = 0.0004) and replicated these findings in Swedish MS/CIS patients (OR 2.18, 95%-CI 1.16-4.02, padjusted = 0.028). In addition, female MS/CIS patients had a significantly higher probability for a positive MRZ reaction than male patients in both cohorts combined (padjusted <0.005). INTERPRETATION: HLA-DRB1*15:01, the strongest genetic risk factor for MS, and female sex, 1 of the most prominent demographic risk factors for developing MS, predispose in MS/CIS patients for a positive MRZ reaction, the most specific CSF biomarker for MS. ANN NEUROL 2024;95:1112-1126.

Microbial peptides activate tumour-infiltrating lymphocytes in glioblastoma.

Microbial organisms have key roles in numerous physiological processes in the human body and have recently been shown to modify the response to immune checkpoint inhibitors1,2. Here we aim to address the role of microbial organisms and their potential role in immune reactivity against glioblastoma. We demonstrate that HLA molecules of both glioblastoma tissues and tumour cell lines present bacteria-specific peptides. This finding prompted us to examine whether tumour-infiltrating lymphocytes (TILs) recognize tumour-derived bacterial peptides. Bacterial peptides eluted from HLA class II molecules are recognized by TILs, albeit very weakly. Using an unbiased antigen discovery approach to probe the specificity of a TIL CD4+ T cell clone, we show that it recognizes a broad spectrum of peptides from pathogenic bacteria, commensal gut microbiota and also glioblastoma-related tumour antigens. These peptides were also strongly stimulatory for bulk TILs and peripheral blood memory cells, which then respond to tumour-derived target peptides. Our data hint at how bacterial pathogens and bacterial gut microbiota can be involved in specific immune recognition of tumour antigens. The unbiased identification of microbial target antigens for TILs holds promise for future personalized tumour vaccination approaches.

Vaccination with Designed Neopeptides Induces Intratumoral, Cross-reactive CD4+ T-cell Responses in Glioblastoma.

PURPOSE: The low mutational load of some cancers is considered one reason for the difficulty to develop effective tumor vaccines. To overcome this problem, we developed a strategy to design neopeptides through single amino acid mutations to enhance their immunogenicity. EXPERIMENTAL DESIGN: Exome and RNA sequencing as well as in silico HLA-binding predictions to autologous HLA molecules were used to identify candidate neopeptides. Subsequently, in silico HLA-anchor placements were used to deduce putative T-cell receptor (TCR) contacts of peptides. Single amino acids of TCR contacting residues were then mutated by amino acid replacements. Overall, 175 peptides were synthesized and sets of 25 each containing both peptides designed to bind to HLA class I and II molecules applied in the vaccination. Upon development of a tumor recurrence, the tumor-infiltrating lymphocytes (TIL) were characterized in detail both at the bulk and clonal level. RESULTS: The immune response of peripheral blood T cells to vaccine peptides, including natural peptides and designed neopeptides, gradually increased with repetitive vaccination, but remained low. In contrast, at the time of tumor recurrence, CD8+ TILs and CD4+ TILs responded to 45% and 100%, respectively, of the vaccine peptides. Furthermore, TIL-derived CD4+ T-cell clones showed strong responses and tumor cell lysis not only against the designed neopeptide but also against the unmutated natural peptides of the tumor. CONCLUSIONS: Turning tumor self-peptides into foreign antigens by introduction of designed mutations is a promising strategy to induce strong intratumoral CD4+ T-cell responses in a cold tumor like glioblastoma.

Characterization of Antigen-Induced CD4+ T-Cell Senescence in Multiple Sclerosis.

Antigen-induced T-cell exhaustion and T-cell senescence are peripheral regulatory mechanisms that control effector T-cell responses. Markers of exhaustion and senescence on T Cells indicate the previous activation by repetitive stimulation with specific antigens. Malignant tumors are accompanied by enhanced T-cell exhaustion and T-cell senescence resulting in immune evasion, while these control mechanisms might be diminished in autoimmune diseases including multiple sclerosis (MS). To better understand the involvement of antigen-induced T-cell senescence in controlling CD4+ T-cell-mediated autoimmune responses in MS, we have analyzed the re-expression of CD45RA and the downregulation of CD28 and CD27 molecules as markers of antigen-induced T-cell senescence in fresh cerebrospinal fluid (CSF)-infiltrating and paired circulating T cells from patients with MS. Patients with different levels of CD4+ T-cell senescence were identified and characterized regarding demographical and clinical features as well as intrathecal markers of neurodegeneration. CD4+ T-cell senescence was also analyzed in control patients to explore a putative deficit of this regulatory mechanism in MS. This study shows heterogeneity of markers of CD4+ T-cell senescence in patients with MS. Patients with high levels of CD4+ T-cell senescence in peripheral blood showed increased frequencies of CSF-infiltrating CD28+ CD27-EM CD4+ T cells with a proinflammatory Th1 functional phenotype. The correlation of these cells with the intrathecal levels of neurofilament light chain, a marker of neurodegeneration, suggests their relevance in disease pathogenesis and the involvement of T-cell senescence in their regulation. Markers of antigen-induced T-senescence, therefore, show promise as a tool to identify pathogenic CD4+ T cells in patients with MS.

T-Cell Specificity Influences Disease Heterogeneity in Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Encouraged by the enormous progress that the identification of specific autoantigens added to the understanding of neurologic autoimmune diseases, we undertook here an in-depth study of T-cell specificities in the autoimmune disease multiple sclerosis (MS), for which the spectrum of responsible autoantigens is not fully defined yet. The identification of target antigens in MS is crucial for therapeutic strategies aimed to induce antigen-specific tolerance. In addition, knowledge of relevant T-cell targets can improve our understanding of disease heterogeneity, a hallmark of MS that complicates clinical management. METHODS: The proliferative response and interferon gamma (IFN-γ) release of CSF-infiltrating CD4+ T cells from patients with MS against several autoantigens was used to identify patients with different intrathecal T-cell specificities. Fresh CSF-infiltrating and paired circulating lymphocytes in these patients were characterized in depth by ex vivo immunophenotyping and transcriptome analysis of relevant T-cell subsets. Further examination of these patients included CSF markers of inflammation and neurodegeneration and a detailed characterization with respect to demographic, clinical, and MRI features. RESULTS: By testing CSF-infiltrating CD4+ T cells from 105 patients with MS against seven long-known myelin and five recently described GDP-l-fucose synthase peptides, we identified GDP-l-fucose synthase and myelin oligodendrocyte glycoprotein (35-55) responder patients. Immunophenotyping of CSF and paired blood samples in these patients revealed a significant expansion of an effector memory (CCR7- CD45RA-) CD27- Th1 CD4+ cell subset in GDP-l-fucose synthase responders. Subsequent transcriptome analysis of this subset demonstrated expression of Th1 and cytotoxicity-associated genes. Patients with different intrathecal T-cell specificities also differ regarding inflammation- and neurodegeneration-associated biomarkers, imaging findings, expression of HLA class II alleles, and seasonal distribution of the time of the lumbar puncture. DISCUSSION: Our observations reveal an association between autoantigen reactivity and features of disease heterogeneity that strongly supports an important role of T-cell specificity in MS pathogenesis. These data have the potential to improve patient classification in clinical practice and to guide the development of antigen-specific tolerization strategies.

Multiple sclerosis: doubling down on MHC.

Human leukocyte antigen (HLA)-encoded surface molecules present antigenic peptides to T lymphocytes and play a key role in adaptive immune responses. Besides their physiological role of defending the host against infectious pathogens, specific alleles serve as genetic risk factors for autoimmune diseases. For multiple sclerosis (MS), an autoimmune disease that affects the brain and spinal cord, an association with the HLA-DR15 haplotype was described in the early 1970s. This short opinion piece discusses the difficulties of disentangling the details of this association and recent observations about the functional involvement of not only one, but also the second gene of the HLA-DR15 haplotype. This information is not only important for understanding the pathomechanism of MS, but also for antigen-specific therapies.

Antigen-Specific Immune Tolerance in Multiple Sclerosis-Promising Approaches and How to Bring Them to Patients.

Antigen-specific tolerance induction aims at treating multiple sclerosis (MS) at the root of its pathogenesis and has the prospect of personalization. Several promising tolerization approaches using different technologies and modes of action have already advanced to clinical testing. The prerequisites for successful tolerance induction include the knowledge of target antigens, core pathomechanisms, and how to pursue a clinical development path that is distinct from conventional drug development. Key aspects including patient selection, outcome measures, demonstrating the mechanisms of action as well as the positioning in the rapidly growing spectrum of MS treatments have to be considered to bring this therapy to patients.

Altered CSF Albumin Quotient Links Peripheral Inflammation and Brain Damage in MS.

OBJECTIVE: CNS damage can increase the susceptibility of the blood-brain barrier (BBB) to changes induced by systemic inflammation. The aim of this study is to better understand BBB permeability in patients with MS and to examine whether compromised BBB integrity in some of these patients is associated with CNS damage and systemic inflammation. METHODS: Routine CSF measurements of 121 patients with MS were analyzed including number and type of infiltrating cells, total protein, lactate, and oligoclonal bands, as well as intrathecal production of immunoglobulins and CSF/serum quotients for albumin, immunoglobulins, and glucose. In addition, in a subcohort of these patients, we performed ex vivo immunophenotyping of CSF-infiltrating and paired circulating lymphocytes using a panel of 13 monoclonal antibodies, we quantified intrathecal neurofilament light chain (NF-L) and chitinase 3-like 1 (CHI3L1), and we performed intrathecal lipidomic analysis. RESULTS: Patients with MS with abnormal high levels of albumin in the CSF showed a distinct CSF cell infiltrate and markers of CNS damage such as increased intrathecal levels of NF-L and CHI3L1 as well as a distinct CSF lipidomic profile. In addition, these patients showed higher numbers of circulating proinflammatory Th1 and Th1* cells compatible with systemic inflammation. Of interest, the abnormally high levels of albumin in the CSF of those patients were preserved over time. CONCLUSIONS: Our results support the hypothesis that CNS damage may increase BBB vulnerability to systemic inflammation in a subset of patients and thus contribute to disease heterogeneity.

Mechanistic and Biomarker Studies to Demonstrate Immune Tolerance in Multiple Sclerosis.

The induction of specific immunological tolerance represents an important therapeutic goal for multiple sclerosis and other autoimmune diseases. Sound knowledge of the target antigens, the underlying pathomechanisms of the disease and the presumed mechanisms of action of the respective tolerance-inducing approach are essential for successful translation. Furthermore, suitable tools and assays to evaluate the induction of immune tolerance are key aspects for the development of such treatments. However, investigation of the mechanisms of action underlying tolerance induction poses several challenges. The optimization of sensitive, robust methods which allow the assessment of low frequency autoreactive T cells and the long-term reduction or change of their responses, the detection of regulatory cell populations and their immune mediators, as well as the validation of specific biomarkers indicating reduction of inflammation and damage, are needed to develop tolerance-inducing approaches successfully to patients. This short review focuses on how to demonstrate mechanistic proof-of-concept in antigen-specific tolerance-inducing therapies in MS.

HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis.

The HLA-DR15 haplotype is the strongest genetic risk factor for multiple sclerosis (MS), but our understanding of how it contributes to MS is limited. Because autoreactive CD4+ T cells and B cells as antigen-presenting cells are involved in MS pathogenesis, we characterized the immunopeptidomes of the two HLA-DR15 allomorphs DR2a and DR2b of human primary B cells and monocytes, thymus, and MS brain tissue. Self-peptides from HLA-DR molecules, particularly from DR2a and DR2b themselves, are abundant on B cells and thymic antigen-presenting cells. Furthermore, we identified autoreactive CD4+ T cell clones that can cross-react with HLA-DR-derived self-peptides (HLA-DR-SPs), peptides from MS-associated foreign agents (Epstein-Barr virus and Akkermansia muciniphila), and autoantigens presented by DR2a and DR2b. Thus, both HLA-DR15 allomorphs jointly shape an autoreactive T cell repertoire by serving as antigen-presenting structures and epitope sources and by presenting the same foreign peptides and autoantigens to autoreactive CD4+ T cells in MS.

When a T cell engages a B cell: novel insights in multiple sclerosis.

Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) in which autoreactive T cells are considered to be the major effector cells in orchestrating and promoting CNS injuries. However, B cells emerged as additional important cellular player in multiple sclerosis immunopathogenesis since B cell depletion therapy has been found to be very effective in reducing new relapses. This short review summarises important new insights into the interaction between these two cell populations and outlines recent observations regarding how memory B cells activate brain-homing autoreactive T cells in multiple sclerosis.

Comparative Analysis of T-Cell Responses to Aquaporin-4 and Myelin Oligodendrocyte Glycoprotein in Inflammatory Demyelinating Central Nervous System Diseases.

Autoantibodies against aquaporin-4 (AQP4-Ab) and myelin oligodendrocyte glycoprotein (MOG-Ab) are associated with rare central nervous system inflammatory demyelinating diseases like neuromyelitis optica spectrum disorders (NMOSD). Previous studies have shown that not only antibodies, but also autoreactive T-cell responses against AQP4 are present in NMOSD. However, no study has yet analyzed the presence of MOG reactive T-cells in patients with MOG antibodies. Therefore, we compared AQP4 and MOG specific peripheral T-cell response in individuals with AQP4-Ab (n = 8), MOG-Ab (n = 10), multiple sclerosis (MS, n = 8), and healthy controls (HC, n = 14). Peripheral blood mononuclear cell cultures were stimulated with eight AQP4 and nine MOG peptides selected from previous studies and a tetanus toxoid peptide mix as a positive control. Antigen-specific T-cell responses were assessed using the carboxyfluorescein diacetate succinimidyl ester proliferation assay and the detection of granulocyte macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-ɤ and interleukin (IL)-4, IL-6, and IL-17A in cell culture supernatants. Additionally, human leukocyte antigen (HLA)-DQ and HLA-DR genotyping of all participants was performed. We classified a T-cell response as positive if proliferation (measured by a cell division index ≥3) was confirmed by the secretion of at least one cytokine. Reactivity against AQP4 peptides was observed in many groups, but the T-cell response against AQP4 p156-170 was present only in patients with AQP4-Ab (4/8, 50%) and absent in patients with MOG-Ab, MS and HC (corrected p = 0.02). This AQP4 p156-170 peptide specific T-cell response was significantly increased in participants with AQP4-Ab compared to those without [Odds ratio (OR) = 59.00, 95% confidence interval-CI 2.70-1,290.86]. Moreover, T-cell responses against at least one AQP4 peptide were also more frequent in participants with AQP4-Ab (OR = 11.45, 95% CI 1.24-106.05). We did not observe any significant differences for the other AQP4 peptides or any MOG peptide. AQP4-Ab were associated with HLA DQB1*02 (OR = 5.71, 95% CI 1.09-30.07), DRB1*01 (OR = 9.33, 95% CI 1.50-58.02) and DRB1*03 (OR = 6.75, 95% CI = 1.19-38.41). Furthermore, HLA DRB1*01 was also associated with the presence of AQP4 p156-170 reactive T-cells (OR = 31.67, 95% CI 1.30-772.98). To summarize, our findings suggest a role of AQP4-specific, but not MOG-specific T-cells, in NMOSD.

Human CD4+ T cell subsets differ in their abilities to cross endothelial and epithelial brain barriers in vitro.

BACKGROUND: The brain barriers establish compartments in the central nervous system (CNS) that significantly differ in their communication with the peripheral immune system. In this function they strictly control T-cell entry into the CNS. T cells can reach the CNS by either crossing the endothelial blood-brain barrier (BBB) or the epithelial blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP). OBJECTIVE: Analysis of the cellular and molecular mechanisms involved in the migration of different human CD4+ T-cell subsets across the BBB versus the BCSFB. METHODS: Human in vitro models of the BBB and BCSFB were employed to study the migration of circulating and CNS-entry experienced CD4+ T helper cell subsets (Th1, Th1*, Th2, Th17) across the BBB and BCSFB under inflammatory and non-inflammatory conditions in vitro. RESULTS: While under non-inflammatory conditions Th1* and Th1 cells preferentially crossed the BBB, under inflammatory conditions the migration rate of all Th subsets across the BBB was comparable. The migration of all Th subsets across the BCSFB from the same donor was 10- to 20-fold lower when compared to their migration across the BBB. Interestingly, Th17 cells preferentially crossed the BCSFB under both, non-inflamed and inflamed conditions. Barrier-crossing experienced Th cells sorted from CSF of MS patients showed migratory characteristics indistinguishable from those of circulating Th cells of healthy donors. All Th cell subsets could additionally cross the BCSFB from the CSF to ChP stroma side. T-cell migration across the BCSFB involved epithelial ICAM-1 irrespective of the direction of migration. CONCLUSIONS: Our observations underscore that different Th subsets may use different anatomical routes to enter the CNS during immune surveillance versus neuroinflammation with the BCSFB establishing a tighter barrier for T-cell entry into the CNS compared to the BBB. In addition, CNS-entry experienced Th cell subsets isolated from the CSF of MS patients do not show an increased ability to cross the brain barriers when compared to circulating Th cell subsets from healthy donors underscoring the active role of the brain barriers in controlling T-cell entry into the CNS. Also we identify ICAM-1 to mediate T cell migration across the BCSFB.

Effects of natalizumab therapy on intrathecal antiviral antibody responses in MS.

OBJECTIVE: To investigate the effects of natalizumab (NAT) treatment on intrathecally produced antiviral antibodies in MS. METHODS: We performed a longitudinal, observational study analyzing both serum and CSF samples collected before and during NAT treatment for antibodies against measles, rubella, mumps, influenza, entero, herpes, and polyoma viruses, including JC polyomavirus (JCV) and its nearest homologue BK polyomavirus (BKV), and bacterial control antigens by ELISA to determine the antigen-specific CSF antibody index (CAI). CAI ≥1.5 indicated intrathecal synthesis of antigen-specific antibodies. Oligoclonal bands (OCBs) by isoelectric focusing and total IgG, IgM, and IgA by immunonephelometry were analyzed additionally. RESULTS: Intrathecal synthesis of JCV- and BKV-specific IgG was detected in 20% of patients with MS at baseline and was lost significantly more frequently during NAT treatment compared with other intrathecal antiviral and antibacterial antibody reactivities. Peripheral JCV- and BKV-specific antibody responses persisted, and no cross-reactivity between JCV- and BKV-specific CSF antibodies was found. Intrathecal production of antibodies against measles, rubella, and zoster antigens (MRZ reaction) was most prevalent and persisted (73.3% before vs 66.7% after 1 year of NAT therapy). CSF OCBs also persisted (93.3% vs 80.0%), but total CSF IgG and IgM levels declined significantly. CONCLUSIONS: These data indicate that JCV-specific antibodies are produced intrathecally in a minority of patients with MS, and NAT treatment affects the intrathecal humoral immune response against JCV relatively specifically compared with other neurotropic viruses. Further studies are needed to determine whether this effect translates to higher risk of progressive multifocal leukoencephalopathy development.

Brain Citrullination Patterns and T Cell Reactivity of Cerebrospinal Fluid-Derived CD4+ T Cells in Multiple Sclerosis.

Immune responses to citrullinated peptides have been described in autoimmune diseases like rheumatoid arthritis (RA) and multiple sclerosis (MS). We investigated the post-translational modification (PTM), arginine to citrulline, in brain tissue of MS patients and controls (C) by proteomics and subsequently the cellular immune response of cerebrospinal fluid (CSF)-infiltrating T cells to citrullinated and unmodified peptides of myelin basic protein (MBP). Using specifically adapted tissue extraction- and combined data interpretation protocols we could establish a map of citrullinated proteins by identifying more than 80 proteins with two or more citrullinated peptides in human brain tissue. We report many of them for the first time. For the already described citrullinated proteins MBP, GFAP, and vimentin, we could identify additional citrullinated sites. The number of modified proteins in MS white matter was higher than control tissue. Citrullinated peptides are considered neoepitopes that may trigger autoreactivity. We used newly identified epitopes and previously reported immunodominant myelin peptides in their citrullinated and non-citrullinated form to address the recognition of CSF-infiltrating CD4+ T cells from 22 MS patients by measuring proliferation and IFN-γ secretion. We did not detect marked responses to citrullinated peptides, but slightly more strongly to the non-modified version. Based on these data, we conclude that citrullination does not appear to be an important activating factor of a T cell response, but could be the consequence of an immune- or inflammatory response. Our approach allowed us to perform a deep proteome analysis and opens new technical possibilities to analyze complex PTM patterns on minute quantities of rare tissue samples.

GDP-l-fucose synthase is a CD4+ T cell-specific autoantigen in DRB3*02:02 patients with multiple sclerosis.

Multiple sclerosis is an immune-mediated autoimmune disease of the central nervous system that develops in genetically susceptible individuals and likely requires environmental triggers. The autoantigens and molecular mimics triggering the autoimmune response in multiple sclerosis remain incompletely understood. By using a brain-infiltrating CD4+ T cell clone that is clonally expanded in multiple sclerosis brain lesions and a systematic approach for the identification of its target antigens, positional scanning peptide libraries in combination with biometrical analysis, we have identified guanosine diphosphate (GDP)-l-fucose synthase as an autoantigen that is recognized by cerebrospinal fluid-infiltrating CD4+ T cells from HLA-DRB3*-positive patients. Significant associations were found between reactivity to GDP-l-fucose synthase peptides and DRB3*02:02 expression, along with reactivity against an immunodominant myelin basic protein peptide. These results, coupled with the cross-recognition of homologous peptides from gut microbiota, suggest a possible role of this antigen as an inducer or driver of pathogenic autoimmune responses in multiple sclerosis.

Memory B Cells Activate Brain-Homing, Autoreactive CD4+ T Cells in Multiple Sclerosis.

Multiple sclerosis is an autoimmune disease that is caused by the interplay of genetic, particularly the HLA-DR15 haplotype, and environmental risk factors. How these etiologic factors contribute to generating an autoreactive CD4+ T cell repertoire is not clear. Here, we demonstrate that self-reactivity, defined as "autoproliferation" of peripheral Th1 cells, is elevated in patients carrying the HLA-DR15 haplotype. Autoproliferation is mediated by memory B cells in a HLA-DR-dependent manner. Depletion of B cells in vitro and therapeutically in vivo by anti-CD20 effectively reduces T cell autoproliferation. T cell receptor deep sequencing showed that in vitro autoproliferating T cells are enriched for brain-homing T cells. Using an unbiased epitope discovery approach, we identified RASGRP2 as target autoantigen that is expressed in the brain and B cells. These findings will be instrumental to address important questions regarding pathogenic B-T cell interactions in multiple sclerosis and possibly also to develop novel therapies.

B cells in multiple sclerosis.

PURPOSE OF REVIEW: Although it is becoming increasingly clear that B cells play important roles in multiple sclerosis (MS) pathogenesis, it is incompletely understood how they contribute. The purpose of this review is to provide an overview of the current knowledge about B cells in MS taking into account MS heterogeneity. RECENT FINDINGS: The efficacy of B cell-depleting therapies has provided strong evidence for the involvement of these cells in MS pathogenesis. Although pathogenic antibodies were found in some MS patients, the observation that plasma cells and antibodies remain largely unchanged after B-cell depletion suggests that B cells are involved in MS by other mechanisms than antibody production. SUMMARY: MS is an autoimmune disease, in which T and B cells play a crucial role. B cells can be involved in MS by different mechanisms such as presentation of antigens to T cells, transport of antigens from tissues to secondary lymphoid organs, secretion of pro-inflammatory or anti-inflammatory cytokines and in a subgroup of patients also production of pathogenic antibodies. As several B-cell/antibody-directed therapies are available, it is important to understand how these different functions of B cells and antibodies vary among patients in order to identify which could benefit best from the different therapies.

Detailed Characterization of T Cell Receptor Repertoires in Multiple Sclerosis Brain Lesions.

The antigen-specific activation of pathogenic T cells is considered essential in the initiation and maintenance of multiple sclerosis (MS). The site of activation, the differential involvement of CD4+, and CD8+ T cells, their functional phenotype, and specificity, are important aspects to understand MS pathogenesis. The analysis of clonal expansions of brain-infiltrating T cells may reveal local antigen-driven activation or specific brain homing and allow the identification of putatively pathogenic T cells. We used high-throughput T cell receptor ß-chain variable gene (TRBV) sequencing (-seq) of genomic (g)DNA, which reflects the quantity and diversity of the TRBV repertoire, to characterize three white matter demyelinating lesions with different location and inflammatory activity, and paired peripheral blood memory CD4+ and CD8+ T cell pools from a secondary progressive (SP)MS patient. Our results revealed an important sharing of clonally expanded T cells with identical TRBV sequence (clonotypes) across MS lesions independently of their proximity or inflammatory activity. Comparison with circulating T cells showed that the most frequent brain-infiltrating CD8+, but not CD4+ clonotypes were also those with highest frequency in the peripheral blood, indicating clonal expansion inside the brain or specific brain homing of CD4+ but not CD8+ T cells. Parallel TRBV-seq of complementary (c)DNA that reflects the activation status of the cells, revealed differences between lesions regarding inflammatory activity and appears to facilitate the identification of putatively pathogenic T cells in active lesions. Approaches to identify pathogenic T cells in brain lesions using TRBV-seq may benefit from focusing on lesions with high inflammatory activity and from combining gDNA and cDNA sequencing.

Phenotypic and functional complexity of brain-infiltrating T cells in Rasmussen encephalitis.

OBJECTIVE: To characterize the brain-infiltrating immune cell repertoire in Rasmussen encephalitis (RE) with special focus on the subsets, clonality, and their cytokine profile. METHODS: The immune cell infiltrate of freshly isolated brain tissue from RE was phenotypically and functionally characterized using immunohistology, flow cytometry, and T-cell receptor (TCR) deep sequencing. Identification of clonally expanded T-cell clones (TCCs) was achieved by combining flow cytometry sorting of CD4+ and CD8+ T cells and high-throughput TCR Vß-chain sequencing. The most abundant brain-infiltrating TCCs were isolated and functionally characterized. RESULTS: We found that CD4+, CD8+, and also γδ T cells infiltrate the brain tissue in RE. Further analysis surprisingly revealed that not only brain-infiltrating CD8+ but also CD4+ T cells are clonally expanded in RE. All 3 subsets exhibited a Tc1/Th1 phenotype characterized by the production of interferon (IFN)-γ and TNF. Broad cytokine profiling at the clonal level showed strong production of IFN-γ and TNF and also secretion of interleukin (IL)-5, IL-13, and granzyme B, both in CD4+ and CD8+ T cells. CONCLUSIONS: CD8+ T cells were until now considered the central players in the immunopathogenesis of RE. Our study adds to previous findings and highlights that CD4+ TCCs and γδ T cells that secrete IFN-γ and TNF are also involved. These findings underline the complexity of T-cell immunity in RE and suggest a specific role for CD4+ T cells in orchestrating the CD8+ T-cell effector immune response.