Immunoglobulin Heavy Chain Research Articles (Page 1)

ABSTRACTIntroductionRecent advancements in risk stratification have greatly improved outcomes in pediatric B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL). Despite favorable prognostic indicators, including the absence of cytogenetic abnormalities and minimal residual disease (MRD) negativity, relapse remains a major clinical concern.Methods and ResultsWe investigated the clinical significance of immunoglobulin heavy chain (IGH) clonality using RNA sequencing data in BCP‐ALL. We analyzed IGH clonality from 136 patients. IGH abundance followed a power law distribution, which enabled us to identify disease clones as outliers based on read count. In total, 330 disease clones were detected, and patients were categorized into three clonotype groups: undetectable disease clone (UDC), incomplete disease clone (IDC), and complete disease clone (CDC). Clinical outcomes were compared across clonotypes, including in subgroups with high hyperdiploidy (HHD) and MRD negativity. Among patients with HHD, significant prognostic differences were observed across clonotypes (event‐free survival [EFS], p = 0.01; overall survival [OS], p = 0.08), even among those who were MRD‐negative (EFS, p = 0.01; OS, p = 0.03). Furthermore, comparisons of IGH sequences between diagnosis and relapse indicated that while initial disease clones often contributed to relapse, newly expanded clones frequently emerged, particularly in patients with HHD.ConclusionsThese findings highlight the importance of analyzing the IGH repertoire in refining risk stratification and underscore the need for advanced sequencing‐based MRD monitoring.

Multifunctional Nanobody Fusion Proteins in Immunoassays: Diverse Strategies for Enhanced Analytical Performance.

Chronic lymphocytic leukemia (CLL) is the most common low-grade B-cell neoplasm worldwide. While diagnostic criteria are well established, prognostication and management of this disease are an evolving field, owing to the biological and clinical heterogeneity of CLL. Molecular, cytogenetic, and immunogenetic workup are key in the management of CLL, and include evaluation for specific gene variants, copy number variants (CNVs), and detailed analysis of the immunoglobulin heavy chain variable region (IGHV) with respect to somatic hypermutation (SHM) status and the presence of recurrent stereotyped IGHV subsets. IGHV status has significant prognostic, predictive, and therapeutic implications in CLL and has been incorporated into multiple risk stratification systems. The advent of both next-generation sequencing (NGS) and novel targeted therapies has added further complexity to immunogenetic analysis in CLL. Owing to the necessity and growing complexity of IGHV analysis, the European Research Initiative on CLL (ERIC) developed guidelines to standardize methods for immunogenetic analysis and provide recommendations for interpretation of challenging cases (including multiple productive IGHV clones and discordant SHM status) and has published multiple updates, revising recommendations and raising new questions. This review discusses the biology and clinical significance of IGHV status in CLL as well as laboratory methodology in immunogenetic analysis, the evolution of the ERIC recommendations leading into the era of NGS, and recent advances and emerging strategies in this field.

Squamate reptiles are a diverse group of terrestrial vertebrates that includes over 10,000 species. Despite their broad distribution, they are relatively understudied immunologically. Fortunately, recent advances in comparative genomics have facilitated studies of squamate immune systems, revealing their unique features. For example, squamates lack of γδ T cells entirely due to genomic deletions. Here we annotated and analyzed the loci encoding the Tiliqua rugosa immunoglobulin heavy and lambda light chains, revealing a remarkably large and complex number of variable region genes in both. Tiliqua rugosa lacks genes encoding a kappa light chain, which can be found in some but not all squamates. We also identified common reptile IgH isotypes including IgM, IgD, and IgY, but no IgA. Overall, T. rugosa, has the germline capability of generating a highly diverse antibody repertoire.

HeavyBuilder: Analysis of High-Throughput of Antibody Heavy Chain Repertoires in the Structural Space.

Chronic lymphocytic leukemia (CLL) is divided into unmutated (UM-CLL) and mutated (M-CLL) subtypes depending on somatic hypermutation (SHM) frequency in their immunoglobulin heavy chain V (IGHV) region. We previously demonstrated that CD27bright memory B cells (MBCs) are germinal center (GC)-dependent with higher mutation rate, whereas CD27dull MBCs accumulate fewer mutations and originate independently from the GC. We conducted a meta-transcriptomic analysis on bulk RNA data from 116 individuals combining four CLL cohorts and healthy B cell subsets (naïve, CD27dull and CD27bright MBCs) to decipher the transcriptional and mechanistic functions of CLL subtypes. CD27bright MBCs showed more transcriptional similarity to M-CLL rather than UM-CLL. Functional enrichment analysis revealed that LPL, ZNF667 and ZNF667-AS1 are potential informative biomarkers for stratification of CLL subtypes. They are part of the mechanistic regulatory pathways of CLL pathology through cholesterol and Epithelial Mesenchymal Transition (EMT) regulation. We applied markers for the GC B-cell substages to map in silico the CLL cohorts to their potential GC B cell counterpart. UM-CLL represented transcriptional mimicry to an early intermediary GC substage whereas M-CLL mimicked later substages in the GC. This could potentially explain the IGHV mutational status of M-CLL as well as hypothesize that CLL subtypes could derive from a GC-dependent pathway.

B cell development in early life sets the stage for how the adaptive immune system will function. Although little is known about B cell biology in children, especially from tissue-residing B cells, it has been previously reported that children generally possess more naive B cell repertoires with less somatic hypermutation and fewer expanded clones compared to adults. In this paper, we move beyond these findings by studying how clonal selection differs between children and adults from both blood and tissue-residing B cells. By integrating deep sequencing data of immunoglobulin heavy chains from genomic DNA from extracted tissues and blood of 15 children and 9 adults across multiple datasets, we demonstrate that B cell repertoires in children are not only less mutated, but also undergo reduced negative selection pressures. Mutated clones in children are less likely to possess a trunk compared to adults, but the relative proportion of mutated clones with trunks increases with age in children. This pattern is not observed in adults, where the fraction of mutated clones that possess a trunk has reached a steady state. Furthermore. clones in children are more likely to contain unmutated germline sequences, exhibit a greater number of viable internal nodes in their lineages, and experience less negative selection in the framework regions of their receptors. Overall, our findings show that B cell development is not merely the accumulation of mutations with age, but rather reflects a shift from flexible, broadly permissive repertoires in childhood to refined, stringently selected repertoires in adulthood. This difference in the structure of pediatric repertoires could underlie their enhanced ability to respond to novel antigens. Together, these findings provide new insights into how selection pressures shape immunity across the human lifespan.

Background and ObjectivesIntrathecal synthesis of immunoglobulin G (IgG) is a key feature of multiple sclerosis (MS) and a prognostic marker for the disease course. Although previous studies identified 2 genetic regions—the major histocompatibility complex (MHC) region on chromosome 6 and the immunoglobulin heavy chain constant (IGHC) locus on chromosome 14—associated with intrathecal IgG synthesis in MS, the genetic underpinnings remain insufficiently understood.MethodsWe conducted a genome-wide association study on intrathecal IgG synthesis using the IgG index to identify individuals with (≥0.7) or without (<0.7) quantitative intrathecal synthesis. We used logistic regression models adjusting for sex, age, and population structure. We performed secondary analyses to examine associations between identified loci and the extent of intrathecal IgG synthesis and the presence and extent of intrathecal immunoglobulin A and M synthesis. We further conducted association analyses for imputed human leukocyte antigen alleles and analyzed whether a higher genetic burden for MS risk—quantified through polygenic risk scores—is associated with intrathecal IgG synthesis.ResultsIn the discovery cohort (n = 3,934), we identified a novel genome-wide significant association of the intronic variant rs844586 (p = 1.48 × 10−8) in the sterile alpha motif domain containing 5 (SAMD5) gene on chromosome 6, with intrathecal IgG synthesis. We could confirm this association in a replication cohort (n = 1,094) and demonstrated that it is independent of a previously described association signal at the MHC region. In a subset (n = 1,413), we further identified rs1407 as a potential causal variant (p = 3.80 × 10−11, posterior inclusion probability = 0.92) for the previously reported association signal at the IGHC locus with the extent of intrathecal IgG synthesis. In addition, we demonstrated that a higher genetic burden for MS susceptibility, both within and outside of the MHC region, is associated with a higher likelihood of and a more pronounced intrathecal IgG synthesis.DiscussionOur study revealed a previously unknown association between an intronic variant in SAMD5 with intrathecal IgG synthesis and identified a potential causal variant within the IGHC locus. It further provides evidence for possible effects of known MS risk variants on disease severity through their effect on the intrathecal humoral immune response, a prognostic marker for the disease course.

IntroductionNanobodies (NBs) are small antibody fragments derived from camelid heavy-chain antibodies, which represent the minimal functional domain capable of antigen recognition and binding. NBs are 10 times smaller than conventional antibodies, exhibit a compact structure, and have high stability, making them ideal for recombinant production. The eukaryotic unicellular system Pichia pastoris provides multiple advantages for protein expression, including the ability to perform several eukaryotic post-translational modifications such as glycosylation.MethodsIn this work, we engineered a modular plasmid sequence that, through specific restriction enzyme cuts and ligations, codes the expression of a secreted anti-mouse kappa chain NB fused with various accessory peptides in P. pastoris. This system enables the incorporation of a plastic binding sequence for immobilization onto polystyrene surfaces, a histidine tag (Hisx6) for purification, the horseradish peroxidase (HRP) enzyme for chemiluminescence detection, or the biotinylatable AviTag sequence for detection using a different method, in multiple combinations.ResultsWe successfully expressed and purified anti-kappa NBs fused to a Hisx6-tag (κNB) and HRP–Hisx6-tag (κNB–HRP), with subsequent structural and functional characterization revealing high affinity and specificity for mouse immunoglobulins. The κNB–kappa light chain domain complex was modeled, showing a fitted surface interaction of the CDR3 domain. The position of a glycan present in κNB CDR3 within the complex was modeled, predicting that glycan addition would not affect the interaction surface. Accordingly, no functional differences were observed in κNB after deglycosylation, indicating that high mannose glycan addition has not interfered with its binding capability. Glycosylated and deglycosylated κNBs fused to HRP were produced with retained HRP activity and proved to be functional as secondary antibodies.DiscussionOur results show the P. pastoris eukaryotic system’s versatility in producing NBs and conjugated NBs with or without post-translational modifications that may be required for diverse biotechnological applications.

The instability of conventional antibodies (ConAbs) in food matrix or target extraction reagents often limits the convenience or sensitivity of immunoassays. In this study, alpacas were immunized with water-soluble adjuvants to induce heavy chain antibodies (HCAbs) against antibacterial synergists (ASGs). Compared to ConAb C5, the HCAb H5 exhibited superior stability (binding activity >80% and IC50 change <2-fold) under harsh conditions (63 °C, pH 3.0-11.0, 6.0 mol L-1 NaCl, 30% methanol, 30% acetonitrile). An indirect competitive ELISA (icELISA) based on H5 showed IC50 values of 1.24, 1.44, 1.60, and 2.11 μg L-1 for trimethoprim (TMP), dimethoprim (DVD), ormetoprim (OMP), and baquiloprim (BQP), with cross-reactivity rates of 100%, 86.3%, 77.7%, and 56%, respectively. Sample preparation was simplified to minimal dilution steps─chicken, egg, pork, and milk samples were diluted only 2-, 4-, 4-, and 8-fold, respectively─and the entire pretreatment could be completed within 1-8 min. The limits of detection (LODs) ranged from 0.14 to 1.36 μg L-1/μg kg-1, with recoveries of 76.4-107.3%, and coefficients of variation (CV) below 14.1%. This is the first HCAb-based icELISA for ASGs, offering high sensitivity, robustness, and minimized pretreatment for complex food matrices.

In pro-B cells, VDJ recombination at the immunoglobulin heavy chain locus is impaired. B cell progenitor recombination implies the formation of DNA double strand breaks (DSBs) by the RAG recombinase, which are subsequently repaired by specific mechanisms. We cultured primary murine pro-B cells with IL-7 to evaluate H2AX histone phosphorylation, a well-established marker of DSB formation (γ-H2AX foci) and the expression of proteins involved in DNA repair. Our results indicated that IL-7 upregulated the expression of several molecules involved in homologous recombination, the most accurate DSB repair mechanism. Quantitative analyses of γ-H2AX foci revealed that IL-7 significantly increased DSB formation in a time-dependent manner. Furthermore, γ-H2AX expression was altered in RAG2-deficient pro-B cells and absent in RAG1-deficient pro-B cells treated with IL-7, demonstrating the requirement of both RAG1 and RAG2 recombinase subunits. CD43 expression inversely correlates with the degree of cell differentiation and its level is often evaluated to assess the B lymphoid developmental stage. We observed that IL-7 upregulated CD43 expression and the percentage of large CD43/γ-H2AX double-positive cells, suggesting an effect on less differentiated, immature cells. Notably, we also found that IL-7 increased radiation-induced DSBs, while simultaneously supporting cell survival. This study uncovers novel effects of IL-7 on B cell differentiation, DSB formation, and DNA repair. It is well established that IL-7 promotes the proliferation and survival of acute lymphoblastic leukemia (ALL) cells. Our data suggest that drugs targeting IL-7 could improve ALL therapeutic protocols.

People living with Human Immunodeficiency Virus (HIV) (PLWH) may develop HIV-associated neurocognitive disorder (HAND) despite the use of antiretroviral therapy. Therefore, more studies are needed to identify novel therapies, which require a better understanding of the molecular and cellular mechanisms underlying HIV neurotoxicity. The HIV envelope protein gp120 causes neuronal degeneration similar to that observed in HAND. One mechanism contributing to gp120-mediated neurotoxicity may involve its ability to inhibit protein processing in the Golgi apparatus and endoplasmic reticulum (ER). To provide data in support to this hypothesis, we have used a variety of experimental approaches to investigate the effect of gp120 on ER dynamics. We first analyzed the levels of ER stress-associated proteins, such as immunoglobulin heavy chain binding protein (BiP) and phosphorylated Inositol-Requiring Enzyme 1 alpha (p-IRE1α) by western blot, as well as ER morphology by electron microscopy in gp120 transgenic (tg) mice. We found that the hippocampus of gp120tg mice exhibits an increase of BiP levels and p-IRE1α, as well as altered ER morphology when compared to wild type mice. We confirmed that gp120 alters ER morphology in neurons by using rat cortical neurons in culture. The effect of gp120 was chemokine-co-receptor dependent because AMD3100, a CXCR4 receptor antagonist, abolished the effect of gp120 on BiP immunoreactivity. Moreover, using Gluc-ASARTDL, a reporter protein for monitoring ER calcium, and live Ca2+ imaging, we show that gp120 induces ER Ca2+ depletion in neurons. Overall, our data suggest that gp120 promotes ER stress in neurons.

Staphylococcus aureus is a major pathogen responsible for staphylococcal food poisoning (SFP), with its pathogenicity primarily dependent on staphylococcal enterotoxins (SEs). Among these, staphylococcal enterotoxin A (SEA) is a critical risk factor due to its high toxicity, high detection rate (accounting for 80% of SFP cases), strong thermal stability, and resistance to hydrolysis. Traditional SEA immunoassays, such as enzyme-linked immunosorbent assay (ELISA), are prone to false-positive results caused by nonspecific binding interference from S. aureus surface protein A (SpA). In recent years, nanobodies (single-domain heavy-chain antibodies) have emerged as an ideal alternative to address SpA interference owing to their small molecular weight (15 kDa), high affinity, robust stability, and lack of Fc regions. In this study, based on a previously developed highly specific monoclonal antibody against SEA (mAb-4C6), four anti-SEA nanobodies paired with mAb-4C6 were obtained through two-part (four-round) of biopanning from a naive nanobody phage display library. Among these, SEA-4-20 and SEA-4-31 were selected as optimal candidates and paired with mAb-4C6 to construct double-antibody sandwich ELISAs. The detection limits for SEA were 0.135 ng/mL and 0.137 ng/mL, respectively, with effective elimination of SpA interference. This approach provides a reliable tool for rapid and accurate detection of SEA in food, clinical, and environmental samples.

Abstract Background Multiple myeloma is a hematological malignancy most prevalent in individuals aged 65 years and older and is characterized by the presence of clonal plasma cells in the bone marrow. Diagnostic testing for multiple myeloma includes serum protein electrophoresis and serum immunofixation/immunotyping to quantify the monoclonal protein and identify the immunoglobulin isotype, respectively. Electrophoresis and immunofixation/immunotyping are performed using either a gel or a capillary separation technique. Gel electrophoresis and immunofixation are the methods used by most clinical laboratories; however, capillary electrophoresis and immunotyping usage have been increasing in recent years due to advances in technology and automation. “Though Quest Diagnostics – Chantilly has many years of experience in performing capillary electrophoresis, serum immunofixation by gel electrophoresis has remained the methodology employed for multiple myeloma diagnostic testing. To streamline processes and improve service, the laboratory transitioned to immunotyping by capillary electrophoresis. Methods To determine the impact of the switch from gel electrophoresis immunofixation (Helena SPIFE TOUCH) to immunotyping by capillary electrophoresis (Capillarys III Tetra), the change in turnaround time (collection to result), percentage of monoclonals identified, and rates of monoclonal identification by both immunofixation/immunotyping and serum protein electrophoresis monoclonal quantification were evaluated for the same 3-month period prior to the change in methodology and after the change. Results After implementation of immunotyping, turnaround time was reduced by 33%, from six days to four days. One or more monoclonal proteins were identified in 57% of samples for immunofixation and in 63% of samples for immunotyping. Two or more clones were identified in 18% of immunofixation samples and 8% of immunotyping samples. IgG kappa (42%-45%) was the predominant clone detected by both methods, followed by IgG lambda (20%-22%), IgM kappa (6%-8%), IgA kappa (6%-8%), IgA lambda (3.5%-5%), IgM lambda (2.2%-3.5%), free light chains only (∼1%) and IgD kappa and lambda (&amp;lt;0.25%). There was no significant difference in the percentages of the immunoglobulin heavy and light chains reported by the 2 methods. Immunofixation confirmed the presence of monoclonal proteins in 99.0% of samples resulting in abnormal protein electrophoresis, while immunotyping confirmed 98.2% of such samples Conclusion Overall, results reported for transition from immunofixation to immunotyping showed concordance in the percentage of monoclonals identified as well as the isotype Additionally, the transition from immunofixation to immunotyping resulted in several improvements for clients and patients, including a reduction in the turnaround time and decrease in multiple clones being reported. Considering, True multiple myeloma cases with 2 or more clones are thought to be rare, making up only 1%-5% of cases, this suggests that immunotyping may yield more accurate results when compared to immunofixation methods.

Abstract Background Using machine learning and artificial intelligence (AI) techniques, we have developed a computer vision tool for the automated interpretation of electrophoresis gel scans. Serum protein electrophoresis (SPE) is a well-established routine laboratory diagnostic technique used to quantify different types of blood proteins, whose composition can be altered by various pathologies. Serum immunofixation electrophoresis (IFE), a type of SPE, can help determine disease type, severity, prognosis, and is useful for disease monitoring. Serum IFE is primarily indicated for patients suspected of having plasma cell dyscrasias/disorders, such as multiple myeloma, a type of blood cell cancer. Anti-sera is added into distinct gel lanes for identification of antibody heavy chains (IgG, IgA, IgM) and light chains (kappa and lambda) which correspond to specific monoclonal immunoglobulins and have clinical significance. Interpretation of IFE gels requires significant time and effort from highly skilled experts in the field of clinical pathology and/or laboratory medicine. Methods Over 4,000 archived serum IFE scan images from UofL Health have been collected and processed for model development, with 45% positive for monoclonal proteins and the remainder negative. Model parameters were tuned using 70% of the data for model training, with the remainder reserved as validation data. Additional data was collected as internal (N = 906) and external (N = 120) test data, both of which were excluded from the training process. Training, validation, and internal test scans were labeled according to routine clinical laboratory reports which were interpreted by board-certified clinical pathologists. External test images were labeled by consensus agreement among 3 independent visual interpreters. Results When predicting if a band corresponding to a monoclonal protein was present or not present in IFE gel scans, the model achieved a sensitivity of 0.96, specificity of 0.98, and AUROC of 0.99, on internal test data, and sensitivity of 0.89, specificity of 1.00, and AUROC of 0.98, on external test data, compared to routine visual interpretation. AUROC was 0.95 and 0.96 for identifying IgG heavy chains and 0.98 and 0.99 for kappa light chains within internal and external test data, respectively. IgM heavy chains had an AUROC of 0.95 and 0.73 within internal and external test data, respectively. Further, heavy chain and light chain combinations were evaluated for monoclonal protein identification. For instance, IgG-kappa monoclonal proteins were detected with a sensitivity of 1.0 in both internal and external test scans. The model pipeline is also practical to implement, with an estimated average computation time of 16 seconds per sample using personal computer class desktop hardware and less than 0.5 seconds per sample with professional workstation class hardware. Conclusion The computer vision tool is poised to be integrated with routine clinical laboratory workflows where it can assist the interpretive efforts of medical staff by triaging patients with potential plasma cell disorders.

The Chronic Lymphocytic Leukemia Epigenome: A Powerful and Stable Framework for Understanding Biological and Clinical Heterogeneity.

The human IG heavy chain constant gene locus is enriched for large structural variants and coding polymorphisms that vary among human populations.

Clinical Validation of Duoseq, a Novel Assay for Clinical DNA and RNA Sequencing.

We compared the immunoglobulin (IG) heavy chain (IGH) leader and FR1 primer sets to measure clone sizes and detect immunoglobulin heavy chain variable (IGHV) region somatic hypermutations (SHMs) in Korean patients with chronic lymphocytic leukemia (CLL). We also analyzed IGH and immunoglobulin kappa (IGK) to identify Korean-specific IGs in CLL. Next-generation sequencing (NGS)-based gene rearrangements and IGHV SHMs were assessed in 40 patients using IGH leader, IGH FR1, and IGK primers. Flow cytometry, karyotyping, interphase FISH, and NGS-based variant analyses were performed for 165 genes. Clonal IGH and IGK rearrangements were detected in 100.0% and 97.5% of patients, respectively. Clonal size was generally smaller per NGS than per flow cytometry, particularly when using the IGH leader (median: 52.5%) versus the IGH FR1 primer set (73.2%). IGHV SHMs occurred in approximately 70% of patients; 10% showed primer set discrepancies. The incidence of IGHV SHMs was low in patients at high risk (i.e., with TP53 abnormalities; complex karyotypes; and ATM, NOTCH1, SF3B1, or BIRC3 variants). IGHV3 was the most common IGHV (58.3%), and IGHV4-34 was most frequently identified (14.6%). IGHV1 and IGHV1-69 usage differed significantly between Koreans and westerners. IGHJ4 was the most common IGHJ (56.3%). A single IGKV-IGKJ gene rearrangement was most frequently observed (18.9%), whereas intron-KDE was the most common rearrangement (30.6%). NGS may underestimate CLL clonal size, particularly when using the IGH leader primer set. IGHV SHMs were inversely associated with negative prognostic factors. Our data suggest ethnic differences in CLL pathogenesis.

The World Health Organization (WHO) recommends combination therapy with two non-overlapping monoclonal antibodies (mAbs) to enhance protection in Rabies post-exposure prophylaxis (PEP). While bispecific antibodies (bsAbs) provide superior protection over monoclonal antibodies (mAbs) by simultaneously targeting two distinct epitopes, their development is hindered by complex and inefficient vector design. In this study, we aimed to develop a novel single-vector expression system to efficiently produce potent anti-rabies virus (RABV) neutralizing bsAbs. We engineered a minimalistic peaSKYA single-vector featuring bidirectional dual-CMV promoters to enable synchronous co-expression of antibody heavy (HC) and light chains (LC) within a single construct. By eliminating redundant backbone elements, this optimized vector increased the expression-cassette ratio by 17% and improves transfection efficiency by 32% compared to dual-vector systems. The platform demonstrated 2-fold greater yields of bispecific antibodies in HEK293F transient expression, with the tetravalent DVD-Ig format antibody HHDVD showing significantly enhanced neutralization potency versus parental monoclonal antibodies in both pseudovirus-based neutralization assays (PBNA, 2193.19 IU/mg) and rapid fluorescent focus inhibition tests (RFFIT, 1679.52 IU/mg). We developed peaSKYA single-vector expression system for robust production of bispecific antibodies, with candidate HHDVD exhibiting potential in Rabies PEP. This streamlined vector design approach exhibited remarkable potential in bispecific neutralizing antibody development, offering a transferable platform for other engineered bispecific antibodies.