Mark J. Kwakkenbos

Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming

The B cell lymphoma-6 (Bcl-6) and Bcl-xL proteins are expressed in germinal center B cells and enable them to endure the proliferative and mutagenic environment of the germinal center. By introducing these genes into peripheral blood memory B cells and culturing these cells with two factors produced by follicular helper T cells, CD40 ligand (CD40L) and interleukin-21 (IL-21), we convert them to highly proliferating, cell surface B cell receptor (BCR)–positive, immunoglobulin-secreting B cells with features of germinal center B cells, including expression of activation-induced cytidine deaminase (AID). We generated cloned lines of B cells specific for respiratory syncytial virus and used these cells as a source of antibodies that effectively neutralized this virus in vivo. This method provides a new tool to study B cell biology and signal transduction through antigen-specific B cell receptors and for the rapid generation of high-affinity human monoclonal antibodies.

STAT3-Mediated Up-Regulation of BLIMP1 Is Coordinated with BCL6 Down-Regulation to Control Human Plasma Cell Differentiation

STAT family members have been implicated in regulating the balance between B cell lymphoma (BCL)6 and B lymphocyte induced maturation protein (BLIMP)1 to control plasma cell differentiation. We previously showed that STAT5 induces BCL6 to block plasma cell differentiation and extend the life span of human B cells. The heterogeneity in STAT activation by cytokines and their effects on B cell differentiation prompted us to investigate the effect of STAT3 activation in plasma cell differentiation. First stimulation with IL-21, which promotes plasma cell differentiation, induced robust and prolonged STAT3 activation in primary human B cells. We then investigated effects of direct STAT3 activation on regulation of plasma cell genes, cellular phenotype, and Ig production. Activation of a tamoxifen-regulated STAT3-estrogen receptor fusion protein triggered BLIMP1 mRNA and protein up-regulation, plasma cell phenotypic features, and Ig secretion. When STAT3 was activated by IL-21 in B cells ectopically expressing BCL6, BLIMP1 was up-regulated, but only partial plasma cell differentiation was achieved. Lastly, through coexpression of BCL6 and STAT3-ER, we verified that STAT3 activation functionally mimicked IL-21 treatment and that STAT3-mediated BLIMP1 up-regulation occurred despite high BCL6 expression levels indicating that BCL6 is not the dominant repressor of BLIMP1. Thus, up-regulation of BLIMP1 alone is not sufficient for differentiation of primary human B cells into plasma cells; concomitant down-regulation of BCL6 is absolutely required for completion of the plasma cell differentiation program.

A common solution to group 2 influenza virus neutralization

Significance The HA surface glycoprotein on influenza A viruses mediates viral entry into host cells. HA is highly variable and classified into 18 divergent subtypes, which cluster into two major phylogenetic groups. Antibody CR8043 has heterosubtypic neutralizing activity against group 2 viruses, including H3 viruses that currently circulate in humans. X-ray and EM structures of CR8043 Fab in complex with H3 HAs reveal that the antibody targets a conserved epitope on the HA stem. Compared with CR8020, the only other structurally characterized group 2 neutralizing antibody, CR8043 binds to HA with a different approach angle using different contact residues. The epitopes of both antibodies are very similar, which suggests that this conserved stem epitope has great potential for design of therapeutics and vaccines. The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.

EMR1, the human homolog of F4/80, is an eosinophil-specific receptor.

The EGF‐TM7 F4/80 is a defining marker of murine macrophage populations. Applying flow cytometric analysis using the newly generated mAb A10, and quantitative real‐time PCR, we here report the surprising observation that the human ortholog of F4/80, EGF‐like module containing mucin‐like hormone receptor (EMR)1, is absent on mononuclear phagocytic cells including monocytes, macrophages, and myeloid dendritic cells. Unexpectedly, we found that EMR1 expression is restricted to eosinophilic granulocytes, where expression is overlapping with the eotaxin receptor CCR3 and the immunoglobulin‐like lectin Siglec‐8. Absence on other leukocytes, including basophils, implies that EMR1 is a highly specific marker for eosinophils in humans.

Expression of the largest CD97 and EMR2 isoforms on leukocytes facilitates a specific interaction with chondroitin sulfate on B cells

The EGF‐TM7 receptors CD97 and EMR2 are heptahelical molecules predominantly expressed on leukocytes. A characteristic of these receptors is their ability to interact with cellular ligands via the N‐terminal epidermal growth factor (EGF)‐like domains. The first two EGF domains of CD97 (but not EMR2) bind CD55 (decay‐accelerating factor), while the fourth EGF domain of both CD97 and EMR2 interacts with the glycosaminoglycan chondroitin sulfate (CS). Using fluorescent beads coated with soluble recombinant CD97 and EMR2 protein, and isoform‐specific monoclonal antibodies, we have determined the cellular and molecular characteristics of the interaction with CS. The fourth EGF domain of CD97 and EMR2 is expressed on activated lymphocytes and myeloid cells, whereas the ligand is specifically found on B cells within the peripheral blood. The interaction between CD97/EMR2 and CS may therefore play a role in the interaction of activated T cells, dendritic cells, and macrophages with B cells.

Proteolytic cleavage of the EMR2 receptor requires both the extracellular stalk and the GPS motif

EMR2 is a human myeloid‐restricted member of the EGF‐TM7 receptor family that contains a highly conserved protein‐coupled receptor roteolysis ite (GPS) in the membrane‐proximal region. Here the post‐translational proteolytic cleavage of EMR2 at GPS was investigated. We show the cleavage occurs at Leu517‐Ser518 and is independent of the transmembrane domains. The non‐covalent association of the resulting extracellular α‐subunit and transmembrane β‐subunit requires a minimum of eight amino acids in the β‐subunit. The GPS motif is necessary, but not sufficient for receptor cleavage, which requires the entire extracellular stalk. Thus, an alternatively spliced EMR2 isoform with a truncated stalk fails to undergo proteolytic cleavage. Alternative splicing therefore provides a means to regulate GPS cleavage, producing receptors with two distinct structures.

An unusual mode of concerted evolution of the EGF-TM7 receptor chimera EMR2

The epidermal growth factor (EGF)‐ TM7 receptors CD97, EMR1, EMR2, EMR3, and EMR4 form a group of adhesion class heptahelical molecules predominantly expressed by cells of the immune system. These receptors bind cellular ligands through EGF‐like domains, localized N‐terminal to a large extracellular region. Remarkably, EMR2 possesses a chimeric structure with a seven‐span transmembrane (TM7) region most related to EMR3 and an EGF domain region nearly identical to CD97. By comparing EGF‐TM7 receptors in primates and dogs, we identified an intriguing pattern of concerted evolution, apparently mediated by gene conversion, among EMR2 and the oppositely orientated and physically adjacent genes CD97 and EMR3. This concerted evolution has continuously maintained the chimeric structure of EMR2 since early mammal radiation. Most highly conserved between EMR2 and CD97 is the fourth EGF domain, which mediates binding to chondroitin sulfate, a ligand specificity shared by both receptors. Another ligand, CD55, is bound effectively only by CD97. We show that different molecular mechanisms (mutations vs. alternative splicing) prevent CD55 binding by EMR2 in hominoids. Our findings illustrate how various and partially opposing evolutionary events have shaped the structure and ligand specificity of a modern mammalian gene family.—Kwakkenbos, M. J., Matmati, M., Madsen, O., Pouwels, W., Wang, Y. Y., Bontrop, R. E., Heidt, P. J., Hoek, R. M., Hamann, J. An unusual mode of concerted evolution of the EGF‐TM7 receptor chimera EMR2. FASEB J. 20, E2026–E2035 (2006)

Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity

Significance Bispecific antibodies expand the function of conventional antibodies. However, therapeutic application of bispecifics is hampered by the reduced physiochemical stability of such molecules. We present a format for bispecific antibodies, fusing two full-sized antibodies via their C termini. This format does not require mutations in the antibody constant domains beyond installation of a five-residue tag, ensuring that the native antibody structure is fully retained in the bispecific product. We have validated the approach by linking two anti-influenza A antibodies, each active against a different subgroup of the virus. The bispecific antibody dimer retains the activity and the stability of the two original antibodies. Bispecific antibodies have therapeutic potential by expanding the functions of conventional antibodies. Many different formats of bispecific antibodies have meanwhile been developed. Most are genetic modifications of the antibody backbone to facilitate incorporation of two different variable domains into a single molecule. Here, we present a bispecific format where we have fused two full-sized IgG antibodies via their C termini using sortase transpeptidation and click chemistry to create a covalently linked IgG antibody heterodimer. By linking two potent anti-influenza A antibodies together, we have generated a full antibody dimer with bispecific activity that retains the activity and stability of the two fusion partners.

Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.

Differential role of CD97 in interleukin-8-induced and granulocyte-colony stimulating factor-induced hematopoietic stem and progenitor cell mobilization

CD97 is a transmembrane receptor involved in neutrophil migration. This study shows that CD97 plays a role in interleukin-8-induced hematopoietic stem cell and progenitor mobilization. CD97 is broadly expressed on hematopoietic cells and is involved in neutrophil migration. Since neutrophils are key regulators in HSC/HPC mobilization, we studied a possible role for CD97 in interleukin-8 and granulocyte-colony stimulating factor-induced HSC/HPC mobilization. Mobilization was absent in mice receiving CD97 mAb followed by interleukin-8, while granulocyte-colony stimulating factor-induced mobilization remained unaltered following anti-CD97 administration. Furthermore, combined administration of CD97 mAb and IL-8 induced a significant reduction in the neutrophilic compartment. We hypothesize that the absence of interleukin-8-induced HSC/HPC mobilization after CD97 mAb administration is due to its effect on neutrophil function.