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BACKGROUND AND OBJECTIVES: After the enormous health burden during the acute stages of the COVID-19 pandemic, we are now facing another important challenge, that is, long-COVID, a clinical condition with often disabling signs and symptoms of the neuropsychiatric, gastrointestinal, respiratory, cardiovascular, and immune systems. While the pathogenesis of this syndrome is still poorly understood, alterations of immune function and the gut microbiota seem to play important roles. Because affected individuals are frequently unable to work for prolonged periods and suffer numerous health compromises, effective treatments represent a major unmet medical need. Multiple potential therapies have been tried, but none is approved yet. Approaches that are able to influence the immune system and gut microbiota such as probiotics and paraprobiotics, i.e., nonviable probiotics, seem promising candidates. We, therefore, evaluated the clinical and immunologic effects of paraprobiotics in a small pilot study. METHODS: A total of 6 patients with long-COVID were followed systematically for more than 12 months after disease onset using standardized validated questionnaires, a smartphone app, and wearable sensors to assess neurocognitive function, fatigue, depressiveness, autonomic nervous system alterations, and quality of life. We then offered patients defined paraprobiotics for 4 weeks and evaluated them at the end of the treatment period using the same questionnaires, smartphone app, and wearable sensors. In addition, a comprehensive immunophenotyping and gut microbiota analysis was performed before and after treatment. RESULTS: Improvements in several of the neurologic symptoms such as dysautonomia, fatigue, and depression were documented using both patient-reported outcomes and data from the smartphone app and wearable sensors. Of interest, the expression of activation markers on some immune cell populations such as B cells and nonclassical monocytes and the expression of toll-like receptor 2 (TLR2) on T cells were reduced after paraprobiotics treatment. DISCUSSION: Our results suggest that paraprobiotics might exert positive effects in patients with long-COVID most likely by modulating immune cell activation and expression of TLR2 on T cells. Further studies with paraprobiotics should confirm the promising observations of this small pilot study and hopefully not only improve the outcome of long-COVID but also unravel the pathomechanisms of this condition. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that paraprobiotics increase the probability of favorable changes of clinical and immunologic markers in patients with long-COVID.
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COVID-19 , Microbioma Gastrointestinal , Síndrome de COVID-19 Pós-Aguda , Probióticos , Humanos , Projetos Piloto , Masculino , COVID-19/imunologia , COVID-19/complicações , COVID-19/terapia , Probióticos/farmacologia , Probióticos/administração & dosagem , Feminino , Pessoa de Meia-Idade , Idoso , Adulto , Qualidade de VidaRESUMO
BACKGROUND AND OBJECTIVES: Multiple sclerosis (MS) is considered a prototypic autoimmune disease of the CNS. It is the leading cause of chronic neurologic disability in young adults. Proinflammatory B cells and autoreactive T cells both play important roles in its pathogenesis. We aimed to study alterations of regulatory T cells (Tregs), which likely also contribute to the disease, but their involvement is less clear. METHODS: By combining multiple experimental approaches, we examined the Treg compartments in 41 patients with relapsing-remitting MS and 17 healthy donors. RESULTS: Patients with MS showed a reduced frequency of CD4+ T cells and Foxp3+ Tregs and age-dependent alterations of Treg subsets. Treg suppressive function was compromised in patients, who were treated with natalizumab, while it was unaffected in untreated and anti-CD20-treated patients. The changes in natalizumab-treated patients included increased proinflammatory cytokines and an altered transcriptome in thymus-derived (t)-Tregs, but not in peripheral (p)-Tregs. DISCUSSION: Treg dysfunction in patients with MS might be related to an altered transcriptome of t-Tregs and a proinflammatory environment. Our findings contribute to a better understanding of Tregs and their subtypes in MS.
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Esclerose Múltipla Recidivante-Remitente , Natalizumab , Linfócitos T Reguladores , Humanos , Linfócitos T Reguladores/imunologia , Adulto , Feminino , Masculino , Esclerose Múltipla Recidivante-Remitente/imunologia , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Natalizumab/farmacologia , Pessoa de Meia-Idade , Timo/imunologia , Fatores Imunológicos/farmacologia , Adulto JovemRESUMO
Clinical, pathological, and imaging evidence in multiple sclerosis (MS) suggests that a smoldering inflammatory activity is present from the earliest stages of the disease and underlies the progression of disability, which proceeds relentlessly and independently of clinical and radiological relapses (PIRA). The complex system of pathological events driving "chronic" worsening is likely linked with the early accumulation of compartmentalized inflammation within the central nervous system as well as insufficient repair phenomena and mitochondrial failure. These mechanisms are partially lesion-independent and differ from those causing clinical relapses and the formation of new focal demyelinating lesions; they lead to neuroaxonal dysfunction and death, myelin loss, glia alterations, and finally, a neuronal network dysfunction outweighing central nervous system (CNS) compensatory mechanisms. This review aims to provide an overview of the state of the art of neuropathological, immunological, and imaging knowledge about the mechanisms underlying the smoldering disease activity, focusing on possible early biomarkers and their translation into clinical practice. ANN NEUROL 2024;96:1-20.
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Biomarcadores , Progressão da Doença , Esclerose Múltipla , Humanos , Biomarcadores/metabolismo , Esclerose Múltipla/patologia , Esclerose Múltipla/diagnóstico por imagem , Esclerose Múltipla/metabolismo , RecidivaRESUMO
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.
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Imunoglobulina G , Esclerose Múltipla , Humanos , Feminino , Masculino , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/líquido cefalorraquidiano , Imunoglobulina G/sangue , Adulto , Pessoa de Meia-Idade , Herpesvirus Humano 3/imunologia , Herpesvirus Humano 3/genética , Cadeias HLA-DRB1/genética , Suécia/epidemiologia , Estudos de Coortes , Adulto Jovem , Vírus da Rubéola/genética , Vírus da Rubéola/imunologia , Antígenos HLA/genética , Anticorpos Antivirais/líquido cefalorraquidiano , Anticorpos Antivirais/sangue , Alelos , Suíça/epidemiologiaRESUMO
T cell recognition of human leukocyte antigen (HLA)-presented tumor-associated peptides is central for cancer immune surveillance. Mass spectrometry (MS)-based immunopeptidomics represents the only unbiased method for the direct identification and characterization of naturally presented tumor-associated peptides, a key prerequisite for the development of T cell-based immunotherapies. This study reports on the implementation of ion mobility separation-based time-of-flight (TOFIMS) MS for next-generation immunopeptidomics, enabling high-speed and sensitive detection of HLA-presented peptides. Applying TOFIMS-based immunopeptidomics, a novel extensive benignTOFIMS dataset was generated from 94 primary benign samples of solid tissue and hematological origin, which enabled the expansion of benign reference immunopeptidome databases with > 150,000 HLA-presented peptides, the refinement of previously described tumor antigens, as well as the identification of frequently presented self antigens and not yet described tumor antigens comprising low abundant mutation-derived neoepitopes that might serve as targets for future cancer immunotherapy development.
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Antígenos de Histocompatibilidade Classe I , Neoplasias , Humanos , Antígenos de Neoplasias , Espectrometria de Massas/métodos , Antígenos HLA , Neoplasias/terapia , Peptídeos/química , Antígenos de Histocompatibilidade Classe IIRESUMO
Background: While substantial progress has been made in the development of disease-modifying medications for multiple sclerosis (MS), a high percentage of treated patients still show progression and persistent inflammatory activity. Autologous haematopoietic stem cell transplantation (AHSCT) aims at eliminating a pathogenic immune repertoire through intense short-term immunosuppression that enables subsequent regeneration of a new and healthy immune system to re-establish immune tolerance for a long period of time. A number of mostly open-label, uncontrolled studies conducted over the past 20 years collected about 4000 cases. They uniformly reported high efficacy of AHSCT in controlling MS inflammatory disease activity, more markedly beneficial in relapsing-remitting MS. Immunological studies provided evidence for qualitative immune resetting following AHSCT. These data and improved safety profiles of transplantation procedures spurred interest in using AHSCT as a treatment option for MS. Objective: To develop expert consensus recommendations on AHSCT in Germany and outline a registry study project. Methods: An open call among MS neurologists as well as among experts in stem cell transplantation in Germany started in December 2021 to join a series of virtual meetings. Results: We provide a consensus-based opinion paper authored by 25 experts on the up-to-date optimal use of AHSCT in managing MS based on the Swiss criteria. Current data indicate that patients who are most likely to benefit from AHSCT have relapsing-remitting MS and are young, ambulatory and have high disease activity. Treatment data with AHSCT will be collected within the German REgistry Cohort of autologous haematopoietic stem CeLl trAnsplantation In MS (RECLAIM). Conclusion: Further clinical trials, including registry-based analyses, are urgently needed to better define the patient characteristics, efficacy and safety profile of AHSCT compared with other high-efficacy therapies and to optimally position it as a treatment option in different MS disease stages.
Autologous haematopoietic stem cell transplantation for multiple sclerosis Substantial progress has been made in the development of disease-modifying medications for multiple sclerosis (MS) during the last 20 years. However, in a relevant percentage of patients, the disease cannot completely be contained. Autologous haematopoietic stem cell transplantation (AHSCT) enables rebuilding of a new and healthy immune system and to potentially stop the autoimmune disease process for a long time. A number of studies documenting 4000 cases cumulatively over the past 20 years reported high efficacy of AHSCT in controlling MS inflammatory disease activity. These data and improved safety profiles of the treatment procedures spurred interest in using AHSCT as a treatment option for MS. An open call among MS neurologists as well as among experts in stem cell transplantation in Germany started in December 2021 to join a series of video calls to develop recommendations and outline a registry study project. We provide a consensus-based opinion paper authored by 25 experts on the up-to-date optimal use of AHSCT in managing MS. Current data indicate that patients are most likely to benefit from AHSCT if they are young, ambulatory, with high disease activity, that is, relapses or new magnetic resonance imaging (MRI) lesions. Treatment data with AHSCT will be collected within the German REgistry Cohort of autoLogous haematopoietic stem cell transplantation MS (RECLAIM). Further clinical trials including registry-based analyses and systematic follow-up are urgently needed to better define the optimal patient characteristics as well as the efficacy and safety profile of AHSCT compared with other high-efficacy therapies. These will help to position AHSCT as a treatment option in different MS disease stages.
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Multiple sclerosis (MS) is an inflammatory disease of the central nervous system, for which and Epstein-Barr virus (EBV) infection is a likely prerequisite. Due to the homology between Epstein-Barr nuclear antigen 1 (EBNA1) and alpha-crystallin B (CRYAB), we examined antibody reactivity to EBNA1 and CRYAB peptide libraries in 713 persons with MS (pwMS) and 722 matched controls (Con). Antibody response to CRYAB amino acids 7 to 16 was associated with MS (OR = 2.0), and combination of high EBNA1 responses with CRYAB positivity markedly increased disease risk (OR = 9.0). Blocking experiments revealed antibody cross-reactivity between the homologous EBNA1 and CRYAB epitopes. Evidence for T cell cross-reactivity was obtained in mice between EBNA1 and CRYAB, and increased CRYAB and EBNA1 CD4+ T cell responses were detected in natalizumab-treated pwMS. This study provides evidence for antibody cross-reactivity between EBNA1 and CRYAB and points to a similar cross-reactivity in T cells, further demonstrating the role of EBV adaptive immune responses in MS development.
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Infecções por Vírus Epstein-Barr , Esclerose Múltipla , alfa-Cristalinas , Animais , Camundongos , Infecções por Vírus Epstein-Barr/complicações , Herpesvirus Humano 4RESUMO
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.
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Antígenos de Neoplasias , Bactérias , Proteínas de Bactérias , Glioblastoma , Linfócitos do Interstício Tumoral , Fragmentos de Peptídeos , Humanos , Antígenos de Neoplasias/imunologia , Proteínas de Bactérias/imunologia , Vacinas Anticâncer/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Microbioma Gastrointestinal/imunologia , Glioblastoma/imunologia , Glioblastoma/patologia , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos HLA/imunologia , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/imunologia , Fragmentos de Peptídeos/imunologia , Simbiose , Bactérias/imunologia , Bactérias/patogenicidadeRESUMO
Autologous hematopoietic stem cell transplantation (aHSCT) is a highly effective treatment of multiple sclerosis (MS). It depletes autoreactive cells and subsequently renews adaptive immune cells. The possible proinflammatory potential of surviving T cells early after aHSCT has not been studied. Here, we examined the dynamics of new and surviving T cells in 27 patients after aHSCT by multidimensional flow cytometry, T cell receptor (TCR) sequencing, specificity testing, telomere length profiling, and HLA genotyping. Early after aHSCT, naïve T cells are barely detectable, whereas effector memory (EM) T cells quickly reconstitute to pre-aHSCT values. EM CD4+ T cells early after aHSCT have shorter telomeres, have higher expression of senescence and exhaustion markers, and proliferate less than those before aHSCT. We find a median TCR repertoire overlap of 26% between the early post-aHSCT EM CD4+ T cells and pre-aHSCT, indicating persistence of EM CD4+ T cells early after transplantation. The EM CD4+ TCR repertoire overlap declines to 15% at 12 months after aHSCT, whereas the naïve TCR repertoire entirely renews. HLA-DR-associated EM CD4+ T cell reactivity toward MS-related antigens decreased after aHSCT, whereas reactivity toward EBV increased. Our data show substantial survival of pre-aHSCT EM CD4+ T cells early after transplantation but complete renewal of the T cell repertoire by nascent T cells later.
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Transplante de Células-Tronco Hematopoéticas , Esclerose Múltipla , Humanos , Esclerose Múltipla/terapia , Transplante Autólogo/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Contagem de LinfócitosRESUMO
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.
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Linfócitos T CD4-Positivos , Glioblastoma , Humanos , Glioblastoma/genética , Glioblastoma/terapia , Recidiva Local de Neoplasia , Linfócitos do Interstício Tumoral , Receptores de Antígenos de Linfócitos T/genética , Vacinação , Peptídeos , Aminoácidos , Linfócitos T CD8-PositivosRESUMO
BACKGROUND AND OBJECTIVES: Autologous hematopoietic stem cell transplantation (aHSCT) is increasingly used to treat aggressive forms of multiple sclerosis (MS). This procedure is believed to result in an immune reset and restoration of a self-tolerant immune system. Immune reconstitution has been extensively studied for T cells, but only to a limited extent for B cells. As increasing evidence suggests an important role of B cells in MS pathogenesis, we sought here to better understand reconstitution and the extent of renewal of the B-cell system after aHSCT in MS. METHODS: Using longitudinal multidimensional flow cytometry and immunoglobulin heavy chain (IgH) repertoire sequencing following aHSCT with BCNU + Etoposide + Ara-C + Melphalan anti-thymocyte globulin, we analyzed the B-cell compartment in a cohort of 20 patients with MS in defined intervals before and up to 1 year after aHSCT and compared these findings with data from healthy controls. RESULTS: Total B-cell numbers recovered within 3 months and increased above normal levels 1 year after transplantation, successively shifting from a predominantly transitional to a naive immune phenotype. Memory subpopulations recovered slowly and remained below normal levels with reduced repertoire diversity 1 year after transplantation. Isotype subclass analysis revealed a proportional shift toward IgG1-expressing cells and a reduction in IgG2 cells. Mutation analysis of IgH sequences showed that highly mutated memory B cells and plasma cells may transiently survive conditioning while the analysis of sequence cluster overlap, variable (IGHV) and joining (IGHJ) gene usage and repertoire diversity suggested a renewal of the late posttransplant repertoire. In patients with early cytomegalovirus reactivation, reconstitution of naive and memory B cells was delayed. DISCUSSION: Our detailed characterization of B-cell reconstitution after aHSCT in MS indicates a reduced reactivation potential of memory B cells up to 1 year after transplantation, which may leave patients susceptible to infection, but may also be an important aspect of its mechanism of action.
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Transplante de Células-Tronco Hematopoéticas , Esclerose Múltipla , Soro Antilinfocitário , Carmustina , Citarabina , Etoposídeo , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Imunoglobulina G , Cadeias Pesadas de Imunoglobulinas , Melfalan , Esclerose Múltipla/terapiaRESUMO
Autologous hematopoietic stem cell transplantation (aHSCT) is a highly efficient treatment of multiple sclerosis (MS), and hence it likely normalizes pathological and/or enhances beneficial processes in MS. The disease pathomechanisms include neuroinflammation, glial cell activation and neuronal damage. We studied biomarkers that in part reflect these, like markers for neuroinflammation (C-X-C motif chemokine ligand (CXCL) 9, CXCL10, CXCL13, and chitinase 3-like 1 (CHI3L1)), glial perturbations (glial fibrillary acidic protein (GFAP) and in part CHI3L1), and neurodegeneration (neurofilament light chain (NfL)) by enzyme-linked immunosorbent assays (ELISA) and single-molecule array assay (SIMOA) in the serum and cerebrospinal fluid (CSF) of 32 MS patients that underwent aHSCT. We sampled before and at 1, 3, 6, 12, 24 and 36 months after aHSCT for serum, as well as before and 24 months after aHSCT for CSF. We found a strong increase of serum CXCL10, NfL and GFAP one month after the transplantation, which normalized one and two years post-aHSCT. CXCL10 was particularly increased in patients that experienced reactivation of cytomegalovirus (CMV) infection, but not those with Epstein-Barr virus (EBV) reactivation. Furthermore, patients with CMV reactivation showed increased Th1 phenotype in effector memory CD4+ T cells. Changes of the other serum markers were more subtle with a trend for an increase in serum CXCL9 early post-aHSCT. In CSF, GFAP levels were increased 24 months after aHSCT, which may indicate sustained astroglia activation 24 months post-aHSCT. Other CSF markers remained largely stable. We conclude that MS-related biomarkers indicate neurotoxicity early after aHSCT that normalizes after one year while astrocyte activation appears increased beyond that, and increased serum CXCL10 likely does not reflect inflammation within the central nervous system (CNS) but rather occurs in the context of CMV reactivation or other infections post-aHSCT.
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Quitinases , Infecções por Citomegalovirus , Infecções por Vírus Epstein-Barr , Transplante de Células-Tronco Hematopoéticas , Esclerose Múltipla , Biomarcadores , Proteína Glial Fibrilar Ácida , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Herpesvirus Humano 4 , Humanos , Ligantes , Transplante AutólogoRESUMO
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), in which pathological T cells, likely autoimmune, play a key role. Despite its central importance, the autoantigen repertoire remains largely uncharacterized. Using a novel in vitro antigen delivery method combined with the Human Protein Atlas library, we screened for T cell autoreactivity against 63 CNS-expressed proteins. We identified four previously unreported autoantigens in MS: fatty acid-binding protein 7, prokineticin-2, reticulon-3, and synaptosomal-associated protein 91, which were verified to induce interferon-γ responses in MS in two cohorts. Autoreactive profiles were heterogeneous, and reactivity to several autoantigens was MS-selective. Autoreactive T cells were predominantly CD4+ and human leukocyte antigen-DR restricted. Mouse immunization induced antigen-specific responses and CNS leukocyte infiltration. This represents one of the largest systematic efforts to date in the search for MS autoantigens, demonstrates the heterogeneity of autoreactive profiles, and highlights promising targets for future diagnostic tools and immunomodulatory therapies in MS.
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Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). Its first clinical presentation (clinically isolated syndrome, CIS) is often followed by the development of relapsing-remitting MS (RRMS). The periphery-to-CNS transmission of inflammatory molecules is a major pathophysiological pathway in MS. This could include signalling via extracellular vesicle (EV) microRNAs (miRNAs). In this study, we investigated the serum EV miRNome in CIS and RRMS patients and matched controls, with the aims to identify MS stage-specific differentially expressed miRNAs and investigate their biomarker potential and pathophysiological relevance. miRNA sequencing was conducted on serum EVs from CIS-remission, RRMS-relapse, and viral inflammatory CNS disorder patients, as well as from healthy and hospitalized controls. Differential expression analysis was conducted, followed by predictive power and target-pathway analysis. A moderate number of dysregulated serum EV miRNAs were identified in CIS-remission and RRMS-relapse patients, especially relative to healthy controls. Some of these miRNAs were also differentially expressed between the two MS stages and had biomarker potential for patient-control and CIS-RRMS separations. For the mRNA targets of the RRMS-relapse-specific EV miRNAs, biological processes inherent to MS pathophysiology were identified using in silico analysis. Study findings demonstrate that specific serum EV miRNAs have MS stage-specific biomarker potential and contribute to the identification of potential targets for novel, efficacious therapies.
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Vesículas Extracelulares/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , MicroRNAs/sangue , Esclerose Múltipla Recidivante-Remitente/sangue , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
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.
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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.
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Esclerose Múltipla/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Linfócitos T/imunologia , Adulto , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Esclerose Múltipla/fisiopatologia , Glicoproteína Mielina-Oligodendrócito/imunologiaRESUMO
BACKGROUND: Multiple sclerosis (MS) is a major health problem, leading to a significant disability and patient suffering. Although chronic activation of the immune system is a hallmark of the disease, its pathogenesis is poorly understood, while current treatments only ameliorate the disease and may produce severe side effects. METHODS: Here, we applied a network-based modeling approach based on phosphoproteomic data to uncover the differential activation in signaling wiring between healthy donors, untreated patients, and those under different treatments. Based in the patient-specific networks, we aimed to create a new approach to identify drug combinations that revert signaling to a healthy-like state. We performed ex vivo multiplexed phosphoproteomic assays upon perturbations with multiple drugs and ligands in primary immune cells from 169 subjects (MS patients, n=129 and matched healthy controls, n=40). Patients were either untreated or treated with fingolimod, natalizumab, interferon-ß, glatiramer acetate, or the experimental therapy epigallocatechin gallate (EGCG). We generated for each donor a dynamic logic model by fitting a bespoke literature-derived network of MS-related pathways to the perturbation data. Last, we developed an approach based on network topology to identify deregulated interactions whose activity could be reverted to a "healthy-like" status by combination therapy. The experimental autoimmune encephalomyelitis (EAE) mouse model of MS was used to validate the prediction of combination therapies. RESULTS: Analysis of the models uncovered features of healthy-, disease-, and drug-specific signaling networks. We predicted several combinations with approved MS drugs that could revert signaling to a healthy-like state. Specifically, TGF-ß activated kinase 1 (TAK1) kinase, involved in Transforming growth factor ß-1 proprotein (TGF-ß), Toll-like receptor, B cell receptor, and response to inflammation pathways, was found to be highly deregulated and co-druggable with all MS drugs studied. One of these predicted combinations, fingolimod with a TAK1 inhibitor, was validated in an animal model of MS. CONCLUSIONS: Our approach based on donor-specific signaling networks enables prediction of targets for combination therapy for MS and other complex diseases.
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Sistema Imunitário/metabolismo , Modelos Biológicos , Esclerose Múltipla/metabolismo , Esclerose Múltipla/terapia , Transdução de Sinais , Adulto , Algoritmos , Biomarcadores , Estudos de Casos e Controles , Terapia Combinada/métodos , Gerenciamento Clínico , Suscetibilidade a Doenças , Feminino , Humanos , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/imunologia , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/etiologia , Fosfoproteínas/metabolismo , Prognóstico , Proteoma , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacos , Resultado do TratamentoRESUMO
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.
Assuntos
Antígenos HLA/genética , Esclerose Múltipla/genética , Estudo de Associação Genômica Ampla , Subtipos Sorológicos de HLA-DR/genética , Haplótipos/genética , Humanos , Esclerose Múltipla/imunologia , Linfócitos T/imunologiaRESUMO
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.
Assuntos
Autoantígenos/imunologia , Tolerância Imunológica/imunologia , Esclerose Múltipla/imunologia , HumanosRESUMO
BACKGROUND: The human leucocyte antigen (HLA) complex controls adaptive immunity by presenting defined fractions of the intracellular and extracellular protein content to immune cells. Understanding the benign HLA ligand repertoire is a prerequisite to define safe T-cell-based immunotherapies against cancer. Due to the poor availability of benign tissues, if available, normal tissue adjacent to the tumor has been used as a benign surrogate when defining tumor-associated antigens. However, this comparison has proven to be insufficient and even resulted in lethal outcomes. In order to match the tumor immunopeptidome with an equivalent counterpart, we created the HLA Ligand Atlas, the first extensive collection of paired HLA-I and HLA-II immunopeptidomes from 227 benign human tissue samples. This dataset facilitates a balanced comparison between tumor and benign tissues on HLA ligand level. METHODS: Human tissue samples were obtained from 16 subjects at autopsy, five thymus samples and two ovary samples originating from living donors. HLA ligands were isolated via immunoaffinity purification and analyzed in over 1200 liquid chromatography mass spectrometry runs. Experimentally and computationally reproducible protocols were employed for data acquisition and processing. RESULTS: The initial release covers 51 HLA-I and 86 HLA-II allotypes presenting 90,428 HLA-I- and 142,625 HLA-II ligands. The HLA allotypes are representative for the world population. We observe that immunopeptidomes differ considerably between tissues and individuals on source protein and HLA-ligand level. Moreover, we discover 1407 HLA-I ligands from non-canonical genomic regions. Such peptides were previously described in tumors, peripheral blood mononuclear cells (PBMCs), healthy lung tissues and cell lines. In a case study in glioblastoma, we show that potential on-target off-tumor adverse events in immunotherapy can be avoided by comparing tumor immunopeptidomes to the provided multi-tissue reference. CONCLUSION: Given that T-cell-based immunotherapies, such as CAR-T cells, affinity-enhanced T cell transfer, cancer vaccines and immune checkpoint inhibition, have significant side effects, the HLA Ligand Atlas is the first step toward defining tumor-associated targets with an improved safety profile. The resource provides insights into basic and applied immune-associated questions in the context of cancer immunotherapy, infection, transplantation, allergy and autoimmunity. It is publicly available and can be browsed in an easy-to-use web interface at https://hla-ligand-atlas.org .