Biology subjects

Reth, M.

Publications and source records attributed to Reth, M..

10 recordsLinked to original sources

bioRxiv2025

Microtubule anchoring and coupling of CD20 to the RhoA/Rock1 pathway

CD20 is a B cell-specific four-helix transmembrane protein and a prominent target of therapeutic anti-CD20 antibodies. CD20 is localized within a membrane nanocluster harboring the IgD class B cell antigen receptor (IgD-BCR) where it functions as a gatekeeper for the resting state of naive B lymphocytes. How CD20 exerts its gatekeeper function is not yet known. Using Ramos and human peripheral blood B cells, we show here that the serine/threonine kinase PKC{delta}, constitutively phosphorylates serine residues at the cytosolic tails of CD20. The phosphorylated CD20 becomes a target for 14-3-3 adaptor proteins that link CD20 to the RhoA GDP/GTP exchange factor GEF-H1 and the microtubule (MT) network controlling the stability of the IgD-BCR nanocluster. The binding of anti-CD20 antibodies results in MT disassembly and the replacement of the GEF-H1/CD20 complex by a RhoA-GTP/ROCK1/CD20 complex which drives actomyosin contractility. Our study suggests that CD20 not only maintains the resting state, but also orchestrates the MT/actin switch in active B lymphocytes. This could have implications for treatment with anti-CD20 antibodies and may help to optimize therapeutic protocols. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/656301v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1cfe15aorg.highwire.dtl.DTLVardef@d3ee14org.highwire.dtl.DTLVardef@1b375eforg.highwire.dtl.DTLVardef@103c9dd_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Class-specific sensing of HIV-1 antigens by the B cell antigen receptor depends on the CH1 domain

How different classes of the B cell antigen receptor (BCR) sense viral antigens used in vaccination protocols is poorly understood. Here we study antigen binding and sensing of Ramos B cells expressing BCRs of either the IgM or IgG1 class with a specificity for the CD4-binding-site of the envelope (Env) protein of the human immune deficiency virus-1 (HIV-1). We find that, in spite of their identical antigen binding site, the two BCR classes differ drastically from each other in that the IgM-BCR and IgG1-BCR bind preferentially to monovalent and polyvalent antigens, respectively. By generating an IgM/IgG1 chimeric BCR we found that the class-specific antigen-sensing behavior can be transferred with the CH1{gamma} domain from the IgG1-BCR to the IgM-BCR. Our results indicate that the class-switching process not only results in the production of antibody classes with different effector functions but also alters the antigen sensing of secondary B lymphocytes. These findings suggest that antigen valency in existing vaccination protocols should be modified and altered between primary versus secondary (booster) immunization.

immunology↗

bioRxiv2023

Discovering Immunoreceptor Coupling and Organization Motifs (ICOMs)

The recently determined cryo-EM structures of the T cell antigen receptor (TCR) and B cell antigen receptor (BCR) show in molecular details the interactions of the ligand-binding part with the signaling subunits but they do not reveal the signaling mechanism of these antigen receptors. Without knowing the molecular basis of antigen sensing by these receptors, a rational design of optimal vaccines is not possible. The existence of conserved amino acids (AAs) that are not involved in the subunit interaction suggests that antigen receptors form higher complexes and/or have lateral interactors that control their activity. Here, I describe evolutionary conserved leucine zipper (LZ) motifs within the transmembrane domains (TMD) of antigen and coreceptor components that are likely to be involved in the oligomerization and lateral interaction of antigen receptor complexes on T and B cells. These immunoreceptor coupling and organization motifs (ICOMs) are also found within the TMDs of other important receptor types and viral envelope proteins. This discovery suggests that antigen receptors do not function as isolated entities but rather as part of an ICOM-based interactome that controls their nanoscale organization on resting cells and their dynamic remodeling on activated lymphocytes.

immunology↗

bioRxiv2022

Structural principles of B-cell antigen receptor assembly

The B-cell antigen receptor (BCR) is composed of a membrane-bound immunoglobulin (mIg) of class M, D, G, A or E for antigen recognition and a disulfide-linked heterodimer between Ig and Ig{beta} (Ig/{beta}, also known as CD79A and CD79B) that functions as the signalling entity. The organizing principle of BCR assembly remains elusive. Here we report the cryo-electron microscopy structures of the intact IgM class BCR at 8.2 [A] resolution and its Fab-deleted form (IgM BCR{Delta}Fab) at 3.6 [A] resolution. At the ectodomain (ECD), Ig and Ig{beta} position their respective Ig folds roughly in parallel with an approximate 2-fold symmetry, which is distinct from structures of Ig{beta}/{beta} homodimers. Unlike previous predictions, the BCR structure displays an asymmetric arrangement, in which the Ig/{beta} ECD heterodimer mainly uses Ig to associate with C{micro}3-C{micro}4 domains of one heavy chain ({micro}HC) while leaving the other heavy chain ({micro}HC) empty. The transmembrane domain (TMD) helices of the two {micro}HCs also deviate from the 2-fold symmetry of the C{micro}3-C{micro}4 domain dimer and form together with the TMD helices of the Ig/{beta} heterodimer a tight 4-helix bundle. The asymmetry at the TMD helices prevents the recruitment of two Ig/{beta} heterodimers. Surprisingly, the connecting peptides (CPs) between the ECD and TMD are braided together through striking charge complementarity, resulting in intervening of the CP of {micro}HC in between those of Ig and Ig{beta} and crossover of the TMD relative to ECD for the Ig/{beta} heterodimer, to guide the TMD assembly. Interfacial analyses suggest that the IgM BCR structure we present here may represent a general organizational architecture of all BCR classes. Our studies thus provide a structural platform for understanding B-cell signalling and for designing rational therapies against BCR-mediated diseases.

immunology↗

bioRxiv2022

Plasma membrane topography governs the three-dimensional dynamic localization of IgM B cell receptor clusters

B lymphocytes recognize bacterial or viral antigens via different classes of the B cell antigen receptor (BCR). Protrusive structures termed microvilli cover lymphocyte surfaces and are thought to perform sensory functions in screening antigen-bearing surfaces. Here, we have studied the cell surface features of Ramos B cells and the spatiotemporal organization of the IgM-BCR using lattice light sheet microscopy in combination with tailored custom-built 4D image analysis. Ramos B cell surfaces were found to form dynamic networks of elevated ridges bridging individual microvilli. A proportion of membrane-localized IgM-BCR was found in clusters, which were associated with the ridges and the microvilli. The dynamic ridge network organization and the IgM-BCR cluster mobility were linked and both were controlled by Arp2/3 complex activity. Our results suggest that topographical features of the cell surface govern the distribution and dynamic localization of IgM-BCR clusters to facilitate antigen screening.

cell biology↗

bioRxiv2022

B cells discriminate HIV-1 Envelope protein affinities by sensing antigen binding association rates

HIV-1 Envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant, KD) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD, and whether B cells discriminate between proteins of similar affinities but that bind with different kinetic rates. Here we used a panel of Env proteins and Ramos B cell lines expressing IgM BCRs with specificity for CD4 binding-site broadly neutralizing (bnAb) or a precursor antibody to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD, but on sensing of association rate and a threshold antigen-BCR half-life.

immunology↗

bioRxiv2021

Engineered chimeric T cell receptor fusion construct (TRuC)-expressing T cells prevent translational shutdown in SARS-CoV-2-infected cells

SARS-CoV-2, the causative agent of Covid-19, is known to evade the immune system by several mechanisms. This includes the shutdown of the host cellular protein synthesis, which abrogates the induction of antiviral interferon responses. The virus initiates the infection of susceptible cells by binding with its spike protein (S) to the host angiotensin-converting enzyme 2 (ACE2). Here we applied the T cell receptor fusion construct (TRuC) technology to engineer T cells against such infected cells. In our TRuCs an S-binding domain is fused to the CD3{varepsilon} component of the T cell receptor (TCR) complex, enabling recognition of S-containing cells in an HLA independent manner. This domain either consists of the S-binding part of ACE2 or a single-chain variable fragment of an anti-S antibody. We show that the TRuC T cells are activated by and kill cells that express S of SARS-CoV-2 and its alpha (B.1.1.7) and beta (B.1.351) variants at the cell surface. Treatment of SARS-CoV-2 infected cells with our engineered T cells did not lead to massive cytotoxicity towards the infected cells, but resulted in a complete rescue of the translational shutdown despite ongoing viral replication. Our data show that engineered TRuC T cell products might be used against SARS-CoV-2 by exposing infected cells to the host innate immune system.

bioengineering↗

bioRxiv2021

Quantitative Proteomics Identifies PTP1B as Modulator of B Cell Antigen Receptor Signaling

B cell antigen receptor (BCR) signaling is initiated by protein kinases and limited by counteracting phosphatases that currently are less well studied in their regulation of BCR signaling. We here used the B cell line Ramos to identify and quantify human B cell signaling components. Specifically, a protein tyrosine phosphatase profiling revealed a high expression of the protein tyrosine phosphatase 1B (PTP1B) in Ramos and human naive B cells. The loss of PTP1B leads to increased B cell activation. Through substrate trapping in combination with quantitative mass spectrometry, we identified 22 putative substrates or interactors of PTP1B. We validated Ig, CD22, PLC{gamma}1/2, CBL, BCAP and APLP2 as specific substrates of PTP1B in Ramos B cells. The tyrosine kinase BTK and the two adaptor proteins GRB2 and VAV1 were identified as direct binding partners and potential substrates of PTP1B. We showed that PTP1B dephosphorylates the inhibitory receptor protein CD22 at phosphotyrosine 807. We conclude that PTP1B negatively modulates BCR signaling by dephosphorylating distinct phosphotyrosines in B cell specific receptor proteins and various downstream signaling components.

biochemistry↗

bioRxiv2020

CD20 as a gatekeeper of the resting stage of human B cells

CD20 is a B cell specific membrane protein and a target of therapeutic antibodies such as rituximab (RTX)1. In spite of the prominent usage of anti-CD20 antibodies in the clinic little is known about the biological function of CD202. Here we show that CD20 controls the nanoscale organization of receptors on the surface of resting B lymphocytes. A CRISPR/Cas-based ablation of CD20 in Ramos B cells results in a relocalisation of the IgM B cell antigen receptor (IgM-BCR) and the co-receptor CD19. The resulting IgM-BCR/CD19 signaling synapse leads to transient B cell activation followed by plasma cell differentiation. Similarly to CD20-deficient Ramos cells, naive human B cells treated with rituximab in vitro or isolated from patients during rituximab administration display hallmarks of transient activation characterized by the formation of the IgM-BCR/CD19 signaling synapse, followed by CD19 and IgM-BCR downregulation. Moreover, increased expression of specific plasma cell genes can be observed after rituximab treatment in relapsed CLL patients. In summary we identify CD20 as a gatekeeper of the resting state on human B cells and demonstrate that a disruption of the nanoscale organization of the B cell surface via CD20 deletion or anti-CD20 treatment profoundly alters B cell fate.

immunology↗

bioRxiv2019

TLR-induced reorganization of the IgM-BCR complex regulates B-1 cell responses to infections

Neonatally-developing, self-reactive B-1 cells generate steady levels natural antibodies throughout life. They can, however, also rapidly respond to infections with increased local antibody production. The mechanisms regulating these two seemingly very distinct functions are poorly understood, but have been linked to expression of CD5, an inhibitor of BCR-signaling. Here we demonstrate that TLR-mediated activation of CD5+ B-1 cells induced the rapid reorganization of the IgM-BCR complex, leading to the eventual loss of CD5 expression, and a concomitant increase in BCR-downstream signaling, both in vitro and in vivo after infections with influenza virus and Salmonella typhimurium. Both, initial CD5 expression and TLR-mediated stimulation, were required for the differentiation of B-1 cells to IgM-producing plasmablasts after infections. Thus, TLR-mediated signals support participation of B-1 cells in immune defense via BCR-complex reorganization.

immunology↗