The immunopathogenesis of autoimmune neurological syndromes (AINS) with antibodies against the 65 kDa isoform of glutamic acid decarboxylase (anti-GAD65 AINS) remains poorly understood. To elucidate underlying disease mechanisms and identify relevant cell populations, we performed single-cell RNA and immune repertoire sequencing of cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMCs) of eight anti-GAD65 AINS individuals compared to eight noninflammatory controls. In addition, PBMCs from 19 anti-GAD65 AINS individuals and 20 healthy controls were analyzed by multidimensional flow cytometry, and brain tissue specimens from four anti-GAD65 AINS individuals were examined histologically. We detected higher frequencies of stem cell-like memory T cells (TSCM) within the PBMCs and a marked enrichment and clonal expansion of activated CD4+TSCM in the CSF of anti-GAD65 AINS individuals. Expanded T cells exhibited increased expression of proinflammatory genes. Histological analyses confirmed intraparenchymal CD8+ TSCM in three of four anti-GAD65 AINS individuals and rare meningeal/intraparenchymal CD4+ TSCM in one person. Although CSF B cell receptors (BCRs) displayed little to no clonal expansion, recombinant expression of 40 CSF BCRs revealed that 25% were GAD65-reactive with increased somatic hypermutations compared to non-GAD65-reactive BCRs. These findings further support the concept of an antigen-specific intrathecal immune response. In summary, we characterize the immune landscape of anti-GAD65 AINS at single-cell resolution and identify clonally expanded TSCM with cytotoxic properties as a hallmark of this disease.

Background Glutamic acid decarboxylase (GAD) antibody-associated autoimmune neurological syndromes (AINS) are a spectrum of autoimmune-mediated CNS disorders. While antibodies targeting the 65 kDa isoform of GAD are of high diagnostic value, T cell mediated cytotoxicity has been identified as a key component of disease pathogenesis. The precise pathophysiological mechanisms by which the disease is triggered and maintained, however, remain incompletely understood. Methods We performed single-cell transcriptome and immune repertoire sequencing (sc-seq) in CSF and blood of 8 anti-GAD65 AINS patients compared to 8 non-inflammatory controls. Monoclonal antibodies (mAbs) were synthesized from B cell receptor (BCR) data to evaluate the B cellular immune response. Findings We identified an increase and expansion of activated CD4+ stem cell-like memory T cells (TSCM) in the CSF of anti-GAD65 AINS patients. Expanded T cells showed increased expression of proinflammatory genes. The mAb analysis revealed a high frequency of GAD65-reactive BCRs in the CSF of anti-GAD65 AINS patients with increased somatic hypermutations compared to non-GAD-reactive BCRs and BCRs from controls. Conclusions Sc-seq identified clonally expanded CD4+ TSCM in the CSF of anti-GAD65 AINS patients harboring cytotoxic properties likely contributing to disease pathogenesis. GAD-reactive B cells circulate in the CSF of anti-GAD65 AINS patients further supporting the concept of an antigen-specific intrathecal immune response. Future studies need to clarify the actual pathogenicity of these immune cells and the link between T and B cellular immune mechanisms in the pathogenesis of anti-GAD65 AINS. Funding German Research Foundation (ERARE18-202 UltraAIE), German Federal Ministry of Education and Research (CONNECT GENERATE (2.0); 01GM1908A and 01GM2208A). ### Competing Interest Statement SB is affiliated with Novartis, Basel, Switzerland; SR received travel grants from Merck Healthcare Germany GmbH, Alexion Pharmaceuticals, Bristol Myers Squibb, the German Neurological Society, and the American Academy of Neurology. She served on a scientific advisory board from Merck Healthcare Germany GmbH and received honoraria for lecturing from Roche and Merck Healthcare Germany GmbH. Her research was supported by Novartis, "Stiftung zur Foerderung junger Neurowissenschaftler", and "Else Kroener-Fresenius-Stiftung"; KE was supported by the "Friedrich-Baur-Stiftung"; EK has received personal honoraria for lectures or advice from UCB, UNEEG, Medtronic, Desitin and Precisis and has participated in trials sponsored by Medtronic, UCB, Ergomed, and Precisis. Her research is supported by the Munich Clinican Scientist Program (MCSP); MR received lecture fees and/or served on advisory boards for AstraZeneca, Echosens, Eli Lilly, Madrigal, Merck-MSD, Novo Nordisk, Synlab and Target RWE and performed investigator-initiated research with support from Boehringer Ingelheim and Novo Nordisk to the German Diabetes Center (DDZ); HW received speaker honoraria from Alexion, Biogen, Bristol Myers Squibb, Genzyme, Merck, Neurodiem, Novartis, Ology, Roche, TEVA, and WebMD Global. He received honoraria for consulting services from Abbvie, Actelion, Argenx, BD, Bristol Myers Squibb, EMD Serono, Fondazione Cariplo, Gossamer Bio, Idorsia, Immunic, Immunovant, INmune Bio_Syneos Health, Janssen, Merck, NexGen, Novartis, Roche, Sanofi, Swiss MS Society, UCB and Worldwide Clinical Trials. His research is supported by the German Myasthenia Gravis Society; NHS declares financial ties to Menarini Silicon Biosystems (CellSearch Assay) in the form of third-party funding for research support, as well as to Illumina (NGS products) in the form of lecture fees; SGM receives honoraria for lecturing, and travel expenses for attending meetings from Academy 2, Argenx, Alexion, Almirall, Amicus Therapeutics Germany, Bayer Health Care, Biogen, BioNtech, BMS, Celgene, Datamed, Demecan, Desitin, Diamed, Diaplan, DIU Dresden, DPmed, Gen Medicine and Healthcare products, Genzyme, Hexal AG, IGES, Impulze GmbH, Janssen Cilag, KW Medipoint, MedDay Pharmaceuticals, Merck Serono, MICE, Mylan, Neuraxpharm, Neuropoint, Novartis, Novo Nordisk, ONO Pharma, Oxford PharmaGenesis, QuintilesIMS, Roche, Sanofi-Aventis, Springer Medizin Verlag, STADA, Chugai Pharma, Teva, UCB, Viatris, Wings for Life international and Xcenda, his research is funded by the German Ministry for Education and Research (BMBF), Bundesinstitut fuer Risikobewertung (BfR), Deutsche Forschungsgemeinschaft (DFG), Else Kroener Fresenius Foundation, Gemeinsamer Bundesausschuss (G-BA), German Academic Exchange Service, Hertie Foundation, Interdisciplinary Center for Clinical Studies (IZKF) Muenster, German Foundation Neurology and Alexion, Almirall, Amicus Therapeutics Germany, Biogen, Diamed, DGM e.v., Fresenius Medical Care, Genzyme, Gesellschaft von Freunden und Foerderern der Heinrich-Heine-Universitaet Duesseldorf e.V., HERZ Burgdorf, Merck Serono, Novartis, ONO Pharma, Roche, and Teva; MJT M.J. Titulaer has received funds for serving on a scientific advisory board of AmGen, ArgenX, Arialys and UCB, received funds from Dioraphte (2001 0403) EpilepsieNL (19-18), filed a patent for methods for typing neurologic disorders and cancer, and devices for use therein, received research funds for consultation at Guidepoint Global LLC, an unrestricted research grant from CSL Behring, and an unrestricted research grant from Euroimmun, was supported by an Interlaken Leadership Award and an E-RARE3 grant (Netherlands Medical Research Foundation [ZonMW], ERARE-JTC2018 202: UltraAIE).; FL is also supported by E-Rare Joint Transnational research support (ERA-Net, LE3064/2-1), Stiftung Pathobiochemie of the German Society for Laboratory Medicine and HORIZON MSCA 2022 Doctoral Network 101119457 - IgG4-TREAT and discloses speaker honoraria from Grifols, Teva, Biogen, Bayer, Roche, Novartis, Fresenius, travel funding from Merck, Grifols and Bayer and serving on advisory boards for Roche, Biogen and Alexion; GMzH received research support from Biogen and Merck Germany; he received honoraria from Alexion and LFB Pharma and participated in Data Safety Monitoring and/or Advisory Boards of LFB Pharma, Roche and Immunovant; FT received grant support from Novartis Pharma GmbH and speaker honoraria from Alexion Pharmaceuticals; NM received honoraria for lecturing and travel expenses for attending meetings from Biogen Idec, GlaxoSmithKline, Teva, Novartis Pharma, Bayer Healthcare, Genzyme, Alexion Pharmaceuticals, Fresenius Medical Care, Diamed, UCB Pharma, AngeliniPharma, BIAL and Sanofi-Aventis, received royalties for consulting from UCB Pharma, Alexion Pharmaceuticals and Sanofi, and received financial research support from Euroimmun, Fresenius Medical Care, Diamed, Alexion Pharmaceuticals, Novartis Pharma, and Sanofi. MS, RM, and LK are employees of EUROIMMUN. The remaining authors declare no conflict of interest. German Research Foundation, ERARE18-202 UltraAIE German Federal Ministry of Education and Research, CONNECT GENERATE (2.0); 01GM1908A and 01GM2208A

Aging is accompanied by considerable changes in the gut microbiome, yet the molecular mechanisms driving aging and the role of the microbiome remain unclear. Here we combined metagenomics, transcriptomics and metabolomics from aging mice with metabolic modelling to characterize host-microbiome interactions during aging. Reconstructing integrated metabolic models of host and 181 mouse gut microorganisms, we show a complex dependency of host metabolism on known and previously undescribed microbial interactions. We observed a pronounced reduction in metabolic activity within the aging microbiome accompanied by reduced beneficial interactions between bacterial species. These changes coincided with increased systemic inflammation and the downregulation of essential host pathways, particularly in nucleotide metabolism, predicted to rely on the microbiota and critical for preserving intestinal barrier function, cellular replication and homeostasis. Our results elucidate microbiome-host interactions that potentially influence host aging processes. These pathways could serve as future targets for the development of microbiome-based anti-aging therapies.

Autoreactive CD4+ cells control the immune response in chronic autoimmune diseases but remain difficult to track. The ex vivo characterization of autoreactive T cells is essential to understand their function and how they adapt to chronic autoimmunity. Neuromyelitis optica spectrum disorder is a prototypic autoimmune disease of the central nervous system targeting aquaporin-4 (AQP4). AQP4-specific CD4+ T cells are centrally involved in disease development. However, their functional and molecular properties are poorly characterized. We combined HLA-tetramer- and antigen-reactive T cell enrichment and scRNA sequencing to characterize ex vivo AQP4-specific CD4+ T cells isolated from blood of patients and healthy donors. Surprisingly, in AQP4-antibody+ NMOSD patients, autoreactive T cells showed reduced proliferative capacity and pro-inflammatory cytokines. Instead, exhaustion-associated co-inhibitory receptors were co-expressed together with FOXP3. The proliferative blockade of autoreactive T cells was reversed in vitro by checkpoint inhibition. The same exhaustion-like phenotype was demonstrated in autoimmune hepatitis and bullous pemphigoid. We identified a new exhaustion-like T cell phenotype and a potential role of FOXP3 for the regulation of chronic autoreactivity in human autoimmune disease. Our data suggest CD4+ T cell exhaustion as a common mechanism of adaptation to chronic self-stimulation across disease types with important implications for their therapeutic targeting Basic Autoimmunity (BA)

Anti-leucine-rich glioma inactivated-1 (LGI1) and anti-contactin-associated-protein-2 (CASPR2) autoimmune encephalitis (AIE) are common and characterized by pathogenic antibodies targeting neuronal autoantigens. However, the drivers of the antibody-secreting cells and involvement of T cells remain unresolved. We performed single-cell RNA sequencing of fresh CSF and parallel blood samples of 15 patients with LGI1-AIE (n = 9) and CASPR2-AIE (n = 6) compared with control patients [multiple sclerosis (n = 15) and idiopathic intracranial hypertension (n = 18)]. We validated our observations in independent cohorts using flow cytometry of CSF and blood. We confirmed autoantibody specificity using recombinant human monoclonal antibodies. In comparison to idiopathic intracranial hypertension and multiple sclerosis controls, we observed clonal CSF-specific antibody-secreting cell expansion in LGI1/CASPR2-AIE despite mostly normal CSF findings. Antibody-secreting cells were dominantly plasmablasts and transcribed IgG4 and IgG1/2 heavy chains. Expanded clones showed signs of affinity maturation and bound the respective neuronal autoantigen. Within CD4 and CD8 T-cell clusters, CD4 and CD8 central memory T cells were activated, clonally restricted and expanded. T-cell clones were often shared between CSF and blood. We also observed a shift of natural killer cells and loss of mucosa-associated invariant T (MAIT) cells in the CSF of LGI1-AIE and the blood of LGI1- and CASPR2-AIE compared with idiopathic intracranial hypertension and multiple sclerosis controls. MAIT-like T cells were detected in autopsied brains of LGI1- and CASPR2-AIE patients, and mice lacking MAIT cells displayed an increased antibody seroconversion and higher titres following active LGI1/CASPR2 immunization. Our data: (i) confirm the intrathecal antigen-specific plasma cell expansion in LGI1- and CASPR2-AIE in a large cohort of untreated AIE patients; (ii) suggest that activated and expanded central memory CD4 and CD8 T cells in the CSF participate in disease pathogenesis; and (iii) implicate invariant T-cell receptor-expressing lymphocytes in the brain, CSF and blood in disease pathogenesis.

Diets that restrict caloric or protein intake offer a variety of benefits, including decreasing the incidence of cancer. However, whether such diets pose a substantial therapeutic benefit as auxiliary cancer treatments remains unclear. We determined the effects of severe protein depletion on tumorigenesis in a Drosophila melanogaster intestinal tumor model, using a human RAF gain-of-function allele. Severe and continuous protein restriction significantly reduced tumor growth but resulted in premature death. Therefore, we developed a diet in which short periods of severe protein restriction alternated cyclically with periods of complete feeding. This nutritional regime reduced tumor mass, restored gut functionality, and rescued the lifespan of oncogene-expressing flies to the levels observed in healthy flies on a continuous, fully nutritious diet. Furthermore, this diet reduced the chemotherapy-induced stem cell activity associated with tumor recurrence. Transcriptome analysis revealed long-lasting changes in the expression of key genes involved in multiple major developmental signaling pathways. Overall, the data suggest that recurrent severe protein depletion effectively mimics the health benefits of continuous protein restriction, without undesired nutritional shortcomings. This provides seminal insights into the mechanisms of the memory effect required to maintain the positive effects of protein restriction throughout the phases of a full diet. Finally, the repetitive form of strict protein restriction is an ideal strategy for adjuvant cancer therapy that is useful in many tumor contexts.

Pro-inflammatory autoantigen-specific CD4+ T helper (auto-Th) cells are central orchestrators of autoimmune diseases (AIDs). We aimed to characterize these cells in human AIDs with defined autoantigens by combining human leukocyte antigen (HLA)-tetramer-based and activation-based multidimensional ex vivo analyses. In aquaporin4-antibody-positive neuromyelitis optica spectrum disorder (AQP4-NMOSD) patients, auto-Th cells expressed CD154, but proliferative capacity and pro-inflammatory cytokines were strongly reduced. Instead, exhaustion-associated co-inhibitory receptors were expressed together with FOXP3, the canonical regulatory T cell (Treg) transcription factor. Auto-Th cells responded in vitro to checkpoint inhibition and provided potent B cell help. Cells with the same exhaustion-like (ThEx) phenotype were identified in soluble liver antigen (SLA)-antibody-autoimmune hepatitis and BP180-antibody-positive bullous pemphigoid, AIDs of the liver and skin, respectively. While originally described in cancer and chronic infection, our data point to T cell exhaustion as a common mechanism of adaptation to chronic (self-)stimulation across AID types and link exhausted CD4+ T cells to humoral autoimmune responses, with implications for therapeutic targeting.

Aging is the predominant cause of morbidity and mortality in industrialized countries. The specific molecular mechanisms that drive aging are poorly understood, especially the contribution of the microbiota in these processes. Here, we combined multi-omics with metabolic modeling in mice to comprehensively characterize host-microbiome interactions and how they are affected by aging. Our findings reveal a complex dependency of host metabolism on microbial functions, including previously known as well as novel interactions. We observed a pronounced reduction in metabolic activity within the aging microbiome, which we attribute to reduced beneficial interactions in the microbial community and a reduction in its metabolic output. These microbial changes coincided with a corresponding downregulation of key host pathways predicted by our model to be dependent on the microbiome that are crucial for maintaining intestinal barrier function, cellular replication, and homeostasis. Our results elucidate microbiome-host interactions that potentially influence host aging processes, focusing on microbial nucleotide metabolism as a pivotal factor in aging dynamics. These pathways could serve as future targets for the development of microbiome-based therapies against aging.

The aggressive features of glioblastoma (GBM) are associated with dormancy. Our previous transcriptome analysis revealed that several genes were regulated during temozolomide (TMZ)-promoted dormancy in GBM. Focusing on genes involved in cancer progression, Chemokine (C-C motif) Receptor-Like (CCRL)1, Schlafen (SLFN)13, Sloan-Kettering Institute (SKI), Cdk5 and Abl Enzyme Substrate (Cables)1, and Dachsous Cadherin-Related (DCHS)1 were selected for further validation. All showed clear expression and individual regulatory patterns under TMZ-promoted dormancy in human GBM cell lines, patient-derived primary cultures, glioma stem-like cells (GSCs), and human GBM ex vivo samples. All genes exhibited complex co-staining patterns with different stemness markers and with each other, as examined by immunofluorescence staining and underscored by correlation analyses. Neurosphere formation assays revealed higher numbers of spheres during TMZ treatment, and gene set enrichment analysis of transcriptome data revealed significant regulation of several GO terms, including stemness-associated ones, indicating an association between stemness and dormancy with the involvement of SKI. Consistently, inhibition of SKI during TMZ treatment resulted in higher cytotoxicity, proliferation inhibition, and lower neurosphere formation capacity compared to TMZ alone. Overall, our study suggests the involvement of CCRL1, SLFN13, SKI, Cables1, and DCHS1 in TMZ-promoted dormancy and demonstrates their link to stemness, with SKI being particularly important.

IntroductionResistance in anti-cancer treatment is a result of clonal evolution and clonal selection. In chronic myeloid leukemia (CML), the hematopoietic neoplasm is predominantly caused by the formation of the BCR::ABL1 kinase. Evidently, treatment with tyrosine kinase inhibitors (TKIs) is tremendously successful. It has become the role model of targeted therapy. However, therapy resistance to TKIs leads to loss of molecular remission in about 25% of CML patients being partially due to BCR::ABL1 kinase mutations, while for the remaining cases, various other mechanisms are discussed.MethodsHere, we established an in vitro-TKI resistance model against the TKIs imatinib and nilotinib and performed exome sequencing.ResultsIn this model, acquired sequence variants in NRAS, KRAS, PTPN11, and PDGFRB were identified in TKI resistance. The well-known pathogenic NRAS p.(Gln61Lys) variant provided a strong benefit for CML cells under TKI exposure visible by increased cell number (6.2-fold, p < 0.001) and decreased apoptosis (-25%, p < 0.001), proving the functionality of our approach. The transfection of PTPN11 p.(Tyr279Cys) led to increased cell number (1.7-fold, p = 0.03) and proliferation (2.0-fold, p < 0.001) under imatinib treatment.DiscussionOur data demonstrate that our in vitro-model can be used to study the effect of specific variants on TKI resistance and to identify new driver mutations and genes playing a role in TKI resistance. The established pipeline can be used to study candidates acquired in TKI-resistant patients, thereby providing new options for the development of new therapy strategies to overcome resistance.

Anti-Leucine-rich glioma inactivated-1 (LGI1) and anti-contactin-associated-protein-2 (CASPR2) associated autoimmune encephalitis (AIE) variants are characterized by directly pathogenic autoantibodies present in serum and CSF. The dynamics and drivers of intrathecal and systemic autoantibody production are incompletely understood. We aimed to elucidate the immunologic basis of the LGI1-/CASPR2-associated AIE variants by performing multi-omic profiling of CSF/blood in untreated patients. We validated findings by flow cytometry in independent cohorts and confirmed functionality using rodent immunization.We identified clonal IgG2 and IgG4 plasma cell expansion and affinity maturation in the CSF together with clonally restricted, activated, antigen-experienced CD8 and CD4 T cells as a hallmark of these encephalitis variants. Using recombinant cloning, we confirmed that expanded CSF plasma cell clones almost exclusively bound the respective neuronal autoantigen. In addition, we found a loss of regulatory mucosa-associated invariant T (MAIT) cells and gamma delta T cells in the CSF and – to a lesser degree – in blood. We validated the functional role of these invariant T cells using a novel murine active immunization paradigm using both autoantigens: MAIT cells suppressed systemic formation of LGI1 and CASPR2-specific anti-neuronal antibodies.We propose that loss of systemic and intrathecal regulatory mechanisms mediated by innate-like T cells promote plasma cell expansion and autoantibody production as a shared mechanism in AIE.One sentence summary Cerebrospinal fluid (CSF) and peripheral blood (PB) single cell transcriptomics of patients with untreated anti-LGI1 and anti-CASPR2 autoimmune encephalitis demonstrated CSF specific expansion of autoantigen-specific plasma cell clones and systemic loss of invariant mucosa-associated T-cells (MAIT).### Competing Interest StatementGMZH has received speaker honoraria from Alexion, LFB pharma and research support from Merck, Biogen. The remaining authors declare no competing interest.FL discloses speaker honoraria from Grifols, Teva, Biogen, Bayer, Roche, Novartis, Fresenius, travel funding from Merck, Grifols and Bayer and serving on advisory boards for Roche, Biogen and Alexion.MT as received research funds for serving on a scientific advisory board of Horizon Therapeutics and UCB; MT has filed a patent for methods for typing neurologic disorders and cancer, and devices for use therein, and has received research funds for consultation at Guidepoint Global LLC and unrestricted research grants from CSL Behring and Euroimmun AG. LMM received travel grants from Alexion. SR received travel grants from Merck Healthcare Germany GmbH, Alexion Pharmaceuticals, Bristol Myers Squibb. She served on a scientific advisory board from Merck Healthcare Germany GmbH and received honoraria for lecturing from Roche. Her research was supported by Novartis. NM has received honoraria for lecturing and travel expenses for attending meetings from Biogen Idec, GlaxoSmith Kline, Teva, Novartis Pharma, Bayer Healthcare, Genzyme, Alexion Pharmaceuticals, Fresenius Medical Care, Diamed, UCB Pharma, AngeliniPharma, BIAL and Sanofi-Aventis, has received royalties for consulting from UCB Pharma, Alexion Pharmaceuticals and Sanofi-Aventis and has received financial research support from Euroimmun, Fresenius Medical Care, Diamed, Alexion Pharmaceuticals, and Novartis Pharma. SM receives honoraria for lecturing, and travel expenses for attending meetings from Academy 2, Argenx, Alexion, Almirall, Amicus Therapeutics Germany, Bayer Health Care, Biogen, BioNtech, BMS, Celgene, Datamed, Demecan, Desitin, Diamed, Diaplan, DIU Dresden, DPmed, Gen Medicine and Healthcare products, Genzyme, Hexal AG, Impulze GmbH, Janssen Cilag, KW Medipoint, MedDay Pharmaceuticals, Merck Serono, MICE, Mylan, Neuraxpharm, Neuropoint, Novartis, Novo Nordisk, ONO Pharma, Oxford PharmaGenesis, Roche, Sanofi-Aventis, Springer Medizin Verlag, STADA, Chugai Pharma, QuintilesIMS ,Teva, Wings for Life international and Xcenda. HW is acting as a paid consultant for AbbVie, Actelion, Biogen, IGES, Johnson & Johnson, Novartis, Roche, Sanofi-Aventis, and the Swiss Multiple Sclerosis Society. Other authors declare that they have no competing interests

To elucidate cross-sectional patterns and longitudinal changes of oral and stool microbiota in multiple sclerosis (MS) patients and the effect of B-cell depletion. We conducted an observational, longitudinal clinical cohort study analysing four timepoints over 12 months in 36 MS patients, of whom 22 initiated B-cell depleting therapy with ocrelizumab and a healthy control group. For microbiota analysis of the oral cavity and the gut, provided stool and oral swab samples underwent 16S rDNA sequencing and subsequent bioinformatic analyses. Oral microbiota-patterns exhibited a reduced alpha-diversity and unique differential microbiota changes compared to stool such as increased levels of Proteobacteria and decreased abundance of Actinobacteria. Following B-cell depletion, we observed increased alpha-diversity in the gut and the oral cavity as well as a long-term sustained reduction of pro-inflammatory Gram-negative bacteria (e.g., Escherichia/Shigella). MS patients have altered stool and oral microbiota diversity patterns compared to healthy controls, which are most pronounced in patients with higher disease activity and disability. Therapeutic B-cell depletion is associated with persisting regression of these changes. Whether these microbial changes are unspecific side-effects of B-cell depletion or indirectly modulate MS disease activity and progression is currently unknown and necessitates further investigations.

Coronavirus disease 2019 (COVID-19) displays high clinical variability but the parameters that determine disease severity are still unclear. Pre-existing T cell memory has been hypothesized as a protective mechanism but conclusive evidence is lacking. Here we demonstrate that all unexposed individuals harbor SARS-CoV-2-specific memory T cells with marginal cross-reactivity to common cold corona and other unrelated viruses. They display low functional avidity and broad protein target specificities and their frequencies correlate with the overall size of the CD4+ memory compartment reflecting the “immunological age” of an individual. COVID-19 patients have strongly increased SARS-CoV-2-specific inflammatory T cell responses that are correlated with severity. Strikingly however, patients with severe COVID-19 displayed lower TCR functional avidity and less clonal expansion. Our data suggest that a low avidity pre-existing T cell memory negatively impacts on the T cell response quality against neoantigens such as SARS-CoV-2, which may predispose to develop inappropriate immune reactions especially in the elderly. We propose the immunological age as an independent risk factor to develop severe COVID-19. Key points ### Competing Interest Statement P.B., A.S. are consultants of Miltenyi Biotec, who own IP rights concerning parts of the ARTE technology. P.K. has received non-financial scientific grants from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany, and the Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany, and received lecture honoraria from or is advisor to Akademie für Infektionsmedizin e.V., Astellas Pharma, the European Confederation of Medical Mycology, Gilead Sciences, GPR Academy Ruesselsheim, MSD Sharp & Dohme GmbH, Noxxon N.V., and University Hospital, LMU Munich outside the submitted work. O.A.C. is supported by the German Federal Ministry of Research and Education, is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy: CECAD, EXC 2030-390661388 and has received research grants from, is an advisor to, or received lecture honoraria from Actelion, Allecra, Amplyx, Astellas, Basilea, Biosys, Cidara, Da Volterra, Entasis, F2G, Gilead, Grupo Biotoscana, IQVIA,Janssen, Matinas, Medicines Company, MedPace, Melinta, Menarini, Merck/MSD, Mylan, Nabriva, Noxxon, Octapharma, Paratek, Pfizer, PSI, Roche Diagnostics, 441 Scynexis, and Shionogi. FL is supported by German Ministry of Education and Research (01GM1908A) and E-Rare Joint Transnational research support (ERA-Net, LE3064/2-1) and discloses speaker honoraria from Grifols, Teva, Biogen, Bayer, Roche, Novartis, Fresenius, travel funding from Merck, Grifols and Bayer and serving on advisory boards for Roche, Biogen and Alexion. ### Funding Statement This research was supported by the German Research Foundation (DFG) under Germanys Excellence Strategy-EXC 2167-390884018 Precision Medicine in Chronic Inflammation (to 413 P.B., A.F., A.S.) and CECAD, EXC 2030-390661388 (to O.A.C.); RU5042-miTarget to P.B., A.F.; DFG grant n. 4096610003 to E.R; FL and DE were supported by the E-Rare Joint Transnational research support (ERA-Net, LE3064/2-415 1). FL is supported by German Ministry of Education and Research (01GM1908A) and E-Rare Joint Transnational research support (ERA-Net, LE3064/2-1) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the Institutional Review board of the UKSH Kiel (Identifier D474/20), the University Hospital Frankfurt (Identifier 11/17) and patients were enrolled in the protocol Coronavirus Disease 19-BioMaSOTA-Genetic factors and longitudinal monitoring of the immune response in COVID-19 (Identifier of the University of Cologne Ethics Committee 20-1295) and Improving Diagnosis of Severe Infections of Immunocompromised Patients (Identifier of the University of Cologne Ethics Committee 08-160) and signed informed consents. All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Authors can confirm that all relevant data are included in the article and/or its supplementary information files

Coronavirus disease 2019 (COVID-19) displays high clinical variability but the parameters that determine disease severity are still unclear. Pre-existing T cell memory has been hypothesized as a protective mechanism but conclusive evidence is lacking. Here we demonstrate that all unexposed individuals harbor SARS-CoV-2-specific memory T cells with marginal cross-reactivity to common cold corona and other unrelated viruses. They display low functional avidity and broad protein target specificities and their frequencies correlate with the overall size of the CD4+ memory compartment reflecting the immunological age of an individual. COVID-19 patients have strongly increased SARS-CoV-2-specific inflammatory T cell responses that are correlated with severity. Strikingly however, patients with severe COVID-19 displayed lower TCR functional avidity and less clonal expansion. Our data suggest that a low avidity pre-existing T cell memory negatively impacts on the T cell response quality against neoantigens such as SARS-CoV-2, which may predispose to develop inappropriate immune reactions especially in the elderly. We propose the immunological age as an independent risk factor to develop severe COVID-19.

CD4+ T cells reactive against SARS-CoV-2 can be found in unexposed individuals, and these are suggested to arise in response to common cold coronavirus (CCCoV) infection. Here, we utilized SARS-CoV-2-reactive CD4+ T cell enrichment to examine the antigen avidity and clonality of these cells, as well as the relative contribution of CCCoV cross-reactivity. SARS-CoV-2-reactive CD4+ memory T cells were present in virtually all unexposed individuals examined, displaying low functional avidity and multiple, highly variable cross-reactivities that were not restricted to CCCoVs. SARS-CoV-2-reactive CD4+ T cells from COVID-19 patients lacked cross-reactivity to CCCoVs, irrespective of strong memory T cell responses against CCCoV in all donors analyzed. In severe but not mild COVID-19, SARS-CoV-2-specific T cells displayed low functional avidity and clonality, despite increased frequencies. Our findings identify low-avidity CD4+ T cell responses as a hallmark of severe COVID-19 and argue against a protective role for CCCoV-reactive T cells in SARS-CoV-2 infection.