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This article discusses the association between cytotoxic effector memory CD8+ T cells and sustained unresponsiveness (SU) after peanut oral immunotherapy (OIT). The study found that a higher frequency of effector memory CD8+ T cells was associated with a higher likelihood of achieving SU. The researchers also identified differentially expressed immune genes and observed that cytolytic molecules such as Granulysin (Gnly) and Granzyme B (GzB) may play a supportive role in the development of SU. However, the study acknowledges the need for further validation using larger cohorts. [Extracted from the article]

A diverse antibody repertoire is essential for humoral immunity. Antibody diversification requires the introduction of deoxyuridine (dU) mutations within immunoglobulin genes to initiate somatic hypermutation (SHM) and class switch recombination (CSR). dUs are normally recognized and excised by the base excision repair (BER) protein uracil-DNA glycosylase 2 (UNG2). However, FAM72A downregulates UNG2 permitting dUs to persist and trigger SHM and CSR. How FAM72A promotes UNG2 degradation is unknown. Here, we show that FAM72A recruits a C-terminal to LisH (CTLH) E3 ligase complex to target UNG2 for proteasomal degradation. Deficiency in CTLH complex components result in elevated UNG2 and reduced SHM and CSR. Cryo-EM structural analysis reveals FAM72A directly binds to MKLN1 within the CTLH complex to recruit and ubiquitinate UNG2. Our study further suggests that FAM72A hijacks the CTLH complex to promote mutagenesis in cancer. These findings show that FAM72A is an E3 ligase substrate adaptor critical for humoral immunity and cancer development. Antibody diversification relies on the intentional mutagenesis of immunoglobulin genes for adaptive immune responses. Here, the authors identified a CTLH E3 ubiquitin ligase complex that co-opts FAM72A to recruit and degrade the UNG2 base excision repair factor to permit mutagenesis. [ABSTRACT FROM AUTHOR]

This article explores missed opportunities in the development of monoclonal antibodies using mice and hybridoma technology. It emphasizes the importance of antigen presentation and the role of major histocompatibility complex (MHC) antigens in determining immunogenicity. The article suggests that using a diverse range of mouse strains or genetically engineered mouse models (GEMMs) could enhance antibody production. It also highlights the success of monoclonal antibody development in mice and proposes alternative approaches to generate a greater number of antibodies for diagnostic and therapeutic purposes. The authors clarify that their views are their own and do not represent their affiliated organizations or the publisher. [Extracted from the article]

Introduction: In systemic sclerosis (SSc), B-cells are activated and present in the skin and lung of patients where they can interact with fibroblasts. The precise impact and mechanisms of the interaction of B-cells and fibroblasts at the tissular level are poorly studied. Objective: We investigated the impact and mechanisms of B-cell/fibroblast interactions in cocultures between B-cells from patients with SSc and 3-dimensional reconstituted healthy skin model including fibroblasts, keratinocytes and extracellular matrix. Methods: The quantification and description of the B-cell infiltration in 3D cocultures were performed using cells imagery strategy and cytometry. The effect of coculture on the transcriptome of B-cells and fibroblasts was studied with bulk and single-cell RNA sequencing approaches. The mechanisms of this interaction were studied by blocking key cytokines like IL-6 and TNF. Results: We showed a significant infiltration of B-cells in the 3D healthy skin model. The amount but not the depth of infiltration was higher with B-cells from SSc patients and with activated B-cells. B-cell infiltrates were mainly composed of naïve and memory cells, whose frequencies differed depending on B-cells origin and activation state: infiltrated B-cells from patients with SSc showed an activated profile and an overexpression of immunoglobulin genes compared to circulating B-cells before infiltration. Our study has shown for the first time that activated B-cells modified the transcriptomic profile of both healthy and SSc fibroblasts, toward a pro-inflammatory (TNF and IL-17 signaling) and interferon profile, with a key role of the TNF pathway. Conclusion: B-cells and 3D skin cocultures allowed the modelization of B-cells infiltration in tissues observed in SSc, uncovering an influence of the underlying disease and the activation state of B-cells. We showed a pro-inflammatory effect on skin fibroblasts and pro-activation effect on infiltrating B-cells during coculture. This reinforces the role of B-cells in SSc and provide potential targets for future therapeutic approach in this disease. [ABSTRACT FROM AUTHOR]

Minimal residual disease (MRD) assessment is a known surrogate marker for survival in multiple myeloma (MM). Here, we present a single institution's experience assessing MRD by NGS of Ig genes and the long-term impact of depth of response as well as clonal diversity on the clinical outcome of a large population of MM patients; 482 MM patients at the University of California, San Francisco (UCSF) diagnosed from 2008 to 2020 were analyzed retrospectively. MRD assessment was performed by NGS. PFS curves were plotted by the Kaplan–Meier method. In the newly diagnosed group, 119 of 304, achieved MRD negativity at the level of 10−6 at least once. These patients had a prolonged PFS versus patients who were persistently MRD positive at different levels (p > 0.0001). In the relapsed disease group, 64 of 178 achieved MRD negativity at 10−6, and PFS was prolonged versus patients who remained MRD positive (p = 0.03). Three categories of MRD dynamics were defined by artificial intelligence: (A) patients with ≥3 consistently MRD negative samples, (B) patients with continuously declining but detectable clones, and (C) patients with either increasing or a stable number of clones. Groups A and B had a more prolonged PFS than group C (p < 10−7). Patients who were MRD positive and had not yet relapsed had a higher clonal diversity than those patients who were MRD positive and had relapsed. MRD dynamics can accurately predict disease evolution and drive clinical decision-making. Clonal Diversity could complement MRD assessment in the prediction of outcomes in MM. [ABSTRACT FROM AUTHOR]

Background: Diagnosis of primary diffuse large B-cell lymphoma of the central nervous system (PCNSL) is challenging and often delayed. MRI imaging, CSF cytology and flow cytometry have a low sensitivity and even brain biopsies can be misleading. We report three cases of PCNSL with various clinical presentation and radiological findings where the diagnosis was suggested by novel CSF biomarkers and subsequently confirmed by brain biopsy or autopsy. Case presentations. The first case is a 79-year-old man with severe neurocognitive dysfunction and static ataxia evolving over 5 months. Brain MRI revealed a nodular ventriculitis. An open brain biopsy was inconclusive. The second case is a 60-year-old woman with progressive sensory symptoms in all four limbs, evolving over 1 year. Brain and spinal MRI revealed asymmetric T2 hyperintensities of the corpus callosum, corona radiata and corticospinal tracts. The third case is a 72-year-old man recently diagnosed with primary vitreoretinal lymphoma of the right eye. A follow-up brain MRI performed 4 months after symptom onset revealed a T2 hyperintense fronto-sagittal lesion, with gadolinium uptake and perilesional edema. In all three cases, CSF flow cytometry and cytology were negative. Mutation analysis on the CSF (either by digital PCR or by next generation sequencing) identified the MYD88 L265P hotspot mutation in all three cases. A B-cell clonality study, performed in case 1 and 2, identified a monoclonal rearrangement of the immunoglobulin light chain lambda (IGL) and kappa (IGK) gene. CSF CXCL-13 and IL-10 levels were high in all three cases, and IL-10/IL-6 ratio was high in two. Diagnosis of PCNSL was later confirmed by autopsy in case 1, and by brain biopsy in case 2 and 3. Conclusions: Taken together, 5 CSF biomarkers (IL-10, IL-10/IL-6 ratio, CXCL13, MYD88 mutation and monoclonal IG gene rearrangements) were strongly indicative of a PCNSL. Using innovative CSF biomarkers can be sensitive and complementary to traditional CSF analysis and brain biopsy in the diagnosis of PCNSL, potentially allowing for earlier diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Introduction: Botulinum neurotoxins (BoNTs) cause botulism and are the most potent natural toxins known. Immunotherapy with neutralizing monoclonal antibodies (MAbs) is considered to be the most effective immediate response to BoNT exposure. Hybridoma technology remains the preferred method for producing MAbs with naturally paired immunoglobulin genes and with preserved innate functions of immune cells. The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against BoNTs. In an effort to develop novel BoNT type A (BoNT/A) immunotherapeutics, sorted by flow cytometry plasmablasts and activated memory B cells from a donor repeatedly injected with BoNT/A for aesthetic botulinum therapy could be used due to obtain hybridomas producing native antibodies. Methods: Plasmablasts and activated memory B-cells were isolated from whole blood collected 7 days after BoNT/A injection and sorted by flow cytometry. The sorted cells were then electrofused with the K6H6/B5 cell line, resulting in a producer of native human monoclonal antibodies (huMAbs). The 3 antibodies obtained were then purified by affinity chromatography, analyzed for binding by Western blot assay and neutralization by FRET assay. Results: We have succeeded in creating 3 hybridomas that secrete huMAbs specific to native BoNT/A and the proteolytic domain (LC) of BoNT/A. The 1B9 antibody also directly inhibited BoNT/A catalytic activity in vitro. Conclusion: The use activated plasmablasts and memory B-cells isolated at the peak of the immune response (at day 7 of immunogenesis) that have not yet completed the terminal stage of differentiation but have undergone somatic hypermutation for hybridization allows us to obtain specific huMAbs even when the immune response of the donor is weak (with low levels of specific antibodies and specific B-cells in blood). A BoNT/A LC-specific antibody is capable of effectively inhibiting BoNT/A by mechanisms not previously associated with antibodies that neutralize BoNT. Antibodies specific to BoNT LC can be valuable components of a mixture of antibodies against BoNT exposure. [ABSTRACT FROM AUTHOR]

Background: Microvascular inflammation (MVI) can occur in biopsies showing T‐cell mediated rejection (TCMR), but it is not well established that T‐cells can directly mediate microvascular injury (TCMR‐MVI). Methods: This was a cross sectional RNAseq based Banff Human Organ Transplant (BHOT) gene expression (GE) analysis. The objective of this study was to probe the molecular signature of TCMR‐MVI in comparison with C4d+, DSA+ antibody mediated rejection (ABMR), stable renal function (STA), and TCMR without MVI. Transcriptome analysis utilized CLC genomic workbench and R‐studio software. Results: No gene set was specific for any diagnostic category, and all were expressed at low levels in STA biopsies. BHOT gene set scores could differentiate ABMR from TCMR and TCMR‐MVI, but not TCMR from TCMR‐MVI. TCMR‐MVI underexpressed several genes associated with ABMR including DSATs, ENDAT, immunoglobulin genes, ADAMDEC1, PECAM1 and NK cell transcripts (MYBL1, GNLY), but overexpressed C3, NKBBIZ, and LTF. On the other hand, there was no significant difference in the expression of these genes in TCMR‐MVI versus TCMR. This indicates that the GE profile of TCMR MVI aligns more closely with TCMR than ABMR. The limitations of classifying biopsies using the binary ABMR‐TCMR algorithm, and the occurrence of common pathogenesis mechanisms amongst different rejection phenotype was highlighted by the frequent presence of molecular mixed rejection. Conclusions: T‐cell mediated mechanisms play a significant role in the pathogenesis of MVI. GE was broadly different between rejection phenotypes, but molecular scores varied substantially between biopsies with the same Banff grade. It was not always possible to achieve precise molecular score‐based diagnostic categorization of individual patients. [ABSTRACT FROM AUTHOR]

Background: The Atlantic cod is a prolific species in the Atlantic, despite its inconsistent specific antibody response. It presents a peculiar case within vertebrate immunology due to its distinct immune system, characterized by the absence of MHCII antigen presentation pathway, required for T cell-dependent antibody responses. Thorough characterisation of immunoglobulin loci and analysis of the antibody repertoire is necessary to further our understanding of the Atlantic cod's immune response on a molecular level. Results: A comprehensive search of the cod genome (gadmor3.0) identified the complete set of IgH genes organized into three sequential translocons on chromosome 2, while IgL genes were located on chromosomes 2 and 5. The Atlantic cod displayed a moderate germline V gene diversity, comprising four V gene families for both IgH and IgL, each with distinct chromosomal locations and organizational structures. 5'RACE sequencing revealed a diverse range of heavy chain CDR3 sequences and relatively limited CDR3 diversity in light chains. The analysis highlighted a differential impact of V-gene germline CDR3 length on receptor CDR3 length between heavy and light chains, underlining different recombination processes. Conclusions: This study reveals that the Atlantic cod, despite its inconsistent antibody response, maintains a level of immunoglobulin diversity comparable to other fish species. The findings suggest that the extensive recent duplications of kappa light chain genes do not result in increased repertoire diversity. This research provides a comprehensive view of the Atlantic cod's immunoglobulin gene organization and repertoire, necessary for future studies of antibody responses at the molecular level. [ABSTRACT FROM AUTHOR]

COVID‐19 induces re‐circulating long‐lived memory B cells (MBC) that, upon re‐encounter with the pathogen, are induced to mount immunoglobulin responses. During convalescence, antibodies are subjected to affinity maturation, which enhances the antibody binding strength and generates new specificities that neutralize virus variants. Here, we performed a single‐cell RNA sequencing analysis of spike‐specific B cells from a SARS‐CoV‐2 convalescent subject. After COVID‐19 vaccination, matured infection‐induced MBC underwent recall and differentiated into plasmablasts. Furthermore, the transcriptomic profiles of newly activated B cells transiently shifted toward the ones of atypical and CXCR3+ B cells and several B‐cell clonotypes massively expanded. We expressed monoclonal antibodies (mAbs) from all B‐cell clones from the largest clonotype that used the VH3‐53 gene segment. The in vitro analysis revealed that some somatic hypermutations enhanced the neutralization breadth of mAbs in a putatively stochastic manner. Thus, somatic hypermutation of B‐cell clonotypes generates an anticipatory memory that can neutralize new virus variants. [ABSTRACT FROM AUTHOR]

Measurable residual disease (MRD) testing, a standard procedure in B-lymphoblastic leukemia (B-ALL) diagnostics, is assessed using multiparametric flow cytometry (MFC) and next-generation sequencing (NGS) analysis of immunoglobulin gene rearrangements. We evaluated the concordance between eight-color, two-tube MFC-MRD the LymphoTrack NGS-MRD assays using 139 follow-up samples from 54 pediatric patients with B-ALL. We also assessed the effect of hemodilution in MFC-MRD assays. The MRD-concordance rate was 79.9% (N=111), with 25 (18.0%) and 3 (2.2%) samples testing positive only by NGSMRD (MFC-NGS+MRD) and MFC-MRD (MFC+NGS-MRD), respectively. We found a significant correlation in MRD values from total nucleated cells between the two methods (r=0.736 [0.647-0.806], P <0.001). The median MRD value of MFC-NGS+MRD samples was estimated to be 0.0012% (0.0001%-0.0263%) using the NGS-MRD assays. Notably, 14.3% of MFC-NGS+MRD samples showed NGS-MRD values below the limit of detection in the MFC-MRD assays. The percentages of hematogones detected in MFC-MRD assays significantly differed between the discordant and concordant cases (P <0.001). MFC and NGS-MRD assays showed relatively high concordance and correlation in MRD assessment, whereas the NGS-MRD assay detected MRD more frequently than the MFC-MRD assay in pediatric B-ALL. Evaluating the hematogone percentages can aid in assessing the impact of sample hemodilution. [ABSTRACT FROM AUTHOR]

Background: Assessment of measurable residual disease (MRD) is an essential prognostic tool for B-lymphoblastic leukaemia (B-ALL). In this study, we evaluated the utility of next-generation sequencing (NGS)–based MRD assessment in real-world clinical practice. Method: The study included 93 paediatric patients with B-ALL treated at our institution between January 2017 and June 2022. Clonality for IGH or IGK rearrangements was identified in most bone marrow samples (91/93, 97.8%) obtained at diagnosis. Results: In 421 monitoring samples, concordance was 74.8% between NGS and multiparameter flow cytometry and 70.7% between NGS and reverse transcription-PCR. Elevated quantities of clones of IGH alone (P < 0.001; hazard ratio [HR], 22.2; 95% confidence interval [CI], 7.1–69.1), IGK alone (P = 0.011; HR, 5.8; 95% CI, 1.5–22.5), and IGH or IGK (P < 0.001; HR, 7.2; 95% CI, 2.6–20.0) were associated with an increased risk of relapse. Detection of new clone(s) in NGS was also associated with inferior relapse-free survival (P < 0.001; HR, 18.1; 95% CI, 3.0–108.6). Multivariable analysis confirmed age at diagnosis, BCR::ABL1-like mutation, TCF3::PBX1 mutation, and increased quantity of IGH or IGK clones during monitoring as unfavourable factors. Conclusion: In conclusion, this study highlights the usefulness of NGS-based MRD as a routine assessment tool for prognostication of paediatric patients with B-ALL. [ABSTRACT FROM AUTHOR]

There is tremendous interindividual and interracial variability in the outcome of SARS-CoV-2 infection, suggesting the involvement of host genetic factors. Here, we investigated whether IgG allotypes GM (γ marker) 3 and GM 17, genetic markers of IgG1, contributed to the severity of COVID-19. IgG1 plays a pivotal role in response against SARS-CoV-2 infection. We also investigated whether these GM alleles synergistically/epistatically with IGHG3 and FCGR2A alleles—which have been previously implicated in COVID-19—modulated the extent of COVID-19 severity. The study population consisted of 316 COVID-19 patients who needed treatment in the intensive care unit of Hospital Universitario Central de Asturias. All individuals were genotyped for GM 3/17, IGHG3 hinge length, and FCGR2A rs1801274 A/G polymorphisms. Among the 316 critical patients, there were 86 deaths. The risk of death among critical patients was significantly higher in subjects with GM 17 (IgG1) and short hinge length (IgG3). GM 17-carriers were at almost three-fold higher risk of death than non-carriers (p < 0.001; OR = 2.86, CI 1.58–5.16). Subjects with short hinge length of IgG3 had a two-fold higher risk of death than those with medium hinge length (p = 0.01; OR = 2.16, CI 1.19–3.90). GM 3/3 and IGHG3 (MM) genotypes were less frequent among death vs. survivors (9% vs 36%, p < 0.001) and associated with protective effect (OR = 0.18, 95% CI = 0.08–0.39). This is the first report implicating IgG1 allotypes in COVID-19-spurred death. It needs to be replicated in an independent study population. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) demands urgent attention for the development of effective treatment strategies due to its aggressiveness and limited therapeutic options [1]. This research is primarily focused on identifying new biomarkers vital for immunotherapy, with the aim of developing tailored treatments specifically for TNBC, such as those targeting the PD-1/PD-L1 pathway. To achieve this, the study places a strong emphasis on investigating Ig genes, a characteristic of immune checkpoint inhibitors, particularly genes expressing Ig-like domains with altered expression levels induced by "cancer deformation," a condition associated with cancer malignancy. Human cells can express approximately 800 Ig family genes, yet only a few Ig genes, including PD-1 and PD-L1, have been developed into immunotherapy drugs thus far. Therefore, we investigated the Ig genes that were either upregulated or downregulated by the artificial metastatic environment in TNBC cell line. As a result, we confirmed the upregulation of approximately 13 Ig genes and validated them using qPCR. In summary, our study proposes an approach for identifying new biomarkers applicable to future immunotherapies aimed at addressing challenging cases of TNBC where conventional treatments fall short. [ABSTRACT FROM AUTHOR]

Public antibody responses have been found against many infectious agents. Structural convergence of public antibodies is usually determined by immunoglobulin V genes. Recently, a human antibody public class against SARS-CoV-2 was reported, where the D gene (IGHD3-22) encodes a common YYDxxG motif in heavy-chain complementarity-determining region 3 (CDR H3), which determines specificity for the receptor-binding domain (RBD). In this review, we discuss the isolation, structural characterization, and genetic analyses of this class of antibodies, which have been isolated from various cohorts of COVID-19 convalescents and vaccinees. All eleven YYDxxG antibodies with available structures target the SARS-CoV-2 RBD in a similar binding mode, where the CDR H3 dominates the interaction with antigen. The antibodies target a conserved site on the RBD that does not overlap with the receptor-binding site, but their particular angle of approach results in direct steric hindrance to receptor binding, which enables both neutralization potency and breadth. We also review the properties of CDR H3-dominant antibodies that target other human viruses. Overall, unlike most public antibodies, which are identified by their V gene usage, this newly discovered public class of YYDxxG antibodies is dominated by a D-gene-encoded motif and uncovers further opportunities for germline-targeting vaccine design. [ABSTRACT FROM AUTHOR]

Recent studies have demonstrated a role for Ten-Eleven Translocation-2 (TET2), an epigenetic modulator, in regulating germinal center formation and plasma cell differentiation in B-2 cells, yet the role of TET2 in regulating B-1 cells is largely unknown. Here, B-1 cell subset numbers, IgM production, and gene expression were analyzed in mice with global knockout of TET2 compared to wildtype (WT) controls. Results revealed that TET2-KO mice had elevated numbers of B-1a and B-1b cells in their primary niche, the peritoneal cavity, as well as in the bone marrow (B-1a) and spleen (B-1b). Consistent with this finding, circulating IgM, but not IgG, was elevated in TET2-KO mice compared to WT. Analysis of bulk RNASeq of sort purified peritoneal B-1a and B-1b cells revealed reduced expression of heavy and light chain immunoglobulin genes, predominantly in B-1a cells from TET2-KO mice compared to WT controls. As expected, the expression of IgM transcripts was the most abundant isotype in B-1 cells. Yet, only in B-1a cells there was a significant increase in the proportion of IgM transcripts in TET2-KO mice compared to WT. Analysis of the CDR3 of the BCR revealed an increased abundance of replicated CDR3 sequences in B-1 cells from TET2-KO mice, which was more clearly pronounced in B-1a compared to B-1b cells. V-D-J usage and circos plot analysis of V-J combinations showed enhanced usage of VH11 and VH12 pairings. Taken together, our study is the first to demonstrate that global loss of TET2 increases B-1 cell number and IgM production and reduces CDR3 diversity, which could impact many biological processes and disease states that are regulated by IgM. [ABSTRACT FROM AUTHOR]

Tertiary lymphoid structures (TLSs) are organized aggregates of immune cells in non-lymphoid tissues and are associated with a favorable prognosis in tumors. However, TLS markers remain inconsistent, and the utilization of machine learning techniques for this purpose is limited. To tackle this challenge, we began by identifying TLS markers through bioinformatics analysis and machine learning techniques. Subsequently, we leveraged spatial transcriptomic data from Gene Expression Omnibus (GEO) and built two support vector classifier models for TLS prediction: one without feature selection and the other using the marker genes. The comparable performances of these two models confirm the efficacy of the selected markers. The majority of the markers are immunoglobulin genes, demonstrating their importance in the identification of TLSs. Our research has identified the markers of TLSs using machine learning methods and constructed a model to predict TLS location, contributing to the detection of TLS and holding the promising potential to impact cancer treatment strategies. [ABSTRACT FROM AUTHOR]

PURPOSE: The estrogen receptor–positive (ER+) breast cancer (BC), which constitutes the majority of BC cases, exhibits highly heterogeneous clinical behavior. To aid precision treatments, we aimed to find molecular subtypes of ER+ BC representing the tumor microenvironment and prognosis. METHODS: We analyzed RNA-seq data of 113 patients with BC and classified them according to the PAM50 intrinsic subtypes using gene expression profiles. Among them, we further focused on 44 patients with luminal-type (ER+) BC for subclassification. The Cancer Genome Atlas (TCGA) data of patients with BC were used as a validation data set to verify the new classification. We estimated the immune cell composition using CIBERSORT and further analyzed its association with clinical or molecular parameters. RESULTS: Principal component analysis clearly divided the patients into two subgroups separately from the luminal A and B classification. The top differentially expressed genes between the subgroups were distinctly characterized by immunoglobulin and B-cell–related genes. We could also cluster a separate cohort of patients with luminal-type BC from TCGA into two subgroups on the basis of the expression of a B-cell–specific gene set, and patients who were predicted to have high B-cell immune activity had better prognoses than other patients. CONCLUSION: Our transcriptomic approach emphasize a molecular phenotype of B-cell immunity in ER+ BC that may help to predict disease prognosis. Although further researches are required, B-cell immunity for patients with ER+ BC may be helpful for identifying patients who are good responders to chemotherapy or immunotherapy. [ABSTRACT FROM AUTHOR]

Vaccine priming immunogens that activate germline precursors for broadly neutralizing antibodies (bnAbs) have promise for development of precision vaccines against major human pathogens. In a clinical trial of the eOD-GT8 60mer germline-targeting immunogen, higher frequencies of vaccine-induced VRC01-class bnAb-precursor B cells were observed in the high dose compared to the low dose group. Through immunoglobulin heavy chain variable (IGHV) genotyping, statistical modeling, quantification of IGHV1-2 allele usage and B cell frequencies in the naive repertoire for each trial participant, and antibody affinity analyses, we found that the difference between dose groups in VRC01-class response frequency was best explained by IGHV1-2 genotype rather than dose and was most likely due to differences in IGHV1-2 B cell frequencies for different genotypes. The results demonstrate the need to define population-level immunoglobulin allelic variations when designing germline-targeting immunogens and evaluating them in clinical trials. [ABSTRACT FROM AUTHOR]

Background: Bone marrow (BM) involvement is an indicator of a poor prognosis in diffuse large B-cell lymphoma (DLBCL); however, few studies have evaluated the role of immunoglobulin gene rearrangement (IgR) in detecting BM involvement. Methods: We evaluated the clinical characteristics and treatment outcomes of patients with DLBCL based on histological BM involvement or positive BM IgR using polymerase chain reaction or next-generation sequencing. We also investigated the role of consolidative upfront autologous hematopoietic stem cell transplantation (ASCT) in patients with DLBCL and BM involvement. Results: Among 624 patients, 123 (19.7%) with histological BM involvement and 88 (17.5%) with positive IgR in histologically negative BM had more advanced disease characteristics. Overall (OS) and progression-free (PFS) survival was better for patients with negative BM histology and negative IgR than that in patients with histological BM involvement (P = 0.050 and P < 0.001, respectively) and positive IgR with negative BM histology (P = 0.001 and P = 0.005, respectively). Survival rates did not differ among 82 (13.1%) patients who were treated with upfront ASCT and had histological BM involvement or positive IgR with negative BM histology. The survival outcomes were worse for patients who were not treated with upfront ASCT and for those with histological BM involvement or positive IgR, than for those with negative BM histology and negative IgR. Conclusion: Patients diagnosed with DLBCL and BM involvement based on histology or IgR had aggressive clinical features and poor survival. Upfront ASCT mitigated poor prognosis due to BM involvement. [ABSTRACT FROM AUTHOR]

Introduction: Analysis of an individual's immunoglobulin (IG) gene repertoire requires the use of high-quality germline gene reference sets. When sets only contain alleles supported by strong evidence, AIRR sequencing (AIRR-seq) data analysis is more accurate and studies of the evolution of IG genes, their allelic variants and the expressed immune repertoire is therefore facilitated. Methods: The Adaptive Immune Receptor Repertoire Community (AIRR-C) IG Reference Sets have been developed by including only human IG heavy and light chain alleles that have been confirmed by evidence from multiple high-quality sources. To further improve AIRR-seq analysis, some alleles have been extended to deal with short 3' or 5' truncations that can lead them to be overlooked by alignment utilities. To avoid other challenges for analysis programs, exact paralogs (e.g. IGHV1-69*01 and IGHV1-69D*01) are only represented once in each set, though alternative sequence names are noted in accompanyingmetadata. Results and discussion: The Reference Sets include less than half the previously recognised IG alleles (e.g. just 198 IGHV sequences), and also include a number of novel alleles: 8 IGHV alleles, 2 IGKV alleles and 5 IGLV alleles. Despite their smaller sizes, erroneous calls were eliminated, and excellent coverage was achieved when a set of repertoires comprising over 4 million V(D)J rearrangements from 99 individuals were analyzed using the Sets. The versiontracked AIRR-C IG Reference Sets are freely available at the OGRDB website (https://ogrdb.airr-community.org/germline_sets/Human) and will be regularly updated to include newly observed and previously reported sequences that can be confirmed by new high-quality data. [ABSTRACT FROM AUTHOR]

In this study, we evaluated the effects of three prebiotics (inulin, β-glucan, and chitosan) on the physiological performance of Totoaba macdonaldi juveniles under culture conditions. The respiratory burst and the leucocyte content were measured in the blood to assess innate immune responses. The intestinal digestive capacity was evaluated by analyzing trypsin, amylase, and lipase activities, whereas the effects of such prebiotics at the transcriptomic level were assessed by implementing the RNA-Seq of liver tissue. After 60 days, fish fed with 0.5% chitosan diets showed the highest respiratory burst, the lowest lipase activity, and the highest number of differentially expressed genes (DEGs), where biological processes related to proteolysis, digestion, and lipid hydroxylation were the most affected. In addition, fish from the chitosan diet showed the highest expression of immunoglobulin genes. In contrast, fish fed with the 1% inulin diet presented the highest diet digestibility and trypsin and lipase activities. These physiological effects align with the highest expression of trypsin-like and chymotrypsin-like genes in the liver of fish from this diet. On the other hand, fish fed the 0.1% β-glucan diets showed the lowest amount of DEGs compared to the control group, most of which were associated with immune response, with an up-regulation of genes related to the complement system and a downregulation of immunoglobulin genes. Based on our results, we propose the inclusion of 1% dietary inulin to improve the digestibility of experimental diets and the addition of 0.5% chitosan to stimulate the immune system of T. macdonaldi juveniles. [ABSTRACT FROM AUTHOR]

Antibody display library is a robust platform for the production of recombinant or monoclonal antibodies that are of great value in diagnostics and therapeutics. A naïve library constructed based on immunoglobulin M (IgM) can reflect the natural immunity of the animal and can offer broad antigen targets. As a natural host of various zoonotic pathogens, the Philippine carabao (Bubalus kerabau Fitzinger, 1860) offers great potential as a surrogate model for molecular biology and biomedical applications. However, studies on its antibody repertoire are very scarce, if not available. Hence, this study was undertaken to design and evaluate degenerate primers targeting the antigen-binding paratope of B. kerabau Fitzinger, 1860 IgM - specifically, lambda variable light chain (VLL), kappa variable light chain (VLK), and variable heavy chain (VH). These primers were mainly based on carabao's close relative in the Bovidae family, the cattle (Bos taurus). A total of 32 degenerate primers, 14 designed and 18 adapted from literature, were used to amplify the target antibody fragments using a high-fidelity DNA polymerase with a modified touchdown-polymerase chain reaction method. Results demonstrate the amplification of VLL, VLK, and VH genes derived from B-cells of carabao splenic tissues using the degenerate primers. The amplicons will be used in the construction of an antibody phage library to elucidate the IgM repertoire of the Philippine carabao. In addition, DNA sequencing surprisingly revealed longer VLL and VLK genes than VH genes in contrast to what is found in other bovines, suggesting a novel organization of immunoglobulin genes of B. kerabau Fitzinger, 1860 that merits further investigation. [ABSTRACT FROM AUTHOR]

Background: Nectin-4 is a novel biomarker overexpressed in various types of cancer, including breast cancer, in which it has been associated with poor prognosis. Current literature suggests that nectin-4 has a role in cancer progression and may have prognostic and therapeutic implications. The present study aims to produce nectin-4-specific single-chain variable fragment (scFv) antibodies and evaluate their applications in breast cancer cell lines and clinical specimens. Methods: We generated recombinant nectin-4 ectodomain fragments as immunogens to immunize chickens and the chickens' immunoglobulin genes were amplified for construction of anti-nectin-4 scFv libraries using phage display. The binding capacities of the selected clones were evaluated with the recombinant nectin-4 fragments, breast cancer cell lines, and paraffin-embedded tissue sections using various laboratory approaches. The binding affinity and in silico docking profile were also characterized. Results: We have selected two clones (S21 and L4) from the libraries with superior binding capacity. S21 yielded higher signals when used as the primry antibody for western blot analysis and flow cytometry, whereas clone L4 generated cleaner and stronger signals in immunofluorescence and immunohistochemistry staining. In addition, both scFvs could diminish attachment-free cell aggregation of nectin-4-positive breast cancer cells. As results from ELISA indicated that L4 bound more efficiently to fixed nectin-4 ectodomain, molecular docking analysis was further performed and demonstrated that L4 possesses multiple polar contacts with nectin-4 and diversity in interacting residues. Conclusion: Overall, the nectin-4-specific scFvs could recognize nectin-4 expressed by breast cancer cells and have the merit of being further explored for potential diagnostic and therapeutic applications. [ABSTRACT FROM AUTHOR]

A 12-week growth trial was conducted to assess the effects of mealworm meals, as a substitution for fishmeal, on the growth, physiobiochemical responses, digesta microbiome, and immune-related genes expression of Atlantic salmon (Salmo salar). Twenty Atlantic salmon parr (38.5 ± 0.1 g, initial weight) were stocked into each of 16 tanks in a recirculating aquaculture system. A fishmeal-based diet (100% FM) was used as the control treatment and was compared with three test diets where: (1) fishmeal was partially (50%) replaced with defatted mealworm meal, Tenebrio molitor (50% DMM), (2) fishmeal was fully replaced with defatted mealworm meal (100% DMM), and (3) fishmeal was partially replaced with whole lesser mealworm meal, Alphitobius diaperinus (50% WMM). All substitutions were done on a crude protein basis. Each of the four experimental diets was evaluated in quadruplicate tanks as part of randomized design. The results indicated that Atlantic salmon showed high survival (greater or equal to 98.8%), and no significant difference in final growth, feed efficiency, feces stability and condition indices. Hepatosomatic index was lower in fish fed 100% DMM and 50% WMM when compared to fish fed the control diet (100% FM). Whole-body proximate and amino acid compositions were not statistically different between treatments, while essential fatty acids, including linolenic, eicosapentaenoic acid, and homo-a-linolenic, were lower in fish fed 100% DMM. Plasma parameters (total protein, alanine aminotransferase, alkaline phosphatase, and total iron-binding capacity), hepatic peroxide, and antioxidant enzymes were not significantly affected by dietary substitutions, whereas plasma immunoglobulin M showed significantly higher levels in fish fed 50% DMM and 100% DMM when compared to fish fed the control diet (100% FM). The inclusion of mealworm meals significantly impacted the overall microbiome composition but not the richness and evenness of the salmon digesta microbiomes compared to control. The most common genus in all treatments was Pseudomonas, which has been previously shown to have both commensal and pathogenic members. The relative expressions of growth (IGF-I) and protein synthesis (TIPRL) were not significantly different between the treatments, whereas immunoglobulin genes (IgM, IgD, and IgT) were significantly upregulated in fish fed the DMM diets when compared to fish fed the control diet. Overall, this study suggests that the mealworm meals tested could be suitable alternatives to fishmeal in the diet of Atlantic salmon. [ABSTRACT FROM AUTHOR]

Lymphoepithelial sialadenitis (LESA), Sjögren's syndrome (SS), and salivary MALT lymphoma are diseases characterized by lymphoepithelial lesions, and the differential diagnosis between them in the salivary glands is challenging. This study aimed to explore clinicopathological and genetic characteristics of the three diseases. We retrospectively analyzed the clinical features, the histomorphology, immunohistochemistry, and genetic profiling by polymerase chain reaction (PCR) and next-generation sequencing (NGS). There included 68 LESAs, 25 SSs, and 62 MALT lymphomas. Ten cases relapsed in total, and 3 of MALT lymphomas died due to high-level transformation. Immunohistochemical double staining showed FCRL4 cells co-expressed Pax-5 and Ki-67, suggesting FCRL4 cells were proliferative B-cells. The expression level of the FCRL4 was significantly higher in MALT lymphoma than LESA and SS. The detection rates of clonal IGH, IGK, and IGL gene rearrangements in MALT lymphoma with a sensitivity of 83.33%. Monoclonal immunoglobulin gene rearrangements were confirmed in five suspected patients by NGS (100%). FCRL4 B cells might play an important role in the formation of lymphoepithelial lesions and might be as a diagnostic positive marker of salivary MALT lymphoma. The application of multiple detection methods could significantly improve the diagnostic accuracy for MALT lymphomas from LESA and SS. [ABSTRACT FROM AUTHOR]

The main pathogen in the Nile tilapia (Oreochromis niloticus) culture, Streptococcus agalactiae, causes economic harm. Infected fish's immune systems worked to eliminate of the infection. This study demonstrated the effect of different bacterial concentrations on tilapia immunity and optimal vaccine concentration to induce immunity in Nile tilapia. The experiment was performed at 102, 104, 106, 108, and 1010 CFU/fish of S. agalactiae compared with the control (PBS) through intraperitoneal injection for 72 h. Fish that survived employed to gather blood, and immune responses were assessed through measures of the survival rate include blood smears, antibody titers, and immunoglobulin gene expression. The vaccine experiment investigated formalin-inactivated S. agalactiae vaccination and administered S. agalactiae injections for 14 days. The statistic revealed a significant difference (p < 0.05) in the 108 and 1010 CFU/fish injections with high survival rates (62.22% and 53.33%, respectively). Immunoglobulin gene expression was highly represented in the 1010 CFU/fish injection; antibody titers were significantly improved from the control group, and antibody levels were high in the 1010 CFU/fish injection. The analysis of blood cell types using the blood smear method revealed a progressive increase in leucocytes, particularly lymphocytes, neutrophils, and monocytes, in the treatment group compared to the control group. Moreover, the erythrocyte/leucocyte ratio decreased significantly in response to the high bacterial injection, indicating an increase in leucocytes. Conversely, the erythrocyte level stayed ed within at the 7.03–9.70 × 102 cell/ml and shown no significant difference (p > 0.05). The lymphocytes were almost two-fold in 1010 CFU/fish compared to 108 CFU/fish. As depicted in the lowest concentration of 106 CFU/fish, the vaccine performance had a high relative percent survival (RPS) at 86.67%. This research suggested that the tilapia infected with high S. agalactiae concentrations did not affect the mortality of the tilapia, and vaccine concentration was effective in 106 CFU/fish. [ABSTRACT FROM AUTHOR]

The anthocyanin biosynthetic pathway is the main pathway regulating floral coloration in Iris germanica, a well-known ornamental plant. We investigated the transcriptome profiles and targeted metabolites to elucidate the relationship between genes and metabolites in anthocyanin biosynthesis in the bitone flower cultivar 'Clarence', which has a deep blue outer perianth and nearly white inner perianth. In this study, delphinidin-, pelargonidin-, and cyanidin-based anthocyanins were detected in the flowers. The content of delphinidin-based anthocyanins increased with the development of the flower. At full bloom (stage 3), delphinidin-based anthocyanins accounted for most of the total anthocyanin metabolites, whereas the content of pelargonidin- and cyanidin-based anthocyanins was relatively low. Based on functional annotations, a number of novel genes in the anthocyanin pathway were identified, which included early biosynthetic genes IgCHS, IgCHI, and IgF3H and late biosynthetic genes Ig F3′5′H, IgANS, and IgDFR. The expression of key structural genes encoding enzymes, such as IgF3H, Ig F3′5′H, IgANS, and IgDFR, was significantly upregulated in the outer perianth compared to the inner perianth. In addition, most structural genes exhibited their highest expression at the half-color stage rather than at the full-bloom stage, which indicates that these genes function ahead of anthocyanins synthesis. Moreover, transcription factors (TFs) of plant R2R3-myeloblastosis (R2R3-MYB) related to the regulation of anthocyanin biosynthesis were identified. Among 56 R2R3-MYB genes, 2 members belonged to subgroup 4, with them regulating the expression of late biosynthetic genes in the anthocyanin biosynthetic pathway, and 4 members belonged to subgroup 7, with them regulating the expression of early biosynthetic genes in the anthocyanin biosynthetic pathway. Quantitative real-time PCR (qRT-PCR) analysis was used to validate the data of RNA sequencing (RNA-Seq). The relative expression profiles of most candidate genes were consistent with the FPKM of RNA-seq. This study identified the key structural genes encoding enzymes and TFs that affect anthocyanin biosynthesis, which provides a basis and reference for the regulation of plant anthocyanin biosynthesis in I. germanica. [ABSTRACT FROM AUTHOR]

Immunoglobulin (Ig) genes carry the unique ability to be reshaped in peripheral B lymphocytes after these cells encounter a specific antigen. B cells can then further improve their affinity, acquire new functions as memory cells and eventually end up as antibody-secreting cells. Ig class switching is an important change that occurs in this context, thanks to local DNA lesions initiated by the enzyme activation-induced deaminase (AID). Several cis -acting elements of the Ig heavy (IgH) chain locus make it accessible to the AID-mediated lesions that promote class switch recombination (CSR). DNA repeats, with a non-template strand rich in G-quadruplexes (G4)-DNA, are prominent cis -targets of AID and define the so-called "switch" (S) regions specifically targeted for CSR. By analyzing the structure of the human IgH locus, we uncover that abundant DNA repeats, some with a putative G4-rich template strand, are additionally present in downstream portions of the IgH coding genes. These like-S (LS) regions stand as 3′ mirror-images of S regions and also show analogies to some previously reported repeats associated with the IgH locus 3' super-enhancer. A regulatory role of LS repeats is strongly suggested by their specific localization close to exons encoding the membrane form of Ig molecules, and by their conservation during mammalian evolution. [Display omitted] • "Switch (S)" regions preceding immunoglobulin (Ig) genes feature G-rich DNA repeats. • Additional "like-S (LS)" highly structured repeats follow Ig heavy (IgH) chain genes. • C-rich LS regions stand downstream of human γ genes as mirror images of S regions. • The symmetric architecture of repeats flanking IgH genes is conserved in many mammals. [ABSTRACT FROM AUTHOR]

Immunoglobulin E, complement, and eosinophils play an important role in host defense, but dysfunction of each of these components can lead to a variety of human disorders. In this review, we summarize how investigators have adapted gene therapy and antisense technology to modulate immunoglobulin E, complement, and/or eosinophil levels to treat these disorders. [ABSTRACT FROM AUTHOR]

Although monoclonal antibodies to the severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) are known, B‐cell receptor repertoire and its change in patients during coronavirus disease‐2019 (COVID‐19) progression is underreported. We aimed to study this molecularly. We used immunoglobulin heavy chain (IGH) variable region (IGHV) spectratyping and next‐generation sequencing of peripheral blood B‐cell genomic DNA collected at multiple time points during disease evolution to study B‐cell response to SARS‐CoV‐2 infection in 14 individuals with acute COVID‐19. We found a broad distribution of responding B‐cell clones. The IGH gene usage was not significantly skewed but frequencies of individual IGH genes changed repeatedly. We found predominant usage of unmutated and low mutation‐loaded IGHV rearrangements characterizing naïve and extrafollicular B cells among the majority of expanded peripheral B‐cell clonal lineages at most tested time points in most patients. IGH rearrangement usage showed no apparent relation to anti‐SARS‐CoV‐2 antibody titers. Some patients demonstrated mono/oligoclonal populations carrying highly mutated IGHV rearrangements indicating antigen experience at some of the time points tested, including even before anti‐SARS‐CoV‐2 antibodies were detected. We present evidence demonstrating that the B‐cell response to SARS‐CoV‐2 is individual and includes different lineages of B cells at various time points during COVID‐19 progression. [ABSTRACT FROM AUTHOR]

Abstract Assessment of minimal residual disease in acute lymphoblastic leukemia by immune repertoire NGS requires spiking CDR3 sequences at known quantities into the patient's sample. Recently, the EuroClonality‐NGS group released one of the most comprehensive protocols for this purpose. ARResT/Interrogate is a closed‐source software for processing these NGS libraries, developed by this same group. Vidjil, an open‐source alternative, currently cannot handle libraries prepared using this protocol. Here, we present a Vidjil add‐on to solve this issue. EuroClonality‐NGS prepared samples analyzed with Vidjil and ARResT/Interrogate were highly concordant (r = 0.998) and presented low error (root‐mean‐square error, RMSE = 0.112).

Secondary immunoglobulin diversification by somatic hypermutation and class switch recombination in B cells is instrumental for an adequate adaptive humoral immune response. These genetic events may, however, also introduce aberrations into other cellular genes and thereby cause B cell malignancies. While the basic mechanism of somatic hypermutation and class switch recombination is now well understood, their regulation and in particular the mechanism of their specific targeting to immunoglobulin genes is still rather mysterious. In this review, we summarize the current knowledge on the mechanism and regulation of secondary immunoglobulin diversification and discuss known mechanisms of physiological targeting to immunoglobulin genes and mistargeting to other cellular genes. We summarize open questions in the field and provide an outlook on future research. [ABSTRACT FROM AUTHOR]

This study proposes a hybrid gene selection method to identify and predict key genes in Arabidopsis associated with various stresses (including salt, heat, cold, high-light, and flagellin), aiming to enhance crop tolerance. An open-source microarray dataset (GSE41935) comprising 207 samples and 30,380 genes was analyzed using several machine learning tools including the synthetic minority oversampling technique (SMOTE), information gain (IG), ReliefF, and least absolute shrinkage and selection operator (LASSO), along with various classifiers (BayesNet, logistic, multilayer perceptron, sequential minimal optimization (SMO), and random forest). We identified 439 differentially expressed genes (DEGs), of which only three were down-regulated (AT3G20810, AT1G31680, and AT1G30250). The performance of the top 20 genes selected by IG and ReliefF was evaluated using the classifiers mentioned above to classify stressed versus non-stressed samples. The random forest algorithm outperformed other algorithms with an accuracy of 97.91% and 98.51% for IG and ReliefF, respectively. Additionally, 42 genes were identified from all 30,380 genes using LASSO regression. The top 20 genes for each feature selection were analyzed to determine three common genes (AT5G44050, AT2G47180, and AT1G70700), which formed a three-gene signature. The efficiency of these three genes was evaluated using random forest and XGBoost algorithms. Further validation was performed using an independent RNA_seq dataset and random forest. These gene signatures can be exploited in plant breeding to improve stress tolerance in a variety of crops. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, the introns are excised from pre-mRNA by the spliceosome. These introns typically have a lariat configuration due to the 2' - 5' phosphodiester bond between an internal branched residue and the 5' terminus of the RNA. The only enzyme known to selectively hydrolyze the 2' -5' linkage of these lariats is the RNA lariat debranching enzyme Dbr1. In humans, Dbr1 is involved in processes such as class-switch recombination of immunoglobulin genes, and its dysfunction is implicated in viral encephalitis, HIV, ALS, and cancer. However, mechanistic details of precisely how Dbr1 affects these processes are missing. Here we show that human Dbr1 contains a disordered C-terminal domain through sequence analysis and nuclear magnetic resonance. This domain stabilizes Dbr1 in vitro by reducing aggregation but is dispensable for debranching activity. We establish that Dbr1 requires Fe2+ for efficient catalysis and demonstrate that the noncatalytic protein Drn1 and the uncharacterized protein trichothiodystrophy nonphotosensitive 1 directly bind to Dbr1. We demonstrate addition of trichothiodystrophy nonphotosensitive 1 to in vitro debranching reactions increases the catalytic efficiency of human Dbr1 19-fold but has no effect on the activity of Dbr1 from the amoeba Entamoeba histolytica, which lacks a disordered C-terminal domain. Finally, we systematically examine how the identity of the branchpoint nucleotide affects debranching rates. These findings describe new aspects of Dbr1 function in humans and further clarify how Dbr1 contributes to human health and disease. [ABSTRACT FROM AUTHOR]

The translocation t(14;18)(q32:q21)/IGH::BCL2 occurs at the pre‐B stage of B‐cell development in the bone marrow and is insufficient for malignant transformation, although it leads to the formation of in situ follicular B‐cell neoplasia (ISFN). Despite that, the translocation is the genetic hallmark of follicular lymphoma (FL), it occurs infrequently in metachronous/synchronous lymphomas, including extranodal marginal zone lymphoma of mucosa‐associated lymphoid tissue (EMZL), mantle cell lymphoma, and Hodgkin's lymphoma. In each of these scenarios, the two lymphomas often appear to be clonally related by analyses of IGH::BCL2 and/or rearranged IG genes. However, it remains largely unknown whether one lymphoma originates from the other or they develop independently. We studied five cases of metachronous EMZL and FL. In four cases, the two lymphomas were clonally related, as shown by identical IGH::BCL2 and/or rearranged IG genes or shared mutations. There were common and unique mutations between the paired EMZL and FL, indicating that they developed independently from a common premalignant cell population, harbouring IGH::BCL2 in three cases. Furthermore, case 1 presented with three metachronous FLs, and all of them originated from a common precursor cell population via divergent evolution. Our findings highlight the multi‐malignant potential of IGH::BCL2‐positive B‐cells. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Antibodies result from the competition of B cell lineages evolving under selection for improved antigen recognition, a process known as affinity maturation. High-affinity antibodies to pathogens such as HIV, influenza, and SARS-CoV-2 are frequently reported to arise from B cells whose receptors, the precursors to antibodies, are encoded by particular immunoglobulin alleles. This raises the possibility that the presence of particular germline alleles in the B cell repertoire is a major determinant of the quality of the antibody response. Alternatively, initial differences in germline alleles' propensities to form high-affinity receptors might be overcome by chance events during affinity maturation. We first investigate these scenarios in simulations: when germline-encoded fitness differences are large relative to the rate and effect size variation of somatic mutations, the same germline alleles persistently dominate the response of different individuals. In contrast, if germline-encoded advantages can be easily overcome by subsequent mutations, allele usage becomes increasingly divergent over time, a pattern we then observe in mice experimentally infected with influenza virus. We investigated whether affinity maturation might nonetheless strongly select for particular amino acid motifs across diverse genetic backgrounds, but we found no evidence of convergence to similar CDR3 sequences or amino acid substitutions. These results suggest that although germline-encoded specificities can lead to similar immune responses between individuals, diverse evolutionary routes to high affinity limit the genetic predictability of responses to infection and vaccination. Author summary: Antibodies arise as B cell receptors encoded by the stochastic recombination of immunoglobulin genes. While those genes evolve over millions of years, the receptors themselves evolve within weeks as B cells compete under selection for improved antigen recognition. This competition shapes the response to infection and vaccination; how much the outcome depends on the initial choice of immunoglobulin genes versus subsequent receptor evolution is an open question that informs the predictability of the immune response and the long-term evolution of immunoglobulins. In simulations, we show that immunoglobulin genes with hardcoded specificity for the antigen can lead to either transient or persistent similarity in the response of different individuals. When the initial advantage is large relative to the effects of mutation, B cells using the same genes consistently dominate the response across individuals. Weaker initial advantages lead to similar responses early on but are later overcome by B cell evolution playing out differently in each individual due to chance events. We observe such increasingly divergent responses in mice infected with influenza virus. While long-term selection might hardcode specificities for particular pathogens on immunoglobulin genes, our results suggest diverse paths to potent antibodies can nonetheless limit the predictability of the response. [ABSTRACT FROM AUTHOR]

Introduction: Normal CD30+ B cells represent a distinct B-cell differentiation stage with features of strong activation. We lack an in depth understanding of these cells, because they are not present in peripheral blood and are typically very rare in reactive lymphoid organs. CD30+ B cells have been discussed as a potential precursor population for the malignant CD30+ Hodgkin and Reed-Sternberg cells in classical Hodgkin lymphoma. As CD30+ B cells can be more numerous in some cases of reactive lymphadenitis, we aimed to characterize these CD30+ B cells in terms of their differentiation stage and clonal composition, also as a means to clarify whether such CD30+ B-cell populations may represent potential precursor lesions of Hodgkin lymphoma. Methods: We microdissected single CD30+ B cells from tissue sections of eight reactive lymph nodes with substantial numbers of such cells and sequenced their rearranged immunoglobulin (Ig) heavy chain V region (IGHV) genes. Results: The CD30+ B cells were polyclonal B cells in all instances, and they not only encompass post-germinal center (GC) B cells with mutated IGHV genes, but also include a substantial fraction of pre-germinal center B cells with unmutated IGHV genes. In five of the lymph nodes, mostly small clonal expansions were detected among the CD30+ B cells. Most of the expanded clones carried somatically mutated IGHV genes and about half of the mutated clones showed intraclonal diversity. Discussion: We conclude that in human reactive lymph nodes with relatively many CD30+ B cells, these cells are a heterogenous population of polyclonal B cells encompassing activated pre-GC B cells as well as GC and post-GC B cells, with some clonal expansions. Because of their polyclonality and frequent pre-GC differentiation stage, there is no indication that such cell-rich CD30+ B-cell populations represent precursor lesions of Hodgkin lymphoma. [ABSTRACT FROM AUTHOR]

The abundant and widely distributed deermice Peromyscus leucopus and P. maniculatus are important reservoirs for several different zoonotic agents in North America. For the pathogens they persistently harbor, these species are also examples of the phenomenon of infection tolerance. In the present study a prior observation of absent expression of the high-affinity Fc immunoglobulin gamma receptor I (FcγRI), or CD64, in P. leucopus was confirmed in an experimental infection with Borreliella burgdorferi, a Lyme disease agent. We demonstrate that the null phenotype is attributable to a long-standing inactivation of the Fcgr1 gene in both species by a deletion of the promoter and coding sequence for the signal peptide for FcγRI. The Fcgr1 pseudogene was also documented in the related species P. polionotus. Six other Peromyscus species, including P. californicus, have coding sequences for a full-length FcγRI, including a consensus signal peptide. An inference from reported phenotypes for null Fcgr1 mutations engineered in Mus musculus is that one consequence of pseudogenization of Fcgr1 is comparatively less inflammation during infection than in animals, including humans, with undisrupted, fully active genes. [ABSTRACT FROM AUTHOR]

Objective: A blood-based biomarker is needed to assess lupus nephritis (LN) disease activity, minimizing the need for invasive kidney biopsies. Long noncoding RNAs (lncRNAs) are known to regulate gene expression, appear to be stable in human plasma, and can serve as non-invasive biomarkers. Methods: Transcriptomic data of whole blood samples from 74 LN patients and 20 healthy subjects (HC) were analyzed to identify differentially expressed (DE) lncRNAs associated with quiescent disease and flares. Weighted gene coexpression network analysis (WGCNA) was performed to uncover lncRNAs with a central role (hub lncRNAs) in regulating key biological processes that drive LN disease activity. The association of hub lncRNAs with disease activity was validated using RT-qPCR on an independent cohort of 15 LN patients and 9 HC. cis- and trans-targets of validated lncRNAs were explored in silico to examine potential mechanisms of their action. Results: There were 444 DE lncRNAs associated with quiescent disease and 6 DE lncRNAs associated with flares (FDR <0.05). WGCNA highlighted IFN signaling and B-cell activity/adaptive immunity as the most significant processes contributing to nephritis activity. Four disease-activity-associated lncRNAs, namely, NRIR, KLHDC7B-DT, MIR600HG, and FAM30A, were detected as hub genes and validated in an independent cohort. NRIR and KLHDC7B-DT emerged as potential key regulators of IFN-mediated processes. Network analysis suggests that FAM30A and MIR600HG are likely to play a central role in the regulation of B-cells in LN through cis-regulation effects and a competing endogenous RNA mechanism affecting immunoglobulin gene expression and the IFNγl pathway. Conclusions: The expression of lncRNAs NRIR, KLHDC7B-DT, FAM30A, and MIR600HG were associated with disease activity and could be further explored as blood-based biomarkers and potential liquid biopsy on LN. [ABSTRACT FROM AUTHOR]

• The IG genes are highly polymorphic, but this variation is not covered in current GWAS. • Definition of IG germline gene diversity in different human population groups is a prioritized topic. • Convergent Ab responses indicate preferential IG gene usage. • Technology platforms to untangle IG genotype-function associations are increasingly available. Immunoglobulin (IG) genes, encoding B cell receptors (BCRs), are fundamental components of the mammalian immune system, which evolved to recognize the diverse antigenic universe present in nature. To handle these myriad inputs, BCRs are generated through combinatorial recombination of a set of highly polymorphic germline genes, resulting in a vast repertoire of antigen receptors that initiate responses to pathogens and regulate commensals. Following antigen recognition and B cell activation, memory B cells and plasma cells form, allowing for the development of anamnestic antibody (Ab) responses. How inherited variation in IG genes impacts host traits, disease susceptibility, and Ab recall responses is a topic of great interest. Here, we consider approaches to translate emerging knowledge about IG genetic diversity and expressed repertoires to inform our understanding of Ab function in health and disease etiology. As our understanding of IG genetics grows, so will our need for tools to decipher preferences for IG gene or allele usage in different contexts, to better understand antibody responses at the population level. [ABSTRACT FROM AUTHOR]

Long-term antibiotics are prescribed for a variety of medical conditions, recently including low back pain with Modic changes. The molecular impact of such treatment is unknown. We conducted longitudinal transcriptome and epigenome analyses in patients (n = 100) receiving amoxicillin treatment or placebo for 100 days in the Antibiotics in Modic Changes (AIM) study. Gene expression and DNA methylation were investigated at a genome-wide level at screening, after 100 days of treatment, and at one-year follow-up. We identified intra-individual longitudinal changes in gene expression and DNA methylation in patients receiving amoxicillin, while few changes were observed in patients receiving placebo. After 100 days of amoxicillin treatment, 28 genes were significantly differentially expressed, including the downregulation of 19 immunoglobulin genes. At one-year follow-up, the expression levels were still not completely restored. The significant changes in DNA methylation (n = 4548 CpGs) were mainly increased methylation levels between 100 days and one-year follow-up. Hence, the effects on gene expression occurred predominantly during treatment, while the effects on DNA methylation occurred after treatment. In conclusion, unrecognized side effects of long-term amoxicillin treatment were revealed, as alterations were observed in both gene expression and DNA methylation that lasted long after the end of treatment. [ABSTRACT FROM AUTHOR]

IntroductionNormal CD30+ B cells represent a distinct B-cell differentiation stage with features of strong activation. We lack an in depth understanding of these cells, because they are not present in peripheral blood and are typically very rare in reactive lymphoid organs. CD30+ B cells have been discussed as a potential precursor population for the malignant CD30+ Hodgkin and Reed-Sternberg cells in classical Hodgkin lymphoma. As CD30+ B cells can be more numerous in some cases of reactive lymphadenitis, we aimed to characterize these CD30+ B cells in terms of their differentiation stage and clonal composition, also as a means to clarify whether such CD30+ B-cell populations may represent potential precursor lesions of Hodgkin lymphoma.MethodsWe microdissected single CD30+ B cells from tissue sections of eight reactive lymph nodes with substantial numbers of such cells and sequenced their rearranged immunoglobulin (Ig) heavy chain V region (IGHV) genes.ResultsThe CD30+ B cells were polyclonal B cells in all instances, and they not only encompass post-germinal center (GC) B cells with mutated IGHV genes, but also include a substantial fraction of pre-germinal center B cells with unmutated IGHV genes. In five of the lymph nodes, mostly small clonal expansions were detected among the CD30+ B cells. Most of the expanded clones carried somatically mutated IGHV genes and about half of the mutated clones showed intraclonal diversity.DiscussionWe conclude that in human reactive lymph nodes with relatively many CD30+ B cells, these cells are a heterogenous population of polyclonal B cells encompassing activated pre-GC B cells as well as GC and post-GC B cells, with some clonal expansions. Because of their polyclonality and frequent pre-GC differentiation stage, there is no indication that such cell-rich CD30+ B-cell populations represent precursor lesions of Hodgkin lymphoma.

Introduction: In mature B cells, activation-induced deaminase reshapes Ig genes through somatic hypermutation and class switch recombination of the Ig heavy chain (IgH) locus under control of its 3’ cis-regulatory region (3’RR). The 3’RR is itself transcribed and can undergo “locus suicide recombination” (LSR), then deleting the constant gene cluster and terminating IgH expression. The relative contribution of LSR to B cell negative selection remains to be determined. Methods: Here, we set up a knock-in mouse reporter model for LSR events with the aim to get clearer insights into the circumstances triggering LSR. In order to explore the consequences of LSR defects, we reciprocally explored the presence of autoantibodies in various mutant mouse lines in which LSR was perturbed by the lack of Sµ or of the 3’RR. Results: Evaluation of LSR events in a dedicated reporter mouse model showed their occurrence in various conditions of B cell activation, notably in antigen-experienced B cells Studies of mice with LSR defects evidenced increased amounts of self-reactive antibodies. Discussion: While the activation pathways associated with LSR are diverse, in vivo as well as in vitro, this study suggests that LSR may contribute to the elimination of self-reactive B cells. [ABSTRACT FROM AUTHOR]

The phenotypes of B lineage cells that produce oligoclonal IgG in multiple sclerosis have not been unequivocally determined. Here, we utilized singlecell RNA-seq data of intrathecal B lineage cells in combination with mass spectrometry of intrathecally synthesized IgG to identify its cellular source. We found that the intrathecally produced IgG matched a larger fraction of clonally expanded antibody-secreting cells compared to singletons. The IgG was traced back to two clonally related clusters of antibody-secreting cells, one comprising highly proliferating cells, and the other consisting of more differentiated cells expressing genes associated with immunoglobulin synthesis. These findings suggest some degree of heterogeneity among cells that produce oligoclonal IgG in multiple sclerosis. [ABSTRACT FROM AUTHOR]

Background: Pregnancy is known to induce extensive biological changes in the healthy mother. Little is known, however, about what these changes are at the molecular level. We have examined systemic expression changes in proteincoding genes and long non-coding (lnc) RNAs during and after pregnancy, compared to before pregnancy, among healthy women with term pregnancies. Methods: Blood samples were collected from 14 healthy women enrolled in our prospective pregnancy cohort at 7 time-points (before, during and after pregnancy). Total RNA from frozen whole blood was used for RNA sequencing. Following raw read alignment and assembly, gene-level counts were obtained for protein-coding genes and long non-coding RNAs. At each time-point, cell type proportions were estimated using deconvolution. To examine associations between pregnancy status and gene expression over time, Generalized Estimating Equation (GEE) models were fitted, adjusting for age at conception, and with and without adjusting for changes in cell type proportions. Fold-changes in expression at each trimester were examined relative to the pre-pregnancy baseline. Results: Numerous immune-related genes demonstrated pregnancy-associated expression, in a time-dependent manner. The genes that demonstrated the largest changes in expression included several that were neutrophil-related (over-expressed) and numerous immunoglobulin genes (under-expressed). Estimated cell proportions revealed a marked increase in neutrophils, and less so of activated CD4 memory T cells, during pregnancy, while most other cell type proportions decreased or remained unchanged. Adjusting for cell type proportions in our model revealed that although most of the expression changes were due to changes in cell type proportions in the bloodstream, transcriptional regulation was also involved, especially in down-regulating expression of type I interferon inducible genes. Conclusion: Compared to a pre-pregnancy baseline, there were extensive systemic changes in cell type proportions, gene expression and biological pathways associated with different stages of pregnancy and postpartum among healthy women. Some were due to changes in cell type proportions and some due to gene regulation. In addition to providing insight into term pregnancy among healthy women, these findings also provide a "normal" reference for abnormal pregnancies and for autoimmune diseases that improve or worsen during pregnancy, to assess deviations from normal. [ABSTRACT FROM AUTHOR]

Sidekick cell adhesion molecule 1 (SDK1) gene belonging to the immunoglobulin superfamily (IgSF) is reported to be associated with disease resistance. A novel missense mutation (XM_015469413.1: c.2678 A > G: p. Asp893Gly) in bovine SDK1 gene was observed in the Bovine Genome Variation Database (BGVD). The purpose of the current study was to determine the allelic frequency distribution of XM_015469413.1: c.2678 A > G: p. Asp893Gly and analyze its association with disease resistance in native Chinese cattle. Here, we explored the allele frequency of the missense mutation (M_015469413.1: c.2678 A > G: p. Asp893Gly) in 542 individuals from 27 Chinese indigenous cattle breeds using PCR and DNA sequencing methods. The frequency of the mutant allele (G) gradually decreased from the southern cattle groups to the northern cattle groups, whereas the frequency of the wild-type allele A showed an opposite pattern, consistent with the distribution of indicine and taurine cattle in China. Thus, our results proved that the bovine SDK1 gene might be candidate genes associated with disease resistance in Chinese cattle. [ABSTRACT FROM AUTHOR]

T‐acute lymphoblastic leukemia/lymphoma (T‐ALL/LBL) is a malignant neoplasm of immature lymphoblasts. Terminal deoxynucleotidyl transferase (TDT) is a template‐independent DNA polymerase that plays an essential role in generating diversity for immunoglobulin genes. T‐ALL/LBL patients with TDT− have a worse prognosis. However, how TDT− promotes the disease progression of T‐ALL/LBL remains unknown. Here we analyzed the prognosis of T‐ALL/LBL patients in Shanghai Children's Medical Center (SCMC) and confirmed that TDT− patients had a higher rate of recurrence and remission failure and worse outcomes. Cellular experiments demonstrated that TDT was involved in DNA damage repair. TDT knockout delayed DNA repair, arrested the cell cycle and decreased apoptosis to induce the accumulation of chromosomal abnormalities and tolerance to abnormal karyotypes. Our study demonstrated that the poor outcomes in TDT− T‐ALL/LBL might be due to the drug resistance (VP16 and MTX) induced by chromosomal abnormalities. Our findings revealed novel functions and mechanisms of TDT in T‐ALL/LBL and supported that hematopoietic stem cell transplantation (HSCT) might be a better choice for these patients. [ABSTRACT FROM AUTHOR]

The need for sensitive monitoring of minimal/measurable residual disease (MRD) in multiple myeloma emerged as novel therapies led to deeper responses. Moreover, the potential benefits of blood-based analyses, the so-called liquid biopsy is prompting more and more studies to assess its feasibility. Considering these recent demands, we aimed to optimize a highly sensitive molecular system based on the rearranged immunoglobulin (Ig) genes to monitor MRD from peripheral blood. We analyzed a small group of myeloma patients with the high-risk t(4;14) translocation, using next-generation sequencing of Ig genes and droplet digital PCR of patient-specific Ig heavy chain (IgH) sequences. Moreover, well established monitoring methods such as multiparametric flow cytometry and RT-qPCR of the fusion transcript IgH::MMSET (IgH and multiple myeloma SET domain-containing protein) were utilized to evaluate the feasibility of these novel molecular tools. Serum measurements of M-protein and free light chains together with the clinical assessment by the treating physician served as routine clinical data. We found significant correlation between our molecular data and clinical parameters, using Spearman correlations. While the comparisons of the Ig-based methods and the other monitoring methods (flow cytometry, qPCR) were not statistically evaluable, we found common trends in their target detection. Regarding longitudinal disease monitoring, the applied methods yielded complementary information thus increasing the reliability of MRD evaluation. We also detected indications of early relapse before clinical signs, although this implication needs further verification in a larger patient cohort. [ABSTRACT FROM AUTHOR]

Activation-induced cytidine deaminase (AID) is the essential enzyme for imprinting immunological memory through class switch recombination (CSR) and somatic hypermutation (SHM) of the immunoglobulin (Ig) gene. AID-dependent reduction of Topoisomerase 1 (Top1) promotes DNA cleavage that occurs upon Ig gene diversification, whereas the mechanism behind AID-induced Top1 reduction remains unclear. Here, we clarified the contribution of the microRNA-Ago2 complex in AID-dependent Top1 decrease. Ago2 binds to Top1 3'UTR with two regions of AID-dependent Ago2-binding sites (5'- and 3'dABs). Top1 3'UTR knockout (3'UTRKO) in B lymphoma cells leads to decreases in DNA break efficiency in the IgH gene accompanied by a reduction in CSR and SHM frequencies. Furthermore, AID-dependent Top1 protein reduction and Ago2-binding to Top1 mRNA are down-regulated in 3'UTRKO cells. Top1 mRNA in the highly translated fractions of the sucrose gradient is decreased in an AID-dependent and Top1 3'UTR-mediated manner, resulting in a decrease in Top1 protein synthesis. Both AID and Ago2 localize in the mRNA-binding protein fractions and they interact with each other. Furthermore, we found some candidate miRNAs which possibly bind to 5'- and 3'dAB in Top1 mRNA. Among them, miR-92a-3p knockdown induces the phenotypes of 3'UTRKO cells to wild-type cells whereas it does not impact on 3'UTRKO cells. Taken together, the Ago2-miR-92a-3p complex will be recruited to Top1 3'UTR in an AID-dependent manner and posttranscriptionally reduces Top1 protein synthesis. These consequences cause the increase in a non-B-DNA structure, enhance DNA cleavage by Top1 in the Ig gene and contribute to immunological memory formation. [ABSTRACT FROM AUTHOR]