Immunoglobulin Variable Region Research Articles

Interpretable deep learning reveals the role of an E-box motif in suppressing somatic hypermutation of AGCT motifs within human immunoglobulin variable regions.

Somatic hypermutation (SHM) of immunoglobulin variable (V) regions by activation induced deaminase (AID) is essential for robust, long-term humoral immunity against pathogen and vaccine antigens. AID mutates cytosines preferentially within WRCH motifs (where W=A or T, R=A or G and H=A, C or T). However, it has been consistently observed that the mutability of WRCH motifs varies substantially, with large variations in mutation frequency even between multiple occurrences of the same motif within a single V region. This has led to the notion that the immediate sequence context of WRCH motifs contributes to mutability. Recent studies have highlighted the potential role of local DNA sequence features in promoting mutagenesis of AGCT, a commonly mutated WRCH motif. Intriguingly, AGCT motifs closer to 5' ends of V regions, within the framework 1 (FW1) sub-region1, mutate less frequently, suggesting an SHM-suppressing sequence context. Here, we systematically examined the basis of AGCT positional biases in human SHM datasets with DeepSHM, a machine-learning model designed to predict SHM patterns. This was combined with integrated gradients, an interpretability method, to interrogate the basis of DeepSHM predictions. DeepSHM predicted the observed positional differences in mutation frequencies at AGCT motifs with high accuracy. For the conserved, lowly mutating AGCT motifs in FW1, integrated gradients predicted a large negative contribution of 5'C and 3'G flanking residues, suggesting that a CAGCTG context in this location was suppressive for SHM. CAGCTG is the recognition motif for E-box transcription factors, including E2A, which has been implicated in SHM. Indeed, we found a strong, inverse relationship between E-box motif fidelity and mutation frequency. Moreover, E2A was found to associate with the V region locale in two human B cell lines. Finally, analysis of human SHM datasets revealed that naturally occurring mutations in the 3'G flanking residues, which effectively ablate the E-box motif, were associated with a significantly increased rate of AGCT mutation. Our results suggest an antagonistic relationship between mutation frequency and the binding of E-box factors like E2A at specific AGCT motif contexts and, therefore, highlight a new, suppressive mechanism regulating local SHM patterns in human V regions.

Abstract 3099: A new HTS ELISA for monitoring NRAS activity to expedite drug discovery

Abstract Oncogenic mutations in the KRAS, HRAS, and NRAS isoforms of the RAS gene family alter GTP binding and hydrolysis leading to errant over activation of the MAPK/ERK pathway. While the total of all such RAS mutations drives some 20-25% of all human cancers, each isoform tends to favor particular mutations that are cancer specific. Many evolving treatment strategies for these cancers target these mutant isoforms to minimize off target effects. NRAS mutations are seen in about one-fifth of cutaneous melanomas and patients with NRAS mutation melanomas have a poorer prognosis due to the high aggressiveness of RAS mutant tumors, a lack of efficient targeted therapies, or rapidly emerging resistance to existing treatments. Furthermore, there is no currently authorized targeted treatment for NRAS-mutated melanoma. As a result, there is a growing need for assays which can quantify activated NRAS. Here we describe the development of a highly specific and sensitive ELISA for determining the active NRAS protein in high throughput compatible (HTS) format. A NRAS specific recombinant antibody was developed via cloning of immunoglobulin variable regions from antigen positive B cells isolated by FACS from rabbits immunized with a NRAS peptide corresponding to a region of low homology between the RAS isoforms. Immunoblotting and a bead-based multiplex ELISA with all three RAS isoforms were used to confirm antibody specificity with recombinant HRAS, KRAS, and NRAS proteins over a wide concentration range. The new immunoassay enables in-well lysis of cells cultivated in 96-well plate. Activated NRAS in the lysate is collected on the test plate and detected using the NRAS specific antibody via chemiluminescence. We anticipate that this assay will further the discovery of novel NRAS specific therapeutics. EGF treatment of MCF7 cells demonstrated activation in cells expressing wildtype NRAS in preliminary assay validation. Evaluation of assay performance in mutant NRAS melanoma cell lines will be presented. Citation Format: Hyun Min Park, Julie Weber, Michelle Carlson, Mary Anne Jelinek. A new HTS ELISA for monitoring NRAS activity to expedite drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3099.

A Novel Heterozygous Variant in AICDA Impairs Ig Class Switching and Somatic Hypermutation in Human B Cells and is Associated with Autosomal Dominant HIGM2 Syndrome

B cells and their secreted antibodies are fundamental for host-defense against pathogens. The generation of high-affinity class switched antibodies results from both somatic hypermutation (SHM) of the immunoglobulin (Ig) variable region genes of the B-cell receptor and class switch recombination (CSR) which alters the Ig heavy chain constant region. Both of these processes are initiated by the enzyme activation-induced cytidine deaminase (AID), encoded by AICDA. Deleterious variants in AICDA are causal of hyper-IgM syndrome type 2 (HIGM2), a B-cell intrinsic primary immunodeficiency characterised by recurrent infections and low serum IgG and IgA levels. Biallelic variants affecting exons 2, 3 or 4 of AICDA have been identified that impair both CSR and SHM in patients with autosomal recessive HIGM2. Interestingly, B cells from patients with autosomal dominant HIGM2, caused by heterozygous variants (V186X, R190X) located in AICDA exon 5 encoding the nuclear export signal (NES) domain, show abolished CSR but variable SHM. We herein report the immunological and functional phenotype of two related patients presenting with common variable immunodeficiency who were found to have a novel heterozygous variant in AICDA (L189X). This variant led to a truncated AID protein lacking the last 10 amino acids of the NES at the C-terminal domain. Interestingly, patients’ B cells carrying the L189X variant exhibited not only greatly impaired CSR but also SHM in vivo, as well as CSR and production of IgG and IgA in vitro. Our findings demonstrate that the NES domain of AID can be essential for SHM, as well as for CSR, thereby refining the correlation between AICDA genotype and SHM phenotype as well as broadening our understanding of the pathophysiology of HIGM disorders.

Phage antibody library technology in tumor therapy: a review

Tumor is a serious threat to human health. At present, surgical resection, chemoradiotherapy, targeted therapy and immunotherapy are the main therapeutic strategies. Monoclonal antibody has gradually become an indispensable drug type in the clinical treatment of cancer due to its high efficiency and low toxicity. Phage antibody library technology (PALT) is a novel monoclonal antibody preparation technique. The recombinant immunoglobulin variable region of heavy chain (VH)/variable region of light chain (VL) gene is integrated into the phage vector, and the antibody is expressed on the phage surface in the form of fusion protein to obtain a diverse antibody library. Through the process of adsorption-elution-amplification, the antibody library can be screened to obtain the antibody molecule with specific binding antigen as well as its gene sequence. PALT has the advantages of short antibody production cycle, strong plasticity of antibody structure, large antibody yield, high diversity and direct production of humanized antibodies. It has been used in screening tumor markers and preparation of antibody drugs for breast cancer, gastric cancer, lung cancer and liver cancer. This article reviews the recent progress and the application of PALT in tumor therapy.

Rheumatoid arthritis specific autoimmunity in the lung before and at the onset of disease.

The lung is implicated as a site for breaching tolerance prior to onset of seropositive RA. To substantiate this, we have investigated lung-resident B cells in bronchoalveolar lavage (BAL) samples from early untreated RA patients (n=9) and anti-citrullinated protein antibody (ACPA)-positive individuals at risk of developing RA (n=3). Single B cells (n=7680) were phenotyped and isolated from BAL of individuals during the risk-RA phase and at RA diagnosis. The Immunoglobulin variable region transcripts were sequenced and selected for expression as monoclonal antibodies (n=141). Monoclonal ACPAs were tested for reactivity patterns and binding to neutrophils. By our single cell approach, we found significantly increased proportions of B lymphocytes in autoantibody-positive as compared to negative individuals. Memory and double negative (DN) B cells were prominent in all subgroups. Upon antibody re-expression, seven highly mutated citrulline autoreactive clones originating from different memory B cell subsets where identified, both in at-risk and early RA patients. Lung IgG variable gene transcripts from ACPA-positive individuals carry frequent mutation-induced N-linked Fab glycosylation sites (p<0.001), often in the framework-3 of the variable region. Two of the lungs ACPAs bound to activated neutrophils, one from an at-risk individual and one from early RA. We conclude that T cell driven B cell differentiation, resulting in local class switching and somatic hypermutation are evident in lungs before, as well as in, early stages of ACPA-positive RA. Our findings add to the notion of lung mucosa being a site for initiation of the citrulline autoimmunity preceding seropositive RA. This article is protected by copyright. All rights reserved.

Abstract 5328: Novel KRAS-specific in-well lysis ELISA for high throughput screening

Abstract RAS is one of the most frequently mutated genes in cancer. Gain–of–function missense mutations promoting oncogenesis cluster at codons 12, 13, and 61 of KRAS, NRAS and HRAS. These result in enhanced GTP binding due to fast exchange of nucleotide and/or impairment of GAP (GTPase Activating Protein) binding. Although these mutants are all activating, they are not equal in their oncogenic potential and differences in patient survival are associated with different RAS mutants and which RAS allele is affected, with KRAS being the most prevalent. Current RAS therapeutic approaches have migrated to be isoform specific. With this in mind, we have developed an activation state specific high throughput screening compatible (HTS) assay that is specific for KRAS. A novel rabbit anti–KRAS recombinant antibody was isolated via cloning of immunoglobulin variable regions from antigen positive B cells isolated from rabbits immunized with a peptide immunogen targeting a region of low homology between the RAS isoforms. Antibody specificity was validated with recombinant HRAS, KRAS and NRAS proteins over a wide concentration range by both immunoblotting and by a bead–based multiplex ELISA where all three RAS isoforms were present. The new assay allows for direct in–well lysis of cells cultured in 96–well plate format, either directly in media or following a PBS wash step. Activated KRAS in the lysate is captured on the assay plate and detected via chemiluminescence using the KRAS specific antibody. Preliminary assay validation has included EGF treatment of HeLa cells to show activation in cells expressing wildtype KRAS and inhibition with the selective non–covalent KRASG12D inhibitor MRTX1133 in Aspc1 cells known to express KRASG12D over a wide range of cell seeding densities. We anticipate that this assay will further the discovery of novel KRAS specific therapeutics. Citation Format: Hyun Min Park, Jason Poole, Mary Anne Jelinek. Novel KRAS-specific in-well lysis ELISA for high throughput screening. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5328.

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children.

Isolated anti-Ro52 identifies a severe subset of Sjögren's syndrome patients.

Serum autoantibodies targeting the SSA/Ro proteins are a key component of the classification criteria for the diagnosis of Sjögren's syndrome (SS). Most patients' serum reacts with both Ro60 and Ro52 proteins. Here we compare the molecular and clinical characteristics of patients diagnosed with SS with anti-Ro52 in the presence or absence of anti-Ro60/La autoantibodies. A cross-sectional study was performed. Patients in the SS biobank at Westmead Hospital (Sydney, Australia) that were positive for anti-Ro52 were included and stratified based on the absence (isolated) or presence (combined) of anti-Ro60/La, measured by line immunoassay. We examined clinical associations and the serological and molecular characteristics of anti-Ro52 using ELISA and mass spectrometry in serological groups. A total of 123 SS patients were included for study. SS patients with isolated anti-Ro52 (12%) identified a severe serological subset characterised by higher disease activity, vasculitis, pulmonary involvement, rheumatoid factor (RhF) and cryoglobulinaemia. Serum antibodies reacting with Ro52 in the isolated anti-Ro52 subset displayed less isotype switching, less immunoglobulin variable region subfamily usage and a lower degree of somatic hypermutation than the combined anti-Ro52 subset. In our cohort of SS patients, isolated anti-Ro52 represents a severe subset of SS, and is associated with the presence of cryoglobulinaemia. We therefore provide clinical relevance to the stratification of SS patients by their sero-reactivities. It is possible that the autoantibody patterns may be immunological epiphenomena of the underlying disease process, and further work is required to unearth the mechanisms of the differential clinical phenotypes.

OBINUTUZUMAB FOR THE TREATMENT OF CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS: SINGLE CENTER EXPERIENCE

Monoclonal antibodies (mAbs) targeting CD20 molecule on B lymphocyte are of great importance in the treatment of B-cell malignancies. In recent years a great effort has been put in developing novel mAbs that can provide greater efficacy than the well-known rituximab. A second class of mAbs obinutuzumab has been presented as a more powerful tool in the treatment of this group of patients. In this retrospective study 70 patients with symptomatic CLL were included. CLL patients were diagnosed and treated at the University Clinic for Hematology between January 2018 and January 2022. All patients were evaluated for traditional clinical and laboratory prognostic factors and newer prognostic factors including IGHV mutation status and CLL prognostic and predictive genetic abnormalities. Most of the patients treated with obinutuzumab had Binet B stage (56%). Mutational status of the immunoglobulin variable region heavy chain (IGHV) in most of CLL patients treated with obinutuzumab was unmated IGHV gene. The most frequently encountered adverse events were tumor lysis syndrome and leukopenia. The analysis of the initial results of the application of obinutuzumab-based therapy allows us to conclude that this therapeutical modality is not associated with severe adverse events that would limit the administration of therapy.

Ancestral reconstruction reveals catalytic inactivation of activation-induced cytidine deaminase concomitant with cold water adaption in the Gadiformes bony fish

BackgroundAntibody affinity maturation in vertebrates requires the enzyme activation-induced cytidine deaminase (AID) which initiates secondary antibody diversification by mutating the immunoglobulin loci. AID-driven antibody diversification is conserved across jawed vertebrates since bony and cartilaginous fish. Two exceptions have recently been reported, the Pipefish and Anglerfish, in which the AID-encoding aicda gene has been lost. Both cases are associated with unusual reproductive behavior, including male pregnancy and sexual parasitism. Several cold water fish in the Atlantic cod (Gadinae) family carry an aicda gene that encodes for a full-length enzyme but lack affinity-matured antibodies and rely on antibodies of broad antigenic specificity. Hence, we examined the functionality of their AID.ResultsBy combining genomics, transcriptomics, immune responsiveness, and functional enzymology of AID from 36 extant species, we demonstrate that AID of that Atlantic cod and related fish have extremely lethargic or no catalytic activity. Through ancestral reconstruction and functional enzymology of 71 AID enzymes, we show that this enzymatic inactivation likely took place relatively recently at the emergence of the true cod family (Gadidae) from their ancestral Gadiformes order. We show that this AID inactivation is not only concordant with the previously shown loss of key adaptive immune genes and expansion of innate and cell-based immune genes in the Gadiformes but is further reflected in the genomes of these fish in the form of loss of AID-favored sequence motifs in their immunoglobulin variable region genes.ConclusionsRecent demonstrations of the loss of the aicda gene in two fish species challenge the paradigm that AID-driven secondary antibody diversification is absolutely conserved in jawed vertebrates. These species have unusual reproductive behaviors forming an evolutionary pressure for a certain loss of immunity to avoid tissue rejection. We report here an instance of catalytic inactivation and functional loss of AID rather than gene loss in a conventionally reproducing vertebrate. Our data suggest that an expanded innate immunity, in addition to lower pathogenic pressures in a cold environment relieved the pressure to maintain robust secondary antibody diversification. We suggest that in this unique scenario, the AID-mediated collateral genome-wide damage would form an evolutionary pressure to lose AID function.

DC-SIGN Binding to the Oligomannose-Type Glycans on the Surface Immunoglobulin Variable Region of Follicular Lymphoma Promotes Selective Adhesion to Microenvironmental VCAM-1

The selective acquisition of oligomannose-type glycans at the antigen-binding site of the surface immunoglobulin (sIg-Mann) is a tumor-specific post-translational modification of all follicular lymphomas (FL). Our immunogenetic and structural analyses have revealed that these oligomannoses at N-glycosylation sites are universally acquired and persist during the entire natural history of FL from the early stages throughout transformation into EZB lymphoma (Chiodin G, Blood 2021). If a FL cell loses these sites, which is rare, the progeny disappears unless a new glycosylation site is acquired (Odabashian M, Blood 2020). These data indicate that sIg-Mann is a fundamental functional requirement for the entire clonal population of all FLs. The low-affinity interaction of sIg-Mann with its specific ligand dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), expressed on interfollicular M2-macrophages and follicular dendritic cells (FDC) of FL (RadtkeA, BioRxiv, 2022), distinguishes from conventional high-affinity antigen:Ig protein interactions for promoting prolonged low-level growth and prosurvival signals via AKT and MYC and not endocytosis (Linley A, Blood 2015). However, the function of DC-SIGN:Ig-Mann remains to be further elucidated. In this study, we investigated the hypothesis that the specific engagement of DC-SIGN:Ig-Mann interaction functions to promote homing, while maintaining selective growth and survival signals. We found that DC-SIGN induced adhesion of sIg-Mann+ve lymphoma cells to VCAM-1 and inhibited migration towards SDF-1 in vitro. Blocking of DC-SIGN with an anti-DC-SIGN antibody specific to the carbohydrate-recognition domain completely abrogated DC-SIGN-induced adhesion, confirming that adhesion was mediated by the selective interaction with the oligomannose of sIg-Mann. Although intracellular signals were significantly lower than those by soluble anti-IgM F(ab)2 (here used to mimic the binding of cross-linking polyvalent antigen to Ig protein), the level of adhesion induced by DC-SIGN was similar to that induced by anti-IgM F(ab)2. Adhesion to VCAM-1 was observed at concentrations from 20 µg/ml down to 20 ng/ml, even when AKT and ERK phosphorylation was not detectable by immunoblotting. However, either proximal inhibition of the PI3K/AKT pathway or distal inhibition of ARP2/3, formin, Rac and Cdc42 for lamellipodium formation at the surface membrane, suppressed adhesion. This suggested that Ig-Mann-mediated adhesion required both low-level intracellular signals and membrane adaptation (Valle-Argos, Sci Rep 2021). Remarkably, unlike anti-IgM F(ab)2, adhesion was specific to VCAM-1 but not to other stromal elements including fibronectin (FN). Instead, using the sIg-Mann+ve (IGHV4-34+ve) WSU-FSCCL cells, DC-SIGN appeared to mimic the interaction of monovalent ligands, including Fab fragments to either the Ig constant (anti-IgM Fab) or Ig variable region (9G4 Fab, which recognizes an epitope in the framework region 1 of IGHV4-34). Like DC-SIGN, Fab fragments induced selective adhesion to VCAM-1 and low-level signals via AKT, but neither endocytosis nor adhesion to FN. These results identify an important functional role of the DC-SIGN:sIg-Mann in mediating a signal that is tuned at the right threshold to promote selective and, in the absence of Ig endocytosis, persistent adhesion to environmental VCAM-1, determining homing of the FL tumor cells in the lymph node. This tumor-specific antigen-independent low-affinity DC:SIGN-Ig-Mann interaction contrasts to conventional high-affinity antigen:Ig protein interaction, which is prevented in FL (Schneider D, Blood 2015) and would instead lead to endocytosis and death. Therapeutic interruption of the interaction of DC-SIGN+ve cells with sIg-Mann+ve FL cells would provide a new way to displace the tumor cells from their prosurvival microenvironment.

Prevalence of Acquired N-Glycosylation Sites at the Single Cell Level in Follicular Lymphoma

Follicular lymphoma (FL), a malignancy derived from germinal center B cells, is characterized by ongoing somatic hypermutation of the immunoglobulin variable region (IGV) genes. This somatic hypermutation can introduce novel N-glycosylation sites (N-sites), consisting of Asn-X-Ser/Thr motifs, in the IGV resulting in the addition of glycans at these sites. Binding of lectins, such as DC-SIGN, to these glycosylated surface immunoglobulins (i.e., B cell receptors) activates the B cell receptor pathway in an antigen-independent manner, promoting FL B cell survival and proliferation. Previous studies have concluded that the acquisition of N-sites is a stable and clonal event during FL evolution (Odabashian et al., Blood 2020). Here, we investigated the dynamics and potential impact of N-site prevalence at a single cell level, in paired heavy and light chains, across sites and over time in 16 patients with FL. We profiled tumor fine needle aspirates from patients enrolled in two clinical trials (NCT02927964, NCT03410901). 15 of 16 patients were previously, but not recently treated, while one was previously untreated. Samples were collected from different tumor sites before treatment, on-treatment, and at tumor progression. Single cell suspensions were subjected to single cell B cell receptor (scBCR) and RNA (scRNA) sequencing using the 10X chromium platform. Only BCR sequences belonging to patient-specific dominant malignant heavy and light chain clones were considered for further analysis. In total, we analyzed more than 250,000 FL tumor cells from 89 tumor samples from 16 FL patients. While most patients acquired at least one N-site as an early and stable event that was conserved across distinct tumor sites and over time, 3 patient cases proved to be exceptions: one patient had no detectable N-sites in either the heavy or light chain IGV genes at any tumor site or timepoint. In a second patient, only one of two tumor sites exhibited a novel N-site, and this was probably acquired after divergence of the tumor populations to distinct tumor sites (Haebe, Shree et al., Blood 2021). Interestingly, clinical tumor progression in this patient arose from selection and expansion of an N-site negative subclone, despite the putative survival advantage conferred by acquired N-sites. In the third patient, N-sites were acquired in a precursor cell before subclonal divergence, but subsequently overtaken by an N-site-negative subpopulation, which comprised up to 90% of tumor cells at a given site (Figure 1A). These results suggest that other acquired features can out-compete the selective advantage of N-glycosylation. To elucidate biological differences between cells with and without N-sites within each sample, we integrated scBCR and scRNA data for the third patient using shared cell barcodes. N-site positive cells exhibited a higher expression of the BCR pathway and inflammatory response, while cells without N-sites had higher activity of metabolic processes, including oxidative phosphorylation and glycolysis (Figure 1B). In conclusion, while acquired N-sites likely support FL pathogenesis through antigen-independent BCR signaling in many FL patients, selection and expansion of N-site negative cells in some patients in our study suggests that N-site negative cells can thrive, and likely depend on other pathways, with our preliminary results pointing to differential metabolic regulation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ASSOCIATION OF THE FREQUENCY OF SF3B1 GENE MUTATIONS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS WITH THE STRUCTURE OF THE B-CELL RECEPTOR

Introduction. The course of chronic lymphocytic leukemia (CLL) varied from indolent to rapidly progressive. Stratification of CLL patients into risk groups is based on the stage of disease, the mutational status of immunoglobulin variable region genes (IGHV genes), and mutations of ТР53, NOTCH1, and SF3B1 genes. The frequency of SF3B1 gene mutations increases significantly in the progression of CLL. Aim: to determine the group of CLL patients with increased risk of developing SF3B1 mutations. Material and methods. Analysis was performed on the group of 261 CLL patients. The SF3B1 mutations and IGHV gene mutational status were studied by polymerase chain reaction (PCR) followed by direct sequencing. Results. SF3B1 mutations were identified in 30 patients (11.5%). The frequency of mutations did not differ among primary patients (8.6%) and patients examined before the start of the first line of therapy (9.9%) but increased in relapsed patients (23.8%), p = 0,01. In cases with the expression of IGHV3-21, IGHV3-30, IGHV4-59, and IGHV4-61 genes, regardless of their mutational status, the risk of developing SF3B1 mutations increased 4.97 times (95% CI = 2.21–11.19; p = 0.000047). In addition, stereotypy of the B-cell receptor (cluster #2), the risk of SF3B1 gene mutations increased in 8.65 times (95% CI = 1.66–45.05; p = 0.0027). Conclusion. The expression of individual IGHV genes and the configuration of the B-cell receptor are significant measure the probability of occurrence of SF3B1 gene mutations. Key words: chronic lymphocytic leukemia, SF3B1 gene mutations, IGHV genes, B-cell receptor stereotype

IgTreeZ, A Toolkit for Immunoglobulin Gene Lineage Tree-Based Analysis, Reveals CDR3s Are Crucial for Selection Analysis.

Somatic hypermutation (SHM) is an important diversification mechanism that plays a part in the creation of immune memory. Immunoglobulin (Ig) variable region gene lineage trees were used over the last four decades to model SHM and the selection mechanisms operating on B cell clones. We hereby present IgTreeZ (Immunoglobulin Tree analyZer), a python-based tool that analyses many aspects of Ig gene lineage trees and their repertoires. Using simulations, we show that IgTreeZ can be reliably used for mutation and selection analyses. We used IgTreeZ on empirical data, found evidence for different mutation patterns in different B cell subpopulations, and gained insights into antigen-driven selection in corona virus disease 19 (COVID-19) patients. Most importantly, we show that including the CDR3 regions in selection analyses - which is only possible if these analyses are lineage tree-based - is crucial for obtaining correct results. Overall, we present a comprehensive lineage tree analysis tool that can reveal new biological insights into B cell repertoire dynamics.

Characterization of SARS-CoV-2 Escape Mutants to a Pair of Neutralizing Antibodies Targeting the RBD and the NTD.

Mutations in the spike protein of SARS-CoV-2 can lead to evasion from neutralizing antibodies and affect the efficacy of passive and active immunization strategies. Immunization of mice harboring an entire set of human immunoglobulin variable region gene segments allowed to identify nine neutralizing monoclonal antibodies, which either belong to a cluster of clonally related RBD or NTD binding antibodies. To better understand the genetic barrier to emergence of SARS-CoV-2 variants resistant to these antibodies, escape mutants were selected in cell culture to one antibody from each cluster and a combination of the two antibodies. Three independently derived escape mutants to the RBD antibody harbored mutations in the RBD at the position T478 or S477. These mutations impaired the binding of the RBD antibodies to the spike protein and conferred resistance in a pseudotype neutralization assay. Although the binding of the NTD cluster antibodies were not affected by the RBD mutations, the RBD mutations also reduced the neutralization efficacy of the NTD cluster antibodies. The mutations found in the escape variants to the NTD antibody conferred resistance to the NTD, but not to the RBD cluster antibodies. A variant resistant to both antibodies was more difficult to select and only emerged after longer passages and higher inoculation volumes. VOC carrying the same mutations as the ones identified in the escape variants were also resistant to neutralization. This study further underlines the rapid emergence of escape mutants to neutralizing monoclonal antibodies in cell culture and indicates the need for thorough investigation of escape mutations to select the most potent combination of monoclonal antibodies for clinical use.

Safety and Immunogenicity of Combined DNA-Polyethylenimine and Oral Bacterial Idiotypic Vaccine for Patients with B-Cell Non-Hodgkin Lymphoma: A Pilot Study.

Simple SummaryImmunoglobulin variable domains, or idiotypes, have been used as lymphoma-specific antigens for therapeutic vaccination against B-cell lymphomas in a number of clinical trials. The effectiveness of DNA vaccines significantly depends on the chosen method of DNA delivery. In this study, we applied the intramuscular injection of a DNA–PEI vaccine followed by an oral vaccine-carrying Salmonella boost for lymphoma patients, which was safe and well tolerated. The observed remission was accompanied by T-cell but not an antibody response to the vaccine in most of the patients.We report, in brief, the results of a phase I, non-randomized study of idiotypic DNA vaccination in patients with B-cell non-Hodgkin’s lymphoma (ISRCTN31090206). The DNA sequence of lymphoma-derived immunoglobulin variable regions was used as a tumor-specific antigen fused to the potato virus X coat protein. A conjugate of plasmid DNA with polyethylenimine was used for the intramuscular injections, followed by a boost with an oral live-attenuated Salmonella vaccine carrying the same plasmid. The patients with a complete or partial response to previous chemotherapy received one or two courses of vaccination, including four injections at monthly intervals. The vaccine was well tolerated, with low-grade adverse events. The T-cell immune responses were assessed by ELISpot, at last vaccine, one week and one month post-vaccination, and were detected in 11/14 (78.6%) of the patients. In cases of progression requiring chemotherapy, or the presence of a positive MRD after the first course of vaccination, the patients underwent a second course of vaccination. At the end point, 6/19 vaccinated patients had disease stabilization, while 13/19 were in complete remission. The overall survival was 100% at follow-up, of a median of 2.3 years.

Development of a fully human anti-GITR antibody with potent antitumor activity using H2L2 mice.

Glucocorticoid‐induced TNF receptor‐related (GITR) can act as a co‐stimulatory receptor, representing a potential target for safely enhancing immunotherapy efficacy. GITR is triggered by a GITR ligand or an agonist antibody and activates CD8+ and CD4+ effector T cells, reducing tumor‐infiltrating Treg numbers and resulting in activation of immune responses and tumor cell destruction by effector T cells. GITR is an attractive target for immunotherapy, especially in combination therapy with immune checkpoint inhibitors, as is being explored in clinical trials. Using H2L2 transgenic mice encoding the human immunoglobulin variable region and hybridoma technology, we generated a panel of fully human antibodies that showed excellent specific affinity and strong activation of human T cells. After conversion to fully human antibodies and engineering modification, we obtained an anti‐GITR antibody hab019e2 with enhanced antitumor activity in a B‐hGITR MC38 mouse model compared to Tab9H6V3, an anti‐GITR antibody that activates T cells and inhibits Treg suppression from XenoMouse. As a fully human antibody with its posttranslational modification hot spot removed, the hab019e2 antibody exerted more potent therapeutic effects, and may have potential as a novel and developable antibody targeting GITR for follow‐up drug studies.

Biological and Clinical Insight from Analysis of the Tumor B-Cell Receptor Structure and Function in Chronic Lymphocytic Leukemia.

Simple SummaryThe goal of this review is to describe the biological and clinical implications associated with the analysis of the immunoglobulin (also known as B-cell receptor) expressed on the surface of the tumor cells of chronic lymphocytic leukemia (CLL). Analysis of the surface immunoglobulin structure, levels, and signaling characteristics has regularly improved our understanding of this leukemia for the last +20 years since the identification of two subsets with unmutated tumor immunoglobulin (U-CLL) and bad prognosis or mutated immunoglobulins (M-CLL) and good prognosis. In this review, the authors summarize how analysis of the tumor immunoglobulin informs origin, maintenance, progression, current therapy choice, and prognosis of CLL while providing clues for future investigations.The B-cell receptor (BCR) is essential to the behavior of the majority of normal and neoplastic mature B cells. The identification in 1999 of the two major CLL subsets expressing unmutated immunoglobulin (Ig) variable region genes (U-IGHV, U-CLL) of pre-germinal center origin and poor prognosis, and mutated IGHV (M-CLL) of post-germinal center origin and good prognosis, ignited intensive investigations on structure and function of the tumor BCR. These investigations have provided fundamental insight into CLL biology and eventually the mechanistic rationale for the development of successful therapies targeting BCR signaling. U-CLL and M-CLL are characterized by variable low surface IgM (sIgM) expression and signaling capacity. Variability of sIgM can in part be explained by chronic engagement with (auto)antigen at tissue sites. However, other environmental elements, genetic changes, and epigenetic signatures also contribute to the sIgM variability. The variable levels have consequences on the behavior of CLL, which is in a state of anergy with an indolent clinical course when sIgM expression is low, or pushed towards proliferation and a more aggressive clinical course when sIgM expression is high. Efficacy of therapies that target BTK may also be affected by the variable sIgM levels and signaling and, in part, explain the development of resistance.

Tracking Immunoglobulin Repertoire and Transcriptomic Changes in Germinal Center B Cells by Single-Cell Analysis.

In response to T-cell-dependent antigens, mature B cells in the secondary lymphoid organs are stimulated to form germinal centers (GCs), which are histological structures deputed to antibody affinity maturation, a process associated with immunoglobulin gene editing by somatic hypermutation (SHM) and class switch recombination (CSR). GC B cells are heterogeneous and transition across multiple stages before being eliminated by apoptosis or committing to post-GC differentiation as memory B cells or plasma cells. In order to explore the dynamics of SHM and CSR during the GC reaction, we identified GC subpopulations by single-cell (sc) transcriptomics and analyzed the load of immunoglobulin variable (V) region mutations as well as the isotype class distribution in each subpopulation. The results showed that the large majority of GC B cells display a quantitatively similar mutational load in the V regions and analogous IGH isotype class distribution, except for the precursors of memory B cells (PreM) and plasma cells (PBL). PreM showed a bimodal pattern with about half of the cells displaying high V region germline identity and enrichment for unswitched IGH, while the rest of the cells carried a mutational load similar to the bulk of GC B cells and showed a switched isotype. PBL displayed a bias toward expression of IGHG and higher V region germline identity compared to the bulk of GC B cells. Genes implicated in SHM and CSR were significantly induced in specific GC subpopulations, consistent with the occurrence of SHM in dark zone cells and suggesting that CSR can occur within the GC.

Deep learning model of somatic hypermutation reveals importance of sequence context beyond hotspot targeting

SummaryB cells undergo somatic hypermutation (SHM) of the Immunoglobulin (Ig) variable region to generate high-affinity antibodies. SHM relies on the activity of activation-induced deaminase (AID), which mutates C>U preferentially targeting WRC (W=A/T, R=A/G) hotspots. Downstream mutations at WA Polymerase η hotspots contribute further mutations. Computational models of SHM can describe the probability of mutations essential for vaccine responses. Previous studies using short subsequences (k-mers) failed to explain divergent mutability for the same k-mer. We developed the DeepSHM (Deep learning on SHM) model using k-mers of size 5–21, improving accuracy over previous models. Interpretation of DeepSHM identified an extended WWRCT motif with particularly high mutability. Increased mutability was further associated with lower surrounding G content. Our model also discovered a conserved AGYCTGGGGG (Y=C/T) motif within FW1 of IGHV3 family genes with unusually high T>G substitution rates. Thus, a wider sequence context increases predictive power and identifies features that drive mutational targeting.