Inhibition of PI3K p110δ activity reduces IgE production in IL-4 and anti-CD40 stimulated human B cell cultures.

Phosphoinositide 3-kinase (PI3K) p110δ signalling negatively regulates the production of mouse IgE. However, there are disparities between the mouse and human IgE biology, and the role of PI3K p110δ in the production of human IgE is yet to be determined. To investigate the effect of PI3K p110δ inhibition in the production of human IgE we isolated human B cells from tonsil tissue and stimulated them with IL-4 and anti-CD40 antibody to induce class switching to IgE and IgG1 in the presence or absence of IC87114, a small molecule inhibitor of PI3K p110δ. Using FACS, RT-PCR and ELISA we examined the effect of PI3K p110δ inhibition on IgE production and determined the mechanisms involved. Unlike in mice, we observed that PI3K p110δ inhibition significantly reduces the number of IgE+ switched cells and the amounts of secreted IgE in IL4 and anti-CD40 cultures. However, the number of IgG1+ cells and secreted IgG1 were largely unaffected by PI3K p110δ inhibition. The expression levels of AID, ε and γ1 germinal transcripts or other factors involved in the regulation of CSR to IgE and IgG1 were also unaffected by IC87114. However, we found that IC87114 significantly decreases the proliferation of tonsil B cells stimulated with IL-4 and anti-CD40, specifically reducing the frequency of cells that had undergone 4 divisions or more. In addition, PI3K p110δ inhibition reduced the levels of IRF4 expression in IgE+ germinal centre-like B cells leading to a block in plasma cell differentiation. In conclusion, PI3K p110δ signalling is required for the production of human IgE, which makes it a pharmacological target for the treatment of allergic disease.

Altered chromatin landscape in circulating T follicular helper and regulatory cells following grass pollen subcutaneous and sublingual immunotherapy.

BACKGROUND: Allergen-specific immunotherapy is a disease-modifying treatment that induces long-term T-cell tolerance. OBJECTIVE: We sought to evaluate the role of circulating CXCR5+PD-1+ T follicular helper (cTFH) and T follicular regulatory (TFR) cells following grass pollen subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) and the accompanying changes in their chromatin landscape. METHODS: Phenotype and function of cTFH cells were initially evaluated in the grass pollen-allergic (GPA) group (n = 28) and nonatopic healthy controls (NAC, n = 13) by mathematical algorithms developed to manage high-dimensional data and cell culture, respectively. cTFH and TFR cells were further enumerated in NAC (n = 12), GPA (n = 14), SCIT- (n = 10), and SLIT- (n = 8) treated groups. Chromatin accessibility in cTFH and TFR cells was assessed by assay for transposase-accessible chromatin sequencing (ATAC-seq) to investigate epigenetic mechanisms underlying the differences between NAC, GPA, SCIT, and SLIT groups. RESULTS: cTFH cells were shown to be distinct from TH2- and TH2A-cell subsets, capable of secreting IL-4 and IL-21. Both cytokines synergistically promoted B-cell class switching to IgE and plasma cell differentiation. Grass pollen allergen induced cTFH-cell proliferation in the GPA group but not in the NAC group (P < .05). cTFH cells were higher in the GPA group compared with the NAC group and were lower in the SCIT and SLIT groups (P < .01). Time-dependent induction of IL-4, IL-21, and IL-6 was observed in nasal mucosa following intranasal allergen challenge in the GPA group but not in SCIT and SLIT groups. TFR and IL-10+ cTFH cells were induced in SCIT and SLIT groups (all, P < .01). ATAC-seq analyses revealed differentially accessible chromatin regions in all groups. CONCLUSIONS: For the first time, we showed dysregulation of cTFH cells in the GPA group compared to NAC, SCIT, and SLIT groups and induction of TFR and IL-10+ cTFH cells following SCIT and SLIT. Changes in the chromatin landscape were observed following allergen-specific immunotherapy in cTFH and TFR cells.

Serial magnetic resonance imaging findings during severe attacks of familial hemiplegic migraine type 2: a case report.

BACKGROUND: Hemiplegic migraines represent a heterogeneous disorder with various presentations. Hemiplegic migraines are classified as sporadic or familial based on the presence of family history, but both subtypes have an underlying genetic etiology. Mutations in the ATP1A2 gene are responsible for Familial Hemiplegic type 2 (FHM2) or the sporadic hemiplegic migraine (SHM) counterpart if there is no family history of the disorder. Manifestations include migraine with aura and hemiparesis along with a variety of other symptoms likely dependent upon the specific mutation(s) present. CASE PRESENTATION: We report the case of an adult man who presented with headache, aphasia, and right-sided weakness. Workup for stroke and various infectious agents was unremarkable during the patient's extended hospital stay. We emphasize the changes in the Magnetic Resonance Imaging (MRI) over time and the delay from onset of symptoms to MRI changes in Isotropic Diffusion Map (commonly referred to as Diffusion Weighted Imaging (DWI)) as well as Apparent Diffusion Coefficient (ADC). CONCLUSIONS: We provide a brief review of imaging findings correlated with signs/symptoms and specific mutations in the ATP1A2 gene reported in the literature. Description of the various mutations and consequential presentations may assist neurologists in identifying cases of Hemiplegic Migraine, which may include transient changes in ADC and DWI imaging throughout the course of an attack.

Activation-Associated Accelerated Apoptosis of Memory B Cells in Critically Ill Patients With Sepsis.

OBJECTIVE: Sepsis is life-threatening organ dysfunction due to dysregulated host responses to infection. Current knowledge of human B-cell alterations in sepsis is sparse. We tested the hypothesis that B-cell loss in sepsis involves distinct subpopulations of B cells and investigated mechanisms of B-cell depletion. DESIGN: Prospective cohort study. SETTING: Critical care units. PATIENTS: Adult sepsis patients without any documented immune comorbidity. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: B-cell subsets were quantified by flow cytometry; annexin-V status identified apoptotic cells and phosphorylation of intracellular kinases identified activation status of B-cell subsets. B cell-specific survival ligand concentrations were measured. Gene expression in purified B cells was measured by microarray. Differences in messenger RNA abundance between sepsis and healthy controls were compared. Lymphopenia present in 74.2% of patients on admission day was associated with lower absolute B-cell counts (median [interquartile range], 0.133 [0.093-0.277] 10 cells/L) and selective depletion of memory B cells despite normal B cell survival ligand concentrations. Greater apoptotic depletion of class-switched and IgM memory cells was associated with phosphorylation of extracellular signal-regulated kinases, implying externally driven lymphocyte stress and activation-associated cell death. This inference is supported by gene expression profiles highlighting mitochondrial dysfunction and cell death pathways, with enriched intrinsic and extrinsic pathway apoptosis genes. CONCLUSIONS: Depletion of the memory B-cell compartment contributes to the immunosuppression induced by sepsis. Therapies targeted at reversing this immune memory depletion warrant further investigation.

Soluble CD23 controls IgE synthesis and homeostasis in human B cells.

CD23, the low-affinity receptor for IgE, exists in membrane and soluble forms. Soluble CD23 (sCD23) fragments are released from membrane (m)CD23 by the endogenous metalloprotease a disintegrin and metalloprotease 10. When purified tonsil B cells are incubated with IL-4 and anti-CD40 to induce class switching to IgE in vitro, mCD23 is upregulated, and sCD23 accumulates in the medium prior to IgE synthesis. We have uncoupled the effects of mCD23 cleavage and accumulation of sCD23 on IgE synthesis in this system. We show that small interfering RNA inhibition of CD23 synthesis or inhibition of mCD23 cleavage by an a disintegrin and metalloprotease 10 inhibitor, GI254023X, suppresses IL-4 and anti-CD40-stimulated IgE synthesis. Addition of a recombinant trimeric sCD23 enhances IgE synthesis in this system. This occurs even when endogenous mCD23 is protected from cleavage by GI254023X, indicating that IgE synthesis is positively controlled by sCD23. We show that recombinant trimeric sCD23 binds to cells coexpressing mIgE and mCD21 and caps these proteins on the B cell membrane. Upregulation of IgE by sCD23 occurs after class-switch recombination, and its effects are isotype-specific. These results suggest that mIgE and mCD21 cooperate in the sCD23-mediated positive regulation of IgE synthesis on cells committed to IgE synthesis. Feedback regulation may occur when the concentration of secreted IgE becomes great enough to allow binding to mCD23, thus preventing further release of sCD23. We interpret these results with the aid of a model for the upregulation of IgE by sCD23.

Extracellular vesicles in COVID-19 convalescence can regulate T cell metabolism and function.

Long-term T cell dysregulation has been reported following COVID-19 disease. Prolonged T cell activation is associated with disease severity and may be implicated in producing long-covid symptoms. Here, we assess the role of extracellular vesicles (EV) in regulating T cell function over several weeks post COVID-19 disease. We find that alterations in cellular origin and protein content of EV in COVID-19 convalescence are linked to initial disease severity. We demonstrate that convalescent donor-derived EV can alter the function and metabolic rewiring of CD4 and CD8 T cells. Of note, EV following mild, but not severe disease, show distinctly immune-suppressive properties, reducing T cell effector cytokine production and glucose metabolism. Mechanistically our data indicate the involvement of EV-surface ICAM-1 in facilitating EV-T cell interaction. Our data demonstrate that circulatory EV are phenotypically and functionally altered several weeks following acute infection, suggesting a role for EV as long-term immune modulators.

SARS-CoV-2-Specific Memory B Cell Responses Are Maintained After Recovery from Natural Infection and Postvaccination.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has resulted in major worldwide disruption and loss of life over the last 2 years. Many research studies have shown waning serological SARS-CoV-2-specific IgG antibody titers over time, yet, it is unclear whether these changes are reflected in the potential functional reactivation of SARS-CoV-2 antigen-specific memory B cells (MBC) populations. This is especially true in the contexts of differing COVID-19 disease severity and after vaccination regimens. This study aimed to investigate these by polyclonal in vitro reactivation of MBC populations followed by analysis using SAR-CoV-2 antigen-specific B cell ELISpots and IgG antibody ELISAs. Natural disease-associated differences were investigated in 52 donors who have recovered from COVID-19 with varying disease severity, from asymptomatic to severe COVID-19 disease, accompanied by a longitudinal evaluation in a subset of donors. Overall, these data showed limited disease severity-associated differences between donor groups but did show that COVID-19 serologically positive donors had strong antigen-specific MBC-associated responses. MBC responses were better maintained 6 months after recovery from infection when compared to serological antigen-specific IgG antibody titers. A similar investigation after vaccination using 14 donors showed robust serological antigen-specific antibody responses against spike protein that waned over time. MBC-associated responses against spike protein were also observed but showed less waning over time, indicating maintenance of a protective response 6 months after vaccination. Further research is required to evaluate these putatively functional SARS-CoV-2-specific responses in the context of long-term protection mediated by vaccination against this pathogen.

Analysis of intergenic transcription and histone modification across the human immunoglobulin heavy-chain locus.

Ig class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID) mediated deamination of the switch (S) regions; the resultant mismatch is processed to yield the DNA breaks required for recombination. Whereas many of the pathways involved in the mechanism of recombination have been identified, little is known about how CSR is regulated. AID action is known to require transcription of the Ig heavy-chain genes. However, it is not understood how AID is restricted to the Ig genes. Many aspects of gene expression are known to be regulated by modification of chromatin structure. In turn, chromatin is known to be regulated by several RNA-dependent activities. We have mapped the transcriptional and chromatin landscape of the human Ig heavy-chain locus to investigate the effect these activities have on CSR. We demonstrate that the Ig heavy-chain constant genes and 3'-regulatory regions are in an active chromatin conformation in unstimulated total human B cells: the locus undergoes both genic and intergenic transcription and possesses histone modifications associated with "active" chromatin (acetylated H3 and H4 and lysine 4 trimethylated H3). However, on cytokine stimulation, these modifications spread into the S regions, demonstrating a chromatin remodeling activity associated with switching. Surprisingly, after stimulation, the S regions also accumulate lysine 9 trimethylated H3, a modification previously associated with gene silencing. These data demonstrates that the Ig locus is maintained with a complex pattern of both positive and negative histone marks and suggest that some of these marks may have dual functions.

Quantitative assessment of NFκB transcription factor activity.

The Nuclear Factor Kappa B (NFκB) pathway is an important signalling pathway in the immune system. Single gene defects in the NFκB pathway are described in a number of immunodeficiency diseases. These conditions provide a unique opportunity to investigate the mechanisms of NFκB function and how genetic mutations that disrupt this function lead to human disease. Here we describe a robust method for quantifying small differences in the functional activity of the NFκB pathway. Peripheral blood mononuclear cells from healthy donors were stimulated over several days, with a combination of anti-IgM antibody and multimeric CD40 ligand. Nuclear proteins were thereafter extracted and tested for the ability of activated transcription factors, to bind known NFκB DNA binding motifs. Repeatability experiments showed that the DNA binding Activity can be quantified with an average inter and intra assay coefficient of variation of less than 10% (RelB and p52) and less than 15% (p50 and RelA). In healthy individuals there is a significant increase in the DNA binding activity of NFκB transcription factors in response to stimulation, although the magnitude of this response varies across individuals. The kinetics of the DNA binding activity also differs between the canonical and non-canonical transcription factors. P50 and RelA DNA binding activity responds within hours of stimulation, whilst RelB and p52 response was delayed to more than a day after stimulation. Activation of NFκB signalling in response to B cell specific stimulation, can be precisely measured to distinguish individuals with differences in the functional activity of this pathway. This test may prove to be an important biomarker for investigating the functional impact of genetic variants on NFκB signalling.

Exosome-mediated RNAi of PAK4 prolongs survival of pancreatic cancer mouse model after loco-regional treatment.

With a dismal survival rate, pancreatic cancer (PC) remains one of the most aggressive and devastating malignancies, predominantly due to the absence of a valid biomarker for diagnosis and limited therapeutic options for advanced diseases. Exosomes (Exo) as cell-derived vesicles, are widely used as natural nanocarriers for drug delivery. P21-activated kinase 4 (PAK4) is oncogenic when overexpressed, promoting cell survival, migration and anchorage-independent growth. Herein we validated PAK4 as a therapeutic target in an in vivo PC tumour mouse model using Exo-mediated RNAi following intra-tumoural administration. PC derived Exo were firstly isolated by ultracentrifugation on sucrose cushion and characterised for their surface marker expression, size, number, purity and morphology. SiRNA was encapsulated into Exo via electroporation and dual uptake of Exo and siRNA was investigated by flow cytometry and confocal microscopy. In vitro siPAK4 silencing in PC cells following uptake was assessed by flow cytometry, western blotting, and in vitro scratch assay. In vivo efficacy (tumour growth delay and mouse survival) of siPAK4 was evaluated in PC bearing NSG mouse model. Ex vivo tumours were examined using Haematoxylin and eosin (H&E) staining and immunohistochemistry. Results showed high quality PC-derived PANC-1 Exo were obtained. SiRNA was incorporated in Exo with 16.5% encapsulation efficiency. In vitro imaging confirmed Exo and siRNA co-localisation in cells. PAK4 knockdown was successful with 30 nM Exo-siPAK4 at 24 h post incubation in vitro. Intra-tumoural administration of Exo-siPAK4 (0.03 mg/kg siPAK4 and 6.1 × 1011 Exo, each dose, two doses) reduced PC tumour growth in vivo and enhanced mice survival (p < 0.001), with minimal toxicity observed compared to polyethylenimine (PEI) used as a commercial transfection reagent. H&E staining of tumours showed significant tissue apoptosis in siPAK4 treated groups. PAK4 knockdown prolongs survival of PC-bearing mice suggesting its potential as a new therapeutic target for PC. PANC-1 Exo demonstrated comparable efficacy but safer profile than PEI as in vivo RNAi transfection reagent.

Human marginal zone B cell development from early T2 progenitors.

B cells emerge from the bone marrow as transitional (TS) B cells that differentiate through T1, T2, and T3 stages to become naive B cells. We have identified a bifurcation of human B cell maturation from the T1 stage forming IgMhi and IgMlo developmental trajectories. IgMhi T2 cells have higher expression of α4ß7 integrin and lower expression of IL-4 receptor (IL4R) compared with the IgMlo branch and are selectively recruited into gut-associated lymphoid tissue. IgMhi T2 cells also share transcriptomic features with marginal zone B cells (MZBs). Lineage progression from T1 cells to MZBs via an IgMhi trajectory is identified by pseudotime analysis of scRNA-sequencing data. Reduced frequency of IgMhi gut-homing T2 cells is observed in severe SLE and is associated with reduction of MZBs and their putative IgMhi precursors. The collapse of the gut-associated MZB maturational axis in severe SLE affirms its existence in health.

Transcriptional Analysis of the Human IgE-Expressing Plasma Cell Differentiation Pathway.

IgE is secreted by plasma cells (PCs) and is central to allergic disease. Using an ex vivo tonsil B cell culture system, which mimics the Th2 responses in vivo, we have recently characterized the development pathway of human IgE-expressing PCs. In this system, as in mice, we reported the predisposition of IgE-expressing B cells to differentiate into PCs. To gain a comprehensive understanding of the molecular events involved in the differentiation of human IgE+ B cells into PCs we have used the Illumina HumanHT-12 v4 Expression BeadChip array to analyse the gene expression profile of ex vivo generated human IgE+ B cells at various stages of their differentiation into PCs. We also compared the transcription profiles of IgE+ and IgG1+ cells to discover isotype-specific patterns. Comparisons of IgE+ and IgG1+ cell transcriptional profiles revealed molecular signatures specific for IgE+ cells, which diverge from their IgG1+ cell counterparts upon differentiation into PCs. At the germinal center (GC) stage of development, unlike in some mouse studies of IgE biology, we observed similar rates of apoptosis and no significant differences in the expression of apoptosis-associated genes between the IgE+ and IgG1+ B cells. We identified a gene interaction network associated with early growth response 1 (EGR1) that, together with the up-regulated IRF4, may account for the predisposition of IgE+ B cells to differentiate into PCs. However, despite their swifter rates of PC differentiation, the transcription profile of IgE+ PCs is more closely related to IgE+ and IgG1+ plasmablasts (PBs) than to IgG1+ PCs, suggesting that the terminal differentiation of IgE+ cells is impeded. We also show that IgE+ PCs have increased levels of apoptosis suggesting that the IgE+ PCs generated in our in vitro tonsil B cell cultures, as in mice, are short-lived. We identified gene regulatory networks as well as cell cycle and apoptosis signatures that may explain the diverging PC differentiation programme of these cells. Overall, our study provides a detailed analysis of the transcriptional pathways underlying the differentiation of human IgE-expressing B cells and points to molecular signatures that regulate IgE+ PC differentiation and function.

miR-29b directly targets activation-induced cytidine deaminase in human B cells and can limit its inappropriate expression in naïve B cells.

Class-switch recombination (CSR) is an essential B cell process that alters the isotype of antibody produced by the B cell, tailoring the immune response to the nature of the invading pathogen. CSR requires the activity of the mutagenic enzyme AID (encoded by AICDA) to generate chromosomal lesions within the immunoglobulin genes that initiate the class switching recombination event. These AID-mediated mutations also participate in somatic-hypermutation of the immunoglobulin variable region, driving affinity maturation. As such, AID poses a significant oncogenic threat if it functions outside of the immunoglobulin locus. We found that expression of the microRNA, miR-29b, was repressed in B cells isolated from tonsil tissue, relative to circulating naïve B cells. Further investigation revealed that miR-29b was able to directly initiate the degradation of AID mRNA. Enforced overexpression of miR-29b in human B cells precipitated a reduction in overall AID protein and a corresponding diminution in CSR to IgE. Given miR-29b's ability to potently target AID, a mutagenic molecule that can initiate chromosomal translocations and "off-target" mutations, we propose that miR-29b acts to silence premature AID expression in naïve B cells, thus reducing the likelihood of inappropriate and potentially dangerous deamination activity.

Spatiotemporal segregation of human marginal zone and memory B cell populations in lymphoid tissue.

Human memory B cells and marginal zone (MZ) B cells share common features such as the expression of CD27 and somatic mutations in their IGHV and BCL6 genes, but the relationship between them is controversial. Here, we show phenotypic progression within lymphoid tissues as MZ B cells emerge from the mature naïve B cell pool via a precursor CD27-CD45RBMEM55+ population distant from memory cells. By imaging mass cytometry, we find that MZ B cells and memory B cells occupy different microanatomical niches in organised gut lymphoid tissues. Both populations disseminate widely between distant lymphoid tissues and blood, and both diversify their IGHV repertoire in gut germinal centres (GC), but nevertheless remain largely clonally separate. MZ B cells are therefore not developmentally contiguous with or analogous to classical memory B cells despite their shared ability to transit through GC, where somatic mutations are acquired.

BET proteins are a key component of immunoglobulin gene expression.

AIM: BET proteins have been shown to regulate gene expression including inflammatory genes. METHODS: In order to investigate the role of the BET proteins in immunoglobulin production we treated the human B-cell line CLNH11.4 and primary human B cells and ozone-exposed mice with BET inhibitors (JQ1 or IBET151). RESULTS: Both proliferation and IgG production were reduced by JQ1 in a concentration-dependent manner. JQ1 significantly reduced immunoglobulin gene transcription. In vivo treatment of ozone-exposed mice with the BET inhibitor IBET151 similarly inhibited ozone-induced immunoglobulin production. JQ1 did not reduce the protein levels of Brd4 or Oct2 per se but reduced the ability of Brd4 and Oct2 to co-immunoprecipitate and of Oct2 to bind to immunoglobulin gene promoters. CONCLUSION: Our results indicate that BET proteins including Brd4 play a crucial role regulation B-cell-specific gene expression and immunoglobulin production.

Global gene regulation during activation of immunoglobulin class switching in human B cells.

Immunoglobulin class switch recombination (CSR) to IgE is a tightly regulated process central to atopic disease. To profile the B-cell transcriptional responses underlying the activation of the germinal centre activities leading to the generation of IgE, naïve human B-cells were stimulated with IL-4 and anti-CD40. Gene expression and alternative splicing were profiled over 12 days using the Affymetrix Human Exon 1.0 ST Array. A total of 1,399 genes, forming 13 temporal profiles were differentially expressed. CCL22 and CCL17 were dramatically induced but followed a temporal trajectory distinct from classical mediators of isotype switching. AICDA, NFIL3, IRF4, XBP1 and BATF3 shared a profile with several genes involved in innate immunity, but with no recognised role in CSR. A transcription factor BHLHE40 was identified at the core of this profile. B-cell activation was also accompanied by variation in exon retention affecting >200 genes including CCL17. The data indicate a circadian component and central roles for the Th2 chemokines CCL22 and CCL17 in the activation of CSR.

IgG subclass switching and clonal expansion in cutaneous melanoma and normal skin.

B cells participate in immune surveillance in human circulation and tissues, including tumors such as melanoma. By contrast, the role of humoral responses in cutaneous immunity is underappreciated. We report circulating skin-homing CD22+CLA+B cells in healthy volunteers and melanoma patients (n = 73) and CD22+ cells in melanoma and normal skin samples (n = 189). Normal and malignant skin featured mature IgG and CD22 mRNA, alongside mRNA for the transiently-expressed enzyme Activation-induced cytidine Deaminase (AID). Gene expression analyses of publically-available data (n = 234 GEO, n = 384 TCGA) confirmed heightened humoral responses (CD20, CD22, AID) in melanoma. Analyses of 51 melanoma-associated and 29 normal skin-derived IgG sequence repertoires revealed lower IgG1/IgGtotal representation compared with antibodies from circulating B cells. Consistent with AID, comparable somatic hypermutation frequencies and class-switching indicated affinity-matured antibodies in normal and malignant skin. A melanoma-associated antibody subset featured shorter complementarity-determining (CDR3) regions relative to those from circulating B cells. Clonal amplification in melanoma-associated antibodies and homology modeling indicated differential potential antigen recognition profiles between normal skin and melanoma sequences, suggesting distinct antibody repertoires. Evidence for IgG-expressing B cells, class switching and antibody maturation in normal and malignant skin and clonally-expanded antibodies in melanoma, support the involvement of mature B cells in cutaneous immunity.

Mechanisms regulating the targeting and activity of activation induced cytidine deaminase.

Activation induced cytidine deaminase (AID) plays a central role in the vertebrate adaptive immune response, initiating immunoglobulin (Ig) somatic hypermutation (SHM) and class-switch recombination (CSR). AID converts deoxycytosine (dC) in the DNA to deoxyuridine (dU), causing a DNA base-pairing mismatch. How this mismatch is recognised and resolved determines whether the site will undergo mutation, recombination or high-fidelity repair. Although AID action is essential for antibody diversification it is also known to act upon many non-Ig genes where it can cause tumourigenic mutations and translocations. Although much is known about the pathways of Ig diversification, there is still very little known about the mechanisms that target AID to its sites of action and regulate the different repair processes that can participate at these sites.

IgG4 subclass antibodies impair antitumor immunity in melanoma.

Host-induced antibodies and their contributions to cancer inflammation are largely unexplored. IgG4 subclass antibodies are present in IL-10-driven Th2 immune responses in some inflammatory conditions. Since Th2-biased inflammation is a hallmark of tumor microenvironments, we investigated the presence and functional implications of IgG4 in malignant melanoma. Consistent with Th2 inflammation, CD22+ B cells and IgG4(+)-infiltrating cells accumulated in tumors, and IL-10, IL-4, and tumor-reactive IgG4 were expressed in situ. When compared with B cells from patient lymph nodes and blood, tumor-associated B cells were polarized to produce IgG4. Secreted B cells increased VEGF and IgG4, and tumor cells enhanced IL-10 secretion in cocultures. Unlike IgG1, an engineered tumor antigen-specific IgG4 was ineffective in triggering effector cell-mediated tumor killing in vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions. IgG4 blockade was mediated through reduction of FcγRI activation. Additionally, IgG4 significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. Furthermore, serum IgG4 was inversely correlated with patient survival. These findings suggest that IgG4 promoted by tumor-induced Th2-biased inflammation may restrict effector cell functions against tumors, providing a previously unexplored aspect of tumor-induced immune escape and a basis for biomarker development and patient-specific therapeutic approaches.

Comprehensive FISH probe design tool applied to imaging human immunoglobulin class switch recombination.

We present a web engine boosted fluorescence in-situ hybridization (webFISH) algorithm using a genome-wide sequence similarity search to design target-specific single-copy and repetitive DNA FISH probes. The webFISH algorithm featuring a user-friendly interface (http://www.webfish2.org/) maximizes the coverage of the examined sequences with FISH probes by considering locally repetitive sequences absent from the remainder of the genome. The highly repetitive human immunoglobulin heavy chain sequence was analyzed using webFISH to design three sets of FISH probes. These allowed direct simultaneous detection of class switch recombination in both immunoglobulin-heavy chain alleles in single cells from a population of cultured primary B cells. It directly demonstrated asynchrony of the class switch recombination in the two alleles in structurally preserved nuclei while permitting parallel readout of protein expression by immunofluorescence staining. This novel technique offers the possibility of gaining unprecedented insight into the molecular mechanisms involved in class switch recombination.