Ig Molecules Research Articles

Generation and Selection of Phage Display Antibody Libraries in Fab Format.

Monoclonal antibodies (mAbs) have exceptional utility as research reagents and pharmaceuticals. As a complement to both traditional and contemporary single-B-cell cloning technologies, the mining of antibody libraries via display technologies-which mimic and simplify B cells by physically linking phenotype (protein) to genotype (protein-encoding DNA or RNA)-has become an important method for mAb discovery. Among these display technologies, phage display has been particularly successful for the generation of mAbs that bind to a wide variety of antigens with exceptional specificities and affinities. Rather than multivalent whole antibodies, phage display typically uses monovalent antibody fragments, such as "fragment antigen binding" (Fab), as the format of choice. The ∼50-kDa Fab format consists of four immunoglobulin (Ig) domains on two polypeptide chains (light chain and shortened heavy chain), and exhibits its antigen binding site in a natural configuration found in bivalent IgG and other multivalent Ig molecules. The Fab fragment has a high melting temperature and a low tendency to aggregate, and can be readily converted to natural and nonnatural Ig formats without affecting antigen binding properties, which has made it a favored format for phage display for more than three decades. Here, I briefly summarize some of the approaches used for the generation and selection of phage display antibody libraries in Fab format, from human and nonhuman antibody repertoires.

Therapeutic Monoclonal Antibodies against Cancer: Present and Future.

A series of monoclonal antibodies with therapeutic potential against cancer have been generated and developed. Ninety-one are currently used in the clinics, either alone or in combination with chemotherapeutic agents or other antibodies, including immune checkpoint antibodies. These advances helped to coin the term personalized medicine or precision medicine. However, it seems evident that in addition to the current work on the analysis of mechanisms to overcome drug resistance, the use of different classes of antibodies (IgA, IgE, or IgM) instead of IgG, the engineering of the Ig molecules to increase their half-life, the acquisition of additional effector functions, or the advantages associated with the use of agonistic antibodies, to allow a broad prospective usage of precision medicine successfully, a strategy change is required. Here, we discuss our view on how these strategic changes should be implemented and consider their pros and cons using therapeutic antibodies against cancer as a model. The same strategy can be applied to therapeutic antibodies against other diseases, such as infectious or autoimmune diseases.

Unique repetitive nucleic acid structures mirror switch regions in the human IgH locus

Immunoglobulin (Ig) genes carry the unique ability to be reshaped in peripheral B lymphocytes after these cells encounter a specific antigen. B cells can then further improve their affinity, acquire new functions as memory cells and eventually end up as antibody-secreting cells. Ig class switching is an important change that occurs in this context, thanks to local DNA lesions initiated by the enzyme activation-induced deaminase (AID). Several cis-acting elements of the Ig heavy (IgH) chain locus make it accessible to the AID-mediated lesions that promote class switch recombination (CSR). DNA repeats, with a non-template strand rich in G-quadruplexes (G4)-DNA, are prominent cis-targets of AID and define the so-called “switch” (S) regions specifically targeted for CSR. By analyzing the structure of the human IgH locus, we uncover that abundant DNA repeats, some with a putative G4-rich template strand, are additionally present in downstream portions of the IgH coding genes. These like-S (LS) regions stand as 3′ mirror-images of S regions and also show analogies to some previously reported repeats associated with the IgH locus 3’ super-enhancer. A regulatory role of LS repeats is strongly suggested by their specific localization close to exons encoding the membrane form of Ig molecules, and by their conservation during mammalian evolution.

Isolation and Analysis of B-cell Progenitors from Bone Marrow by Flow Cytometry.

B cells play a critical role in host defense, producing antibodies in response to microbial infection. An inability to produce an effective antibody response leaves affected individuals prone to serious infection; therefore, proper B-cell development is essential to human health. B-cell development begins in the bone marrow and progresses through various stages until maturation occurs in the spleen. This process involves several sequential, complex events, starting with pre- and pro-B cells, which rearrange the heavy and light chain genes responsible for producing clonally diverse immunoglobulin (Ig) molecules. These cells then differentiate into immature B cells, followed by mature B cells. The bone marrow is a complex ecological niche of supporting stromal cells, extracellular matrix components, macrophages, and hematopoietic precursor cells influencing B-cell development, maturation, and differentiation. Once fully mature, B cells circulate in peripheral lymphoid organs and can respond to antigenic stimuli. As specific cell surface markers are expressed during each stage of B-cell development, researchers use flow cytometry as a powerful tool to evaluate developmental progression. In this protocol, we provide a step-by-step method for bone marrow isolation, cell staining, and data analysis. This tool will help researchers gain a deeper understanding of the progression of B-cell development and provide a pertinent flow gating strategy.

Cryomicroscopy reveals the structural basis for a flexible hinge motion in the immunoglobulin M pentamer

Immunoglobulin M (IgM) is the most ancient of the five isotypes of immunoglobulin (Ig) molecules and serves as the first line of defence against pathogens. Here, we use cryo-EM to image the structure of the human full-length IgM pentamer, revealing antigen binding domains flexibly attached to the asymmetric and rigid core formed by the Cμ4 and Cμ3 constant regions and the J-chain. A hinge is located at the Cμ3/Cμ2 domain interface, allowing Fabs and Cμ2 to pivot as a unit both in-plane and out-of-plane. This motion is different from that observed in IgG and IgA, where the two Fab arms are able to swing independently. A biased orientation of one pair of Fab arms results from asymmetry in the constant domain (Cμ3) at the IgM subunit interacting most extensively with the J-chain. This may influence the multi-valent binding to surface-associated antigens and complement pathway activation. By comparison, the structure of the Fc fragment in the IgM monomer is similar to that of the pentamer, but is more dynamic in the Cμ4 domain.

In Vitro Stimulation of IRE1α/XBP1-Deficient B Cells with LPS.

During immune responses, pathogen-specific B cells differentiate into plasma cells. Plasma cells synthesize and secrete large amounts of immunoglobulin (Ig) molecules which play a central role in immunity against pathogens. The synthesis, proper folding, and secretion of these Ig molecules require expansion of the extensive endoplasmic reticulum (ER) network. Accumulation of unfolded or misfolded proteins in the ER is sensed by three sensors: IRE1/XBP1, PERK, and ATF6, which coordinate with each other and initiate the unfolded protein response (UPR) pathway to expand the ER network and its protein folding and secretion capability. The expansion and maintenance of the ER network in plasma cells is triggered by activation of the IRE1/XBP1 branch of the UPR pathway. Here, we discuss the methods to stimulate the differentiation of B cells into plasma cells, measure the activation of the XBP1 pathway, and quantify the ER network.

M-Protein Sequencing and Monitoring in Serum of LC-Only Multiple Myeloma Patients

M-Protein Sequencing and Monitoring in Serum of LC-Only Multiple Myeloma Patients

Single B Cell Immunoglobulin Sequencing Identifies Distinct Features of Monoclonal Antibodies in Patients with Immmune Thrombotic Thrombocytoepenic Purpura

Introduction. Immune thrombotic thrombocytopenic purpura (iTTP) is a potentially fatal blood disorder, resulting from autoantibodies against ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor. However, the structural feature, binding epitope, and the mechanism of action of these autoantibodies in patients with acute iTTP are not fully understood.Methods. To further understand the pathogenesis of iTTP, single B cell immunoglobulin (Ig) sequencing using 10xChromium in 4 patients experiencing an acute episode of iTTP was performed; the expression and preliminary functional characterizations of selected clones were also carried out.Results. Approximately 2,631 viable and fluoresceinated ADAMTS13 labeled B cells (e.g., 7AAD -CD19 +CD20 +ADAMTS13 +) were sorted out from peripheral blood mononuclear cells of four patients with acute iTTP. These enriched ADAMTS13 antibody-producing B cells were then used for single cell analysis using 10xGenomics 5'-VDJ kit following the manufacturer's instruction. The single-cell gene expression libraries and VDJ libraries were constructed and sequenced by Hiseq at 20,000 reads/cell for gene expression and 5,000 reads/cell for VDJ sequences. Sequencing FASTQ files were mapped and counted by running through the Cell Ranger pipeline, and the final data were then further analyzed by the Loupe browser.We showed for the first time that the most frequent VJ combinations in the anti-ADAMTS13 IgG were: IGHV4-39:ILGJ4, IGHV3-48:ILGJ4, IGLV1-44:ILGLJ2, GLV5-45:ILGLJ3, IGLV2-14:ILGJ2, and IGLV3-21:ILGJ3 as shown in Figure 1. Of the top ten clones, the most frequently observed CDR3 (complementarity-determining region-3) sequences of these antibodies were CARDQLGISETQGSDLW on the heavy chain and CVIWHNSAWVF on the light chain as shown in Figure 2 and Table 1.The variable region sequences from the heavy and the light chains of Ig molecules were cloned into a human IgHG1 and a human IgL vector, respectively, which was then cotransfected in HEK293 cells. Western blotting, ELISA, immunoprecipitation, and functional assays were used to determine the expression and the function of human monoclonal IgG antibodies. Our preliminary results demonstrated the human monoclonal IgG antibodies bound and/or inhibited plasma ADAMTS13 activity.Conclusions. We conclude that there is clonal expansion of ADAMTS13 antibody producing B cells in acute iTTP and the cloned human monoclonal antibodies using the single B cell sequencing approach are functional. Our ongoing analysis on the structural and functional relationship of a large number of isolated human monoclonal antibodies may shed new light on the pathogenesis of iTTP. These antibodies may be useful to explore structural elements required for allosteric regulation of ADAMTS13 activity. [Display omitted] DisclosuresZheng: AJMC: Honoraria; Clotsolution: Other: Co-founder; Takeda: Consultancy, Honoraria; Sanofi-Genzyme: Honoraria, Speakers Bureau; Alexion: Speakers Bureau.

A chimeric HLA-A2:β2M:Ig fusion protein for the study of virus-specific CD8+ T-cells

IntroductionThe response mediated by CD8+ T-cells in the context of infection and vaccination has been thoroughly investigated and represents one of the most important branches that allow for the development of immunity against intracellular pathogens and, thus, the establishment of robust antiviral responses. However, there is a lack of methods to assess antigen-specific CD8+ T-cells. ObjectiveSearch for the ideal assays to assess the function of antigen-specific CD8+ T-cells. MethodsIn the present study a chimeric HLA-A2:β2M:Ig fusion protein was produced, purified, and evaluated in functional CD8+ T-cell response studies using samples from Influenza A patients and humanized mice upon adenoviral vaccination. ResultsThe HLA-A2:β2M:Ig molecule, bound to immunodominant viral peptides by passive transfer, was able to induce robust antiviral CD8+ T-cell responses mediated by IFN-γ. The in vitro IFN-γ release assay using the chimeric HLA-A2:β2M:Ig fusion protein detected bona fide human CD8+ T-cells, demonstrating superior production of IFN-γ by human CD8+ T-cells induced by Influenza A immunodominant GILGFVFTL peptide. Removal of antigen-presenting cells and CD8+ T-cell enrichment improved significantly the IFN-γ production. The chimeric HLA-A2:β2M:Ig fusion protein also triggered HLA-A2-restricted CD8+ T-cell response in a humanized mouse model upon vaccination with adenovirus encoding HLA-A2-restricted HIV p24 antigen. The results strongly suggest the use of tailor-made assays for detecting HLA-A2-restricted CD8+ T-cell Responses in the Humanized Mouse Model. ConclusionThe chimeric HLA-A2:β2M:Ig fusion protein-based assays provided a sensitive tool that may be paramount to measure virus-specific CD8+ T-cell response in a range of viral infections of clinical relevance.

Specific Targeting of Lymphoma Cells Using Semisynthetic Anti-Idiotype Shark Antibodies.

The B-cell receptor (BCR) is a key player of the adaptive immune system. It is a unique part of immunoglobulin (Ig) molecules expressed on the surface of B cells. In case of many B-cell lymphomas, the tumor cells express a tumor-specific and functionally active BCR, also known as idiotype. Utilizing the idiotype as target for lymphoma therapy has emerged to be demanding since the idiotype differs from patient to patient. Previous studies have shown that shark-derived antibody domains (vNARs) isolated from a semi-synthetic CDR3-randomized library allow for the rapid generation of anti-idiotype binders. In this study, we evaluated the potential of generating patient-specific binders against the idiotype of lymphomas. To this end, the BCRs of three different lymphoma cell lines SUP-B8, Daudi, and IM-9 were identified, the variable domains were reformatted and the resulting monoclonal antibodies produced. The SUP-B8 BCR served as antigen in fluorescence-activated cell sorting (FACS)-based screening of the yeast-displayed vNAR libraries which resulted after three rounds of screening in the enrichment of antigen-binding vNARs. Five vNARs were expressed as Fc fusion proteins and consequently analyzed for their binding to soluble antigen using biolayer interferometry (BLI) revealing binding constants in the lower single-digit nanomolar range. These variants showed specific binding to the parental SUP-B8 cell line confirming a similar folding of the recombinantly expressed proteins compared with the native cell surface-presented BCR. First initial experiments to utilize the generated vNAR-Fc variants for BCR-clustering to induce apoptosis or ADCC/ADCP did not result in a significant decrease of cell viability. Here, we report an alternative approach for a personalized B-cell lymphoma therapy based on the construction of vNAR-Fc antibody-drug conjugates to enable specific killing of malignant B cells, which may widen the therapeutic window for B-cell lymphoma therapy.

T Cells from NOD-PerIg Mice Target Both Pancreatic and Neuronal Tissue.

It has become increasingly appreciated that autoimmune responses against neuronal components play an important role in type 1 diabetes (T1D) pathogenesis. In fact, a large proportion of islet-infiltrating B lymphocytes in the NOD mouse model of T1D produce Abs directed against the neuronal type III intermediate filament protein peripherin. NOD-PerIg mice are a previously developed BCR-transgenic model in which virtually all B lymphocytes express the H and L chain Ig molecules from the intra-islet-derived anti-peripherin-reactive hybridoma H280. NOD-PerIg mice have accelerated T1D development, and PerIg B lymphocytes actively proliferate within islets and expand cognitively interactive pathogenic T cells from a pool of naive precursors. We now report adoptively transferred T cells or whole splenocytes from NOD-PerIg mice expectedly induce T1D in NOD.scid recipients but, depending on the kinetics of disease development, can also elicit a peripheral neuritis (with secondary myositis). This neuritis was predominantly composed of CD4+ and CD8+ T cells. Ab depletion studies showed neuritis still developed in the absence of NOD-PerIg CD8+ T cells but required CD4+ T cells. Surprisingly, sciatic nerve-infiltrating CD4+ cells had an expansion of IFN-γ- and TNF-α- double-negative cells compared with those within both islets and spleen. Nerve and islet-infiltrating CD4+ T cells also differed by expression patterns of CD95, PD-1, and Tim-3. Further studies found transitory early B lymphocyte depletion delayed T1D onset in a portion of NOD-PerIg mice, allowing them to survive long enough to develop neuritis outside of the transfer setting. Together, this study presents a new model of peripherin-reactive B lymphocyte-dependent autoimmune neuritis.

CD32a receptor in health and disease

Low-affinity Fcγ-receptors that recognize the Fc portion of immunoglobulin (Ig) molecules, usually being in antigen-bound state, thus representing a link between innate and adaptive immunity. They play a significant role in inflammatory and infectious diseases. Among them, a separate FcγRII family (CD32) is discerned, which is characterized by transmission of intracellular signal independently of the common γ-chain, they have one α-chain containing two extracellular immunoglobulin-like domains. FcγRII receptors are present in almost all cells of the innate immune system: monocytes and macrophages, neutrophils, eosinophils, dendritic cells, as well as on B-lymphocytes and platelets. They perform two main functions: target recognition, facilitation of phagocytosis and destruction of antibody-opsonized cells by monocytes/ macrophages (including pathogenic cells). In parallel, the phagocytes are activated via the cytokine synthesis stimulation. The FcγRIIA (CD32a) and FcγRIIC (CD32c) activating receptors, like as FcγRIIB (CD32b) inhibiting receptors are present among the members of the FcγRII family. The low-affinity FcγRII receptors bind to IgG, with immune complexes being their natural ligands. High levels of immune complexes are usually found in both chronic viral infections and autoimmune diseases. There are shown polymorphic variants of the CD32a gene, which can affect the receptor function, and, thereby, causing susceptibility for different infections, influence the development of autoimmune diseases and primary immunodeficiencies. Activation of the CD32a receptor induces the production of pro-inflammatory cytokines, including TNFα and interferons, that are involved into inflammation in systemic lupus erythematosus, Kawasaki disease, Graves’ disease and rheumatoid arthritis. It has been shown that antibacterial activity of platelets is carried out via the CD32a receptor. The study of CD32a expression in people The CD32a receptor is considered a biomarker of cells that are a reservoir of HIV infection. At the present time, however, many questions remain regarding the mechanisms of CD32a expression of on HIV-infected cells and the role of CD32a in the formation of an HIV reservoir and/or development of appropriate resistance. In addition to HIV infection, the significance of FcγR receptors is shown in other infectious diseases, for example, with influenza and dengue virus infections. Better understanding of the CD32a structure and function will help to assess its role in immunopathogenesis of different conditions. This review focuses on the role of CD32a in development of the normal immune response in normal state and various diseases.

Recombination may occur in the absence of transcription in the immunoglobulin heavy chain recombination centre.

Developing B cells undergo V(D)J recombination to generate a vast repertoire of Ig molecules. V(D)J recombination is initiated by the RAG1/RAG2 complex in recombination centres (RCs), where gene segments become accessible to the complex. Whether transcription is the causal factor of accessibility or whether it is a side product of other processes that generate accessibility remains a controversial issue. At the IgH locus, V(D)J recombination is controlled by Eμ enhancer, which directs the transcriptional, epigenetic and recombinational events in the IgH RC. Deletion of Eμ enhancer affects both transcription and recombination, making it difficult to conclude if Eμ controls the two processes through the same or different mechanisms. By using a mouse line carrying a CpG-rich sequence upstream of Eμ enhancer and analyzing transcription and recombination at the single-cell level, we found that recombination could occur in the RC in the absence of detectable transcription, suggesting that Eμ controls transcription and recombination through distinct mechanisms. Moreover, while the normally Eμ-dependent transcription and demethylating activities were impaired, recruitment of chromatin remodeling complexes was unaffected. RAG1 was efficiently recruited, thus compensating for the defective transcription-associated recruitment of RAG2, and providing a mechanistic basis for RAG1/RAG2 assembly to initiate V(D)J recombination.

JNK/AP1 Pathway Regulates MYC Expression and BCR Signaling through Ig Enhancers in Burkitt Lymphoma Cells.

In Burkitt lymphoma (BL), a chromosomal translocation by which the MYC gene is fused to an immunoglobulin (Ig) gene locus is frequently found. The translocated MYC gene is overexpressed, which is the major driver of BL tumorigenesis. Studies have shown that Ig enhancers are essential for MYC overexpression, but the involved mechanisms are not fully understood. In addition, the survival of BL cells relies on B-cell receptor (BCR) signaling, which is determined by the levels of Ig molecules expressed on the cell surface. However, whether MYC has any impact on Ig expression and its functional relevance in BL has not been investigated. Herein, we show that MYC upregulates Ig kappa (Igκ) expression in BL cells through two Igκ enhancers, the intronic enhancer (Ei) and the 3ʹ enhancer (E3ʹ). Mechanistically, by activating the JNK pathway, MYC induces the phosphorylation of c-Fos/c-Jun and their recruitment to AP1 binding sites in the Igκ enhancers, leading to the activation of the enhancers and subsequent Igκ upregulation. The AP1-mediated activation of the Igκ enhancers is also required for the expression of the translocated MYC gene, indicating positive feedback for the MYC overexpression in BL cells. Importantly, interrupting the JNK pathway inhibits both Igκ and MYC gene expression and suppresses BL cell proliferation. Our study not only reveals a novel mechanism underlying MYC overexpression in BL but also suggests that targeting the JNK pathway may provide a unique strategy to suppress BL tumorigenesis.

A symmetric geometry of transmembrane domains inside the B cell antigen receptor complex

B lymphocytes have the ability to sense thousands of structurally different antigens and produce cognate antibodies against these molecules. For this they carry on their surface multiple copies of the B cell antigen receptor (BCR) comprising the membrane-bound Ig (mIg) molecule and the Igα/Igβ heterodimer functioning as antigen binding and signal transducing components, respectively. The mIg is a symmetric complex of 2 identical membrane-bound heavy chains (mHC) and 2 identical light chains. How the symmetric mIg molecule is asymmetrically associated with only one Igα/Igβ heterodimer has been a puzzle. Here we describe that Igα and Igβ both carry on one side of their α-helical transmembrane domain a conserved amino acid motif. By a mutational analysis in combination with a BCR rebuilding approach, we show that this motif is required for the retention of unassembled Igα or Igβ molecules inside the endoplasmic reticulum and the binding of the Igα/Igβ heterodimer to the mIg molecule. We suggest that the BCR forms within the lipid bilayer of the membrane a symmetric Igα-mHC:mHC-Igβ complex that is stabilized by an aromatic proline-tyrosine interaction. Outside the membrane this symmetry is broken by the disulfide-bridged dimerization of the extracellular Ig domains of Igα and Igβ. However, symmetry of the receptor can be regained by a dimerization of 2 BCR complexes as suggested by the dissociation activation model.

A hypomorphic Ephb2 gene variant contributes to autoimmune diabetes in NOD mice associated with altered Mapk3 activities in B-lymphocytes

Abstract β-cell destruction in type 1 diabetes (T1D) is considered a T-cell mediated autoimmune disease, but based on studies in the NOD mouse model, B-lymphocytes also play a critical pathogenic role. They do so by serving as a preferential subset of pathogenic APC that expand diabetogenic T cells. Immunological tolerance induction mechanisms that normally eliminate or inactivate autoreactive B-lymphocytes are defective in NOD mice and also in T1D patients. We previously mapped a faulty NOD B-lymphocyte tolerance phenotype to a 6.22Mb region on Chromosome 4. With a combination of differential gene expression and ingenuity pathway analyses (IPA) we identified a hypomorphic Ephb2 allelic variant within this region on Chromosome 4 as a potential candidate gene interacting with human T1D susceptibility genes in a common pathway modulating Mapk3 activities. Ephrin-B1 ligand engagement of Ephb2 attenuated B-cell receptor (BCR) induced Mapk3 phosphorylation in vitro. We tested the candidacy of Ephb2 by introducing into NOD mice a transgenic higher expression variant of Ephb2 derived from the B6 strain (NOD-Ephb2B6). Elevated expression of Ephb2 afforded significant T1D protection due to changes in hematopoietic cells. Despite having enhanced proliferative capacity, B-lymphocytes from NOD-Ephb2B6 mice had diminished diabetogenic activity associated with attenuated Mek-Erk signaling. Introduction of the Ephb2 transgene into NOD BCR transgenic models expressing autoreactive peripherin (Per) specific Ig molecules also inhibited the accelerated T1D development characterizing these stocks. Thus, signaling via Ephb2 in B-lymphocytes can fine-tune BCR signaling that ultimately determine their diabetogenic activities.

Identification and expression analysis of Fc receptor-like proteins in Japanese flounder (Paralichthys olivaceus)

Fc receptors (FcRs) are specific to the Fc portion of immunoglobulin (Ig) molecules. Here, four Fc receptor-like proteins, JF-FcR-like protein 1-4, were identified in Japanese flounder. Their open reading frames encoded 358, 255, 519 and 441 amino acid residues, respectively. JF-FcR-like protein mRNAs were mainly detected in kidney and spleen of healthy fish. Injection of formalin-killed cells (FKCs) of Edwardsiella tarda significantly increased the spleen mRNA levels of JF-FcR-like protein 1 but not the other JF-FcR-like proteins. Injection of FKC of Streptococcus iniae did not significantly affect any of the JF-FcR-like protein mRNAs. These findings suggest that the FcR-like proteins have different involvements in pathogen responses.

Nonsecretory and Light Chain Escape in Patients With Multiple Myeloma

BackgroundMultiple myeloma (MM) is characterized by the secretion of monoclonal protein by malignant plasma cells in the vast majority of cases. We identified and analyzed patterns of disease relapse and progression associated with disappearance of the paraprotein (“nonsecretory [NS] escape”), or conversion from production of intact Ig molecule to its associated light chain (“LC escape”). Patients and MethodsWe retrospectively reviewed medical records and a database of 791 consecutive patients with symptomatic MM. ResultsTwenty-eight (3.5%) patients had disease evolution associated with either NS (n = 13) or LC (n = 15) escape. The event occurred at a median of 37 months (range, 3-156 months) after the diagnosis of MM, and after a median of 3 chemotherapy regimens (range, 1-8 regimens). Presence of extramedullary disease at progression was detected in 8 (29%) patients. Sensitivity to chemotherapy before and after escape was present in 21 (75%) and 14 (50%) patients, respectively. After a median follow-up of 55 months, 19 (68%) patients died, and progressive MM was the cause of death in 18 patients. The median overall survival after escape was 20 months (95% confidence interval, 9-25 months), and no significant difference was found between the NS and LC groups (P = .44). The median overall survival after diagnosis of MM was worse in patients with NS/LC escape than in those without escape (52 vs. 94 months; P = .018). ConclusionsOur study describes the largest series of NS and LC escape in MM to date. The development of this phenomenon is associated with more aggressive clinical features, frequent resistance to chemotherapy, and worse clinical outcome.

Lung squamous cell carcinoma cells express non-canonically glycosylated IgG that activates integrin-FAK signaling

It is increasingly recognized that many human carcinomas express immunoglobulin (Ig) molecules that are distinct from B-cell-derived Ig and play important roles in cancer initiation, progression, and metastasis. However, the molecular mechanisms underlying the functions of cancer-derived Ig remain elusive. Here, we report that lung squamous cell carcinoma (LSCC) cells frequently express high levels of cancer IgG (CIgG) that is specifically recognized by a monoclonal antibody RP215. RP215 recognizes CIgG via a novel epitope that involves an N-glycan modification at a non-consensus site within the CH1 domain. We demonstrate that RP215 recognized CIgG (RP215-CIgG) promotes survival, migration and in vivo growth of LSCC cells, and these oncogenic activities are strongly inhibited by RP215. Mechanistically, RP215-CIgG executes its oncogenic function through interacting with the integrin α6β4 complex and activating the FAK and Src pathways. Notably, the CIgG-integrin-FAK signaling depends on the N-glycan epitope, which is inhibited by RP215. Together, our studies identified a novel CIgG molecule that activates the oncogenic integrin-FAK signaling in LSCC cells. In addition, the activity of CIgG is inhibited by RP215, providing an attractive target for antibody-based therapy of LSCC.

Heterogeneity of Memory Marginal Zone B Cells.

The marginal zone (MZ) is largely composed of a unique subpopulation of B cells, the so-called MZ-B cells. At a molecular level, memory B cells are characterized by the presence of somatically mutated IGV genes. The earliest studies in the rat have documented the presence of hapten-specific MZ-B cells after immunization in the MZ. This work later received experimental support demonstrating that the IGHV-Cµ transcripts expressed by phenotypically defined splenic MZ-B cells (defined as CD90negIgMhighIgDlow B cells) can carry somatic hypermutation. However, only a minor fraction (< 10%-20%) of these MZ-B cells is mutated and is considered to represent memory B cells. Memory B cells can either be class-switched (IgG, IgA, IgE), or non-class-switched (IgM) B cells. B cells in the MZ are a heterogeneous population of cells and both naïve MZ-B cells; class switched and unswitched memory MZ-B cells are present at this unique site in the spleen. Naïve MZ-B cells carry unmutated Ig genes, produce low-affinity IgM molecules and constitute a first line of defense against invading pathogens. Memory MZ-B cells express high-affinity Ig molecules, directed to (microbial) antigens that have been encountered. In this review, we report on the memory compartment of splenic MZ-B cells in the rat to provide insights into the origin and function of these memory MZ-B cells.