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1.
Leukemia ; 38(1): 168-180, 2024 01.
Article in English | MEDLINE | ID: mdl-38049509

ABSTRACT

Patients with multiple myeloma (MM) routinely receive mRNA-based vaccines to reduce COVID-19-related mortality. However, whether disease- and therapy-related alterations in immune cells and cytokine-responsiveness contribute to the observed heterogeneous vaccination responses is unclear. Thus, we analyzed peripheral blood mononuclear cells from patients with MM during and after SARS-CoV-2 vaccination and breakthrough infection (BTI) using combined whole-transcriptome and surface proteome single-cell profiling with functional serological and T-cell validation in 58 MM patients. Our results demonstrate that vaccine-responders showed a significant overrepresentation of cytotoxic CD4+ T- and mature CD38+ NK-cells expressing FAS+/TIM3+ with a robust cytokine-responsiveness, such as type-I-interferon-, IL-12- and TNF-α-mediated signaling. Patients with MM experiencing BTI developed strong serological and cellular responses and exhibited similar cytokine-responsive immune cell patterns as vaccine-responders. This study can expand our understanding of molecular and cellular patterns associated with immunization responses and may benefit the design of improved vaccination strategies in immunocompromised patients.


Subject(s)
COVID-19 , Multiple Myeloma , Humans , COVID-19 Vaccines , Cytokines , Leukocytes, Mononuclear , Multiple Myeloma/therapy , SARS-CoV-2 , Vaccination
2.
Mol Syst Biol ; 18(8): e10874, 2022 08.
Article in English | MEDLINE | ID: mdl-35904277

ABSTRACT

Wnt pathways are important for the modulation of tissue homeostasis, and their deregulation is linked to cancer development. Canonical Wnt signaling is hyperactivated in many human colorectal cancers due to genetic alterations of the negative Wnt regulator APC. However, the expression levels of Wnt-dependent targets vary between tumors, and the mechanisms of carcinogenesis concomitant with this Wnt signaling dosage have not been understood. In this study, we integrate whole-genome CRISPR/Cas9 screens with large-scale multi-omic data to delineate functional subtypes of cancer. We engineer APC loss-of-function mutations and thereby hyperactivate Wnt signaling in cells with low endogenous Wnt activity and find that the resulting engineered cells have an unfavorable metabolic equilibrium compared with cells which naturally acquired Wnt hyperactivation. We show that the dosage level of oncogenic Wnt hyperactivation impacts the metabolic equilibrium and the mitochondrial phenotype of a given cell type in a context-dependent manner. These findings illustrate the impact of context-dependent genetic interactions on cellular phenotypes of a central cancer driver mutation and expand our understanding of quantitative modulation of oncogenic signaling in tumorigenesis.


Subject(s)
Colorectal Neoplasms , Wnt Signaling Pathway , Carcinogenesis/genetics , Colorectal Neoplasms/metabolism , Homeostasis , Humans , Wnt Signaling Pathway/genetics , beta Catenin/genetics , beta Catenin/metabolism
3.
Genome Biol ; 21(1): 53, 2020 03 02.
Article in English | MEDLINE | ID: mdl-32122365

ABSTRACT

Pooled CRISPR screens are a powerful tool to probe genotype-phenotype relationships at genome-wide scale. However, criteria for optimal design are missing, and it remains unclear how experimental parameters affect results. Here, we report that random decreases in gRNA abundance are more likely than increases due to bottle-neck effects during the cell proliferation phase. Failure to consider this asymmetry leads to loss of detection power. We provide a new statistical test that addresses this problem and improves hit detection at reduced experiment size. The method is implemented in the R package gscreend, which is available at http://bioconductor.org/packages/gscreend.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Gene-Environment Interaction , Genome-Wide Association Study/methods , Genomics/methods , Software , Animals , Cell Proliferation , Genotype , HCT116 Cells , HEK293 Cells , Humans , Mice , Models, Genetic , RNA, Guide, Kinetoplastida/chemistry , RNA, Guide, Kinetoplastida/genetics
4.
Proc Natl Acad Sci U S A ; 117(8): 4320-4327, 2020 02 25.
Article in English | MEDLINE | ID: mdl-32047037

ABSTRACT

The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ∼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21-derived heavy chains (HCs) with IGLV3-21-derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110-expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR-BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01-expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110-expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors.


Subject(s)
Immunoglobulin lambda-Chains/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Receptors, Antigen, B-Cell/immunology , B-Lymphocytes/immunology , Cohort Studies , Complementarity Determining Regions/genetics , Complementarity Determining Regions/immunology , Genetic Predisposition to Disease , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin lambda-Chains/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Point Mutation , Receptors, Antigen, B-Cell/genetics
5.
Science ; 360(6395): 1358-1362, 2018 06 22.
Article in English | MEDLINE | ID: mdl-29880723

ABSTRACT

Affinity maturation selects B cells expressing somatically mutated antibody variants with improved antigen-binding properties to protect from invading pathogens. We determined the molecular mechanism underlying the clonal selection and affinity maturation of human B cells expressing protective antibodies against the circumsporozoite protein of the malaria parasite Plasmodium falciparum (PfCSP). We show in molecular detail that the repetitive nature of PfCSP facilitates direct homotypic interactions between two PfCSP repeat-bound monoclonal antibodies, thereby improving antigen affinity and B cell activation. These data provide a mechanistic explanation for the strong selection of somatic mutations that mediate homotypic antibody interactions after repeated parasite exposure in humans. Our findings demonstrate a different mode of antigen-mediated affinity maturation to improve antibody responses to PfCSP and presumably other repetitive antigens.


Subject(s)
Antibodies, Protozoan/immunology , Antibody Affinity/immunology , Antibody Formation/immunology , Antigens, Protozoan/immunology , B-Lymphocytes/immunology , Epitopes, B-Lymphocyte/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Antibodies, Protozoan/chemistry , Antibodies, Protozoan/genetics , Antibody Affinity/genetics , Antibody Formation/genetics , Antigens, Protozoan/chemistry , Antigens, Protozoan/genetics , Humans , Lymphocyte Activation , Mutation , Protozoan Proteins/genetics , Repetitive Sequences, Nucleic Acid/genetics , Repetitive Sequences, Nucleic Acid/immunology , Selection, Genetic
6.
Immunol Rev ; 284(1): 51-66, 2018 07.
Article in English | MEDLINE | ID: mdl-29944762

ABSTRACT

A hallmark of the adaptive immune system is the specificity of B cell and T cell responses. Mechanistically, this feature relies on the fact that the two genes that encode B cell and T cell antigen receptors are not germline encoded and instead are assembled from a large number of small gene segments during lymphocyte development. The underlying somatic gene recombination process can generate a quasi-unlimited repertoire of antigen receptors. The high degree of diversity is essential to guarantee recognition of nearly any antigenic structure to protect from the large variety of potential invading pathogens and to keep the balance with commensals. Due to the enormous complexity of the antigen receptor repertoire, our understanding of its actual size and functional convergence at the level of the individual and the population is still limited. A better understanding of the actual degree of diversity could help to predict adaptive immune responses and would have wide implications for the development of preventive and therapeutic measures against infectious and autoimmune diseases as well as cancer. Here, we discuss the recent advances in the field with a specific focus on B cells and the function of antibodies.


Subject(s)
Adaptive Immunity/immunology , Antibodies/genetics , Antibodies/immunology , B-Lymphocytes/immunology , Receptors, Antigen, B-Cell/genetics , Antigens/immunology , Genetic Variation/genetics , High-Throughput Nucleotide Sequencing , Humans , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/immunology , V(D)J Recombination/genetics
7.
Sci Immunol ; 3(20)2018 02 16.
Article in English | MEDLINE | ID: mdl-29453292

ABSTRACT

Affinity maturation, the clonal selection and expansion of antigen-activated B cells expressing somatically mutated antibody variants that develop during T cell-dependent germinal center reactions, is considered pivotal for efficient development of protective B cell memory responses to infection and vaccination. Repeated antigen exposure promotes affinity maturation but each time also recruits antigen-reactive naïve B cells into the response. Here, we determined the relative impact of affinity maturation versus antigen-mediated clonal selection of naïve B cells to mount potent B cell memory responses in humans after repeated exposure to a complex pathogen, the malaria parasite Plasmodium falciparum (Pf). Using single-cell immunoglobulin (Ig) gene sequencing and production of recombinant monoclonal antibodies, we analyzed the origin, development, and quality of memory B cell responses to Pf circumsporozoite protein (PfCSP), the major sporozoite surface protein. We show that after repeated immunization of Pf-naïve volunteers with infectious Pf sporozoites (PfSPZ Challenge) under chloroquine prophylaxis (PfSPZ-CVac), the clonal selection of potent germline and memory B cell precursors against the central PfCSP NANP repeat outpaces affinity maturation because the majority of Ig gene mutations are affinity-neutral. Mathematical modeling explains how the efficiency of affinity maturation decreases strongly with antigen complexity. Thus, in the absence of long-term exposure, the frequency of antigen-reactive precursors and likelihood of their activation rather than affinity maturation will determine the quality of anti-PfCSP memory B cell responses. These findings have wide implications for the design of vaccination strategies to induce potent B cell memory responses against PfCSP and presumably other structurally complex antigens.


Subject(s)
B-Lymphocytes/cytology , B-Lymphocytes/immunology , Clonal Selection, Antigen-Mediated/immunology , Malaria/immunology , Animals , Female , Humans , Malaria Vaccines/immunology , Mice , Mice, Inbred C57BL , Plasmodium falciparum/immunology , Plasmodium falciparum/pathogenicity , Protozoan Proteins/immunology
8.
BMC Bioinformatics ; 17: 67, 2016 Feb 04.
Article in English | MEDLINE | ID: mdl-26847109

ABSTRACT

BACKGROUND: The sequencing of immunoglobulin (Ig) transcripts from single B cells yields essential information about Ig heavy:light chain pairing, which is lost in conventional bulk sequencing experiments. The previously limited throughput of single-cell approaches has recently been overcome by the introduction of multiple next-generation sequencing (NGS)-based platforms. Furthermore, single-cell techniques allow the assignment of additional data types (e.g. cell surface marker expression), which are crucial for biological interpretation. However, the currently available computational tools are not designed to handle single-cell data and do not provide integral solutions for linking of sequence data to other biological data. RESULTS: Here we introduce sciReptor, a flexible toolkit for the processing and analysis of antigen receptor repertoire sequencing data at single-cell level. The software combines bioinformatics tools for immunoglobulin sequence annotation with a relational database, where raw data and analysis results are stored and linked. sciReptor supports attribution of additional data categories such as cell surface marker expression or immunological metadata. Furthermore, it comprises a quality control module as well as basic repertoire visualization tools. CONCLUSION: sciReptor is a flexible framework for standardized sequence analysis of antigen receptor repertoires on single-cell level. The relational database allows easy data sharing and downstream analyses as well as immediate comparisons between different data sets.


Subject(s)
Computational Biology/methods , Genes, Immunoglobulin , High-Throughput Nucleotide Sequencing/methods , Immunoglobulins/genetics , Single-Cell Analysis/methods , Software , Humans , Molecular Sequence Annotation , Receptors, Immunologic/genetics
9.
Cell Microbiol ; 18(1): 125-36, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26243691

ABSTRACT

Viral glycoproteins are highly variable in their primary structure, but on the other hand feature a high functional conservation to fulfil their versatile tasks during the pathogenic life cycle. Typically, all protein domains are optimized in that indispensable functions can be assigned to small conserved motifs or even individual amino acids. The cytoplasmic tail of many viral spike proteins, although of particular relevance for the virus biology, is often only insufficiently characterized. Hemagglutinin (HA), the receptor-binding protein of the influenza virus comprises a short cytoplasmic tail of 13 amino acids that exhibits three highly conserved palmitoylation sites. However, the particular importance of these modifications and the tail in general for intracellular trafficking and lateral membrane organization remains elusive. In this study, we generated HA core proteins consisting of transmembrane domain, cytoplasmic tail and a minor part of the ectodomain, tagged with a yellow fluorescent protein. Different mutation and truncation variants of these chimeric proteins were investigated using confocal microscopy, to characterize the role of cytoplasmic tail and palmitoylation for the intracellular trafficking to plasma membrane and Golgi apparatus. In addition, we assessed raft partitioning of the variants by Foerster resonance energy transfer with an established raft marker. We revealed a substantial influence of the cytoplasmic tail length on the intracellular distribution and surface exposure of the proteins. A complete removal of the tail hampers a physiological trafficking of the protein, whereas a partial truncation can be compensated by cytoplasmic palmitoylations. Plasma membrane raft partitioning on the other hand was found to imperatively require palmitoylations, and the cysteine at position 551 turned out to be of most relevance. Our data shed further light on the tight interconnection between cytoplasmic elements and intracellular trafficking and suggest a function of HA palmitoylations in both lateral sorting and anterograde trafficking of the glycoprotein.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Host-Pathogen Interactions , Membrane Microdomains/metabolism , Orthomyxoviridae/physiology , Animals , Bacterial Proteins/analysis , Bacterial Proteins/genetics , CHO Cells , Cricetulus , Golgi Apparatus/metabolism , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Microscopy, Confocal , Protein Processing, Post-Translational , Protein Transport , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics
11.
J R Soc Interface ; 12(108): 20150282, 2015 Jul 06.
Article in English | MEDLINE | ID: mdl-26136227

ABSTRACT

The circadian clock coordinates daily physiological, metabolic and behavioural rhythms. These endogenous oscillations are synchronized with external cues ('zeitgebers'), such as daily light and temperature cycles. When the circadian clock is entrained by a zeitgeber, the phase difference ψ between the phase of a clock-controlled rhythm and the phase of the zeitgeber is of fundamental importance for the fitness of the organism. The phase of entrainment ψ depends on the mismatch between the intrinsic period τ and the zeitgeber period T and on the ratio of the zeitgeber strength to oscillator amplitude. Motivated by the intriguing complexity of empirical data and by our own experiments on temperature entrainment of mouse suprachiasmatic nucleus (SCN) slices, we present a theory on how clock and zeitgeber properties determine the phase of entrainment. The wide applicability of the theory is demonstrated using mathematical models of different complexity as well as by experimental data. Predictions of the theory are confirmed by published data on Neurospora crassa strains for different period mismatches τ - T and varying photoperiods. We apply a novel regression technique to analyse entrainment of SCN slices by temperature cycles. We find that mathematical models can explain not only the stable asymptotic phase of entrainment, but also transient phase dynamics. Our theory provides the potential to explore seasonal variations of circadian rhythms, jet lag and shift work in forthcoming studies.


Subject(s)
Circadian Clocks/physiology , Models, Biological , Neurospora crassa/physiology , Photoperiod , Suprachiasmatic Nucleus/physiology , Animals , Mice
12.
J Biol Chem ; 288(19): 13705-17, 2013 May 10.
Article in English | MEDLINE | ID: mdl-23525100

ABSTRACT

BACKGROUND: cAMP research in intracellular parasites remains underappreciated, and it requires a specific method for cyclic nucleotide regulation. RESULTS: Optogenetic induction of cAMP in T. gondii affects host-cell invasion, stage-specific expression, and parasite differentiation. The underlying method allows a versatile control of parasite cAMP. CONCLUSIONS: Optogenetic parasite strains offer valuable tools for dissecting cAMP-mediated processes. SIGNIFICANCE: The method is applicable to other gene-tractable intertwined systems. Successful infection and transmission of the obligate intracellular parasite Toxoplasma gondii depends on its ability to switch between fast-replicating tachyzoite (acute) and quiescent bradyzoite (chronic) stages. Induction of cAMP in the parasitized host cells has been proposed to influence parasite differentiation. It is not known whether the parasite or host cAMP is required to drive this phenomenon. Other putative roles of cAMP for the parasite biology also remain to be identified. Unequivocal research on cAMP-mediated signaling in such intertwined systems also requires a method for an efficient and spatial control of the cAMP pool in the pathogen or in the enclosing host cell. We have resolved these critical concerns by expressing a photoactivated adenylate cyclase that allows light-sensitive control of the parasite or host-cell cAMP. Using this method, we reveal multiple roles of the parasite-derived cAMP in host-cell invasion, stage-specific expression, and asexual differentiation. An optogenetic method provides many desired advantages such as: (i) rapid, transient, and efficient cAMP induction in extracellular/intracellular and acute/chronic stages; (ii) circumvention of the difficulties often faced in cultures, i.e. poor diffusion, premature degradation, steady activation, and/or pleiotropic effects of cAMP agonists and antagonists; (iii) genetically encoded enzyme expression, thus inheritable to the cell progeny; and (iv) conditional and spatiotemporal control of cAMP levels. Importantly, a successful optogenetic application in Toxoplasma also illustrates its wider utility to study cAMP-mediated signaling in other genetically amenable two-organism systems such as in symbiotic and pathogen-host models.


Subject(s)
Adenylyl Cyclases/biosynthesis , Cyclic AMP/physiology , Toxoplasma/metabolism , Adenylyl Cyclases/genetics , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Cells, Cultured , Fibroblasts/metabolism , Fibroblasts/parasitology , Gene Expression Regulation, Enzymologic , Host-Parasite Interactions , Humans , Optogenetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Second Messenger Systems , Toxoplasma/genetics , Toxoplasma/physiology
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