Galectin-Glycan Interactions as Regulators of B Cell Immunity

Cell surface glycans and their glycan-binding partners (lectins) have generally been recognized as adhesive assemblies with neighbor cells or matrix scaffolds in organs and the blood stream. However, our understanding of the roles for glycan-lectin interactions in immunity has expanded substantially to include regulation of nearly every stage of an immune response, from pathogen sensing to immune contraction. In this Mini-Review, we discuss the role of the ß-galactoside-binding lectins known as galectins specifically in the regulation of B-lymphocyte (B cell) development, activation, and differentiation. In particular, we highlight several recent studies revealing new roles for galectin (Gal)-9 in the modulation of B cell receptor-mediated signaling and activation in mouse and man. The roles for cell surface glycosylation, especially I-branching of N-glycans synthesized by the glycosyltransferase GCNT2, in the regulation of Gal-9 binding activity are also detailed. Finally, we consider how dysregulation of these factors may contribute to aberrant immune activation and autoimmune disease

N-Linked Glycosylation Regulates CD22 Organization and Function

The organization and clustering of cell surface proteins plays a critical role in controlling receptor signaling; however, the biophysical mechanisms regulating these parameters are not well understood. Elucidating these mechanisms is highly significant to our understanding of immune function in health and disease, given the importance of B cell receptor (BCR) signaling in directing B cells to produce antibodies for the clearance of pathogens, and the potential deleterious effects of dysregulated BCR signaling, such as in B cell malignancies or autoimmune disease. One of main inhibitory co-receptors on B cells is CD22, a sialic-acid binding protein, which interacts homotypically with other sialylated CD22 molecules, as well as heterotypically with IgM and CD45. Although the importance of CD22 in attenuating BCR signaling is well established, we still do not fully understand what mediates CD22 organization and association to BCRs. CD22 is highly glycosylated, containing 12 N-linked glycosylation sites on its extracellular domain, the function of which remain to be resolved. We were interested in how these glycosylation sites mediate homotypic vs. heterotypic interactions. To this end, we mutated five out of the six N-linked glycosylation residues on CD22 localized closest to the sialic acid binding site. Glycan site N101 was not mutated as this resulted in lack of CD22 expression. We used dual-color super-resolution imaging to investigate the impact of altered glycosylation of CD22 on the nanoscale organization of CD22 and its association with BCR. We show that mutation of these five glycosylation sites increased the clustering tendency of CD22 and resulted in higher density CD22 nanoclusters. Consistent with these findings of altered CD22 organization, we found that mutation of N-glycan sites attenuated CD22 phosphorylation upon BCR stimulation, and consequently, increased BCR signaling. Importantly, we identified that these sites may be ligands for the soluble secreted lectin, galectin-9, and are necessary for galectin-9 mediated inhibition of BCR signaling. Taken together, these findings implicate N-linked glycosylation in the organization and function of CD22, likely through regulating heterotypic interactions between CD22 and its binding partners

Phospholipase C-γ2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen

B cell receptor (BCR) recognition of membrane-bound antigen initiates a spreading and contraction response, the extent of which is controlled through the formation of signaling-active BCR-antigen microclusters and ultimately affects the outcome of B cell activation. We followed a genetic approach to define the molecular requirements of BCR-induced spreading and microcluster formation. We identify a key role for phospholipase C-γ2 (PLCγ2), Vav, B cell linker, and Bruton's tyrosine kinase in the formation of highly coordinated “microsignalosomes,” the efficient assembly of which is absolutely dependent on Lyn and Syk. Using total internal reflection fluorescence microscopy, we examine at high resolution the recruitment of PLCγ2 and Vav to microsignalosomes, establishing a novel synergistic relationship between the two. Thus, we demonstrate the importance of cooperation between components of the microsignalosome in the amplification of signaling and propagation of B cell spreading, which is critical for appropriate B cell activation

Dynamic cortical actin remodeling by ERM proteins controls BCR microcluster organization and integrity

By dynamically remodeling the cortical actin network, ezrin and moesin control BCR microcluster formation, organization, and integrity

Microclusters of inhibitory killer immunoglobulin–like receptor signaling at natural killer cell immunological synapses

We report the supramolecular organization of killer Ig–like receptor (KIR) phosphorylation using a technique applicable to imaging phosphorylation of any green fluorescent protein–tagged receptor at an intercellular contact or immune synapse. Specifically, we use fluorescence lifetime imaging (FLIM) to report Förster resonance energy transfer (FRET) between GFP-tagged KIR2DL1 and a Cy3-tagged generic anti-phosphotyrosine monoclonal antibody. Visualization of KIR phosphorylation in natural killer (NK) cells contacting target cells expressing cognate major histocompatibility complex class I proteins revealed that inhibitory signaling is spatially restricted to the immune synapse. This explains how NK cells respond appropriately when simultaneously surveying susceptible and resistant target cells. More surprising, phosphorylated KIR was confined to microclusters within the aggregate of KIR, contrary to an expected homogeneous distribution of KIR signaling across the immune synapse. Also, yellow fluorescent protein–tagged Lck, a kinase important for KIR phosphorylation, accumulated in a multifocal distribution at inhibitory synapses. Spatial confinement of receptor phosphorylation within the immune synapse may be critical to how activating and inhibitory signals are integrated in NK cells

Engineering pan–HIV-1 neutralization potency through multispecific antibody avidity

Deep mining of B cell repertoires of HIV-1-infected individuals has resulted in the isolation of dozens of HIV-1 broadly neutralizing antibodies (bNAbs). Yet, it remains uncertain whether any such bNAbs alone are sufficiently broad and potent to deploy therapeutically. Here, we engineered HIV-1 bNAbs for their combination on a single multispecific and avid molecule via direct genetic fusion of their Fab fragments to the human apoferritin light chain. The resulting molecule demonstrated a remarkable median IC50 value of 0.0009 g/mL and 100% neutralization coverage of a broad HIV-1 pseudovirus panel (118 isolates) at a 4 g/mL cutoff-a 32-fold enhancement in viral neutralization potency compared to a mixture of the corresponding HIV-1 bNAbs. Importantly, Fc incorporation on the molecule and engineering to modulate Fc receptor binding resulted in IgG-like bioavailability invivo. This robust plug-and-play antibody design is relevant against indications where multispecificity and avidity are leveraged simultaneously to mediate optimal biological activity.The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases: TZM-bl cells (ARP-8129; contributed by Dr. John C. Kappes and Dr. Xiaoyun Wu); anti–HIV-1 gp160 monoclonal antibody (N6/ PGDM1400x10E8v4) (ARP-13390; contributed by Drs. Ling Xu and Gary Nabel); HIV-1 NL4-3 ΔEnv Vpr luciferase reporter vector (pNL4-3.Luc.R-E-) (ARP-3418; contributed by Dr. Nathaniel Landau and Aaron Diamond); plasmids pcDNA3.1 D/V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11017, ARP-11018, ARP-11024, and ARP-11022; contributed by Drs. David Montefiori, Feng Gao, and Ming Li); plasmid pcDNA3.1(+)-expressing HIV-1 Env/Rev (ARP-11037; contributed by Drs. B. H. Hahn and D. L. Kothe); plasmid pcDNA3.1 D/V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11308; contributed by Drs. D. Montefiori, F. Gao, C. Wil- liamson, and S. Abdool Karim); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11309; contributed by Drs. B. H. Hahn, Y. Li, and J. F. Sala- zar-Gonzalez); HIV-1 BG505 Env expression vector (BG505.W6M.ENV.C2) (ARP- 11518; contributed by Dr. Julie Overbaugh); HIV-1 Env expression vector (CRF02_AG clone 257) (ARP-11599; contributed by Drs. D. Ellenberger, B. Li, M. Callahan, and S. Butera); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 CNE8 Env (ARP-12653; contributed by Drs. Linqi Zhang, Hong Shang, David Montefiori, Tsinghua University (Beijing, China), China Medical University (Bei- jing, China), and Duke University (Durham, NC); HIV-1 SF162 gp160 expression vector (ARP-10463; contributed by Drs. Leonidas Stamatatos and Cecilia Cheng- Mayer); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11034; contributed by Drs. B. H. Hahn, X. Wei, and G. M. Shaw); plasmid pcDNA3.1/V5- His TOPO-expressing HIV Env/Rev (ARP-11038; contributed by Drs. B. H. Hahn and D. L. Kothe); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11310; contributed by Drs. B. H. Hahn, Y. Li, and J. F. Salazar-Gonzalez); HIV-1 Env expression vector (p16845 env) (ARP-11503; contributed by Drs. R. Paranjape, S. Kulkarni, and D. Montefiori); HIV-1 1054 Env expression vector (p1054.TC4.1499) (ARP-11561) and 6244 Env expression vector (p6244_13.B5.4576) (ARP-11566; contributed by Drs. Beatrice H. Hahn, Brandon F. Keele, and George M. Shaw); HIV-1 ZM246F Env expression vector (pZM246F_C1G) (ARP-11830; contributed by Dr. Beatrice Hahn); HIV-1 Env expression vector (CRF02_AG clone 278) (ARP-11605; contributed by Drs. Michael Thomson, Ana Revilla, Elena Delgado, David Montefiori, Sonia P erez Castro, Centro Nacional de Microbiologia, Instituto de Salud Carlos III (Majada- honda, Madrid, Spain), Complejo Hospitalario Santa Mar ıa Madre (Orense, Spain), Duke University (Durham, NC), and the CAVD; and NL4-3 Env expression vector (pDOLHIVenv) (from Dr. Eric Freed and Dr. Rex Risser). The following reagents were kindly provided by CAVD: X2988, ZM106.9, and 3817. We thank S. Tabruyn and F. Arbogast for their assistance with in vivo studies. We thank the SickKids-University Health Network Flow Cytometry Facility. This work wassupported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant 6280100058 (J.-P.J.) and by Operating Grant PJ4- 169662 from the Canadian Institutes of Health Research (CIHR; B.T. and J.-P.J.). This research was also supported by the European Union’s Horizon 2020 research and innovation program under Marie Sklodowska-Curie Grant 790012 (E.R.), a Hospital for Sick Children Restracomp Postdoctoral Fellowship (C.B.A.), an NSERC postgraduate doctoral scholarship (T.Z.), a predoctoral fel- lowship from the Basque Government (PRE_2019_2_0046) (S.I.), the Canadian Institute for Advanced Research (CIFAR) Azrieli Global Scholar program (J.-P.J.), the Ontario Early Researcher Awards program (J.-P.J.), and the CanadaResearch Chairs program (B.T. and J.-P.J.). This work was supported, in part, by NSERC Discovery Grant RGPIN-2019-06442 and CIHR Project Grant–Priority Announcement PJH-175379 to C.G., and a CIHR Canada Graduate Scholarship (CGS-M) to J.B. Further support was obtained from the Spanish Ministry of Sci- ence, Innovation and Universities (MCIU) with the support of the Spanish Research Agency/The European Regional Development Fund (AEI/FEDER) (RTI2018-095624-B-C21) (J.L.N.) and the Basque Government (IT1196-19) (J.L.N.). Biophysical data were collected at the Structural & Biophysical Core facility supported by the Canada Foundation for Innovation and Ontario Research Fun

Engineering pan-HIV-1 neutralization potency through multispecific antibody avidity

Deep mining of B cell repertoires of HIV-1–infected individuals has resulted in the isolation of dozens of HIV-1 broadly neutralizing antibodies (bNAbs). Yet, it remains uncertain whether any such bNAbs alone are sufficiently broad and potent to deploy therapeutically. Here, we engineered HIV-1 bNAbs for their combination on a single multispecific and avid molecule via direct genetic fusion of their Fab fragments to the human apoferritin light chain. The resulting molecule demonstrated a remarkable median IC50 value of 0.0009 µg/mL and 100% neutralization coverage of a broad HIV-1 pseudovirus panel (118 isolates) at a 4 µg/mL cutoff—a 32-fold enhancement in viral neutralization potency compared to a mixture of the corresponding HIV-1 bNAbs. Importantly, Fc incorporation on the molecule and engineering to modulate Fc receptor binding resulted in IgG-like bioavailability in vivo. This robust plug-and-play antibody design is relevant against indications where multispecificity and avidity are leveraged simultaneously to mediate optimal biological activity.This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant 6280100058 (J.-P.J.) and by Operating Grant PJ4-169662 from the Canadian Institutes of Health Research (CIHR; B.T. and J.-P.J.). This research was also supported by the European Union’s Horizon 2020 research and innovation program under Marie Sklodowska-Curie Grant 790012 (E.R.), a Hospital for Sick Children Restracomp Postdoctoral Fellowship (C.B.A.), an NSERC postgraduate doctoral scholarship (T.Z.), a predoctoral fellowship from the Basque Government (PRE_2019_2_0046) (S.I.), the Canadian Institute for Advanced Research (CIFAR) Azrieli Global Scholar program (J.-P.J.), the Ontario Early Researcher Awards program (J.-P.J.), and the Canada Research Chairs program (B.T. and J.-P.J.). This work was supported, in part, by NSERC Discovery Grant RGPIN-2019-06442 and CIHR Project Grant–Priority Announcement PJH-175379 to C.G., and a CIHR Canada Graduate Scholarship (CGS-M) to J.B. Further support was obtained from the Spanish Ministry of Science, Innovation and Universities (MCIU) with the support of the Spanish Research Agency/The European Regional Development Fund (AEI/FEDER) (RTI2018-095624-B-C21) (J.L.N.) and the Basque Government (IT1196-19) (J.L.N.). Biophysical data were collected at the Structural & Biophysical Core facility supported by the Canada Foundation for Innovation and Ontario Research Fund.Peer reviewe

A multi-specific, multi-affinity antibody platform neutralizes sarbecoviruses and confers protection against SARS-CoV-2 in vivo

© 2023 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY). https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has been responsible for a global pandemic. Monoclonal antibodies (mAbs) have been used as antiviral therapeutics; however, these therapeutics have been limited in efficacy by viral sequence variability in emerging variants of concern (VOCs) and in deployment by the need for high doses. In this study, we leveraged the multi-specific, multi-affinity antibody (Multabody, MB) platform, derived from the human apoferritin protomer, to enable the multimerization of antibody fragments. MBs were shown to be highly potent, neutralizing SARS-CoV-2 at lower concentrations than their corresponding mAb counterparts. In mice infected with SARS-CoV-2, a tri-specific MB targeting three regions within the SARS-CoV-2 receptor binding domain was protective at a 30-fold lower dose than a cocktail of the corresponding mAbs. Furthermore, we showed in vitro that mono-specific MBs potently neutralize SARS-CoV-2 VOCs by leveraging augmented avidity, even when corresponding mAbs lose their ability to neutralize potently, and that tri-specific MBs expanded the neutralization breadth beyond SARS-CoV-2 to other sarbecoviruses. Our work demonstrates how avidity and multi-specificity combined can be leveraged to confer protection and resilience against viral diversity that exceeds that of traditional monoclonal antibody therapies.This work was supportedby Natural Sciences and Engineering Research Council of Canada discovery grant 6280100058 (to J.-P.J.), operating grant PJ4-169662 from the Canadian Institutes of Health Research (CIHR; to B.T. and J.-P.J.), COVID-19 Research Fund C-094-2424972-JULIEN (to J.-P.J.) from the Province of Ontario Ministry of Colleges and Universities, the Bill and Melinda Gates Foundation INV-023398 (to J.-P.J.), and the Hospital for Sick Children Foundation. This research was also supported by Hospital for Sick Children Restracomp Postdoctoral Fellowships (to C.B.A.and I.K.), an Ontario Graduate Scholarship (OGS; to K.M.), a Banting Postdoctoral Fellowship (to C.B.A.), the CIFAR Azrieli Global Scholar program (to J.-P.J.), the Ontario Early Researcher Awards program (to J.-P.J.), and the Canada Research Chairs program (to J.L.R., B.T., and J.-P.J.). Cryo-EM data were collected at the Toronto High-Resolution High-Throughput cryo-EM facility, and biophysical data were collected at the Structural and Biophysical Core Facility, both supported by the Canada Foundation for Innovation and Ontario Research Fund. X-ray diffraction experiments were performed at GM/CA@APS,which has been funded in wholeor in part with federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). The EIGER16M detector at GM/CA-XSD was funded by NIH grant S10OD012289.This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science user facility operated for the U.S. DOE Office of Science by Argonne National Laboratory under contract DE-AC02-06CH11357.Peer reviewe

Probing signalling events at the natural killer cell immunological synapse by fluorescence lifetime imaging

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Probing signalling events at the natural killer cell immunological synapse by fluorescence lifetime imaging

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