Individual Isotypes Research Articles

Tubb4b is required for multi-ciliogenesis in the mouse.

Cilia are microtubule (MT)-based organelles present on the surface of nearly all vertebrate cells. MTs are polymers of α- and β-tubulins that are each encoded by multiple, individual isotype genes. Tubulin isotype composition is thought to influence MT behaviors. Ciliary MTs differ from other MTs in the cell in terms of organization, stability and post-translational modifications. However, little is known about the tubulin isotypes that build ciliary MTs and the functional requirements for tubulin isotypes in cilia have not been examined in vertebrates. Here, we have tested the role of the β-tubulin isotype genes in the mouse that harbor a conserved amino acid motif associated with ciliated organisms. We found that Tubb4b localizes to cilia in multi-ciliated cells (MCCs) specifically. In respiratory and oviduct MCCs, Tubb4b is asymmetrically localized within multi-cilia, indicating that the tubulin isotype composition changes along the length of the ciliary axonemal MTs. Deletion of Tubb4b resulted in striking structural defects within the axonemes of multi-cilia, without affecting primary cilia. These studies show that Tubb4b is essential for the formation of a specific MT-based subcellular organelle and sheds light on the requirements of tubulin isotypes in cilia.

Effect of posttranslational modifications and subclass on IgG activity: from immunity to immunotherapy.

Humoral immune responses are characterized by complex mixtures of polyclonal antibody species varying in their isotype, target epitope specificity and affinity. Posttranslational modifications occurring during antibody production in both the antibody variable and constant domain create further complexity and can modulate antigen specificity and antibody Fc-dependent effector functions, respectively. Finally, modifications of the antibody backbone after secretion may further impact antibody activity. An in-depth understanding of how these posttranslational modifications impact antibody function, especially in the context of individual antibody isotypes and subclasses, is only starting to emerge. Indeed, only a minute proportion of this natural variability in the humoral immune response is currently reflected in therapeutic antibody preparations. In this Review, we summarize recent insights into how IgG subclass and posttranslational modifications impact IgG activity and discuss how these insights may be used to optimize therapeutic antibody development.

Diagnostic performance of four lateral flow immunoassays for COVID-19 antibodies in Peruvian population.

Serological assays have been used in seroprevalence studies to inform the dynamics of COVID-19. Lateral flow immunoassay (LFIA) tests are a very practical technology to use for this objective; however, one of their challenges may be variable diagnostic performance. Given the numerous available LFIA tests, evaluation of their accuracy is critical before real-world implementation. We performed a retrospective diagnostic evaluation study to independently determine the diagnostic accuracy of 4 different antibody-detection LFIA tests: Now Check (Bionote), CareStart (Access bio), Covid-19 BSS (Biosynex) and OnSite (CTK Biotech). The sample panel was comprised of specimens collected and stored in biobanks; specifically, specimens that were RT-PCR positive for SARS-CoV-2 collected at various times throughout the COVID-19 disease course and those that were collected before the pandemic, during 2018 or earlier, from individuals with upper respiratory symptoms but were negative for tuberculosis. Clinical performance (sensitivity and specificity) was analyzed overall, and subset across individual antibody isotypes, and days from symptoms onset. A very high specificity (98% - 100%) was found for all four tests. Overall sensitivity was variable, ranging from 29% [95% CI: 21%-39%] to 64% [95% CI: 54%-73%]. When considering detection of IgM only, the highest sensitivity was 42% [95% CI: 32%-52%], compared to 57% [95% CI: 47%-66%] for IgG only. When the analysis was restricted to at least 15 days since symptom onset, across any isotype, the sensitivity reached 90% for all four brands. All four LFIA tests proved effective for identifying COVID-19 antibodies when two conditions were met: 1) at least 15 days have elapsed since symptom onset and 2) a sample is considered positive when either IgM or IgG is present. With these considerations, the use of this assays could help in seroprevalence studies or further exploration of its potential uses.

Tubb3 expression levels are sensitive to neuronal activity changes and determine microtubule growth and kinesin-mediated transport

Microtubules are dynamic polymers of α/β-tubulin. They regulate cell structure, cell division, cell migration, and intracellular transport. However, functional contributions of individual tubulin isotypes are incompletely understood. The neuron-specific β-tubulin Tubb3 displays highest expression around early postnatal periods characterized by exuberant synaptogenesis. Although Tubb3 mutations are associated with neuronal disease, including abnormal inhibitory transmission and seizure activity in patients, molecular consequences of altered Tubb3 levels are largely unknown. Likewise, it is unclear whether neuronal activity triggers Tubb3 expression changes in neurons. In this study, we initially asked whether chemical protocols to induce long-term potentiation (cLTP) affect microtubule growth and the expression of individual tubulin isotypes. We found that growing microtubules and Tubb3 expression are sensitive to changes in neuronal activity and asked for consequences of Tubb3 downregulation in neurons. Our data revealed that reduced Tubb3 levels accelerated microtubule growth in axons and dendrites. Remarkably, Tubb3 knockdown induced a specific upregulation of Tubb4 gene expression, without changing other tubulin isotypes. We further found that Tubb3 downregulation reduces tubulin polyglutamylation, increases KIF5C motility and boosts the transport of its synaptic cargo N-Cadherin, which is known to regulate synaptogenesis and long-term potentiation. Due to the large number of tubulin isotypes, we developed and applied a computational model based on a Monte Carlo simulation to understand consequences of tubulin expression changes in silico. Together, our data suggest a feedback mechanism with neuronal activity regulating tubulin expression and consequently microtubule dynamics underlying the delivery of synaptic cargoes.

Antiviral antibody responses to systemic administration of an oncolytic RNA virus: the impact of standard concomitant anticancer chemotherapies

BackgroundOncolytic reovirus therapy for cancer induces a typical antiviral response to this RNA virus, including neutralizing antibodies. Concomitant treatment with cytotoxic chemotherapies has been hypothesized to improve the therapeutic potential...

Isotype-Specific Detection of Serum Immunoglobulins Against Allergens.

Type-I hypersensitivity is commonly characterized by increased levels of antigen-specific immunoglobulin (Ig) E. Therefore, it is important for clinical and research investigators to reliably measure serum levels of IgE in allergic patients and animal models. While current ELISA-based methods are simple and commonly performed for the detection of allergen-specific IgE using serum or plasma, they may produce misleading results. This is in part due to decreased sensitivity for IgE in the presence of other Ig isotypes in the same sample, such as IgG, that are typically more abundant than IgE. When assessment of multiple Ig isotypes is necessary, performing optimized assays for individual isotypes requires high sample volumes. Here, we describe an approach to increase the sensitivity for IgE detection while conserving the sample volume needed. This method not only improves the accuracy of serum IgE measurements but also allows simultaneous analysis of other allergen-specific immunoglobulins.

Rapid Reduction of Anti-Sars-Cov-2 Antibodies in Convalescent Plasma Donors; Results of a Phase 2 Clinical Study

Introduction: Convalescent plasma is a promising therapeutic option for corona virus disease 2019 (COVID-19). A recent study in 34 COVID-19 patients showed a reduction of recovered patients antibodies within 3 months of infection. The aim on this analysis was to evaluate the antibody titers and explore possible correlations with disease characteristics in volunteer donors, who participated in a phase 2 study for the use of convalescent plasma for the treatment of severe COVID-19 infection.Patients and Methods: This in an ongoing phase 2 study (NCT04408209) for the use of convalescent plasma for severe COVID-19. This analysis reports the results of the first part of the study, regarding the presence of anti-SARS-CoV-2 antibodies in volunteer plasma donors and their correlation with disease characteristics. The main Inclusion criteria for plasma donors included: (i) confirmed SARS-CoV-2 infection by PCR of the nasal/pharyngeal swab; (ii) interval of at least 14 days after complete recovery from COVID-19; (iii) presence of anti-SARS-CoV-2 antibodies; (iv) two negative SARS-CoV-2 PCR results (the second at least 7 days prior to plasmapheresis). For the detection of anti-SARS-CoV-2 antibodies we used two commercially developed assays: one ELISA assay (Euroimmun Medizinische Labordiagnostika AG, Lubeck, Germany), which detects antibodies against the recombinant Spike protein of the virus (S1 domain) and a multiplex assay (ProtATonce Ltd, Athens, Greece) based on the Luminex® xMAP™ technology that detects total antibodies (IgG/IgM/IgA) and individual antibody isotypes IgG, IgM and IgA against 3 SARS-CoV-2 antigens (S1, basic nucleocapsid (N) protein and receptor-binding domain (RBD).Results: To-date, 260 (137M/123F) possible plasma donors were tested for the presence of anti-SARS-CoV-2 antibodies. At the time of their COVID-19 diagnosis, 20 (7.7%) were asymptomatic, 157 (60.3%) were symptomatic but did not need hospitalization and 83 (32%) were hospitalized. Median time from the day of their first symptom or PCR+ (for asymptomatic patients) till the day of screening was 62 (range: 14-104) days. Anti-SARS-CoV-2 antibodies were detected in 229 (88%) donors with the Euroimmun assay and in 238 (91.5%) with the multiplex assay (including the 229 who had antibodies with the Euroimmun method). Univariate analysis showed that donors who had asymptomatic COVID-19 had lower antibody titer compared to those who had symptomatic disease but did not need hospitalization or those who hospitalized (Fig. A-D). Donors <50 years had lower antibody titer compared with older patients [for Euroimmun method, median (IQR): 3.94 (5.10) vs. 7.34 (6.16); p<0.0001], while patients who were tested within 60 days from the first day of symptom or PCR+ (for asymptomatic patients) had higher antibody titer [6.09 (6.52) vs. 4.68 (6.12); p=0.024]. The multivariate analysis showed that age ≥50 years (OR 2.88, 95% CI:1.60-5.18; p<0.001) and need for hospitalization (OR 4.11, 95% CI:2.13-7.90; p<0.001) correlated with higher antibody titers, while asymptomatic phase (OR 0.10, 95% CI:0,01-0.82; p<0.001) and testing within ≥60 days post symptoms onset (OR 0.36, 95% CI:0.20-0.66; p=0.001) correlated with lower antibody titers. In the multivariate logistic regression analysis examining associations between individual symptoms and antibody levels, there was strong correlation between anti-SARS-CoV-2 antibodies and anosmia (OR 11.14, 95% CI:3.92-31.67; p<0.001), loss of taste (OR 5.50, 95% CI:2.23-13.56; p<0.001), fever (OR 4.25, 95% CI:1.90-9.51; p<0.001), and headache (OR 2.34, 95% CI:1.09-5.03; p=0.029). To-date, plasmapheresis was performed in 74 patients with anti-SARS-CoV-2 antibodies, within a median time of 12 (8-19) days after screening; the respective median time (range) from the first day of symptoms or PCR+ was 52 (14-84) days. Interestingly, there was a significant reduction in the antibody titers between the day of screening and the day of plasmapheresis [Fig. E].Conclusion: Lower anti-SARS-CoV-2 antibody titers, against all studied epitopes, are found in asymptomatic patients, in patients <50 years and in those who were tested ≥60 days post onset of first symptoms. The rapid reduction of anti-SARS-CoV-2 antibodies in our cohort reveals a time pattern of reduction, although we do not know if neutralizing antibodies share the same trend or if this reduction affects the host immunity against SARS-CoV-2.DisclosuresTerpos:Amgen: Honoraria, Research Funding; BMS: Honoraria; Genesis pharma SA: Honoraria, Other: travel expenses , Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Other: travel expenses , Research Funding; Celgene: Honoraria; Sanofi: Honoraria. Pappa:Genesis pharma SA: Research Funding. Dimopoulos:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Speakers Bureau.

The Interplay Between Non-Esterified Fatty Acids and Bovine Peroxisome Proliferator-Activated Receptors: Results of an In Vivo-In Vitro Hybrid Approach

Abstract Objectives Metabolic stressors and energy deficiency related to parturition induce lipolysis in dairy cows with a subsequent increase in circulating non-esterified fatty acids (NEFA). We hypothesized that circulating NEFA directly activate the Peroxisome Proliferator-Activated Receptors (PPAR), a transcription factor with known nutrigenomics response to fatty acids, in a dose-dependent manner. Methods Whole-blood samples were collected from five cows at different times between −40 and +10 days relative to parturition to isolate serum. NEFA were extracted from said samples through solid-phase extraction. A bioluminescent reported, bound to a PPAR response element, was used to assess PPAR activation in response to the treatments. To estimate individual PPAR isotype activation, PPAR isotype-specific antagonists were used, along with the treatments. Results Isolated NEFA activate PPAR linearly up to 400 μM, with a decrease in their effect when albumin is introduced. A hybrid approach, treating cells with bovine serum from different stages of parturition, revealed that much of the PPAR activation can be explained by the amount of NEFA in the serum, and that its activation follows a quadratic tendency (calculated maximum activation at 1.47 mM NEFA of 4.8-fold vs. DMEM control, R2 = 0.91). Further analysis of the effect of serum NEFA on PPAR revealed that they mostly activate PPARδ and PPARγ, but not PPARα. However, addition of palmitic acid to the media activate PPARδ and PPARα, but not PPARγ, and only when circulating NEFA are low (0.16 mM, prepartum), while additional palmitate did not increase PPAR activation in serum with high NEFA (0.71 mM, postpartum). Finally, the addition of lipoprotein lipase to the serum, in order to mimic release of NEFA from VLDL, increased PPAR activation, recapitulating the role of dietary lipids in the regulation of PPAR. Conclusions Taken together, our results support the activation of PPAR by NEFA, helping to explain prior observations on PPAR activation in peripartum cows. Additionally, we outlined the role of dietary fatty acids in activating PPAR and their interaction with serum NEFA. Our results aid in setting the foundation for nutrigenomics approaches in ruminant nutrition. Funding Sources This study was funded by the Oregon Beef Council.

Superiority of high sensitivity cardiac troponin T vs. I for long-term prognostic value in patients with chest pain; data from the Akershus cardiac Examination (ACE) 3 study

BackgroundCardiac troponins (cTn) are essential in the diagnostic assessment of non-ST-segment-elevation acute coronary syndrome (NSTE-ACS). Elevated concentrations of cTnT and cTnI predict cardiovascular events in non-acute settings, but the individual troponin isotype association with long-term mortality in patients with suspected unstable angina pectoris (UAP) is less clear. MethodsPatients hospitalized with chest pain between June 2009 and December 2010 were included in the Akershus Cardiac Examination 3 Study and followed for median 6.6 (IQR 6.2-7.1) years. The index diagnosis was adjudicated by an independent committee as NSTE-myocardial infarction (NSTEMI), UAP or non-ACS. Blood samples were collected within 24 h of admission and analyzed with high sensitivity assays for cTnT (hs-cTnT, Roche) and cTnI (hs-cTnI, Singulex). ResultsOf 402 patients included, 74 (18%) were classified as NSTEMI, 88 (22%) UAP and 240 (60%) non-ACS. hs-cTnI concentrations were detectable in all patients (median 3 [IQR 1–11] ng/L), while hs-cTnT concentrations were above the level of blank in 205 (51%) (median 3 [IQR 3–16] ng/L). In patients with UAP, both log2-transformed hs-cTnT and hs-cTnI were associated with all-cause mortality in analyses that adjusted for other risk factors: HR 2.40 [95% CI 1.75–3.30], p < 0.001 and HR 1.44 [1.14–1.81], p = 0.002. There were no significant sex-dependent differences in the association between hs-cTnT or hs-cTnI and outcome. Time dependent receiver-operating characteristics area under the curve was 0.85 (95% CI 0.79–0.92) for hs-cTnT and 0.74 (0.64–0.84) for hs-cTnI, p = 0.008 for difference between values. ConclusionsHigher concentrations of hs-cTnT and hs-cTnI were both associated with all-cause mortality in patients with UAP, but the association with outcome was stronger for hs-cTnT than for hs-cTnI.

Anticyclic Citrullinated Peptide Antibodies 3.1 and Anti-CCP-IgA Are Associated with Increasing Age in Individuals Without Rheumatoid Arthritis.

We investigated the association of age and anticyclic citrullinated peptide antibodies (anti-CCP) in subjects without rheumatoid arthritis (RA). Serum was tested for anti-CCP3.1 (IgG/IgA) in 678 first-degree relatives (FDR) of patients with RA and 330 patients with osteoarthritis (OA). Individual isotypes (anti-CCP-IgA and anti-CCP-IgG) were also tested in all FDR. In FDR, increasing age was significantly associated with positivity for anti-CCP3.1 (per year, OR 1.03) and anti-CCP-IgA (per year, OR 1.05) but not anti-CCP-IgG. In FDR and OA subjects, anti-CCP3.1 prevalence was significantly increased after age 50 years. Increasing age in individuals without RA should be considered in the interpretation of anti-CCP3.1 positivity.

PMD8 - Serum Autoantibodies Multi-Testing in Rheumatoid Arthritis in Primary Care can Reduce Avoidable Costs Associated to False Positives. A Simulation Study in the United States of America

Rheumatoid arthritis (RA) diagnosis requires a combination of clinical, laboratory and imaging investigations. The consequences of wrong serology test results are costly: False Positive results (FPs) are initially managed as RA-patients, bringing about extra costs until correct diagnosis is made. The study goals were to: simulate the diagnostic performance of Rheumatoid Factor (RF)-IgA, RF-IgM, and cyclic citrullinated peptides (CCP2), used alone or in multi-testing parallel/sequential combinations; quantify the economic consequences of serology FPs in the United States. 190 established RA patients and 197 controls were used to assess the diagnostic performance of mono-/multi-serology testing. A 12-month Markov model simulated 500,000 RA-suspected individuals tested in Primary Care with mono- or multi-testing. Costs came from the published literature. Uncertainty was addressed with sensitivity analysis. The mono-testing diagnostic performance was: 1=RF-IgA: sensitivity[95%CI]=40.5%[33.5%-47.9%], specificity=92.4%[87.8%-95.7%]; 2=RF-IgM: sensitivity=59.0%[51.6%-66.0%], specificity=89.3%[84.2%-93.3%]; 3=CCP2: sensitivity=59.5%[52.1%-66.5%], specificity=96.5%[92.8%-98.6%]. Defining a “positive result” as “positivity to at least one test” increased sensitivity; “positivity to all the tests” increased specificity: multi-parallel testing: multi.parallel.1) using CCP2+RF-IgM increased specificity to 99.5[97.2-100]; multi.parallel.2) the 3 tests used simultaneously maximized specificity (100[98.1-100]), reducing sensitivity (35.8[29.0-43.1]); multi-sequential testing: testing positive to RF IgA + RF IgM followed by CCP testing led to 90.7[81.7-96.2] sensitivity and to 0[54.1-100.0] specificity. Multi-testing simulation results reduced the number of FPs: mono.1=32,908, mono.2=46,331, mono.3=14,722, multi.1.parallel.1=2,165, multi.parallel.2=0, multi.sequential=0. Multi-testing allowed for important cost savings due to reduced clinical procedures and resource utilization of FPs. In all the scenarios considered, the cost-savings of multi-testing ranged between $9.488.406-$55.032.756-$ compared with CCP mono-testing, and ranged between $56,930,438-$64,521,163 compared with RF-IgM-mono-testing. Simulation results show that multi-serology testing can improve the diagnostic accuracy of serology in RA: the inclusion of CCP2/RF-IgA/RF-IgM individual isotypes minimizes the number of FPs, reducing avoidable costs. Consequently, multi-testing for RA demonstrates superior value from patient and payer perspective.

Immunogenicity induced by biologicals in the treatment of psoriasis and psoriatic arthritis: View of the problem

The present-day views of the immunogenicity of biological agents (BAs) used to in the treatment of psoriasis and psoriatic arthritis are analyzed. The immunogenicity of these medicaments is noted to depend on their molecular structure, individual patient characteristics, and used treatment regimens. As this takes place, the primary structure of the drug and its posttranslation modifications during manufacture are key factors. It is pointed out that a number of antigenic structures may give rise to the body's BA antibodies – murine epitopes, idiotopes, and allotropes, neoantigens forming in the coupling area of hybrid proteins, nonlinear epitopes present in the aggregated preparations. BAs that tend to form large immune complexes with these antibodies are most immunogenic. The antibodies to most BAs, except drugs based on soluble tumor necrosis factor-α receptors (etanercept), are neutralizing, i.e. they affect the efficiency of therapy, particularly when used over a long period of time. The results of trials evaluating the impact of antibodies to BAs on their clinical value are considered. It is believed that immunogenicity is itself of great importance in respect to the occurrence of the escape phenomenon of a response to BA therapy and to its safety. Attention is drawn to immunogenicity diagnostic problems; at the same it is noted that none of the used laboratory diagnostic techniques can reveal individual BA antibody forms and isotypes. It is concluded that there is a need for further investigations to standardize optimal methods for diagnosing neutralizing antibodies, to elaborate criteria for predicting a response to therapy in terms of an immunogenicity factor, and to reveal pathogenetic mechanisms responsible for the production of antibodies to BAs. The design of novel medicaments with minimal immunogenicity will depend on whether these mechanisms are common to all drugs or specific.

Generation of IgA plasma cells during a secondary response in the absence of IgA memory B cells (LYM6P.800)

Abstract IgA antibodies are important for protection against infection at mucosal surfaces. Despite this importance in protective immunity, the differentiation of IgA B cells is not well understood. To address this issue, an antigen-based enrichment method was used to track polyclonal B cells specific for the model antigen R-Phycoerythrin (PE) during the course of a T-dependent immune response. While 10-40% of the PE-specific short lived plasma cells expressed IgA, less than 0.05% of the PE-specific germinal center and memory B cells expressed IgA. Despite the lack of IgA memory B cells, secondary antigen challenge resulted in the generation of large numbers of PE-specific IgA plasma cells that appeared at times prior to the appearance of IgA plasma cells during the primary response. Since IgA memory B cells were infrequently detected, we hypothesize that IgA plasma cells generated during the secondary challenge are the result of a secondary switch from memory B cells expressing other isotypes. We are currently examining the role that individual isotypes of memory B cells play in the generation of IgA plasma cells by looking for IgG switch remnants in the PE-specific IgA plasma cells generated after secondary challenge, and by testing the ability of purified isotypes of memory B cells to produce IgA plasma cells after transfer and challenge of recipient mice.

Modelling of feather pecking behavior in beak-trimmed and non-beak-trimmed crossbred laying hens: Variance component and trait-based approach

Because of a ban on the use of beak trimming in some European countries, feather pecking is becoming a substantial problem in the layer industry, both from animal welfare and economic points of view. The feather condition score (FCS) as a measure of feather damage has been shown to be closely related to feather pecking behavior in laying hens housed in groups. To obtain a better understanding of genetic and other biological mechanisms underlying feather pecking behavior, data on FCS of a population of 2,724 female offspring from crossing 50 male W1 and 907 female WB purebred lines were used. The offspring of 25 sires were beak-trimmed, and the offspring of another 25 sires were non-beak-trimmed. Titers of plasma natural antibody (NAb) isotypes IgM and IgG binding keyhole limpet hemocyanin at 24 wk of age were measured. Feather condition was scored at 53 wk of age. In the first part of the present study, we estimated genetic parameters for FCS with 2 variance components models: a traditional linear animal model and a model combining direct and associative genetic effects. In the second part of the present study, a trait-based analysis for FCS was conducted to investigate whether NAb isotype titers can explain variation in FCS among individuals, by fitting a linear mixed model. Though the estimated associative genetic variance was substantial, associative effects for FCS were not statistically significant in both populations (P = 0.09 in beak-trimmed birds, and P = 0.08 in non-beak-trimmed birds). This suggests an insufficient number of records on FCS. Individual's NAb isotypes titers did not show direct effect for FCS of itself, but individual's IgG titers showed a suggestive effect on the FCS of cage mates (associative effect) in beak-trimmed laying hens, which need further confirmation.

Variant B Cell Receptor Isotype Functions Differ in Hairy Cell Leukemia with Mutated BRAF and IGHV Genes

A functional B-cell receptor (BCR) is critical for survival of normal B-cells, but whether it plays a comparable role in B-cell malignancy is as yet not fully delineated. Typical Hairy Cell Leukemia (HCL) is a rare B-cell tumor, and unique in expressing multiple surface immunoglobulin (sIg) isotypes on individual tumor cells (mult-HCL), to raise questions as to their functional relevance. Typical mult-HCL also displays a mutated BRAF V(600)E lesion. Since wild type BRAF is a primary conduit for transducing normal BCR signals, as revealed by deletion modelling studies, it is as yet not apparent if mutated BRAF alters BCR signal transduction in mult-HCL. To address these questions, we examined BCR signalling in mult-HCL cases uniformly displaying mutated BRAF and IGHV genes. Two apparent functional sets were delineated by IgD co-expression. In sIgD+ve mult-HCL, IgD mediated persistent Ca2+ flux, also evident via >1 sIgH isotype, linked to increased ERK activation and BCR endocytosis. In sIgD−ve mult-HCL however, BCR-mediated signals and downstream effects were restricted to a single sIgH isotype, with sIgM notably dysfunctional and remaining immobilised on the cell surface. These observations reveal discordance between expression and function of individual isotypes in mult-HCL. In dual sIgL expressing cases, only a single sIgL was fully functional. We examined effects of anti-BCR stimuli on mult-HCL survival ex-vivo. Significantly, all functional non-IgD isotypes increased ERK1/2 phosphorylation but triggered apoptosis of tumor cells, in both subsets. IgD stimuli, in marked contrast retained tumor viability. Despite mutant BRAF, BCR signals augment ERK1/2 phosphorylation, but isotype dictates functional downstream outcomes. In mult-HCL, sIgD retains a potential to transduce BCR signals for tumor survival in-vivo. The BCR in mult-HCL emerges as subject to complex regulation, with apparent conflicting signalling by individual isotypes when co-expressed with sIgD. This suggests the possibility that mutant BRAF by-passes BCR constraints in mult-HCL.

B Cell Receptor with Variant Surface Isotypes Transduce Functional Signals by Elevating Phospho-ERK1/2 Levels in Hairy Cell Leukemia with Mutant BRAF

A functional BCR is essential for survival of normal B-cells, in response to cognate antigen or tonic, antigen-independent stimuli. Cross-linking of BCR by antigen triggers key phosphorylation events in the early signalosome. Mammalian wild type BRAF, a member of the RAF cytosolic kinase family (A-RAF, B-RAF, C-RAF) is critical to BCR function, mediating RAS-RAF-MEK-ERK or mitogen-activated protein kinase (MAPK) pathway signal transduction, via phosphorylation of ERK1/2. The MAPK pathway is a central conduit for survival and proliferation. Importantly, use of inducible deletion models have shown that wild type BRAF is the predominant kinase that transduces BCR signals to activate ERK1/2, with C-RAF playing an accessory role and A-RAF displaying a x20-30 fold lower basal activity in ERK1/2 stimulation. Tonic BCR survival signals in normal B-cells are also transduced via phosphorylation of ERK1/2, but do not require antigen stimuli. Whether the BCR plays a comparable role in sustaining survival in malignant B-cells is less well characterized and remains a central question in understanding the origins and progression of mature B-cell neoplasms.Hairy cell leukaemia (HCL) is a rare B cell leukaemia with characteristic hair-like cytoplasmic projections on tumor cells, displaying distinctive activation markers. A striking feature of monoclonal HCL tumors is expression of multiple variant immunoglobulin heavy chain (sIgH) isotypes as components of the BCR on individual tumor cells, defining the major subset of disease (mult-HCL). Most mult-HCL tumors exhibit IGV somatic hypermutation with a low level of on-going mutations and AID expressed, implicating initial contact with antigen via the BCR. The functional relevance of the BCR to transformation and survival however has as yet not been mapped in HCL. Seminal exome sequencing data in typical HCL identified mutant BRAF V(600)E as almost universal in this tumor. Consistent with mutant BRAFV(600)E, levels of phosphorylated ERK (p-ERK) are raised in hairy cells. As there is an essential requirement for wild type BRAF in transducing BCR signals, the question arises how mutant BRAF may affect BCR function in HCL, given requirements for dimerization of wild type BRAF for ERK1/2 activation, and whether ERK1/2 activation can be enhanced by functional BCR signals for downstream effector signals in HCL.To examine this, we evaluated BCR signalling in mult-HCL (n=10), in cases uniformly displaying mutated BRAF and IGHV genes. Two apparent functional sets were delineated by IgD co-expression. In sIgD+ mult-HCL, IgD mediated persistent Ca2+ flux, which was also evident via >1 sIgH isotype, linked to increased ERK1/2 activation and BCR endocytosis (4/4 cases). In sIgD-vemult-HCL however (6/6 cases), BCR-mediated signals activating ERK1/2 for downstream effects were restricted to a single sIgH isotype, with sIgM notably dysfunctional and remaining immobilised on the cell surface. These observations reveal a clear discordance between expression and function of individual isotypes in mult-HCL. In dual sIgL expressing mult-HCL (3/3 cases), only a single sIgL was fully functional.We next examined effects of anti-BCR stimuli on mult-HCL tumor cell survival ex-vivo. Significantly, all functional non-IgD isotypes increased ERK1/2 phosphorylation but triggered apoptosis of tumor cells, in both sets (4/4 cases) to establish a consistent outcome in these replicate cases. IgD stimuli, in marked contrast retained tumor viability with no evidence for pre-apoptotic effects (2 cases; 1 mult-HCL and 1 sIgD only-HCL).Despite mutant BRAF, BCR signals amplify ERK1/2 phosphorylation, but isotype dictates functional downstream outcomes. In mult-HCL lacking IgD, a role for antigen in stimulating the BCR for tumor persistence appears unlikely. It remains feasible that mutant BRAF in these cases retains tonic signals through activated ERK1/2 for survival, but this remains speculative at present. Surface IgD emerges with potential to transduce BCR signals for tumor survival and persistence in-vivo, but given its enigmatic role even in normal B-cells, its relevance to HCL progression is unclear.These observations raise the possibility that mutant BRAF may be a mechanism to bypass any antigen-dependent BCR signalling constraints in mult-HCL. Disclosures:No relevant conflicts of interest to declare.

Production of seven monoclonal equine immunoglobulins isotyped by multiplex analysis

Horses have 11 immunoglobulin isotypes: IgM, IgD, IgA, IgE, and seven IgG subclasses designated as IgG1–IgG7, each of which are distinguished by separate genes encoding the constant heavy chain regions. Immunoglobulin (Ig) isotypes have different functions during the immune response and pathogen-specific isotypes can be used as indicators for immunity and protection from disease. In addition to existing monoclonal antibodies to various equine Igs, quantification of the individual isotypes requires pure isotype standards. In this report, we describe a fusion between X63-Ag8.653 mouse myeloma cells and horse PBMC to create equine-murine heterohybridomas. Initial screening for Ig production was performed by ELISA. Further testing was performed by a new 5-plex fluorescent bead-based assay able to simultaneously detect equine IgM, IgG1, IgG4/7, IgG5, and IgG6. Production of IgG3 and IgE was tested by separate bead assays. Seven stable heterohybridoma clones producing monoclonal equine IgM, IgG1, IgG3, IgG4/7, IgG5, IgG6 and IgE were created. Purified Ig isotypes were then tested by SDS–PAGE. The pure, monoclonal equine Ig isotypes and the new equine Ig multiplex testing developed here are valuable tools to quantify antibody responses and to accurately determine individual isotypes concentrations in horses.

Are IgM Hevylite® Immunoglobulin Heavy Chain/Light Chain Analysis a Useful Tool to Differentiate IgM MGUS From Waldenström Macroglobulinemia?

Abstract 5097 Background:Monoclonal IgM is the biomarker that characterize to Waldenstrom's macroglobulinemia (WM), a rare low grade B-cell lymphoma derived of the lymphoplasmacytic cell, even serum IgM component also is presented in MGUS. Recently new determinations of heavy chain/light chain immunoglobulins pairs (HLC) have been developed as biomarkers to apply to every day clinical practice. The diagnostic and prognostic potential of these biomarkers is under investigation. The aim of this study is to present our experience in the use of free light chain assay (sFLC) and HLC as biomarkers at diagnostic in order to discriminate between MGUS and WM, and to evaluate their potential prognostic value during disease course. Patients and Methods:A total of 43 patients (pts) were detected as having a serum monoclonal IgM in the Hematology Department of MSUH. Pts were classified as MGUS or WM according to the morphological, immunophenotype characteristics of lymphoplasmacitic bone marrow cells and CT-scan data. Pts were examined every 3–6 months following our clinical protocol in order to detect progression or transformation. Serum samples were collected prior and during treatment and were kept frozen at −70°C since collection and incorporate to our regional Biobank. Analysis of IgM were performed with the sFLC, (Freelite® test, the Binding Site, Birmingham, UK) and the HLC (Hevylite® immunoassay the Binding Site). Freelite® test is a nephelometric measurement of kappa and lambda light chains that circulate not bound to immunoglobulin heavy chain. Hevylite® immunoassay is based on specific polyclonal antibodies that recognize epitopes spanning the junction of the heavy and light chains of the individual immunoglobulin isotypes, it measures specifically IgMkappa and IgMlambda, separately. A normal range of IgM Hevylite assay was produced from normal (blood donor) sera, median (CI 95%) were: IgMkappa, 0. 634 g/L (0. 29–1. 82); IgMlambda, 0. 42g/L (0. 17–0. 94); HLC ratio (HLCR), 1. 6 (0. 95–2. 3). For ease of comparison IgM HLCR was expressed as the involved monoclonal immunoglobulin (iHLC)/uninvolved polyclonal immunoglobulin (uHLC). Results:The study included a series of 25 WM, 18 IgM-MGUS (included 2 IgM-cryoglobulinemia), 36 at diagnosis and 7 at relapse/refractory. The median age was 67. 1 years (13–85); IgM HLCR was 114. 68 (1. 02 – 353) in WM symptomatic, 71. 55 (1. 02 – 286. 43) in WM asymptomatic and 9. 5 (0. 45 – 50. 74) in IgM MGUS (p=0. 003). HLCR was higher in WM patients requiring treatment (n=13) at diagnosis than in pts (n=30) not requiring treatment (113 v 15. 77 p=0. 019) and also HLCR was significantly higher at relapse/refractory (n=9) than in pts (n=34) not relapse (113 v 17. 17 p=0. 012) uHLC was significantly higher in IgM-MGUS than WM to IgMkappa (n=28) and IgMlambda (n=15) iHLC: 0. 37 g/L (0. 11–1. 25) v 0. 1 g/L (0. 02–4. 03), p=0. 022; and 0. 69 g/L (0. 08–4. 09) v 0. 32 g/L (0. 22–0. 63), p=0. 05 respectively. sFLC level was 64 mg/L (10. 88–993) in WM and 31. 7 mg/L (6. 08–141) in IgM MGUS (p=0. 05). sFLC level was higher in WM requiring treatment at diagnosis than in pts not requiring treatment (73. 7 v 36. 85 p=0. 039). sFLC level was not significative in relapse/refractory (p=0. 168), it was not separate between WM asymptomatic and WM symptomatic (p=0. 092). There was a good correlation between HLCR and sFLC ratio (r=0. 3, p=0. 044) but not with sFLC level, HLCR, sFLC level and sFLC ratio did not predict for overall survival (OS) and progression free survival (PFS) in our study. Mean estimated OS was 78. 3 months (95% CI: 53. 67–102. 94) and PFS 69. 7 months (95% CI: 47. 28–92. 13). Conclusion:It seems that HLCR and sFLC could be good biomarkers to differentiate between IgM-MGUS and WM at diagnostic. High levels of HLC and sFLC were also seen in pts requiring treatment. HLCR discriminates WM symptomatic/asymptomatic and progressing pts uHLC levels were significantly higher in IgM-MGUS than WM pts showing that IgM-MGUS have a more robust immune system. Further studies are needed to evaluate their prognostic value.This work has been partially sponsored by a grant from FEHHA Disclosures:No relevant conflicts of interest to declare.

Hairy Cell Leukaemia Displaying Multiple Surface Immunoglobulin Isotypes Reveal a Functional B-Cell Receptor In Which Isotype Roles Differ

Abstract 1567The B-cell receptor (BCR) is critical to survival of normal B-cells, and regulates key aspects of cellular behavior. Of these, response to antigen determines pathways of normal B-cell maturation, including isotype switch events that occur by deletional class switch recombination (CSR), an irrevocable event, to yield IgG/A memory B-cells. Less frequently, CSR via a cryptic site generates IgD+ B-cells whereas IgM+IgD+ antigen experienced B-cells synthesize each isotype by an alternative transcript splicing mechanism. The role of the BCR in survival of malignant B-cells however is less well defined, in particular in response to antigen. Intriguingly, in Hairy cell leukemia (HCL), BCR assembly occurs with multiple surface immunoglobulin (sIg) isotypes (mult-HCL), many co-expressed on individual hairy cells (HCs) in an otherwise monoclonal tumor. Multiple isotypes appear to exclude deletional CSR events, and suggests a RNA processing mechanism of molecular assembly. This phenotype is rare even amongst malignant B-cells, and raises the question of the functional relevance of individual variant isotypes. It also potentially presents a model to dissect roles of multiple isotypes on single B-cells. To examine this, we investigated the BCR in CD19+CD11c+CD103+ mult-HCL cases (n=10), in which 2–4 differing sIg isotypes were present on most HCs, with single or, in 3 cases, dual sIgL expression. In all cases, IGHV genes were mutated, and confirmed monoclonality. Phenotype revealed 2 distinct subsets by sIg isotype co-expression, IgD+ve and IgD-ve. Using Ca2+ flux and ERK phosphorylation assays after cross-linking with specific anti-sIg antibodies, we observed a functional BCR in all mult-HCL examined, in both subsets (10/10 cases Ca2+, 6/6 cases ERK). However, striking differences emerged between the two subsets. In sIgD+ve mult-HCL, IgD mediated persistent Ca2+ flux, with flux also evident via >1 sIgH isotype. In marked contrast, in sIgD-ve mult-HCL Ca2+ flux was restricted to a single sIgH isotype, but not via IgM. Flux signals in this subset were transient. In most cases only a single sIgL transduced flux. We next evaluated BCR endocytosis after cross-linking individual isotypes and IgL. In 2 sIgD+ve cases, anti-IgD and anti-Igλ stimulation led to endocytosis of both sIgD and sIgλ, and in 1 case, where examined, anti-IgM stimulation endocytosed both sIgM and sIgλ. In 3 sIgD-ve cases, functional sIgH and sIgL induced endocytosis of the stimulated isotype, but again sIgM was dysfunctional, remaining immobilized on the cell surface. Ca2+ flux through endocytosed isotypes was correspondingly either significantly reduced or ablated in both subsets. In HCs, BCR endocytosis is clearly dependent on functional isotypes and IgL, and parallels events in normal B-cells. Lastly, we examined downstream effects of BCR signalling on cell viability, using soluble (sAb) and bound (bAb) anti-sIg antibodies. In a single IgD+ve mult-HCL case, both sAb and bAb anti-IgM yielded a significant level of apoptosis compared to control antibodies, whereas anti-IgD sAb resulted in no appreciable difference to level of spontaneous apoptosis, suggesting a disengagement of signals from this pathway. This disengagement was also observed in a separate HCL case expressing only IgD, and not in the mult-HCL cohort initially selected, where anti-IgD signals again did not increase levels of apoptosis. In IgD-ve mult-HCL (n=4), sAb and bAb specific cross-linking of IgG/A triggered significant apoptosis. These data demonstrate, for the first time, that mult-HCL retains a functional responsiveness via the BCR, suggesting an absence of anergic effects that may follow chronic antigen exposure in-vivo to self-antigen. Signals via sIgM/G/A isotypes, where functional, induce apoptosis in mult-HCL, whereas sIgD opposes such effects. Despite an apparently unique molecular mechanism of IgD expression in mult-HCL, this isotype appears to be hardwired in B-cells to mediate responses that differ from IgM. The persistent flux observed here indicates a more sustained and robust IgD signaling cascade, as also observed in B-cell models. These data reveal distinctive and opposing effects of individual isotypes on BCR mediated behavior in mult-HCL. While apoptotic responses appear to negate a role for antigen in tumor drive in-vivo, potential antigen engagement via IgD, if dominant leaves this question open. Disclosures:No relevant conflicts of interest to declare.

Experimental Demonstration of III-Nitride Hot-Electron Transistor With GaN Base

In this letter, we demonstrate for the first time a III-nitride hot-electron transistor using an AlGaN (24%) emitter, 10-nm GaN base, and an AlGaN (8%) collector. Individual isotype heterojunctions were characterized by I- V measurements. For the device biased in a common base configuration, a common base transfer ratio of 0.97-0.98 was measured. The hot-electron distribution was obtained by plotting the differential of the collector current with respect to the applied base-collector voltage and demonstrated a Maxwellian shape suggesting near-ballistic transport through the 10-nm base.