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Article ; Online: Applying next-generation sequencing to unravel the mutational landscape in viral quasispecies.

Lu, I-Na / Muller, Claude P / He, Feng Q

2020 Volume 283, Page(s) 197963

Abstract: Next-generation sequencing (NGS) has revolutionized the scale and depth of biomedical sciences. Because of its unique ability for the detection of sub-clonal variants within genetically diverse populations, NGS has been successfully applied to analyze ... ...

Abstract	Next-generation sequencing (NGS) has revolutionized the scale and depth of biomedical sciences. Because of its unique ability for the detection of sub-clonal variants within genetically diverse populations, NGS has been successfully applied to analyze and quantify the exceptionally-high diversity within viral quasispecies, and many low-frequency drug- or vaccine-resistant mutations of therapeutic importance have been discovered. Although many works have intensively discussed the latest NGS approaches and applications in general, none of them has focused on applying NGS in viral quasispecies studies, mostly due to the limited ability of current NGS technologies to accurately detect and quantify rare viral variants. Here, we summarize several error-correction strategies that have been developed to enhance the detection accuracy of minority variants. We also discuss critical considerations for preparing a sequencing library from viral RNAs and for analyzing NGS data to unravel the mutational landscape.
MeSH term(s)	Genetic Variation ; Genome, Viral ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Mutation ; Quasispecies/genetics ; RNA, Viral/genetics ; Viruses/genetics
Chemical Substances	RNA, Viral
Keywords	covid19
Language	English
Publishing date	2020-04-09
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	605780-9
ISSN	1872-7492 ; 0168-1702
ISSN (online)	1872-7492
ISSN	0168-1702
DOI	10.1016/j.virusres.2020.197963
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Article ; Online: Alemtuzumab treatment exemplifies discordant immune effects of blood and cerebrospinal fluid in multiple sclerosis.

Müller-Miny, Louisa / Heming, Michael / Lautwein, Tobias / Ruck, Tobias / Lu, I-Na / Wiendl, Heinz / Meyer Zu Hörste, Gerd

Journal of neuroimmunology

2023 Volume 378, Page(s) 578088

Abstract: Background and objectives: Immune responses in the central nervous system (CNS) are highly compartmentalized and cerebrospinal fluid (CSF) in particular often reflects CNS pathology better than peripheral blood. While CSF leukocytes are known to be ... ...

Abstract	Background and objectives: Immune responses in the central nervous system (CNS) are highly compartmentalized and cerebrospinal fluid (CSF) in particular often reflects CNS pathology better than peripheral blood. While CSF leukocytes are known to be distinct from blood, the immediate effects of peripheral leukocyte depletion on CSF leukocytes have not been studied in humans. Methods: We here analyzed CSF and blood from two relapsing-remitting multiple sclerosis (RRMS) patients early after peripheral leukocyte depletion with the anti-CD52 antibody alemtuzumab compared to untreated RRMS and control patients using single cell RNA-sequencing. Results: As expected for alemtuzumab, most leukocyte lineages including T cells were synchronously depleted from CSF and blood, while - surprisingly - pDCs were maintained in CSF but depleted from blood by alemtuzumab. Transcriptionally, genes associated with migration were elevated only in the CSF after alemtuzumab. Predicted cellular interactions indicated a central role of pDCs and enhanced migration signaling in the CSF after alemtuzumab. Discussion: The CSF and blood compartments are thus partially uncoupled, emphasizing that the CNS is only partially accessible even for treatments profoundly affecting the blood.
MeSH term(s)	Humans ; Alemtuzumab/adverse effects ; Multiple Sclerosis/drug therapy ; Multiple Sclerosis/chemically induced ; Multiple Sclerosis, Relapsing-Remitting ; Central Nervous System
Chemical Substances	Alemtuzumab (3A189DH42V)
Language	English
Publishing date	2023-04-11
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	8335-5
ISSN	1872-8421 ; 0165-5728
ISSN (online)	1872-8421
ISSN	0165-5728
DOI	10.1016/j.jneuroim.2023.578088
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Article: Applying next-generation sequencing to unravel the mutational landscape in viral quasispecies

Lu, I-Na / Muller, Claude P / He, Feng Q

Virus research. 2020 July 02, v. 283

2020

Abstract	Next-generation sequencing (NGS) has revolutionized the scale and depth of biomedical sciences. Because of its unique ability for the detection of sub-clonal variants within genetically diverse populations, NGS has been successfully applied to analyze and quantify the exceptionally-high diversity within viral quasispecies, and many low-frequency drug- or vaccine-resistant mutations of therapeutic importance have been discovered. Although many works have intensively discussed the latest NGS approaches and applications in general, none of them has focused on applying NGS in viral quasispecies studies, mostly due to the limited ability of current NGS technologies to accurately detect and quantify rare viral variants. Here, we summarize several error-correction strategies that have been developed to enhance the detection accuracy of minority variants. We also discuss critical considerations for preparing a sequencing library from viral RNAs and for analyzing NGS data to unravel the mutational landscape.
Keywords	DNA libraries ; RNA ; biomedical research ; drugs ; high-throughput nucleotide sequencing ; mutation ; therapeutics ; covid19
Language	English
Dates of publication	2020-0702
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	605780-9
ISSN	1872-7492 ; 0168-1702
ISSN (online)	1872-7492
ISSN	0168-1702
DOI	10.1016/j.virusres.2020.197963
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Article ; Online: Cell-mediated cytotoxicity within CSF and brain parenchyma in spinal muscular atrophy unaltered by nusinersen treatment.

Nature communications

2024 Volume 15, Issue 1, Page(s) 4120

Abstract: 5q-associated spinal muscular atrophy (SMA) is a motoneuron disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Adaptive immunity may contribute to SMA as described in other motoneuron diseases, yet mechanisms remain elusive. ... ...

Abstract	5q-associated spinal muscular atrophy (SMA) is a motoneuron disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Adaptive immunity may contribute to SMA as described in other motoneuron diseases, yet mechanisms remain elusive. Nusinersen, an antisense treatment, enhances SMN2 expression, benefiting SMA patients. Here we have longitudinally investigated SMA and nusinersen effects on local immune responses in the cerebrospinal fluid (CSF) - a surrogate of central nervous system parenchyma. Single-cell transcriptomics (SMA: N = 9 versus Control: N = 9) reveal NK cell and CD8+ T cell expansions in untreated SMA CSF, exhibiting activation and degranulation markers. Spatial transcriptomics coupled with multiplex immunohistochemistry elucidate cytotoxicity near chromatolytic motoneurons (N = 4). Post-nusinersen treatment, CSF shows unaltered protein/transcriptional profiles. These findings underscore cytotoxicity's role in SMA pathogenesis and propose it as a therapeutic target. Our study illuminates cell-mediated cytotoxicity as shared features across motoneuron diseases, suggesting broader implications.
MeSH term(s)	Humans ; Oligonucleotides ; Muscular Atrophy, Spinal/drug therapy ; Muscular Atrophy, Spinal/pathology ; Muscular Atrophy, Spinal/genetics ; Motor Neurons/drug effects ; Motor Neurons/pathology ; Motor Neurons/metabolism ; Killer Cells, Natural/immunology ; Killer Cells, Natural/drug effects ; Brain/pathology ; Brain/drug effects ; Female ; Male ; Survival of Motor Neuron 2 Protein/genetics ; CD8-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/drug effects ; Survival of Motor Neuron 1 Protein/genetics ; Survival of Motor Neuron 1 Protein/metabolism ; Single-Cell Analysis ; Cytotoxicity, Immunologic/drug effects ; Infant ; Child, Preschool ; Child ; Transcriptome
Language	English
Publishing date	2024-05-15
Publishing country	England
Document type	Journal Article
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-024-48195-3
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Article ; Online: Identification of a CD4 T-cell epitope in the hemagglutinin stalk domain of pandemic H1N1 influenza virus and its antigen-driven TCR usage signature in BALB/c mice.

Lu, I-Na / Farinelle, Sophie / Sausy, Aurélie / Muller, Claude P

Cellular & molecular immunology

2017 Volume 14, Issue 6, Page(s) 511–520

Abstract: The stalk region of the influenza virus hemagglutinin is relatively well conserved compared with the globular head domain, which makes it a potential target for use as a universal vaccine against influenza. However, the role of CD4 T cells in the ... ...

Abstract	The stalk region of the influenza virus hemagglutinin is relatively well conserved compared with the globular head domain, which makes it a potential target for use as a universal vaccine against influenza. However, the role of CD4 T cells in the hemagglutinin stalk-specific immune response is not clear. Here we identified a mouse CD4 T-cell epitope that encompasses residues HA2
Language	English
Publishing date	2017-06
Publishing country	China
Document type	Journal Article
ZDB-ID	2435097-7
ISSN	2042-0226 ; 1672-7681
ISSN (online)	2042-0226
ISSN	1672-7681
DOI	10.1038/cmi.2016.20
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Article: Single-cell profiling reveals preferential reduction of memory B cell subsets in cladribine patients that correlates with treatment response.

Teschner, Valerie E / Fleck, Ann-Katrin / Walter, Carolin / Schwarze, Anna-Sophie / Eschborn, Melanie / Wirth, Timo / Steinberg, Olga V / Schulte-Mecklenbeck, Andreas / Lu, I-Na / Herrera-Rivero, Marisol / Janoschka, Claudia / Lünemann, Jan D / Schwab, Nicholas / Meyer Zu Hörste, Gerd / Varghese, Julian / Gross, Catharina C / Pul, Refik / Kleinschnitz, Christoph / Mader, Simone /

Meinl, Edgar / Stoll, Monika / Wiendl, Heinz / Klotz, Luisa

Therapeutic advances in neurological disorders

2023 Volume 16, Page(s) 17562864231211077

Abstract: Background: Cladribine is a highly effective immunotherapy that is applied in two short-term courses over 2 years and reduces relapse rate and disease progression in patients with relapsing multiple sclerosis (MS). Despite the short treatment period, ... ...

Abstract	Background: Cladribine is a highly effective immunotherapy that is applied in two short-term courses over 2 years and reduces relapse rate and disease progression in patients with relapsing multiple sclerosis (MS). Despite the short treatment period, cladribine has a long-lasting effect on disease activity even after recovery of lymphocyte counts, suggesting a yet undefined long-term immune modulating effect. Objectives: Our aim was to provide a more profound understanding of the detailed effects of cladribine, also with regard to the patients' therapy response. Design: We performed an open-labeled, explorative, prospective, single-arm study, in which we examined the detailed lymphocyte subset development of MS patients who received cladribine treatment over 2 years. Methods: We performed in-depth profiling of the effects of cladribine on peripheral blood lymphocytes by flow cytometry, bulk RNA sequencing of sorted CD4 Results: Flow cytometry revealed a predominant and sustained reduction of memory B cells compared to other B cell subsets after cladribine treatment, whereas T cell subsets were slightly reduced in a more uniform pattern. The overall transcriptional profile of total blood B cells exhibited reduced expression of proinflammatory and T cell activating genes, while single-cell transcriptomics revealed that gene expression within each B cell cluster did not change over time. Stable patients displayed stronger reductions of selected memory B cell clusters as compared to patients with clinical or cerebral MRI disease activity. Conclusion: We describe a pronounced and sustained effect of cladribine on the memory B cell compartment, and the resulting change in B cell subset composition causes a significant alteration of B cell transcriptional profiles resulting in reduced proinflammatory and T cell activating capacities. The extent of reduction in selected memory B cell clusters by cladribine may predict treatment response.
Language	English
Publishing date	2023-12-09
Publishing country	England
Document type	Journal Article
ZDB-ID	2442245-9
ISSN	1756-2864 ; 1756-2856
ISSN (online)	1756-2864
ISSN	1756-2856
DOI	10.1177/17562864231211077
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Article ; Online: A single-cell analysis framework allows for characterization of CSF leukocytes and their tissue of origin in multiple sclerosis.

Ostkamp, Patrick / Deffner, Marie / Schulte-Mecklenbeck, Andreas / Wünsch, Christian / Lu, I-Na / Wu, Gregory F / Goelz, Susan / De Jager, Philip L / Kuhlmann, Tanja / Gross, Catharina C / Klotz, Luisa / Meyer Zu Hörste, Gerd / Wiendl, Heinz / Schneider-Hohendorf, Tilman / Schwab, Nicholas

Science translational medicine

2022 Volume 14, Issue 673, Page(s) eadc9778

Abstract: Peripheral central nervous system (CNS)-infiltrating lymphocytes are a hallmark of relapsing-remitting multiple sclerosis. Tissue-resident memory T cells ( ... ...

Abstract	Peripheral central nervous system (CNS)-infiltrating lymphocytes are a hallmark of relapsing-remitting multiple sclerosis. Tissue-resident memory T cells (T
MeSH term(s)	Humans ; Multiple Sclerosis ; Single-Cell Analysis ; Leukocytes ; Central Nervous System ; Multiple Sclerosis, Relapsing-Remitting
Language	English
Publishing date	2022-11-30
Publishing country	United States
Document type	Journal Article
ZDB-ID	2518854-9
ISSN	1946-6242 ; 1946-6234
ISSN (online)	1946-6242
ISSN	1946-6234
DOI	10.1126/scitranslmed.adc9778
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Article ; Online: Structure and applications of novel influenza HA tri-stalk protein for evaluation of HA stem-specific immunity.

Lu, I-Na / Kirsteina, Anna / Farinelle, Sophie / Willieme, Stéphanie / Tars, Kaspars / Muller, Claude P / Kazaks, Andris

PloS one

2018 Volume 13, Issue 9, Page(s) e0204776

Abstract: Long alpha helix (LAH) from influenza virus hemagglutinin (HA) stem or stalk domain is one of the most conserved influenza virus antigens. Expression of N-terminally extended LAH in E. coli leads to assembly of α-h elical homotrimer which is structurally ...

Abstract	Long alpha helix (LAH) from influenza virus hemagglutinin (HA) stem or stalk domain is one of the most conserved influenza virus antigens. Expression of N-terminally extended LAH in E. coli leads to assembly of α-h elical homotrimer which is structurally nearly identical to the corresponding region of post-fusion form of native HA. This novel tri-stalk protein was able to differentiate between group 1 and 2 influenza in ELISA with virus-infected mice sera. It was also successfully applied for enzyme-linked immunospot assay to estimate the number of HA stem-reactive antibody (Ab)-secreting cells in mice. An in-house indirect ELISA was developed using a HA tri-stalk protein as a coating antigen for evaluation of HA stem-specific Ab levels in human sera collected in Luxembourg from 211 persons with occupational exposure to swine before the pandemic H1N1/09 virus had spread to Western Europe. Our results show that 70% of these pre-pandemic sera are positive for HA stem-specific Abs. In addition, levels of HA stem-specific Abs have positive correlation with the corresponding IgG titers and neutralizing activities against pandemic H1N1/09 virus.
MeSH term(s)	Animals ; Antibodies, Viral/immunology ; Female ; Hemagglutinin Glycoproteins, Influenza Virus/chemistry ; Hemagglutinin Glycoproteins, Influenza Virus/immunology ; Humans ; Influenza A Virus, H1N1 Subtype/chemistry ; Influenza A Virus, H1N1 Subtype/immunology ; Influenza, Human/epidemiology ; Influenza, Human/immunology ; Influenza, Human/pathology ; Male ; Mice ; Mice, Inbred BALB C ; Pandemics ; Plasma Cells/immunology ; Plasma Cells/pathology ; Protein Structure, Secondary
Chemical Substances	Antibodies, Viral ; H1N1 virus hemagglutinin ; Hemagglutinin Glycoproteins, Influenza Virus
Language	English
Publishing date	2018-09-27
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0204776
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Article ; Online: Tumor-associated hematopoietic stem and progenitor cells positively linked to glioblastoma progression.

Lu, I-Na / Dobersalske, Celia / Rauschenbach, Laurèl / Teuber-Hanselmann, Sarah / Steinbach, Anita / Ullrich, Vivien / Prasad, Shruthi / Blau, Tobias / Kebir, Sied / Siveke, Jens T / Becker, Jürgen C / Sure, Ulrich / Glas, Martin / Scheffler, Björn / Cima, Igor

Nature communications

2021 Volume 12, Issue 1, Page(s) 3895

Abstract: Brain tumors are typically immunosuppressive and refractory to immunotherapies for reasons that remain poorly understood. The unbiased profiling of immune cell types in the tumor microenvironment may reveal immunologic networks affecting therapy and ... ...

Abstract	Brain tumors are typically immunosuppressive and refractory to immunotherapies for reasons that remain poorly understood. The unbiased profiling of immune cell types in the tumor microenvironment may reveal immunologic networks affecting therapy and course of disease. Here we identify and validate the presence of hematopoietic stem and progenitor cells (HSPCs) within glioblastoma tissues. Furthermore, we demonstrate a positive link of tumor-associated HSPCs with malignant and immunosuppressive phenotypes. Compared to the medullary hematopoietic compartment, tumor-associated HSPCs contain a higher fraction of immunophenotypically and transcriptomically immature, CD38- cells, such as hematopoietic stem cells and multipotent progenitors, express genes related to glioblastoma progression and display signatures of active cell cycle phases. When cultured ex vivo, tumor-associated HSPCs form myeloid colonies, suggesting potential in situ myelopoiesis. In experimental models, HSPCs promote tumor cell proliferation, expression of the immune checkpoint PD-L1 and secretion of tumor promoting cytokines such as IL-6, IL-8 and CCL2, indicating concomitant support of both malignancy and immunosuppression. In patients, the amount of tumor-associated HSPCs in tumor tissues is prognostic for patient survival and correlates with immunosuppressive phenotypes. These findings identify an important element in the complex landscape of glioblastoma that may serve as a target for brain tumor immunotherapies.
MeSH term(s)	Brain Neoplasms/genetics ; Brain Neoplasms/metabolism ; Brain Neoplasms/pathology ; Cell Line, Tumor ; Cell Proliferation/genetics ; Cells, Cultured ; Disease Progression ; Gene Expression Profiling/methods ; Gene Expression Regulation, Neoplastic ; Glioblastoma/genetics ; Glioblastoma/metabolism ; Glioblastoma/pathology ; Hematopoietic Stem Cells/metabolism ; Humans ; Kaplan-Meier Estimate ; Neoplastic Stem Cells/metabolism ; RNA-Seq/methods ; Signal Transduction/genetics ; Single-Cell Analysis/methods ; Tumor Microenvironment/genetics
Language	English
Publishing date	2021-06-23
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-021-23995-z
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Article ; Online: Bcl6 controls meningeal Th17-B cell interaction in murine neuroinflammation.

Hartlehnert, Maike / Börsch, Anna-Lena / Li, Xiaolin / Burmeister, Miriam / Gerwien, Hanna / Schafflick, David / Heming, Michael / Lu, I-Na / Narayanan, Venu / Strecker, Jan-Kolja / Kolz, Anna / Peters, Anneli / Wu, Gregory F / Wiendl, Heinz / Sorokin, Lydia / Meyer Zu Horste, Gerd

Proceedings of the National Academy of Sciences of the United States of America

2021 Volume 118, Issue 36

Abstract: Ectopic lymphoid tissue containing B cells forms in the meninges at late stages of human multiple sclerosis (MS) and when neuroinflammation is induced by interleukin (IL)-17 producing T helper (Th17) cells in rodents. B cell differentiation and the ... ...

Abstract	Ectopic lymphoid tissue containing B cells forms in the meninges at late stages of human multiple sclerosis (MS) and when neuroinflammation is induced by interleukin (IL)-17 producing T helper (Th17) cells in rodents. B cell differentiation and the subsequent release of class-switched immunoglobulins have been speculated to occur in the meninges, but the exact cellular composition and underlying mechanisms of meningeal-dominated inflammation remain unknown. Here, we performed in-depth characterization of meningeal versus parenchymal Th17-induced rodent neuroinflammation. The most pronounced cellular and transcriptional differences between these compartments was the localization of B cells exhibiting a follicular phenotype exclusively to the meninges. Correspondingly, meningeal but not parenchymal Th17 cells acquired a B cell-supporting phenotype and resided in close contact with B cells. This preferential B cell tropism for the meninges and the formation of meningeal ectopic lymphoid tissue was partially dependent on the expression of the transcription factor Bcl6 in Th17 cells that is required in other T cell lineages to induce isotype class switching in B cells. A function of Bcl6 in Th17 cells was only detected in vivo and was reflected by the induction of B cell-supporting cytokines, the appearance of follicular B cells in the meninges, and of immunoglobulin class switching in the cerebrospinal fluid. We thus identify the induction of a B cell-supporting meningeal microenvironment by Bcl6 in Th17 cells as a mechanism controlling compartment specificity in neuroinflammation.
MeSH term(s)	Animals ; B-Lymphocytes/immunology ; Cell Communication ; Cytokines/metabolism ; Encephalomyelitis, Autoimmune, Experimental/metabolism ; Female ; Germinal Center/immunology ; Inflammation/metabolism ; Lymphocyte Activation ; Male ; Meninges/immunology ; Meninges/metabolism ; Mice ; Mice, Inbred C57BL ; Multiple Sclerosis/metabolism ; Neuroinflammatory Diseases/immunology ; Neuroinflammatory Diseases/metabolism ; Neuroinflammatory Diseases/physiopathology ; Parenchymal Tissue/immunology ; Parenchymal Tissue/metabolism ; Proto-Oncogene Proteins c-bcl-6/metabolism ; Proto-Oncogene Proteins c-bcl-6/physiology ; Th17 Cells/immunology ; Th17 Cells/metabolism ; Th17 Cells/physiology
Chemical Substances	Bcl6 protein, mouse ; Cytokines ; Proto-Oncogene Proteins c-bcl-6
Language	English
Publishing date	2021-09-01
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.2023174118
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