Publications by authors named "Victor Greiff"

33 Publications

Immunoglobulin germline gene variation and its impact on human disease.

Genes Immun 2021 Jun 26. Epub 2021 Jun 26.

Department of Immunology, University of Oslo, Oslo, Norway.

Immunoglobulins (Ig) play an important role in the immune system both when expressed as antigen receptors on the cell surface of B cells and as antibodies secreted into extracellular fluids. The advent of high-throughput sequencing methods has enabled the investigation of human Ig repertoires at unprecedented depth. This has led to the discovery of many previously unreported germline Ig alleles. Moreover, it is becoming clear that convergent and stereotypic antibody responses are common where different individuals recognise defined antigenic epitopes with the use of the same Ig V genes. Thus, germline V gene variation is increasingly being linked to the differential capacity of generating an effective immune response, which might lead to varying disease susceptibility. Here, we review recent evidence of how germline variation in Ig genes impacts the Ig repertoire and its subsequent effects on the adaptive immune response in vaccination, infection, and autoimmunity.
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http://dx.doi.org/10.1038/s41435-021-00145-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234759PMC
June 2021

Single-cell BCR and transcriptome analysis after influenza infection reveals spatiotemporal dynamics of antigen-specific B cells.

Cell Rep 2021 Jun;35(12):109286

Department of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden. Electronic address:

B cell responses are critical for antiviral immunity. However, a comprehensive picture of antigen-specific B cell differentiation, clonal proliferation, and dynamics in different organs after infection is lacking. Here, by combining single-cell RNA and B cell receptor (BCR) sequencing of antigen-specific cells in lymph nodes, spleen, and lungs after influenza infection in mice, we identify several germinal center (GC) B cell subpopulations and organ-specific differences that persist over the course of the response. We discover transcriptional differences between memory cells in lungs and lymphoid organs and organ-restricted clonal expansion. Remarkably, we find significant clonal overlap between GC-derived memory and plasma cells. By combining BCR-mutational analyses with monoclonal antibody (mAb) expression and affinity measurements, we find that memory B cells are highly diverse and can be selected from both low- and high-affinity precursors. By linking antigen recognition with transcriptional programming, clonal proliferation, and differentiation, these finding provide important advances in our understanding of antiviral immunity.
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http://dx.doi.org/10.1016/j.celrep.2021.109286DOI Listing
June 2021

TCR repertoire diversity in Multiple Sclerosis: High-dimensional bioinformatics analysis of sequences from brain, cerebrospinal fluid and peripheral blood.

EBioMedicine 2021 Jun 11;68:103429. Epub 2021 Jun 11.

Dipartimento di Medicina Sperimentale e Clinica (DMSC), Laboratory of Neuroimmunology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy. Electronic address:

Background: T cells play a key role in the pathogenesis of multiple sclerosis (MS), a chronic, inflammatory, demyelinating disease of the central nervous system (CNS). Although several studies recently investigated the T-cell receptor (TCR) repertoire in cerebrospinal fluid (CSF) of MS patients by high-throughput sequencing (HTS), a deep analysis on repertoire similarities and differences among compartments is still missing.

Methods: We performed comprehensive bioinformatics on high-dimensional TCR Vβ sequencing data from published and unpublished MS and healthy donors (HD) studies. We evaluated repertoire polarization, clone distribution, shared CDR3 amino acid sequences (CDR3s-a.a.) across repertoires, clone overlap with public databases, and TCR similarity architecture.

Findings: CSF repertoires showed a significantly higher public clones percentage and sequence similarity compared to peripheral blood (PB). On the other hand, we failed to reject the null hypothesis that the repertoire polarization is the same between CSF and PB. One Primary-Progressive MS (PPMS) CSF repertoire differed from the others in terms of TCR similarity architecture. Cluster analysis splits MS from HD.

Interpretation: In MS patients, the presence of a physiological barrier, the blood-brain barrier, does not impact clone prevalence and distribution, but impacts public clones, indicating CSF as a more private site. We reported a high Vβ sequence similarity in the CSF-TCR architecture in one PPMS. If confirmed it may be an interesting insight into MS progressive inflammatory mechanisms. The clustering of MS repertoires from HD suggests that disease shapes the TCR Vβ clonal profile.

Funding: This study was partly financially supported by the Italian Multiple Sclerosis Foundation (FISM), that contributed to Ballerini-DB data collection (grant #2015 R02).
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http://dx.doi.org/10.1016/j.ebiom.2021.103429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8245901PMC
June 2021

Modeling the Dynamics of T-Cell Development in the Thymus.

Entropy (Basel) 2021 Apr 8;23(4). Epub 2021 Apr 8.

Institute for Molecular Medicine, Goethe University, 60590 Frankfurt, Germany.

The thymus hosts the development of a specific type of adaptive immune cells called T cells. T cells orchestrate the adaptive immune response through recognition of antigen by the highly variable T-cell receptor (TCR). T-cell development is a tightly coordinated process comprising lineage commitment, somatic recombination of Tcr gene loci and selection for functional, but non-self-reactive TCRs, all interspersed with massive proliferation and cell death. Thus, the thymus produces a pool of T cells throughout life capable of responding to virtually any exogenous attack while preserving the body through self-tolerance. The thymus has been of considerable interest to both immunologists and theoretical biologists due to its multi-scale quantitative properties, bridging molecular binding, population dynamics and polyclonal repertoire specificity. Here, we review experimental strategies aimed at revealing quantitative and dynamic properties of T-cell development and how they have been implemented in mathematical modeling strategies that were reported to help understand the flexible dynamics of the highly dividing and dying thymic cell populations. Furthermore, we summarize the current challenges to estimating cellular dynamics and to reaching a next-generation multi-scale picture of T-cell development.
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http://dx.doi.org/10.3390/e23040437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069328PMC
April 2021

Animal Immunization, in Vitro Display Technologies, and Machine Learning for Antibody Discovery.

Trends Biotechnol 2021 Mar 25. Epub 2021 Mar 25.

Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.

For years, a discussion has persevered on the benefits and drawbacks of antibody discovery using animal immunization versus in vitro selection from non-animal-derived recombinant repertoires using display technologies. While it has been argued that using recombinant display libraries can reduce animal consumption, we hold that the number of animals used in immunization campaigns is dwarfed by the number sacrificed during preclinical studies. Thus, improving quality control of antibodies before entering in vivo studies will have a larger impact on animal consumption. Both animal immunization and recombinant repertoires present unique advantages for discovering antibodies that are fit for purpose. Furthermore, we anticipate that machine learning will play a significant role within discovery workflows, refining current antibody discovery practices.
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http://dx.doi.org/10.1016/j.tibtech.2021.03.003DOI Listing
March 2021

A compact vocabulary of paratope-epitope interactions enables predictability of antibody-antigen binding.

Cell Rep 2021 Mar;34(11):108856

Department of Immunology, University of Oslo, Oslo, Norway. Electronic address:

Antibody-antigen binding relies on the specific interaction of amino acids at the paratope-epitope interface. The predictability of antibody-antigen binding is a prerequisite for de novo antibody and (neo-)epitope design. A fundamental premise for the predictability of antibody-antigen binding is the existence of paratope-epitope interaction motifs that are universally shared among antibody-antigen structures. In a dataset of non-redundant antibody-antigen structures, we identify structural interaction motifs, which together compose a commonly shared structure-based vocabulary of paratope-epitope interactions. We show that this vocabulary enables the machine learnability of antibody-antigen binding on the paratope-epitope level using generative machine learning. The vocabulary (1) is compact, less than 10 motifs; (2) distinct from non-immune protein-protein interactions; and (3) mediates specific oligo- and polyreactive interactions between paratope-epitope pairs. Our work leverages combined structure- and sequence-based learning to demonstrate that machine-learning-driven predictive paratope and epitope engineering is feasible.
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http://dx.doi.org/10.1016/j.celrep.2021.108856DOI Listing
March 2021

Extended plasma half-life of albumin-binding domain fused human IgA upon pH-dependent albumin engagement of human FcRn and .

MAbs 2021 Jan-Dec;13(1):1893888

Department of Immunology, Oslo University Hospital Rikshospitalet and University of Oslo, Oslo, Norway.

Albumin has a serum half-life of 3 weeks in humans. This feature can be used to improve the pharmacokinetics of shorter-lived biologics. For instance, an albumin-binding domain (ABD) can be used to recruit albumin. A prerequisite for such design is that the ABD-albumin interaction does not interfere with pH-dependent binding of albumin to the human neonatal Fc receptor (FcRn), as FcRn acts as the principal regulator of the half-life of albumin. Thus, there is a need to know how ABDs act in the context of fusion partners and human FcRn. Here, we studied the binding and transport properties of human immunoglobulin A1 (IgA1), fused to a protein G-derived engineered ABD, in and systems harboring human FcRn. IgA has great potential as a therapeutic protein, but its short half-life is a major drawback. We demonstrate that ABD-fused IgA1 binds human FcRn pH-dependently and is rescued from cellular degradation in a receptor-specific manner in the presence of albumin. This occurs when ABD is fused to either the light or the heavy chain. In human FcRn transgenic mice, IgA1-ABD in complex with human albumin, gave 4-6-fold extended half-life compared to unmodified IgA1, where the light chain fusion showed the longest half-life. When the heavy chain-fused protein was pre-incubated with an engineered human albumin with improved FcRn binding, cellular rescue and half-life was further enhanced. Our study reveals how an ABD, which does not interfere with albumin binding to human FcRn, may be used to extend the half-life of IgA. ABD - Albumin binding domain, ADA - anti-drug-antibodies, ADCC - Antibody-dependent cellular cytotoxicity, ELISA - Enzyme-linked Immunosorbent assay, FcαRI - Fcα receptor, FcγR - Fcγ receptor, FcRn - The neonatal Fc receptor, GST - Glutathione S-transferase, HC - Heavy chain, HERA - Human endothelial cell-based recycling assay, Her2 - Human epidermal growth factor 2, HMEC - Human microvascular endothelial cells, IgG - Immunoglobulin G, IgA - Immunoglobulin A, LC - Light chain, QMP - E505Q/T527M/K573P, WT - Wild type.
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http://dx.doi.org/10.1080/19420862.2021.1893888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954421PMC
March 2021

Mucosal or systemic microbiota exposures shape the B cell repertoire.

Nature 2020 08 5;584(7820):274-278. Epub 2020 Aug 5.

Maurice Müller Laboratories (DBMR), Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Bern, Switzerland.

Colonization by the microbiota causes a marked stimulation of B cells and induction of immunoglobulin, but mammals colonized with many taxa have highly complex and individualized immunoglobulin repertoires. Here we use a simplified model of defined transient exposures to different microbial taxa in germ-free mice to deconstruct how the microbiota shapes the B cell pool and its functional responsiveness. We followed the development of the immunoglobulin repertoire in B cell populations, as well as single cells by deep sequencing. Microbial exposures at the intestinal mucosa generated oligoclonal responses that differed from those of germ-free mice, and from the diverse repertoire that was generated after intravenous systemic exposure to microbiota. The IgA repertoire-predominantly to cell-surface antigens-did not expand after dose escalation, whereas increased systemic exposure broadened the IgG repertoire to both microbial cytoplasmic and cell-surface antigens. These microbial exposures induced characteristic immunoglobulin heavy-chain repertoires in B cells, mainly at memory and plasma cell stages. Whereas sequential systemic exposure to different microbial taxa diversified the IgG repertoire and facilitated alternative specific responses, sequential mucosal exposure produced limited overlapping repertoires and the attrition of initial IgA binding specificities. This shows a contrast between a flexible response to systemic exposure with the need to avoid fatal sepsis, and a restricted response to mucosal exposure that reflects the generic nature of host-microbial mutualism in the mucosa.
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http://dx.doi.org/10.1038/s41586-020-2564-6DOI Listing
August 2020

IgM Antibody Repertoire Fingerprints in Mice Are Personalized but Robust to Viral Infection Status.

Front Cell Infect Microbiol 2020 28;10:254. Epub 2020 May 28.

Department of Biosystems and Engineering, ETH Zurich, Basel, Switzerland.

Antibody repertoire sequencing provides a molecular fingerprint of current and past pathogens encountered by the immune system. Most repertoire studies in humans require measuring the B cell response in the blood, resulting in a large bias to the IgM isotype. The extent to which the circulating IgM antibody repertoire correlates to lymphoid tissue-resident B cells in the setting of viral infection remains largely uncharacterized. Therefore, we compared the IgM repertoires from both blood and bone marrow (BM) plasma cells (PCs) following acute or chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. Despite previously reported serum alterations between acute and chronic infection, IgM repertoire signatures based on clonal diversity metrics, public clones, network, and phylogenetic analysis were largely unable to distinguish infection cohorts. Our findings, however, revealed mouse-specific repertoire fingerprints between the blood and PC repertoires irrespective of infection status.
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http://dx.doi.org/10.3389/fcimb.2020.00254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270205PMC
June 2021

Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow.

Nat Commun 2020 05 22;11(1):2570. Epub 2020 May 22.

Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, 10117, Berlin, Germany.

At present, it is not clear how memory B lymphocytes are maintained over time, and whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1 stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility.
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http://dx.doi.org/10.1038/s41467-020-16464-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244721PMC
May 2020

Inter- and intraspecies comparison of phylogenetic fingerprints and sequence diversity of immunoglobulin variable genes.

Immunogenetics 2020 07 4;72(5):279-294. Epub 2020 May 4.

Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.

Protection and neutralization of a vast array of pathogens is accomplished by the tremendous diversity of the B cell receptor (BCR) repertoire. For jawed vertebrates, this diversity is initiated via the somatic recombination of immunoglobulin (Ig) germline elements. While it is clear that the number of these germline segments differs from species to species, the extent of cross-species sequence diversity remains largely uncharacterized. Here we use extensive computational and statistical methods to investigate the sequence diversity and evolutionary relationship between Ig variable (V), diversity (D), and joining (J) germline segments across nine commonly studied species ranging from zebrafish to human. Metrics such as guanine-cytosine (GC) content showed low redundancy across Ig germline genes within a given species. Other comparisons, including amino acid motifs, evolutionary selection, and sequence diversity, revealed species-specific properties. Additionally, we showed that the germline-encoded diversity differs across antibody (recombined V-D-J) repertoires of various B cell subsets. To facilitate future comparative immunogenomics analysis, we created VDJgermlines, an R package that contains the germline sequences from multiple species. Our study informs strategies for the humanization and engineering of therapeutic antibodies.
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http://dx.doi.org/10.1007/s00251-020-01164-8DOI Listing
July 2020

The TCR Repertoire Reconstitution in Multiple Sclerosis: Comparing One-Shot and Continuous Immunosuppressive Therapies.

Front Immunol 2020 9;11:559. Epub 2020 Apr 9.

Dipartimento di Medicina Sperimentale e Clinica (DMSC), University of Florence, Florence, Italy.

Natalizumab (NTZ) and autologous hematopoietic stem cell transplantation (AHSCT) are two successful treatments for relapsing-remitting multiple sclerosis (RRMS), an autoimmune T-cell-driven disorder affecting the central nervous system that is characterized by relapses interspersed with periods of complete or partial recovery. Both RRMS treatments have been documented to impact T-cell subpopulations and the T-cell receptor (TCR) repertoire in terms of clone frequency, but, so far, the link between T-cell naive and memory populations, autoimmunity, and treatment outcome has not yet been established hindering insight into the post-treatment TCR landscape of MS patients. To address this important knowledge gap, we tracked peripheral T-cell subpopulations (naïve and memory CD4+ and CD8+) across 15 RRMS patients before and after two years of continuous treatment (NTZ) and a single treatment course (AHSCT) by high-throughput TCRß sequencing. We found that the two MS treatments left treatment-specific multidimensional traces in patient TCRß repertoire dynamics with respect to clonal expansion, clonal diversity and repertoire architecture. Comparing MS TCR sequences with published datasets suggested that the majority of public TCRs belonged to virus-associated sequences. In summary, applying multi-dimensional computational immunology to a TCRß dataset of treated MS patients, we show that qualitative changes of TCRß repertoires encode treatment-specific information that may be relevant for future clinical trials monitoring and personalized MS follow-up, diagnosis and treatment regimes. Natalizumab (NTZ) and autologous hematopoietic stem cell transplantation (AHSCT) are two successful treatments for relapsing-remitting multiple sclerosis (RRMS), an autoimmune T-cell-driven disorder affecting the central nervous system that is characterized by relapses interspersed with periods of complete or partial recovery. Both RRMS treatments have been documented to impact T-cell subpopulations and the T-cell receptor (TCR) repertoire in terms of clone frequency, but, so far, the link between T-cell naive and memory populations, autoimmunity, and treatment outcome has not yet been established hindering insight into the posttreatment TCR landscape of MS patients. To address this important knowledge gap, we tracked peripheral T-cell subpopulations (naive and memory CD4+ and CD8+) across 15 RRMS patients before and after 2 years of continuous treatment (NTZ) and a single treatment course (AHSCT) by high-throughput TCRβ sequencing. We found that the two MS treatments left treatment-specific multidimensional traces in patient TCRβ repertoire dynamics with respect to clonal expansion, clonal diversity, and repertoire architecture. Comparing MS TCR sequences with published datasets suggested that the majority of public TCRs belonged to virus-associated sequences. In summary, applying multidimensional computational immunology to a TCRβ dataset of treated MS patients, we show that qualitative changes of TCRβ repertoires encode treatment-specific information that may be relevant for future clinical trials monitoring and personalized MS follow-up, diagnosis, and treatment regimens.
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http://dx.doi.org/10.3389/fimmu.2020.00559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160336PMC
March 2021

immuneSIM: tunable multi-feature simulation of B- and T-cell receptor repertoires for immunoinformatics benchmarking.

Bioinformatics 2020 06;36(11):3594-3596

Department of Immunology, University of Oslo, 0372 Oslo, Norway.

Summary: B- and T-cell receptor repertoires of the adaptive immune system have become a key target for diagnostics and therapeutics research. Consequently, there is a rapidly growing number of bioinformatics tools for immune repertoire analysis. Benchmarking of such tools is crucial for ensuring reproducible and generalizable computational analyses. Currently, however, it remains challenging to create standardized ground truth immune receptor repertoires for immunoinformatics tool benchmarking. Therefore, we developed immuneSIM, an R package that allows the simulation of native-like and aberrant synthetic full-length variable region immune receptor sequences by tuning the following immune receptor features: (i) species and chain type (BCR, TCR, single and paired), (ii) germline gene usage, (iii) occurrence of insertions and deletions, (iv) clonal abundance, (v) somatic hypermutation and (vi) sequence motifs. Each simulated sequence is annotated by the complete set of simulation events that contributed to its in silico generation. immuneSIM permits the benchmarking of key computational tools for immune receptor analysis, such as germline gene annotation, diversity and overlap estimation, sequence similarity, network architecture, clustering analysis and machine learning methods for motif detection.

Availability And Implementation: The package is available via https://github.com/GreiffLab/immuneSIM and on CRAN at https://cran.r-project.org/web/packages/immuneSIM. The documentation is hosted at https://immuneSIM.readthedocs.io.

Contact: [email protected] or [email protected]

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334888PMC
June 2020

Quantitative and Qualitative Analysis of Humoral Immunity Reveals Continued and Personalized Evolution in Chronic Viral Infection.

Cell Rep 2020 01;30(4):997-1012.e6

Institute of Microbiology, ETH Zürich, Vladimir-Prelog-Weg 1-5/10, 8093 Zürich, Switzerland. Electronic address:

Control of established chronic lymphocytic choriomeningitis virus (LCMV) infection requires the production of neutralizing antibodies, but it remains unknown how the ensemble of antibodies evolves during ongoing infection. Here, we analyze the evolution of antibody responses during acute or chronic LCMV infection, combining quantitative functional assays and time-resolved antibody repertoire sequencing. We establish that antibody responses initially converge in both infection types on a functional and repertoire level, but diverge later during chronic infection, showing increased clonal diversity, the appearance of mouse-specific persistent clones, and distinct phylogenetic signatures. Chronic infection is characterized by a longer-lasting germinal center reaction and a continuous differentiation of plasma cells, resulting in the emergence of higher-affinity plasma cells exhibiting increased antibody secretion rates. Taken together, our findings reveal the emergence of a personalized antibody response in chronic infection and support the concept that maintaining B cell diversity throughout chronic LCMV infection correlates with the development of infection-resolving antibodies.
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http://dx.doi.org/10.1016/j.celrep.2019.12.088DOI Listing
January 2020

Chronic Viral Infection Promotes Efficient Germinal Center B Cell Responses.

Cell Rep 2020 01;30(4):1013-1026.e7

Department of Biomedicine, Division of Experimental Virology, University of Basel, Haus Petersplatz, 4009 Basel, Switzerland. Electronic address:

Persistent viral infections subvert key elements of adaptive immunity. To compare germinal center (GC) B cell responses in chronic and acute lymphocytic choriomeningitis virus infection, we exploit activation-induced deaminase (AID) fate-reporter mice and perform adoptive B cell transfer experiments. Chronic infection yields GC B cell responses of higher cellularity than acute infections do, higher memory B cell and antibody secreting cell output for longer periods of time, a better representation of the late B cell repertoire in serum immunoglobulin, and higher titers of protective neutralizing antibodies. GC B cells of chronically infected mice are similarly hypermutated as those emerging from acute infection. They efficiently adapt to viral escape variants and even in hypermutation-impaired AID mutant mice, chronic infection selects for GC B cells with hypermutated B cell receptors (BCRs) and neutralizing antibody formation. These findings demonstrate that, unlike for CD8 T cells, chronic viral infection drives a functional, productive, and protective GC B cell response.
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http://dx.doi.org/10.1016/j.celrep.2019.12.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996002PMC
January 2020

Benchmarking immunoinformatic tools for the analysis of antibody repertoire sequences.

Bioinformatics 2020 03;36(6):1731-1739

Institute of Biomedical Engineering and Medical Informatics, School of Life Sciences, FHNW University of Applied Sciences and Arts Northwestern Switzerland, Muttenz 4132, Switzerland.

Summary: Antibody repertoires reveal insights into the biology of the adaptive immune system and empower diagnostics and therapeutics. There are currently multiple tools available for the annotation of antibody sequences. All downstream analyses such as choosing lead drug candidates depend on the correct annotation of these sequences; however, a thorough comparison of the performance of these tools has not been investigated. Here, we benchmark the performance of commonly used immunoinformatic tools, i.e. IMGT/HighV-QUEST, IgBLAST and MiXCR, in terms of reproducibility of annotation output, accuracy and speed using simulated and experimental high-throughput sequencing datasets.We analyzed changes in IMGT reference germline database in the last 10 years in order to assess the reproducibility of the annotation output. We found that only 73/183 (40%) V, D and J human genes were shared between the reference germline sets used by the tools. We found that the annotation results differed between tools. In terms of alignment accuracy, MiXCR had the highest average frequency of gene mishits, 0.02 mishit frequency and IgBLAST the lowest, 0.004 mishit frequency. Reproducibility in the output of complementarity determining three regions (CDR3 amino acids) ranged from 4.3% to 77.6% with preprocessed data. In addition, run time of the tools was assessed: MiXCR was the fastest tool for number of sequences processed per unit of time. These results indicate that immunoinformatic analyses greatly depend on the choice of bioinformatics tool. Our results support informed decision-making to immunoinformaticians based on repertoire composition and sequencing platforms.

Availability And Implementation: All tools utilized in the paper are free for academic use.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075533PMC
March 2020

Editorial: Methods and Applications of Computational Immunology.

Front Immunol 2019 29;10:2818. Epub 2019 Nov 29.

Faculty of Engineering, Bar-Ilan University, Ramat Gan, Israel.

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http://dx.doi.org/10.3389/fimmu.2019.02818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896948PMC
November 2020

Resident memory CD8 T cells persist for years in human small intestine.

J Exp Med 2019 10 23;216(10):2412-2426. Epub 2019 Jul 23.

Department of Pathology, Oslo University Hospital and University of Oslo, Oslo, Norway

Resident memory CD8 T (Trm) cells have been shown to provide effective protective responses in the small intestine (SI) in mice. A better understanding of the generation and persistence of SI CD8 Trm cells in humans may have implications for intestinal immune-mediated diseases and vaccine development. Analyzing normal and transplanted human SI, we demonstrated that the majority of SI CD8 T cells were bona fide CD8 Trm cells that survived for >1 yr in the graft. Intraepithelial and lamina propria CD8 Trm cells showed a high clonal overlap and a repertoire dominated by expanded clones, conserved both spatially in the intestine and over time. Functionally, lamina propria CD8 Trm cells were potent cytokine producers, exhibiting a polyfunctional (IFN-γ IL-2 TNF-α) profile, and efficiently expressed cytotoxic mediators after stimulation. These results suggest that SI CD8 Trm cells could be relevant targets for future oral vaccines and therapeutic strategies for gut disorders.
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http://dx.doi.org/10.1084/jem.20190414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781004PMC
October 2019

Persistence of intrathecal oligoclonal B cells and IgG in multiple sclerosis.

J Neuroimmunol 2019 08 16;333:576966. Epub 2019 May 16.

Department of Immunology and Transfusion Medicine, Faculty of Medicine, University of Oslo and Oslo University Hospital Rikshospitalet, Oslo, Norway; Department of Neurology, Akershus University Hospital, Lørenskog, Norway. Electronic address:

In multiple sclerosis (MS), B cells are trafficking across the blood-brain barrier, but it is not known how this relates to the synthesis of oligoclonal IgG. We used quantitative mass spectrometry of oligoclonal bands and high-throughput sequencing of immunoglobulin heavy-chain variable transcripts to study the longitudinal B cell response in the cerebrospinal fluid (CSF) and blood of two MS patients. Twenty of 22 (91%) and 25 of 28 (89%) of oligoclonal band peptides persisted in samples collected 18 months apart, in spite of a dynamic exchange across the blood-CSF barrier of B lineage cells connecting to oligoclonal IgG.
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http://dx.doi.org/10.1016/j.jneuroim.2019.576966DOI Listing
August 2019

Inferred Allelic Variants of Immunoglobulin Receptor Genes: A System for Their Evaluation, Documentation, and Naming.

Front Immunol 2019 18;10:435. Epub 2019 Mar 18.

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.

Immunoglobulins or antibodies are the main effector molecules of the B-cell lineage and are encoded by hundreds of variable (V), diversity (D), and joining (J) germline genes, which recombine to generate enormous IG diversity. Recently, high-throughput adaptive immune receptor repertoire sequencing (AIRR-seq) of recombined V-(D)-J genes has offered unprecedented insights into the dynamics of IG repertoires in health and disease. Faithful biological interpretation of AIRR-seq studies depends upon the annotation of raw AIRR-seq data, using reference germline gene databases to identify the germline genes within each rearrangement. Existing reference databases are incomplete, as shown by recent AIRR-seq studies that have inferred the existence of many previously unreported polymorphisms. Completing the documentation of genetic variation in germline gene databases is therefore of crucial importance. Lymphocyte receptor genes and alleles are currently assigned by the Immunoglobulins, T cell Receptors and Major Histocompatibility Nomenclature Subcommittee of the International Union of Immunological Societies (IUIS) and managed in IMGT, the international ImMunoGeneTics information system (IMGT). In 2017, the IMGT Group reached agreement with a group of AIRR-seq researchers on the principles of a streamlined process for identifying and naming inferred allelic sequences, for their incorporation into IMGT. These researchers represented the AIRR Community, a network of over 300 researchers whose objective is to promote all aspects of immunoglobulin and T-cell receptor repertoire studies, including the standardization of experimental and computational aspects of AIRR-seq data generation and analysis. The Inferred Allele Review Committee (IARC) was established by the AIRR Community to devise policies, criteria, and procedures to perform this function. Formalized evaluations of novel inferred sequences have now begun and submissions are invited via a new dedicated portal (https://ogrdb.airr-community.org). Here, we summarize recommendations developed by the IARC-focusing, to begin with, on human IGHV genes-with the goal of facilitating the acceptance of inferred allelic variants of germline IGHV genes. We believe that this initiative will improve the quality of AIRR-seq studies by facilitating the description of human IG germline gene variation, and that in time, it will expand to the documentation of TR and IG genes in many vertebrate species.
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http://dx.doi.org/10.3389/fimmu.2019.00435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431624PMC
September 2020

Large-scale network analysis reveals the sequence space architecture of antibody repertoires.

Nat Commun 2019 03 21;10(1):1321. Epub 2019 Mar 21.

Department of Biosystems Science and Engineering, ETH Zurich, 4058, Basel, Switzerland.

The architecture of mouse and human antibody repertoires is defined by the sequence similarity networks of the clones that compose them. The major principles that define the architecture of antibody repertoires have remained largely unknown. Here, we establish a high-performance computing platform to construct large-scale networks from comprehensive human and murine antibody repertoire sequencing datasets (>100,000 unique sequences). Leveraging a network-based statistical framework, we identify three fundamental principles of antibody repertoire architecture: reproducibility, robustness and redundancy. Antibody repertoire networks are highly reproducible across individuals despite high antibody sequence dissimilarity. The architecture of antibody repertoires is robust to the removal of up to 50-90% of randomly selected clones, but fragile to the removal of public clones shared among individuals. Finally, repertoire architecture is intrinsically redundant. Our analysis provides guidelines for the large-scale network analysis of immune repertoires and may be used in the future to define disease-associated and synthetic repertoires.
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http://dx.doi.org/10.1038/s41467-019-09278-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428871PMC
March 2019

High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis.

Nucleic Acids Res 2018 08;46(14):7436-7449

Department of Biosystems Science and Engineering, ETH Zürich, Basel 4058, Switzerland.

Antibody engineering is often performed to improve therapeutic properties by directed evolution, usually by high-throughput screening of phage or yeast display libraries. Engineering antibodies in mammalian cells offer advantages associated with expression in their final therapeutic format (full-length glycosylated IgG); however, the inability to express large and diverse libraries severely limits their potential throughput. To address this limitation, we have developed homology-directed mutagenesis (HDM), a novel method which extends the concept of CRISPR/Cas9-mediated homology-directed repair (HDR). HDM leverages oligonucleotides with degenerate codons to generate site-directed mutagenesis libraries in mammalian cells. By improving HDR to a robust efficiency of 15-35% and combining mammalian display screening with next-generation sequencing, we validated this approach can be used for key applications in antibody engineering at high-throughput: rational library construction, novel variant discovery, affinity maturation and deep mutational scanning (DMS). We anticipate that HDM will be a valuable tool for engineering and optimizing antibodies in mammalian cells, and eventually enable directed evolution of other complex proteins and cellular therapeutics.
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http://dx.doi.org/10.1093/nar/gky550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101513PMC
August 2018

Computational Strategies for Dissecting the High-Dimensional Complexity of Adaptive Immune Repertoires.

Front Immunol 2018 21;9:224. Epub 2018 Feb 21.

Department for Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.

The adaptive immune system recognizes antigens an immense array of antigen-binding antibodies and T-cell receptors, the immune repertoire. The interrogation of immune repertoires is of high relevance for understanding the adaptive immune response in disease and infection (e.g., autoimmunity, cancer, HIV). Adaptive immune receptor repertoire sequencing (AIRR-seq) has driven the quantitative and molecular-level profiling of immune repertoires, thereby revealing the high-dimensional complexity of the immune receptor sequence landscape. Several methods for the computational and statistical analysis of large-scale AIRR-seq data have been developed to resolve immune repertoire complexity and to understand the dynamics of adaptive immunity. Here, we review the current research on (i) diversity, (ii) clustering and network, (iii) phylogenetic, and (iv) machine learning methods applied to dissect, quantify, and compare the architecture, evolution, and specificity of immune repertoires. We summarize outstanding questions in computational immunology and propose future directions for systems immunology toward coupling AIRR-seq with the computational discovery of immunotherapeutics, vaccines, and immunodiagnostics.
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http://dx.doi.org/10.3389/fimmu.2018.00224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826328PMC
March 2019

Comparison of methods for phylogenetic B-cell lineage inference using time-resolved antibody repertoire simulations (AbSim).

Bioinformatics 2017 Dec;33(24):3938-3946

Department of Biosystems Science and Engineering, ETH Zürich, 4058 Basel, Switzerland.

Motivation: The evolution of antibody repertoires represents a hallmark feature of adaptive B-cell immunity. Recent advancements in high-throughput sequencing have dramatically increased the resolution to which we can measure the molecular diversity of antibody repertoires, thereby offering for the first time the possibility to capture the antigen-driven evolution of B cells. However, there does not exist a repertoire simulation framework yet that enables the comparison of commonly utilized phylogenetic methods with regard to their accuracy in inferring antibody evolution.

Results: Here, we developed AbSim, a time-resolved antibody repertoire simulation framework, which we exploited for testing the accuracy of methods for the phylogenetic reconstruction of B-cell lineages and antibody molecular evolution. AbSim enables the (i) simulation of intermediate stages of antibody sequence evolution and (ii) the modeling of immunologically relevant parameters such as duration of repertoire evolution, and the method and frequency of mutations. First, we validated that our repertoire simulation framework recreates replicates topological similarities observed in experimental sequencing data. Second, we leveraged Absim to show that current methods fail to a certain extent to predict the true phylogenetic tree correctly. Finally, we formulated simulation-validated guidelines for antibody evolution, which in the future will enable the development of accurate phylogenetic methods.

Availability And Implementation: https://cran.r-project.org/web/packages/AbSim/index.html.

Contact: [email protected]

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btx533DOI Listing
December 2017

Learning the High-Dimensional Immunogenomic Features That Predict Public and Private Antibody Repertoires.

J Immunol 2017 10 18;199(8):2985-2997. Epub 2017 Sep 18.

Department of Biosystems Science and Engineering, Swiss Federal Institute of Technology Zurich, CH-4058 Basel, Switzerland;

Recent studies have revealed that immune repertoires contain a substantial fraction of public clones, which may be defined as Ab or TCR clonal sequences shared across individuals. It has remained unclear whether public clones possess predictable sequence features that differentiate them from private clones, which are believed to be generated largely stochastically. This knowledge gap represents a lack of insight into the shaping of immune repertoire diversity. Leveraging a machine learning approach capable of capturing the high-dimensional compositional information of each clonal sequence (defined by CDR3), we detected predictive public clone and private clone-specific immunogenomic differences concentrated in CDR3's N1-D-N2 region, which allowed the prediction of public and private status with 80% accuracy in humans and mice. Our results unexpectedly demonstrate that public, as well as private, clones possess predictable high-dimensional immunogenomic features. Our support vector machine model could be trained effectively on large published datasets (3 million clonal sequences) and was sufficiently robust for public clone prediction across individuals and studies prepared with different library preparation and high-throughput sequencing protocols. In summary, we have uncovered the existence of high-dimensional immunogenomic rules that shape immune repertoire diversity in a predictable fashion. Our approach may pave the way for the construction of a comprehensive atlas of public mouse and human immune repertoires with potential applications in rational vaccine design and immunotherapeutics.
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http://dx.doi.org/10.4049/jimmunol.1700594DOI Listing
October 2017

Systems Analysis Reveals High Genetic and Antigen-Driven Predetermination of Antibody Repertoires throughout B Cell Development.

Cell Rep 2017 05;19(7):1467-1478

Department of Biosystems Science and Engineering, ETH Zürich, Basel 4058, Switzerland. Electronic address:

Antibody repertoire diversity and plasticity is crucial for broad protective immunity. Repertoires change in size and diversity across multiple B cell developmental stages and in response to antigen exposure. However, we still lack fundamental quantitative understanding of the extent to which repertoire diversity is predetermined. Therefore, we implemented a systems immunology framework for quantifying repertoire predetermination on three distinct levels: (1) B cell development (pre-B cell, naive B cell, plasma cell), (2) antigen exposure (three structurally different proteins), and (3) four antibody repertoire components (V-gene usage, clonal expansion, clonal diversity, repertoire size) extracted from antibody repertoire sequencing data (400 million reads). Across all three levels, we detected a dynamic balance of high genetic (e.g., >90% for V-gene usage and clonal expansion in naive B cells) and antigen-driven (e.g., 40% for clonal diversity in plasma cells) predetermination and stochastic variation. Our study has implications for the prediction and manipulation of humoral immunity.
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http://dx.doi.org/10.1016/j.celrep.2017.04.054DOI Listing
May 2017

Bioinformatic and Statistical Analysis of Adaptive Immune Repertoires.

Trends Immunol 2015 Nov 25;36(11):738-749. Epub 2015 Oct 25.

Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH Zürich), Mattenstrasse 26, Basel 4058, Switzerland. Electronic address:

High-throughput sequencing (HTS) of immune repertoires has enabled the quantitative analysis of adaptive immune responses and offers the potential to revolutionize research in lymphocyte biology, vaccine profiling, and monoclonal antibody engineering. Advances in sequencing technology coupled to an exponential decline in sequencing costs have fueled the recent overwhelming interest in immune repertoire sequencing. This, in turn, has sparked the development of numerous methods for bioinformatic and statistics-driven interpretation and visualization of immune repertoires. Here, we review the current literature on bioinformatic and statistical analysis of immune repertoire HTS data and discuss underlying assumptions, applicability, and scope. We further highlight important directions for future research, which could propel immune repertoire HTS to becoming a standard method for measuring adaptive immune responses.
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http://dx.doi.org/10.1016/j.it.2015.09.006DOI Listing
November 2015

A bioinformatic framework for immune repertoire diversity profiling enables detection of immunological status.

Genome Med 2015 28;7(1):49. Epub 2015 May 28.

ETH Zürich, Department of Biosystems Science and Engineering, Basel, 4058 Switzerland.

Background: Lymphocyte receptor repertoires are continually shaped throughout the lifetime of an individual in response to environmental and pathogenic exposure. Thus, they may serve as a fingerprint of an individual's ongoing immunological status (e.g., healthy, infected, vaccinated), with far-reaching implications for immunodiagnostics applications. The advent of high-throughput immune repertoire sequencing now enables the interrogation of immune repertoire diversity in an unprecedented and quantitative manner. However, steadily increasing sequencing depth has revealed that immune repertoires vary greatly among individuals in their composition; correspondingly, it has been reported that there are few shared sequences indicative of immunological status ('public clones'). Disconcertingly, this means that the wealth of information gained from repertoire sequencing remains largely unused for determining the current status of immune responses, thereby hampering the implementation of immune-repertoire-based diagnostics.

Methods: Here, we introduce a bioinformatics repertoire-profiling framework that possesses the advantage of capturing the diversity and distribution of entire immune repertoires, as opposed to singular public clones. The framework relies on Hill-based diversity profiles composed of a continuum of single diversity indices, which enable the quantification of the extent of immunological information contained in immune repertoires.

Results: We coupled diversity profiles with unsupervised (hierarchical clustering) and supervised (support vector machine and feature selection) machine learning approaches in order to correlate patients' immunological statuses with their B- and T-cell repertoire data. We could predict with high accuracy (greater than or equal to 80 %) a wide range of immunological statuses such as healthy, transplantation recipient, and lymphoid cancer, suggesting as a proof of principle that diversity profiling can recover a large amount of immunodiagnostic fingerprints from immune repertoire data. Our framework is highly scalable as it easily allowed for the analysis of 1000 simulated immune repertoires; this exceeds the size of published immune repertoire datasets by one to two orders of magnitude.

Conclusions: Our framework offers the possibility to advance immune-repertoire-based fingerprinting, which may in the future enable a systems immunogenomics approach for vaccine profiling and the accurate and early detection of disease and infection.
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http://dx.doi.org/10.1186/s13073-015-0169-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4489130PMC
July 2015

Influenza vaccine response profiles are affected by vaccine preparation and preexisting immunity, but not HIV infection.

Hum Vaccin Immunother 2015 ;11(2):391-6

a Department of Biomedicine ; University Hospital Basel ; Basel , Switzerland.

Vaccines dramatically reduce infection-related morbidity and mortality. Determining factors that modulate the host response is key to rational vaccine design and demands unsupervised analysis. To longitudinally resolve influenza-specific humoral immune response dynamics we constructed vaccine response profiles of influenza A- and B-specific IgM and IgG levels from 42 healthy and 31 HIV infected influenza-vaccinated individuals. Pre-vaccination antibody levels and levels at 3 predefined time points after vaccination were included in each profile. We performed hierarchical clustering on these profiles to study the extent to which HIV infection associated immune dysfunction, adaptive immune factors (pre-existing influenza-specific antibodies, T cell responses), an innate immune factor (Mannose Binding Lectin, MBL), demographic characteristics (gender, age), or the vaccine preparation (split vs. virosomal) impacted the immune response to influenza vaccination. Hierarchical clustering associated vaccine preparation and pre-existing IgG levels with the profiles of healthy individuals. In contrast to previous in vitro and animal data, MBL levels had no impact on the adaptive vaccine response. Importantly, while HIV infected subjects with low CD4 T cell counts showed a reduced magnitude of their vaccine response, their response profiles were indistinguishable from those of healthy controls, suggesting quantitative but not qualitative deficits. Unsupervised profile-based analysis ranks factors impacting the vaccine-response by relative importance, with substantial implications for comparing, designing and improving vaccine preparations and strategies. Profile similarity between HIV infected and HIV negative individuals suggests merely quantitative differences in the vaccine response in these individuals, offering a rationale for boosting strategies in the HIV infected population.
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http://dx.doi.org/10.1080/21645515.2015.1008930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4514422PMC
December 2015
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