Frequency Of Somatic Hypermutation Research Articles

Qualitative Immunoglobulin Deficiency Causes Bacterial Infections in Patients with STAT1 Gain-of-Function Mutations.

STAT1 is a transduction and transcriptional regulator that functions within the classical JAK/STAT pathway. In addition to chronic mucocutaneous candidiasis, bacterial infections are a common occurrence in patients with STAT1 gain-of-function (GOF) mutations. These patients often exhibit skewing of B cell subsets; however, the impact of STAT1-GOF mutations on B cell-mediated humoral immunity remains largely unexplored. It is also unclear whether these patients with IgG within normal range require regular intravenous immunoglobulin (IVIG) therapy. Eleven patients (harboring nine different STAT1-GOF mutations) were enrolled. Reporter assays and immunoblot analyses were performed to confirm STAT1 mutations. Flow cytometry, deep sequencing, ELISA, and ELISpot were conducted to assess the impact of STAT1-GOF on humoral immunity. All patients exhibited increased levels of phospho-STAT1 and total STAT1 protein, with two patients carrying novel mutations. In vitro assays showed that these two novel mutations were GOF mutations. Three patients with normal total IgG levels received regular IVIG infusions, resulting in effective control of bacterial infections. Four cases showed impaired affinity and specificity of pertussis toxin-specific antibodies, accompanied by reduced generation of class-switched memory B cells. Patients also had a disrupted immunoglobulin heavy chain (IGH) repertoire, coupled with a marked reduction in the somatic hypermutation frequency of switched Ig transcripts. STAT1-GOF mutations disrupt B cell compartments and skew IGH characteristics, resulting in impaired affinity and antigen-specificity of antibodies and recurrent bacterial infections. Regular IVIG therapy can control these infections in patients, even those with normal total IgG levels.

Causative mechanisms and clinical impact of immunoglobulin deficiencies in ataxia telangiectasia

BackgroundAtaxia Telangiectasia (AT) is characterized by cerebellar ataxia, telangiectasia, immunodeficiency, and increased cancer susceptibility, caused by mutations in the ataxia telangiectasia mutated (ATM) gene. The immunodeficiency comprises predominantly immunoglobulin (Ig)-deficiency, mainly IgA and IgG2, with a variable severity. So far, the exact mechanisms underlying the Ig-deficiency, especially the variable severity, remain unelucidated. ObjectiveTo characterize the clinical impact of Ig-deficiencies in AT and to elucidate the mechanisms of Ig-deficiencies in AT. MethodsWe analyzed long-term Ig-levels, immunophenotyping, and survival time in our cohort (n=87, median age 16 years; maximum 64 years). Somatic hypermutation (SHM) and class-switch junctions in B-cells were analyzed by next-generation sequencing. Furthermore, an in vitro class-switching induction assay was performed followed by RNAseq, to assess the effect of ATM-inhibition. ResultsOnly the hyper-IgM AT phenotype significantly worsened survival time, while IgA or IgG2-deficiencies did not. The Ig-levels showed predominantly decreased IgG2 and IgA. Moreover, flowcytometric analysis demonstrated reduced naïve B- and T-lymphocytes and a deficiency of class-switched IgG2 and IgA memory B-cells. SHM frequencies were lowered in IgA and IgG2-deficient patients, indicating a hampered germinal center (GC) reaction. In addition, the microhomology of switch junctions was elongated, suggesting alternative end-joining during class-switch DNA-repair. The in vitro class-switching and proliferation were negatively affected by ATM-inhibition. RNA-sequencing analysis showed that ATM-inhibitor influenced expression of GC reaction genes. ConclusionIg-deficiency in AT is caused by disturbed development of class-switched memory B-cells. This study suggests that ATM-deficiency affects both the GC reaction and the choice of DNA-repair pathways in class-switching.

High-throughput single-cell profiling of B cell responses following inactivated influenza vaccination in young and older adults.

Seasonal influenza contributes to a substantial disease burden, resulting in approximately 10 million hospital visits and 50 thousand deaths in a typical year in the United States. 70 - 85% of the mortality occurs in people over the age of 65. Influenza vaccination is the best protection against the virus, but it is less effective for the elderly, which may be in part due to differences in the quantity or type of B cells induced by vaccination. To investigate this possibility, we sorted pre- and post-vaccination peripheral blood B cells from three young and three older adults with strong antibody responses to the inactivated influenza vaccine and employed single-cell technology to simultaneously profile the gene expression and the B cell receptor (BCR) of the B cells. Prior to vaccination, we observed a higher somatic hypermutation frequency and a higher abundance of activated B cells in older adults than in young adults. Following vaccination, young adults mounted a more clonal response than older adults. The expanded clones included a mix of plasmablasts, activated B cells, and resting memory B cells in both age groups, with a decreased proportion of plasmablasts in older adults. Differential abundance analysis identified additional vaccine-responsive cells that were not part of expanded clones, especially in older adults. We observed broadly consistent gene expression changes in vaccine-responsive plasmablasts and greater heterogeneity among activated B cells between age groups. These quantitative and qualitative differences in the B cells provide insights into age-related changes in influenza vaccination response.

Ago2 and a miRNA reduce Topoisomerase 1 for enhancing DNA cleavage in antibody diversification by activation-induced cytidine deaminase

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.

Convergent antibody responses are associated with broad neutralization of hepatitis C virus.

Early development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known. To identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects. We found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing. Together, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.

The Single-Cell Immunogenomic Landscape of B and Plasma Cells in Early-Stage Lung Adenocarcinoma.

While TIBs are highly enriched in LUADs, they are poorly characterized. This study provides a much-needed understanding of the transcriptional, clonotypic states and phenotypes of TIBs, unraveling their potential roles in the immunopathology of early-stage LUADs and constituting a road map for the development of TIB-targeted immunotherapies for the treatment of this morbid malignancy. This article is highlighted in the In This Issue feature, p. 2483.

Altered Immunoglobulin Repertoire and Decreased IgA Somatic Hypermutation in the Gut during Chronic HIV-1 Infection.

ABSTRACTHumoral immune perturbations contribute to pathogenic outcomes in persons with HIV-1 infection (PWH). Gut barrier dysfunction in PWH is associated with microbial translocation and alterations in microbial communities (dysbiosis), and IgA, the most abundant immunoglobulin (Ig) isotype in the gut, is involved in gut homeostasis by interacting with the microbiome. We determined the impact of HIV-1 infection on the antibody repertoire in the gastrointestinal tract by comparing Ig gene utilization and somatic hypermutation (SHM) in colon biopsies from PWH (n = 19) versus age and sex-matched controls (n = 13). We correlated these Ig parameters with clinical, immunological, microbiome and virological data. Gene signatures of enhanced B cell activation were accompanied by skewed frequencies of multiple Ig Variable genes in PWH. PWH showed decreased frequencies of SHM in IgA and possibly IgG, with a substantial loss of highly mutated IgA sequences. The decline in IgA SHM in PWH correlated with gut CD4+ T cell loss and inversely correlated with mucosal inflammation and microbial translocation. Diminished gut IgA SHM in PWH was driven by transversion mutations at A or T deoxynucleotides, suggesting a defect not at the AID/APOBEC3 deamination step but at later stages of IgA SHM. These results expand our understanding of humoral immune perturbations in PWH that could have important implications in understanding mucosal immune defects in individuals with chronic HIV-1 infection.IMPORTANCE The gut is a major site of early HIV-1 replication and pathogenesis. Extensive CD4+ T cell depletion in this compartment results in a compromised epithelial barrier that facilitates the translocation of microbes into the underlying lamina propria and systemic circulation, resulting in chronic immune activation. To date, the consequences of microbial translocation on the mucosal humoral immune response (or vice versa) remains poorly integrated into the panoply of mucosal immune defects in PWH. We utilized next-generation sequencing approaches to profile the Ab repertoire and ascertain frequencies of somatic hypermutation in colon biopsies from antiretroviral therapy-naive PWH versus controls. Our findings identify perturbations in the Ab repertoire of PWH that could contribute to development or maintenance of dysbiosis. Moreover, IgA mutations significantly decreased in PWH and this was associated with adverse clinical outcomes. These data may provide insight into the mechanisms underlying impaired Ab-dependent gut homeostasis during chronic HIV-1 infection.

A Human MSH6 Germline Variant Associated With Systemic Lupus Erythematosus Induces Lupus-like Disease in Mice.

ObjectiveTo determine if single‐nucleotide polymorphisms (SNPs) in DNA repair genes are enriched in individuals with systemic lupus erythematosus (SLE) and if they are sufficient to confer a disease phenotype in a mouse model.MethodsHuman exome chip data of 2499 patients with SLE and 1230 healthy controls were analyzed to determine if variants in 10 different mismatch repair genes (MSH4, EXO1, MSH2, MSH6, MLH1, MSH3, POLH, PMS2, ML3, and APEX2) were enriched in individuals with SLE. A mouse model of the MSH6 SNP, which was found to be enriched in individuals with SLE, was created using CRISPR/Cas9 gene targeting. Wildtype mice and mice heterozygous and homozygous for the MSH6 variant were then monitored for 2 years for the development of autoimmune phenotypes, including the presence of high levels of antinuclear antibodies (ANA). Additionally, somatic hypermutation frequencies and spectra of the intronic region downstream of the VHJ558‐rearranged JH4 immunoglobulin gene was characterized from Peyer's patches.ResultsBased on the human exome chip data, the MSH6 variant (rs63750897, p.Ser503Cys) is enriched among patients with SLE versus controls after we corrected for ancestry (odds ratio = 8.39, P = 0.0398). Mice homozygous for the MSH6 variant (Msh6 S502C/S502C ) harbor significantly increased levels of ANA. Additionally, the Msh6 S502C/S502C mice display a significant increase in the infiltration of CD68+ cells (a marker for monocytes and macrophages) into the lung alveolar space as well as apoptotic cells. Furthermore, characterization of somatic hypermutation in these mice reveals an increase in the DNA polymerase η mutational signature.ConclusionAn MSH6 mutation that is enriched in humans diagnosed with lupus was identified. Mice harboring this Msh6 mutation develop increased autoantibodies and an inflammatory lung disease. These results suggest that the human MSH6 variant is linked to the development of SLE.

Abstract 2940: Somatic hypermutation analysis of chronic lymphocytic leukemia research samples by IGH chain next-generation sequencing

Abstract Chronic lymphocytic leukemia (CLL) is a common form of leukemia characterized by clonal expansion of neoplastic B cells and a heterogenous disease course. Somatic hypermutation (SHM) status of the IGHV gene in CLL clinical research samples is important as SHM frequency is an established prognostic biomarker. Conventionally, SHM analysis is performed via Sanger sequencing which is limited by the inability to evaluate more than one rearrangement due to mutations, multiplex constraints, and template input requirements. Ion AmpliSeq™ next generation sequencing (NGS) assays for research in IGH chain SHM are evaluated using multiple cohorts of CLL research samples. These NGS assays employ multiplex primers that target the Leader or FR1 regions of the IGHV gene and the IGHJ gene in either RNA or DNA templates. The robustness of these assays was demonstrated by an evaluation across multiple labs, including comparisons between DNA and RNA input and correlation to orthogonal NGS testing. Ion AmpliSeq™ multiplex primer sets were designed to target the Leader or Framework 1 (FR1) regions of the IGHV gene with joining gene (J) or constant region (C) primers. The Oncomine™ IGHV Leader-J Assay uses Leader primers, while the RNA-input Oncomine™ IGH-LR Assay, and the DNA-input Oncomine™ IGH FR1-J Assay use FR1 variable gene primers in combination with C and J region primers, respectively. Primer panels were designed to amplify all variable gene families, including all individual alleles, listed in the IMGT database. To evaluate accuracy, each assay was used to measure SHM from a panel of gDNA and RNA rearrangements extracted from CLL research samples for clonality testing and SHM analysis. To test the Leader-J and FR1-J DNA input assays, 24 orthogonally characterized CLL samples were evaluated on the Ion Torrent S5 platform. Stereotypy and confirmation of individual software findings assessed by placing FASTA file results into the online ARResT tool. Both RNA and DNA input assays correctly determined the SHM status of CLL rearrangements, with IGHV SHM values concordant between both RNA and DNA approaches. SHM values derived from FR1 variable gene primers delivered concordant results compared to Leader variable gene primers across a range of SHM frequencies, with FR1 primer coverage of the IGHV gene leading to an expected over-estimation of SHM percentage. SHM status was concordant between NGS assays evaluated in over 90% (21/24) of CLL samples tested. Mutational frequency showed excellent concordance, with an R2=0.97, and V-gene usage was 100% concordant. These results support the robustness of long-read NGS assays to quantify SHM in either DNA or RNA samples. Concordant results were shown between FR1 and Leader-targeting primers using DNA input showing the utility in both priming locations. Orthogonal testing of the Leader-J assay showed excellent concordance for mutation rate, SHM status, and stereotypy. Citation Format: Michelle Toro, Loni Pickle, Shrutii Sarda, Robyn Marshall, Bonnie Pieterse, Tania Venter, Chenchen Yang, Theo Gerdener, Geoffrey Marc Lowman. Somatic hypermutation analysis of chronic lymphocytic leukemia research samples by IGH chain next-generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2940.

Bruton's Tyrosine Kinase Supports Gut Mucosal Immunity and Commensal Microbiome Recognition in Autoimmune Arthritis.

Bruton’s tyrosine kinase (Btk) deficiency preferentially eliminates autoreactive B cells while sparing normal humoral responses, but has not been studied in mucosal immunity. Commensal microbes and intact BTK signaling have been independently shown to be essential for arthritis development in K/BxN mice. Here, we examine how BTK-mediated signaling interfaces with the gut microbiome. Btk-deficient K/BxN mice were found to have small Peyer’s Patches with reduced germinal center and IgA class-switched B cells. IgA-switched plasma cells in small intestines were reduced, especially in villi of Btk-deficient mice. IgH CDR3 sequencing showed similar V gene diversity and somatic hypermutation frequency despite Btk deficiency but showed reduced CDR3 amino acid polarity, suggesting potential qualitative differences in the gut plasma cell repertoire. Small intestinal IgA was low and IgA coating of commensal bacteria was reduced. IgA-seq showed a shift in small intestinal microbes that are normally IgA-coated into the uncoated fraction in Btk-deficient mice. Overall, this study shows that BTK supports normal intestinal IgA development in response to commensals.This manuscript was previously published as a preprint at: https://www.biorxiv.org/content/10.1101/2021.03.10.434762v2.

Single-cell multi-omics reveals dyssynchrony of the innate and adaptive immune system in progressive COVID-19

Dysregulated immune responses against the SARS-CoV-2 virus are instrumental in severe COVID-19. However, the immune signatures associated with immunopathology are poorly understood. Here we use multi-omics single-cell analysis to probe the dynamic immune responses in hospitalized patients with stable or progressive course of COVID-19, explore V(D)J repertoires, and assess the cellular effects of tocilizumab. Coordinated profiling of gene expression and cell lineage protein markers shows that S100Ahi/HLA-DRlo classical monocytes and activated LAG-3hi T cells are hallmarks of progressive disease and highlights the abnormal MHC-II/LAG-3 interaction on myeloid and T cells, respectively. We also find skewed T cell receptor repertories in expanded effector CD8+ clones, unmutated IGHG+ B cell clones, and mutated B cell clones with stable somatic hypermutation frequency over time. In conclusion, our in-depth immune profiling reveals dyssynchrony of the innate and adaptive immune interaction in progressive COVID-19.

FAM72A antagonizes UNG2 to promote mutagenic repair during antibody maturation.

Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination1,2. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells3-6. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determinant for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a-/- mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a-/- mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2(UNG2)7, which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels ofUNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers8 could promote mutagenesis.

Abstract 511: Clonal lineage and somatic hypermutation analysis comparing DNA and RNA as input material by long amplicon IGH chain sequencing

Abstract Background Traditional next generation sequencing methods for quantifying somatic hypermutation (SHM) rely on multiplex primers targeting either the framework 1 (FR1) or Leader regions of the IGH variable gene in combination with joining gene primers to amplify rearranged IGH chains from gDNA templates. Ion AmpliSeq primer panels for SHM evaluation were compared using both DNA and RNA input. Performance was compared using SHM values obtained from RNA samples amplified using FR1 variable gene primers in combination with constant gene primers to determine each IGH isotype and subtype in a single PCR reaction. Comparison of SHM frequencies measured from matched RNA and DNA samples were used to determine the feasibility for use of RNA in the study of SHM as well as comparison of the performance of Leader and FR1 gene primers in DNA studies. Methods Two multiplex primer panels were developed for DNA templates. These panels target the Leader or FR1 regions of the IGHV gene and the IGHJ gene. RNA samples were surveyed using FR1 and constant region primers. Comparisons of SHM frequency using 200ng of gDNA and 25ng of RNA from B cell lines and clinical research samples. Sequencing was accomplished using the Ion Gene Studio S5, while clonotyping, isotype determination, and somatic hypermutation analysis was performed using Ion Reporter 5.16 analysis software. Results Both RNA and DNA input assay workflows were able to correctly determine the SHM status of all rearrangements tested. IGHV SHM values were highly concordant between both RNA and DNA approaches. Additionally, SHM values derived from FR1 targeting variable gene primers delivered concordant results compared to Leader targeting variable gene primers when using DNA input across a wide range of SHM frequencies tested. Conclusions These results support the ability of highly multiplexed long-read NGS assays to accurately quantify SHM in either DNA or RNA samples. Concordant results were shown between FR1 and Leader targeting primers using DNA input. RNA based NGS methods benefit from lower sample requirements as well as the addition of isotype (and subtype) identification, opening new research areas for study of the B cell immune repertoire. Citation Format: Michelle Toro, Geoffrey Lowman, Loni Pickle, Stephanie Ostresh, Mark Andersen, Shrutii Sarda, Chenchen Yang. Clonal lineage and somatic hypermutation analysis comparing DNA and RNA as input material by long amplicon IGH chain sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 511.

The Prognostic Significance of the Complete IgH Rearrangement Pattern Using the BIOMED-2 Protocol in Diffuse Large B-Cell Lymphoma

▪Introduction: In several B-cell malignancies, such as chronic lymphocytic leukemia and mantle cell lymphoma, the somatic hypermutation (SHM) status of immunoglobulin heavy chain (IgH) variable region (VH) significantly correlates with clinical outcome. Diffuse large B-cell lymphoma (DLBCL) usually contains a number of VH SHMs, with considerably variable frequency. However, there have been few reports about the association between VHSHM frequency and clinical outcome in DLBCL.The BIOMED-2 multiplex PCR protocol is one of the standardized methods for detecting IgH gene rearrangements, and has been used to prove clonality in B-cell lymphomas. VH SHMs in the germinal center prevent proper annealing of the applied PCR primers, rendering clonal VH rearrangements undetectable by this protocol. Thus, the complete IgH rearrangement pattern produced by the BIOMED-2 protocol is expected to reflect the frequency of VH SHMs. In this study, we investigated the prognostic impact of complete IgH rearrangement pattern on patients with DLBCL, and analyzed the correlation between complete IgH rearrangement pattern and VH SHM frequency by Sanger sequencing.Materials and Methods: We retrospectively identified 386 patients diagnosed with de novo DLBCL that received anthracycline-based immunochemotherapy at Kobe City Medical Center General Hospital from January 2005 to June 2015. We excluded 83 patients because fresh samples were not available for PCR analysis, and a total of 303 patients were enrolled. Based on the BIOMED-2 protocol, complete IgH rearrangement was detected by three PCR reactions using three VH forward primer sets: framework region (FR) 1, FR2, and FR3. The primary endpoint was 5-year overall survival (5-yr OS). The secondary endpoint was 5-year progression-free survival (5-yr PFS). OS and PFS were estimated using the log-rank test. Multivariate analysis using the Cox proportional hazard ratio was performed for 5-yr OS. For VH sequencing analysis, the cryopreserved PCR products of 42 available samples were sequenced directly using both forward and reverse primers, and for each sequenced sample, the percentage of VH identity to the closest germline gene was calculated, using the immunoglobulin gene database (IMGT/V-QUEST).Results: Of the 303 patients enrolled (median age: 69 years old), 183 patients (60.3%) had advanced stage disease and 148 (48.8%) had a high-intermediate or high International Prognostic Index (IPI) score. The median follow-up was 60.6 months (2.0-133.8 months). Complete IgH rearrangement was detected in 176 (58.1%), 172 (56.8%), and 113 (37.2%) of patients using the FR1, FR2, and FR3 primer sets, respectively. In total, 80 patients (26.4%) were positive for all three PCR reactions, 82 (27.1%) for two, 57 (18.8%) for one, and 84 (27.7%) had completely negative results. We compared the 5-yr OS and PFS among these subgroups (Figure 1), and observed that the probability of survival decreased with accumulation of positive PCR results. As shown in Figure 2, both the 5-yr OS and PFS were significantly worse for patients with all three reactions positive for complete IgH rearrangement (OS: 54.2% vs 73.2% p=0.002; PFS: 34.4% vs. 59.3%, p<0.001). In multivariate analysis adjusted by IPI score, complete IgH rearrangement detected by all three PCR reactions was significantly correlated with poor prognosis (hazard ratio: 2.13; 95% CI: 1.38-3.26; p<0.001).This classification also allowed subgroups with unfavorable prognosis to be identified, even among patients with a high-intermediate or high IPI score (5-yr OS: 28.0% vs. 62.9%, p<0.001). Moreover, VH sequencing analysis of 42 available samples confirmed that patients with complete IgH rearrangement in all three PCR reactions had significantly lower VH gene SHMs than other groups (median value of VH identity; 91.9% vs 84.0%, p<0.001).Conclusion: Complete IgH rearrangement detected by all three primer sets in the BIOMED-2 PCR protocol was associated with poor prognosis in patients with DLBCL. Combined with the additional sequencing analysis, this suggests that the lower levels of VH SHMs may be associated with unfavorable prognosis in DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Human germinal centres engage memory and naive B cells after influenza vaccination.

SummaryInfluenza viruses remain a major public health threat. Seasonal influenza vaccination in humans primarily stimulates pre-existing memory B cells, leading to a transient wave of circulating antibody-secreting plasmablasts1–3. This recall response contributes to “original antigenic sin,” the selective boosting of antibody specificities from prior exposures to influenza virus antigens4. It remains unclear whether such vaccination can also induce germinal centre (GC) reactions in the draining lymph node (LN) where diversification and maturation of recruited B cells can occur5. Here we used ultrasound-guided fine needle aspiration to serially sample the draining LNs and investigate the dynamics and specificity of GC B cell responses after influenza vaccination in humans. We show that influenza vaccine-binding GC B cells can be detected as early as 1 week after vaccination. In 3 out of 8 participants, we detected vaccine-binding GC B cells up to 9 weeks after vaccination. Between 12% and 88% of the responding GC B cell clones overlapped with those detected among early circulating plasmablasts. These shared B cell clones had high frequencies of somatic hypermutation (SHM) and encoded broadly cross-reactive monoclonal antibodies (mAbs). In contrast, vaccine-induced B cell clones detected only in the GC compartment exhibited significantly lower SHM frequencies and predominantly encoded strain-specific mAbs, suggesting a naïve B cell origin. Electron microscopy-based epitope mapping revealed that some of these strain-specific mAbs recognized epitopes that were not targeted by the early plasmablast response. Our results indicate that influenza virus vaccination of humans can elicit a GC reaction to which B cell clones targeting novel epitopes are more likely to be recruited, thereby broadening the spectrum of vaccine-induced protective antibodies against this rapidly mutating pathogen.

LPL deletion is associated with poorer response to ibrutinib-based treatments and overall survival in TP53-deleted chronic lymphocytic leukemia.

Ibrutinib-based therapy represents a recent success in managing high-risk CLL patients with 17p/TP53 deletion. However, a subset of CLL patients are resistant to therapy. Deletion of lipoprotein lipase (LPL) has been postulated as a potential evasion mechanism to ibrutinib-based therapy. In this study, we assessed for LPL deletion by fluorescence in situ hybridization in 176 consecutive CLL patients with 17p/TP53 deletion. LPL deletion was detected in 35 (20%) of CLL patients. Patients with LPL deletion (del) showed a higher frequency of CD38 expression but have comparable frequencies of somatic hypermutation and ZAP-70 expression compared with patients with normal (nml) LPL. Gene mutation analysis showed that TP53 was mutated in 68% of LPL-del versus 91% of LPL-nml patients. The overall response to ibrutinib-based therapy was 57%, including 37% complete remission (CR) and 20% partial remission (PR) in patients with LPL-del versus 90% (56% CR and 34% PR) in patients with LPL-nml (p < 0.001). LPL-del patients also showed a poorer overall survival (OS) compared with patients with LPL-nml (median OS, 236months versus undefined, p < 0.001). In summary, the data presented establish an association between LPL deletion, resistance to ibrutinib-based therapy, and poorer overall survival in TP53-deleted CLL patients. We suggest that LPL deletion might be utilized as a biomarker for risk stratification and to predict therapeutic response in this high-risk group of CLL patients.

Long-Amplicon Sequencing of Rearranged IGH Chains Enables Simultaneous Measurement of Somatic Hypermutation and Isotype in Chronic Lymphocytic Leukemia

Background Chronic lymphocytic leukemia (CLL) is a common form of leukemia characterized by clonal expansion of neoplastic mature B cells and a heterogenous disease course ranging from aggressive clonal expansion requiring early intervention to indolent disease that does not require treatment. Accumulating evidence suggests that the somatic hypermutation (SHM) status of the IGHV gene of the malignant clone may serve as a prognostic marker of clinical outcome, where a SHM frequency of &gt;2% indicates a favorable outcome (SHM-M) while &lt; 2% SHM (SHM-U) indicates comparatively poor prognosis. Current next-generation sequencing (NGS) approaches for analyzing SHM commonly rely on multiplex primers targeting the framework 1 (FR1) or leader region of the IGH variable gene in combination with joining gene primers to amplify rearranged IGH chains from gDNA template. Limitations include the potential for joining gene mutations to interfere with primer binding and an inability to evaluate isotype, which could potentially serve as an additional prognostic marker given the mechanistic link between somatic hypermutation and class-switch recombination. Here we present a novel method for translational research investigations of IGH chain SHM employing multiplex FR1 and isotype (constant gene) specific primers to amplify IGH chains from RNA template. We demonstrate performance of the assay via sequencing of synthesized CLL IGH chains derived from literature. Methods Multiplex PCR primers were designed to target all IGH variable genes and constant genes and alleles in the IMGT database (Oncomine IGH-LR assay). Primers were used to amplify IGH chains in samples consisting of 25ng healthy donor peripheral blood leukocyte (PBL) total RNA spiked with one of 7 synthesized monoclonal CLL rearrangements including three germline rearrangements (0% SHM), and four mutated rearrangements (SHM ranging from 3-9%), each expressed with a different isotype. Resultant libraries were sequenced on the Gene Studio S5 and analyzed via Ion Reporter to identify clonotypes, quantify SHM, and identify B cell clonal lineages. Automated downsampling analysis was used to confirm that libraries had been sequenced to an appropriate depth. Results 7 of 7 synthesized rearrangements were correctly classified as SHM-M or SHM-U, and in each instance the isotype was correctly identified by the software. Clonal lineage analysis indicated that each spike in rearrangement was monoclonal, consistent with expectation. In silico analysis revealed a high correlation between SHM estimates obtained using the entire v-gene sequence and those obtained from FR1-targeting primers (Pearson's correlation &gt;.99). Conclusions These results support the robustness and reliability of multiplex FR1 and constant gene based IGH chain amplification, combined with clonotyping and lineage analysis, for the translational research characterization of somatic hypermutation in CLL and other B cell neoplasms, including efforts to understand the potential prognostic value of isotype. Disclosures Looney: Thermo Fisher Scientific: Employment. Lowman:Thermo Fisher Scientific: Employment. Pickle:Thermo Fisher Scientific: Employment. Chang:Thermo Fisher Scientific: Employment. Topacio-Hall:Thermo Fisher Scientific: Employment. Toro:Thermo Fisher Scientific: Employment. Hyland:Thermo Fisher Scientific: Employment.

Chicken MBD4 Regulates Immunoglobulin Diversification by Somatic Hypermutation.

Immunoglobulin (Ig) diversification occurs via somatic hypermutation (SHM) and class switch recombination (CSR), and is initiated by activation-induced deaminase (AID), which converts cytosine to uracil. Variable (V) region genes undergo SHM to create amino acid substitutions that produce antibodies with higher affinity for antigen. The conversion of cytosine to uracil in DNA promotes mutagenesis. Two distinct DNA repair mechanisms regulate uracil processing in Ig genes. The first involves base removal by the uracil DNA glycosylase (UNG), and the second detects uracil via the mismatch repair (MMR) complex. Methyl binding domain protein 4 (MBD4) is a uracil glycosylase and an intriguing candidate for involvement in somatic hypermutation because of its interaction with the MMR MutL homolog 1 (MLH1). We found that the DNA uracil glycosylase domain of MBD4 is highly conserved among mammals, birds, shark, and insects. Conservation of the human and chicken MBD4 uracil glycosylase domain structure is striking. Here we examined the function of MBD4 in chicken DT40 B cells which undergo constitutive SHM. We constructed structural variants of MBD4 DT40 cells using CRISPR/Cas9 genome editing. Disruption of the MBD4 uracil glycosylase catalytic region increased SHM frequency in IgM loss assays. We propose that MBD4 plays a role in SHM.

Skewed B cell receptor repertoire and reduced antibody avidity in patients with DOCK8 deficiency.

DOCK8 immunodeficiency syndrome (DIDS) is a combined immunodeficiency characterized by recurrent viral infections, severe atopy and early onset malignancy. Immunological abnormalities include lymphopenia, CD8+ T-cell cytoskeleton dysfunction, defective B cell memory and variable serum immunoglobulin levels. Here, we analyse the B cell receptor repertoire (BCR) characteristics and antibody avidity of four DIDS patients, attempt to understand the dysregulated humoral immunity in DIDS patients with a normal antibody titre and suggest a scientific basis for intravenous immunoglobulin (IVIG) replacement therapy for these patients. We analysed BCR characteristics, including somatic hypermutation (SHM) frequency, using deep sequencing of multiplex PCR products derived from BCR heavy chain CDR3 regions from DIDS patients and controls. The antibody avidity of human tetanus and hemophilus influenza B antibodies was determined by ELISA using thiocyanate elution. IVIG replacement treatment and infection conditions were investigated retrospectively. We found skewing of the BCR repertoire and decreased antibody avidity in patients with DIDS. DIDS patients had fewer negatively charged amino acids than healthy controls. The SHM frequency of the IGHV3 gene was lower in patients with DIDS. Patients received regular IVIG therapy, resulting in fewer and less severe infections. We conclude that although IgG levels are normal in most DIDS patients, IVIG replacement therapy is still necessary.

Apurinic/apyrimidinic endonuclease 1 (APE1) is dispensable for activation-induced cytidine deaminase (AID)-dependent somatic hypermutation in the immunoglobulin gene.

Activation-induced cytidine deaminase (AID) initiates DNA breakage in the variable (V) and switch (S) regions of the immunoglobulin gene, which results in somatic hypermutation (SHM) and class switch recombination (CSR), respectively. Apurinic/apyrimidinic endonuclease 1 (APE1) has been shown to be important for CSR, and is supposed to cleave at abasic sites when AID-dependently deaminated cytidine is removed by uracil DNA glycosylase. However, APE1 is unexpectedly dispensable for SHM in the S region and translocation between immunoglobulin heavy chain (IgH) and c-myc genes in the mouse B lymphoma cell line, CH12F3-2A. This suggested that APE1 is not involved in AID-dependent DNA breakage, but rather, in DNA repair. In order to investigate detailed molecular mechanisms underlying APE1's involvement in CSR and SHM, we measured apurinic/apyrimidinic (AP) sites via aldehyde reactive probe labeling. Results indicated that the frequencies of AP sites in the S regions were not different between APE1-/-/-CH12F3-2A and wild-type CH12F3-2A cells. To carry out similar experiments in SHM of the V region, we generated an APE1 knockout (APE1-/-) human Burkitt's lymphoma cell line, and compared SHM between APE1-proficient and -deficient BL2 lymphoma cells. SHM frequencies in the V regions of APE1-/-BL2 and APE1-proficient cells were also similar. Taken together, we showed that AID does not induce AP sites in the S region of the IgH gene, and that APE1 is not necessary for SHM in the V and S regions; however, it is required for DNA repair following DNA breakage in CSR.