CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning.

RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RCs from upstream VHs in a chromatin loop anchored by CTCF-binding elements (CBEs). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and thereby promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function.

Butyrophilin-like 3 Directly Binds a Human Vγ4+ T Cell Receptor Using a Modality Distinct from Clonally-Restricted Antigen.

Butyrophilin (BTN) and butyrophilin-like (BTNL/Btnl) heteromers are major regulators of human and mouse γδ T cell subsets, but considerable contention surrounds whether they represent direct γδ T cell receptor (TCR) ligands. We demonstrate that the BTNL3 IgV domain binds directly and specifically to a human Vγ4+ TCR, "LES" with an affinity (∼15-25 µM) comparable to many αß TCR-peptide major histocompatibility complex interactions. Mutations in germline-encoded Vγ4 CDR2 and HV4 loops, but not in somatically recombined CDR3 loops, drastically diminished binding and T cell responsiveness to BTNL3-BTNL8-expressing cells. Conversely, CDR3γ and CDR3δ loops mediated LES TCR binding to endothelial protein C receptor, a clonally restricted autoantigen, with minimal CDR1, CDR2, or HV4 contributions. Thus, the γδ TCR can employ two discrete binding modalities: a non-clonotypic, superantigen-like interaction mediating subset-specific regulation by BTNL/BTN molecules and CDR3-dependent, antibody-like interactions mediating adaptive γδ T cell biology. How these findings might broadly apply to γδ T cell regulation is also examined.

Diversity, cellular origin and autoreactivity of antibody-secreting cell population expansions in acute systemic lupus erythematosus.

Acute systemic lupus erythematosus (SLE) courses with surges of antibody-secreting cells (ASCs) whose origin, diversity and contribution to serum autoantibodies remain unknown. Here, deep sequencing, proteomic profiling of autoantibodies and single-cell analysis demonstrated highly diversified ASCs punctuated by clones expressing the variable heavy-chain region VH4-34 that produced dominant serum autoantibodies. A fraction of ASC clones contained autoantibodies without mutation, a finding consistent with differentiation outside the germinal centers. A substantial ASC segment was derived from a distinct subset of newly activated naive cells of considerable clonality that persisted in the circulation for several months. Thus, selection of SLE autoreactivities occurred during polyclonal activation, with prolonged recruitment of recently activated naive B cells. Our findings shed light on the pathogenesis of SLE, help explain the benefit of agents that target B cells and should facilitate the design of future therapies.

Sequential Enhancer Sequestration Dysregulates Recombination Center Formation at the IgH Locus.

Immunoglobulin heavy-chain (IgH) genes are assembled by DNA rearrangements that juxtapose a variable (VH), a diversity (DH), and a joining (JH) gene segment. Here, we report that in the absence of intergenic control region 1 (IGCR1), the intronic enhancer (Eµ) associates with the next available CTCF binding site located close to VH81X via putative heterotypic interactions involving YY1 and CTCF. The alternate Eµ/VH81X loop leads to formation of a distorted recombination center and altered DH rearrangements and disrupts chromosome conformation that favors distal VH recombination. Cumulatively, these features drive highly skewed, Eµ-dependent recombination of VH81X. Sequential deletion of CTCF binding regions on IGCR1-deleted alleles suggests that they influence recombination of single proximal VH gene segments. Our observations demonstrate that Eµ interacts differently with IGCR1- or VH-associated CTCF binding sites and thereby identify distinct roles for insulator-like elements in directing enhancer activity.

Contribution of the IGCR1 regulatory element and the 3'Igh CTCF-binding elements to regulation of Igh V(D)J recombination.

Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from VH, D, and JH gene segments located in separate clusters across the Igh locus. RAG endonuclease initiates V(D)J recombination from a JH-based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to JHs to form a DJH-RC. Igh has a provocative number and organization of CTCF-binding elements (CBEs) that can impede loop extrusion. Thus, Igh has two divergently oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the VH and D/JH domains, over 100 CBEs across the VH domain convergent to CBE1, and 10 clustered 3'Igh-CBEs convergent to CBE2 and VH CBEs. IGCR1 CBEs segregate D/JH and VH domains by impeding loop extrusion-mediated RAG-scanning. Downregulation of WAPL, a cohesin unloader, in progenitor-B cells neutralizes CBEs, allowing DJH-RC-bound RAG to scan the VH domain and perform VH-to-DJH rearrangements. To elucidate potential roles of IGCR1-based CBEs and 3'Igh-CBEs in regulating RAG-scanning and elucidate the mechanism of the ordered transition from D-to-JH to VH-to-DJH recombination, we tested effects of inverting and/or deleting IGCR1 or 3'Igh-CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments RAG-scanning impediment activity and suggest that 3'Igh-CBEs reinforce ability of the RC to function as a dynamic loop extrusion impediment to promote optimal RAG scanning activity. Finally, our findings indicate that ordered V(D)J recombination can be explained by a gradual WAPL downregulation mechanism in progenitor-B cells as opposed to a strict developmental switch.

Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages.

Most immunoglobulin (Ig) domains bear only a single highly conserved canonical intradomain, inter-ß-sheet disulfide linkage formed between Cys23-Cys104, and incorporation of rare noncanonical disulfide linkages at other locations can enhance Ig domain stability. Here, we exhaustively surveyed the sequence tolerance of Ig variable (V) domain framework regions (FRs) to noncanonical disulfide linkages. Starting from a destabilized VH domain lacking a Cys23-Cys104 disulfide linkage, we generated and screened phage-displayed libraries of engineered VHs, bearing all possible pairwise combinations of Cys residues in neighboring ß-strands of the Ig fold FRs. This approach identified seven novel Cys pairs in VH FRs (Cys4-Cys25, Cys4-Cys118, Cys5-Cys120, Cys6-Cys119, Cys22-Cys88, Cys24-Cys86, and Cys45-Cys100; the international ImMunoGeneTics information system numbering), whose presence rescued domain folding and stability. Introduction of a subset of these noncanonical disulfide linkages (three intra-ß-sheet: Cys4-Cys25, Cys22-Cys88, and Cys24-Cys86, and one inter-ß-sheet: Cys6-Cys119) into a diverse panel of VH, VL, and VHH domains enhanced their thermostability and protease resistance without significantly impacting expression, solubility, or binding to cognate antigens. None of the noncanonical disulfide linkages identified were present in the natural human VH repertoire. These data reveal an unexpected permissiveness of Ig V domains to noncanonical disulfide linkages at diverse locations in FRs, absent in the human repertoire, whose presence is compatible with antigen recognition and improves domain stability. Our work represents the most complete assessment to date of the role of engineered noncanonical disulfide bonding within FRs in Ig V domain structure and function.

Role of Dot1L and H3K79 methylation in regulating somatic hypermutation of immunoglobulin genes.

Somatic hypermutation (SHM) and class-switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation-induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome, and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt's lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than on the constant (C) region, which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sµ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of polymerase II (Pol II) and its S2 phosphorylated form at the IgH locus. Knockout of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of the P-TEFb complex) on the V region, and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreases SHM and the abundance of Pol II S2P at the IgH locus. Since all these factors play a role in transcription elongation, our studies reinforce the idea that the chromatin context and dynamics of transcription are critical for SHM.

The molecular characterization of antibody binding to a superantigen-like protein from a commensal microbe.

Microorganisms have coevolved diverse mechanisms to impair host defenses. A major one, superantigens, can result in devastating effects on the immune system. While all known superantigens induce vast immune cell proliferation and come from opportunistic pathogens, recently, proteins with similar broad specificity to antibody variable (V) domain families were identified in a commensal microbiota. These proteins, identified in the human commensal Ruminococcus gnavus, are called immunoglobulin-binding protein (Ibp) A and B and have been shown to activate B cells in vitro expressing either human VH3 or murine VH5/6/7. Here, we provide molecular and functional studies revealing the basis of this Ibp/immunoglobulin (Ig) interaction. The crystal structure and biochemical assays of a truncated IbpA construct in complex with mouse VH5 antigen-binding fragment (Fab) shows a binding of Ig heavy chain framework residues to the Ibp Domain D and the C-terminal heavy chain binding domain (HCBD). We used targeted mutagenesis of contact residues and affinity measurements and performed studies of the Fab-IbpA complex to determine the stoichiometry between Ibp and VH domains, suggesting Ibp may serve to cluster full-length IgA antibodies in vivo. Furthermore, in vitro stimulation experiments indicate that binding of the Ibp HCBD alone is sufficient to activate responsive murine B cell receptors. The presence of these proteins in a commensal microbe suggest that binding a broad repertoire of immunoglobulins, particularly in the gut/microbiome environment, may provide an important function in the maintenance of host/microbiome homeostasis contrasting with the pathogenic role of structurally homologous superantigens expressed by pathogens.

Elevated N-Linked Glycosylation of IgG V Regions in Myasthenia Gravis Disease Subtypes.

Elevated N-linked glycosylation of IgG V regions (IgG-VN-Glyc) is an emerging molecular phenotype associated with autoimmune disorders. To test the broader specificity of elevated IgG-VN-Glyc, we studied patients with distinct subtypes of myasthenia gravis (MG), a B cell-mediated autoimmune disease. Our experimental design focused on examining the B cell repertoire and total IgG. It specifically included adaptive immune receptor repertoire sequencing to quantify and characterize N-linked glycosylation sites in the circulating BCR repertoire, proteomics to examine glycosylation patterns of the total circulating IgG, and an exploration of human-derived recombinant autoantibodies, which were studied with mass spectrometry and Ag binding assays to respectively confirm occupation of glycosylation sites and determine whether they alter binding. We found that the frequency of IgG-VN-Glyc motifs was increased in the total BCR repertoire of patients with MG when compared with healthy donors. The elevated frequency was attributed to both biased V gene segment usage and somatic hypermutation. IgG-VN-Glyc could be observed in the total circulating IgG in a subset of patients with MG. Autoantigen binding, by four patient-derived MG autoantigen-specific mAbs with experimentally confirmed presence of IgG-VN-Glyc, was not altered by the glycosylation. Our findings extend prior work on patterns of Ig V region N-linked glycosylation in autoimmunity to MG subtypes.

T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice.

Evaluation of the therapeutic potential of RNAi for HIV infection has been hampered by the challenges of siRNA delivery and lack of suitable animal models. Using a delivery method for T cells, we show that siRNA treatment can dramatically suppress HIV infection. A CD7-specific single-chain antibody was conjugated to oligo-9-arginine peptide (scFvCD7-9R) for T cell-specific siRNA delivery in NOD/SCIDIL2rgamma-/- mice reconstituted with human lymphocytes (Hu-PBL) or CD34+ hematopoietic stem cells (Hu-HSC). In HIV-infected Hu-PBL mice, treatment with anti-CCR5 (viral coreceptor) and antiviral siRNAs complexed to scFvCD7-9R controlled viral replication and prevented the disease-associated CD4 T cell loss. This treatment also suppressed endogenous virus and restored CD4 T cell counts in mice reconstituted with HIV+ peripheral blood mononuclear cells. Moreover, scFvCD7-9R could deliver antiviral siRNAs to naive T cells in Hu-HSC mice and effectively suppress viremia in infected mice. Thus, siRNA therapy for HIV infection appears to be feasible in a preclinical animal model.

IGHV sequencing reveals acquired N-glycosylation sites as a clonal and stable event during follicular lymphoma evolution.

Follicular lymphoma B cells undergo continuous somatic hypermutation (SHM) of their immunoglobulin variable region genes, generating a heterogeneous tumor population. SHM introduces DNA sequences encoding N-glycosylation sites asparagine-X-serine/threonine (N-gly sites) within the V-region that are rarely found in normal B-cell counterparts. Unique attached oligomannoses activate B-cell receptor signaling pathways after engagement with calcium-dependent lectins expressed by tissue macrophages. This novel interaction appears critical for tumor growth and survival. To elucidate the significance of N-gly site presence and loss during ongoing SHM, we tracked site behavior during tumor evolution and progression in a diverse group of patients through next-generation sequencing. A hierarchy of subclones was visualized through lineage trees based on SHM semblance between subclones and their discordance from the germline sequence. We observed conservation of N-gly sites in more than 96% of subclone populations within and across diagnostic, progression, and transformation events. Rare N-gly-negative subclones were lost or negligible from successive events, in contrast to N-gly-positive subclones, which could additionally migrate between anatomical sites. Ongoing SHM of the N-gly sites resulted in subclones with different amino acid compositions across disease events, yet the vast majority of resulting DNA sequences still encoded for an N-gly site. The selection and expansion of only N-gly-positive subclones is evidence of the tumor cells' dependence on sites, despite the changing genomic complexity as the disease progresses. N-gly sites were gained in the earliest identified lymphoma cells, indicating they are an early and stable event of pathogenesis. Targeting the inferred mannose-lectin interaction holds therapeutic promise.

Anti-idiotypic single-chain variable fragment antibody partially mimic the functionally spatial structure of Cry2Aa toxin.

The anti-idiotypic antibody is widely used in the field of immunology to simulate structural features or even induce the biological activity of antigens. In this study, we obtained seven anti-idiotypic single-chain variable fragments (scFv) antibodies of Cry2Aa toxin from a phage-displayed mutant library constructed using error-prone PCR technique. A mutant designated 2-12B showed the best binding ability amongst all anti-idiotypic scFv isolates to Plutella xylostella brush border membrane vesicles (BBMVs). 2-12B and Cry2Aa toxin shared a potential receptor of polycalin in P. xylostella BBMVs. Homology modeling and molecular docking demonstrated that 2-12B and Cry2Aa toxin have seven common binding amino acid residues in polycalin. Insect bioassay results suggested that 2-12 had insecticidal efficacy against P. xylostella larvae. These results indicated that the Cry2Aa anti-idiotypic scFv antibody 2-12B partially mimicked the structure and function of Cry2Aa toxin. The anti-idiotypic scFv antibody provides the basic material for the future study of surrogate molecules or new insecticidal materials.

A Germline-Encoded Structural Arginine Trap Underlies the Anti-DNA Reactivity of a Murine V Gene Segment.

Autoimmunity may have its origins of early repertoire selection in developmental B cells. Such a primary repertoire is probably shaped by selecting B cells that can efficiently perform productive signaling, stimulated by self-antigens in the bone marrow, such as DNA. In support of that idea, we previously found a V segment from VH10 family that can form antibodies that bind to DNA independent of CDR3 usage. In this paper we designed four antibody fragments in a novel single-chain pre-BCR (scpre-BCR) format containing germinal V gene segments from families known to bind DNA (VH10) or not (VH4) connected to a murine surrogate light chain (SLC), lacking the highly charged unique region (UR), by a hydrophilic peptide linker. We also tested the influence of CDR2 on DNA reactivity by shuffling the CDR2 loop. The scpre-BCRs were expressed in bacteria. VH10 bearing scpre-BCR could bind DNA, while scpre-BCR carrying the VH4 segment did not. The CDR2 loop shuffling hampered VH10 reactivity while displaying a gain-of-function in the nonbinding VH4 germline. We modeled the binding sites demonstrating the conservation of a positivity charged pocket in the VH10 CDR2 as the possible cross-reactive structural element. We presented evidence of DNA reactivity hardwired in a V gene, suggesting a structural mechanism for innate autoreactivity. Therefore, while autoreactivity to DNA can lead to autoimmunity, efficiently signaling for B cell development is likely a trade-off mechanism leading to the selection of potentially autoreactive repertoires.

Conformational changes in a Vernier zone region: Implications for antibody dual specificity.

Understanding the determinants of antibody specificity is one of the challenging tasks in antibody development. Monospecific antibodies are still dominant in approved antibody therapeutics but there is a significant body of work to show that multispecific antibodies can increase the overall therapeutic effect. Dual-specific or "Two-in-One" antibodies can bind to two different antigens separately with the same antigen-binding site as opposed to bispecifics, which simultaneously bind to two different antigens through separate antigen-binding units. These nonstandard dual-specific antibodies were recently shown to be promising for new antibody-based therapeutics. Here, we physicochemically and structurally analyzed six different antibodies of which two are monospecific and four are dual-specific antibodies derived from monospecific templates to gain insight about dual-specificity determinants. These dual-specific antibodies can target both human epidermal growth factor receptor 2 and vascular endothelial growth factor at different binding affinities. We showed that a particular region of clustered Vernier zone residues might play key roles in gaining dual specificity. While there are minimal intramolecular interactions between a certain Vernier zone region, namely LV4 and LCDR1 of monospecific template, there is a significant structural change and consequently close contact formation between LV4-LCDR1 loops of derived dual-specific antibodies. Although Vernier zone residues were previously shown to be important for humanization applications, they are mostly underestimated in the literature. Here, we also aim to resurrect Vernier zone residues for antibody engineering efforts.

Histone H3.3 promotes IgV gene diversification by enhancing formation of AID-accessible single-stranded DNA.

Immunoglobulin diversification is driven by activation-induced deaminase (AID), which converts cytidine to uracil within the Ig variable (IgV) regions. Central to the recruitment of AID to the IgV genes are factors that regulate the generation of single-stranded DNA (ssDNA), the enzymatic substrate of AID Here, we report that chicken DT40 cells lacking variant histone H3.3 exhibit reduced IgV sequence diversification. We show that this results from impairment of the ability of AID to access the IgV genes due to reduced formation of ssDNA during IgV transcription. Loss of H3.3 also diminishes IgV R-loop formation. However, reducing IgV R-loops by RNase HI overexpression in wild-type cells does not affect IgV diversification, showing that these structures are not necessary intermediates for AID access. Importantly, the reduction in the formation of AID-accessible ssDNA in cells lacking H3.3 is independent of any effect on the level of transcription or the kinetics of RNAPII elongation, suggesting the presence of H3.3 in the nucleosomes of the IgV genes increases the chances of the IgV DNA becoming single-stranded, thereby creating an effective AID substrate.

Nanobody­horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays.

BACKGROUND: Antibodies are an important reagent to determine the specificity and accuracy of diagnostic immunoassays for various diseases. However, traditional antibodies have several shortcomings due to their limited abundance, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies, which are derived from single-chain camelid antibodies, can circumvent many of these limitations and, thus, appear to be a promising substitute. In the presented study, a sandwich ELISA-like immunoassay and direct fluorescent assay with high sensitivity, good specificity, and easy operation were the first time to develop for detecting porcine parvovirus (PPV). After screening PPV viral particles 2 (VP2) specific nanobodies, horseradish peroxidase (HRP) and enhanced green fluorescent protein (EGFP) fusions were derived from the nanobodies by recombinant technology. Finally, using the nanobody-HRP and -EGFP fusions as probes, the developed immunoassays demonstrate specific, sensitive, and rapid detection of PPV. RESULTS: In the study, five PPV-VP2 specific nanobodies screened from an immunised Bactrian camel were successfully expressed with the bacterial system and purified with a Ni-NTA column. Based on the reporter-nanobody platform, HRP and EGFP fusions were separately produced by transfection of HEK293T cells. A sandwich ELISA-like assay for detecting PPV in the samples was firstly developed using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusions as the detection antibody. The assay showed 92.1% agreement with real-time PCR and can be universally used to surveil PPV infection in the pig flock. In addition, a direct fluorescent assay using PPV-VP2-Nb12-EGFP fusion as a probe was developed to detect PPV in ST cells. The assay showed 81.5% agreement with real-time PCR and can be used in laboratory tests. CONCLUSIONS: For the first time, five PPV-VP2 specific nanobody-HRP and -EGFP fusions were produced as reagents for developing immunoassays. A sandwich ELISA-like immunoassay using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusion as the detection antibody was the first time to develop for detecting PPV in different samples. Results showed that the immunoassay can be universally used to surveil PPV infection in pig flock. A direct fluorescent assay using PPV-VP2-Nb12-EGFP as a probe was also developed to detect PPV in ST cells. The two developed immunoassays eliminate the use of commercial secondary antibodies and shorten detection time. Meanwhile, both assays display great developmental prospect for further commercial production and application.

Deacetylase activity of histone deacetylase 3 is required for productive VDJ recombination and B-cell development.

Histone deacetylase 3 (HDAC3) is the catalytic component of NCoR/SMRT corepressor complexes that mediate the actions of transcription factors implicated in the regulation of B-cell development and function. We crossed Hdac3 conditional knockout mice with Mb1-Cre knockin animals to delete Hdac3 in early progenitor B cells. The spleens of Hdac3F/-Mb1-Cre+/- mice were virtually devoid of mature B cells, and B220+CD43+ B-cell progenitors accumulated within the bone marrow. Quantitative deep sequencing of the Ig heavy chain locus from B220+CD43+ populations identified a defect in VHDJH recombination with a severe reduction in productive rearrangements, which directly corresponded to the loss of pre-B cells from Hdac3Δ/- bone marrow. For Hdac3Δ/- B cells that did show productive VDJ rearrangement, there was significant skewing toward the incorporation of proximal VH gene segments and a corresponding reduction in distal VH gene segment use. Although transcriptional effects within these loci were modest, Hdac3Δ/- progenitor cells displayed global changes in chromatin structure that likely hindered effective distal V-DJ recombination. Reintroduction of wild-type Hdac3 restored normal B-cell development, whereas an Hdac3 point mutant lacking deacetylase activity failed to complement this defect. Thus, the deacetylase activity of Hdac3 is required for the generation of mature B cells.

The Neutrophil: The Underdog That Packs a Punch in the Fight against Cancer.

The advent of immunotherapy has had a major impact on the outcome and overall survival in many types of cancer. Current immunotherapeutic strategies typically aim to (re)activate anticancer T cell immunity, although the targeting of macrophage-mediated anticancer innate immunity has also emerged in recent years. Neutrophils, although comprising ≈ 60% of all white blood cells in the circulation, are still largely overlooked in this respect. Nevertheless, neutrophils have evident anticancer activity and can induce phagocytosis, trogocytosis, as well as the direct cytotoxic elimination of cancer cells. Furthermore, therapeutic tumor-targeting monoclonal antibodies trigger anticancer immune responses through all innate Fc-receptor expressing cells, including neutrophils. Indeed, the depletion of neutrophils strongly reduced the efficacy of monoclonal antibody treatment and increased tumor progression in various preclinical studies. In addition, the infusion of neutrophils in murine cancer models reduced tumor progression. However, evidence on the anticancer effects of neutrophils is fragmentary and mostly obtained in in vitro assays or murine models with reports on anticancer neutrophil activity in humans lagging behind. In this review, we aim to give an overview of the available knowledge of anticancer activity by neutrophils. Furthermore, we will describe strategies being explored for the therapeutic activation of anticancer neutrophil activity.

Variable domain N-linked glycosylation and negative surface charge are key features of monoclonal ACPA: Implications for B-cell selection.

Autoreactive B cells have a central role in the pathogenesis of rheumatoid arthritis (RA), and recent findings have proposed that anti-citrullinated protein autoantibodies (ACPA) may be directly pathogenic. Herein, we demonstrate the frequency of variable-region glycosylation in single-cell cloned mAbs. A total of 14 ACPA mAbs were evaluated for predicted N-linked glycosylation motifs in silico, and compared to 452 highly-mutated mAbs from RA patients and controls. Variable region N-linked motifs (N-X-S/T) were strikingly prevalent within ACPA (100%) compared to somatically hypermutated (SHM) RA bone marrow plasma cells (21%), and synovial plasma cells from seropositive (39%) and seronegative RA (7%). When normalized for SHM, ACPA still had significantly higher frequency of N-linked motifs compared to all studied mAbs including highly mutated HIV broadly-neutralizing and malaria-associated mAbs. The Fab glycans of ACPA-mAbs were highly sialylated, contributed to altered charge, but did not influence antigen binding. The analysis revealed evidence of unusual B-cell selection pressure and SHM-mediated decrease in surface charge and isoelectric point in ACPA. It is still unknown how these distinct features of anti-citrulline immunity may have an impact on pathogenesis. However, it is evident that they offer selective advantages for ACPA+ B cells, possibly through non-antigen driven mechanisms.

Therapeutic blockade of the interleukin-6 receptor (IL-6R) allows sIL-6R generation by proteolytic cleavage.

Blockade of the interleukin-6 receptor (IL-6R) is a successful therapeutic strategy in various inflammatory diseases. IL-6 can signal via membrane-bound (classic signaling) and soluble forms (sIL-6R, trans-signaling) of the IL-6R. Trans-signaling is causative for the pro-inflammatory properties of IL-6, and the selective inhibition of this pathway holds the promise to cause less side effects than the global blockade of IL-6 signaling. We have recently shown that the majority of sIL-6R in humans is generated by proteolytic cleavage of the membrane-bound IL-6R, but whether this process is influenced by therapeutic blockade of the IL-6R is unknown. In this study, we show that the monoclonal antibody tocilizumab and a single chain antibody directed against the IL-6R efficiently block IL-6 signaling, but do not prevent the proteolytic generation of sIL-6R.