Levels Of Somatic Hypermutation Research Articles

The origin and fate of malaria-associated atypical memory B cells

Abstract Chronic and frequently recurring infectious diseases, such as malaria, are commonly associated with expanded populations of atypical memory B cells (atMBCs, here defined as CD19+CD20+CD21−CD27− B cells). While the phenotype and conditions leading to the generation of atMBCs in malaria-experienced individuals have extensively been studied, the origin and fate of these cells remain largely elusive. Using B cell receptor (BCR) sequencing to track clonally related cells present before and after a malaria episode in 5-year-old Ugandan children, we observed that IgM+ atMBCs were predominantly derived from naïve B cells, while IgG+ atMBCs present after a malaria episode mainly originated from FcRL5+IgG+ classical MBCs (cMBCs, CD19+CD20+CD21+CD27+ B cells). IgM+ atMBCs showed fewer connections with other B cell subsets and higher turnover than IgG+ atMBCs. Single-cell transcriptomics analysis revealed that IgG+ atMBCs could be divided into two distinct clusters, one of which was maintained up to 6 months after infection. These results underscore the heterogeneity among atMBCs and highlight differences in their longevity. Finally, we observed that atMBCs from malaria-naïve and malaria-experienced donors showed many similarities in BCR characteristics, such as levels of somatic hypermutation, suggesting that atMBCs undergo similar differentiation pathways in response to different types of pathogens. In terms of BCR characteristics, IgM+ atMBCs of all donors closely resembled NBCs, while IgG+ atMBCs were more similar to IgG+ cMBCs. Collectively, our results highlight differences between unswitched and switched atMBC populations and shed light on the origin and fate of atMBCs in malaria-experienced individuals.

Lupus-like extrafollicular B cell responses and autoreactivity are hallmarks of severe COVID-19

Abstract In late 2019, the emergence of the coronavirus SARS-CoV-2 resulted in a global pandemic, costing over 2 million lives. A hallmark of the disease resulting from SARS-CoV-2 infection, COVID-19, has been it’s heterogeneity in presentation. To understand the immunological underpinnings of this variability in symptoms, we performed a deep characterization of the B cell responses generated in patients within the NIH-defined mild/moderate or severe/critical categories. Somewhat surprisingly, patients with severe disease had higher neutralizing antibody titers than their mild/moderate counterparts, but showed strong evidence of extrafollicular B cell activation by flow cytometry – previously described in humans primarily in autoimmune disease. Further analysis of the B cell repertoire through single cell VDJ sequencing revealed low (or fully absent) levels of somatic hypermutation in the antibody secreting cell compartment, phenocopying the low selective pressures previously associated with extrafollicular responses. Together, these findings suggested the possible relaxation of peripheral tolerance in severe patients. This was confirmed through clinical autoreactivity testing, revealing more than 70% of the most severe COVID-19 patients, despite no history of autoimmunity, test positive for anti-nuclear antibodies or rheumatoid factor. Altogether, these findings provide the immunological context for an emerging consensus in the literature whereby severe COVID-19 is characterized by robust extrafollicular B cell activation, resulting not only in neutralizing antibody production, but also autoreactive B cell targeting with the potential for direct involvement in severe disease pathology.

The Ubiquitin Ligase Itch Regulates Germinal Center Selection

Abstract More than 23 million Americans suffer from autoimmune diseases driven by somatically mutated autoantibodies. High levels of somatic hypermutation of immunoglobulin genes occurs in germinal center (GC) B cells. Some of these mutations render autoreactivity, but cells bearing autoreactive receptors are eliminated due to strict selection of GC B cells by mechanisms that are still poorly defined. Inappropriate positive selection signals can rescue autoreactive cells from elimination and drive autoimmune disease. The E3 ubiquitin ligase Itch prevents the emergence of autoimmune disease and autoantibodies in humans and mice, and patients lacking Itch develop debilitating, multi-faceted, potentially fatal, autoimmune disease; yet how Itch regulates GC B cell fate or function is not well understood. By studying Itch deficient mice, we have recently shown that Itch directly limits B cell activity to shape antibody responses. While Itch-deficient mice displayed normal numbers of pre-immune B cell populations, they showed elevated numbers of GC B cells. Mixed bone marrow chimeras revealed that Itch acts within B cells to skew the proportions of light zone and dark zone GC B cells. Proteomic profiling of GC B cells uncovered that Itch deficient cells exhibit hallmarks of positive selection. These results support a novel role for Itch in limiting positive selection of GC B cells to restrict GC size. In this role, Itch may shape the GC-derived antibody repertoire to prevent autoimmunity.

Unbiased interrogation of memory B cells from convalescent COVID-19 patients reveals a broad antiviral humoral response targeting SARS-CoV-2 antigens beyond the spike protein

Patients who recover from SARS-CoV-2 infections produce antibodies and antigen-specific T cells against multiple viral proteins. Here, an unbiased interrogation of the anti-viral memory B cell repertoire of convalescent patients has been performed by generating large, stable hybridoma libraries and screening thousands of monoclonal antibodies to identify specific, high-affinity immunoglobulins (Igs) directed at distinct viral components. As expected, a significant number of antibodies were directed at the Spike (S) protein, a majority of which recognized the full-length protein. These full-length Spike specific antibodies included a group of somatically hypermutated IgMs. Further, all but one of the six COVID-19 convalescent patients produced class-switched antibodies to a soluble form of the receptor-binding domain (RBD) of S protein. Functional properties of anti-Spike antibodies were confirmed in a pseudovirus neutralization assay. Importantly, more than half of all of the antibodies generated were directed at non-S viral proteins, including structural nucleocapsid (N) and membrane (M) proteins, as well as auxiliary open reading frame-encoded (ORF) proteins. The antibodies were generally characterized as having variable levels of somatic hypermutations (SHM) in all Ig classes and sub-types, and a diversity of VL and VH gene usage. These findings demonstrated that an unbiased, function-based approach towards interrogating the COVID-19 patient memory B cell response may have distinct advantages relative to genomics-based approaches when identifying highly effective anti-viral antibodies directed at SARS-CoV-2.

Single-cell immune repertoire and transcriptome sequencing reveals that clonally expanded and transcriptionally distinct lymphocytes populate the aged central nervous system in mice.

Neuroinflammation plays a crucial role during ageing and various neurological conditions, including Alzheimer's disease, multiple sclerosis and infection. Technical limitations, however, have prevented an integrative analysis of how lymphocyte immune receptor repertoires and their accompanying transcriptional states change with age in the central nervous system. Here, we leveraged single-cell sequencing to simultaneously profile B cell receptor and T cell receptor repertoires and accompanying gene expression profiles in young and old mouse brains. We observed the presence of clonally expanded B and T cells in the central nervous system of aged male mice. Furthermore, many of these B cells were of the IgM and IgD isotypes, and had low levels of somatic hypermutation. Integrating gene expression information additionally revealed distinct transcriptional profiles of these clonally expanded lymphocytes. Our findings implicate that clonally related T and B cells in the CNS of elderly mice may contribute to neuroinflammation accompanying homeostatic ageing.

Functional Profiling of Antibody Immune Repertoires in Convalescent Zika Virus Disease Patients.

The re-emergence of Zika virus (ZIKV) caused widespread infections that were linked to Guillain-Barré syndrome in adults and congenital malformation in fetuses, and epidemiological data suggest that ZIKV infection can induce protective antibody responses. A more detailed understanding of anti-ZIKV antibody responses may lead to enhanced antibody discovery and improved vaccine designs against ZIKV and related flaviviruses. Here, we applied recently-invented library-scale antibody screening technologies to determine comprehensive functional molecular and genetic profiles of naturally elicited human anti-ZIKV antibodies in three convalescent individuals. We leveraged natively paired antibody yeast display and NGS to predict antibody cross-reactivities and coarse-grain antibody affinities, to perform in-depth immune profiling of IgM, IgG, and IgA antibody repertoires in peripheral blood, and to reveal virus maturation state-dependent antibody interactions. Repertoire-scale comparison of ZIKV VLP-specific and non-specific antibodies in the same individuals also showed that mean antibody somatic hypermutation levels were substantially influenced by donor-intrinsic characteristics. These data provide insights into antiviral antibody responses to ZIKV disease and outline systems-level strategies to track human antibody immune responses to emergent viral infections.

Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV.

Human cytomegalovirus (HCMV) causes serious complications to immune compromised hosts. Dendritic cells (iDCgB) expressing granulocyte-macrophage colony-stimulating factor, interferon-alpha and HCMV-gB were developed to promote de novo antiviral adaptive responses. Mice reconstituted with a human immune system (HIS) were immunized with iDCgB and challenged with HCMV, resulting into 93% protection. Immunization stimulated the expansion of functional effector memory CD8+ and CD4+ T cells recognizing gB. Machine learning analyses confirmed bone marrow T/CD4+, liver B/IgA+ and spleen B/IgG+ cells as predictive biomarkers of immunization (≈87% accuracy). CD8+ and CD4+ T cell responses against gB were validated. Splenic gB-binding IgM-/IgG+ B cells were sorted and analyzed at a single cell level. iDCgB immunizations elicited human-like IgG responses with a broad usage of various IgG heavy chain V gene segments harboring variable levels of somatic hypermutation. From this search, two gB-binding human monoclonal IgGs were generated that neutralized HCMV infection in vitro. Passive immunization with these antibodies provided proof-of-concept evidence of protection against HCMV infection. This HIS/HCMV in vivo model system supported the validation of novel active and passive immune therapies for future clinical translation.

Adjuvanted H5N1 influenza vaccine enhances both cross-reactive memory B cell and strain-specific naive B cell responses in humans

There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.

Comparisons of the antibody repertoires of a humanized rodent and humans by high throughput sequencing

The humanization of animal model immune systems by genetic engineering has shown great promise for antibody discovery, tolerance studies and for the evaluation of vaccines. Assessment of the baseline antibody repertoires of unimmunized model animals will be useful as a benchmark for future immunization experiments. We characterized the heavy chain and kappa light chain antibody repertoires of a model animal, the OmniRat, by high throughput antibody sequencing and made use of two novel datasets for comparison to human repertoires. Intra-animal and inter-animal repertoire comparisons reveal a high level of conservation in antibody diversity between the lymph node and spleen and between members of the species. Multiple differences were found in both the heavy and kappa chain repertoires between OmniRats and humans including gene segment usage, CDR3 length distributions, class switch recombination, somatic hypermutation levels and in features of V(D)J recombination. The Inference and Generation of Repertoires (IGoR) software tool was used to model recombination in VH regions which allowed for the quantification of some of these differences. Diversity estimates of the OmniRat heavy chain repertoires almost reached that of humans, around two orders of magnitude less. Despite variation between the species repertoires, a high frequency of OmniRat clonotypes were also found in the human repertoire. These data give insights into the development and selection of humanized animal antibodies and provide actionable information for use in vaccine studies.

Extensive dissemination and intraclonal maturation of HIV Env vaccine-induced B cell responses.

Well-ordered HIV-1 envelope glycoprotein (Env) trimers are prioritized for clinical evaluation, and there is a need for an improved understanding about how elicited B cell responses evolve following immunization. To accomplish this, we prime-boosted rhesus macaques with clade C NFL trimers and identified 180 unique Ab lineages from ∼1,000 single-sorted Env-specific memory B cells. We traced all lineages in high-throughput heavy chain (HC) repertoire (Rep-seq) data generated from multiple immune compartments and time points and expressed several as monoclonal Abs (mAbs). Our results revealed broad dissemination and high levels of somatic hypermutation (SHM) of most lineages, including tier 2 virus neutralizing lineages, following boosting. SHM was highest in the Ab complementarity determining regions (CDRs) but also surprisingly high in the framework regions (FRs), especially FR3. Our results demonstrate the capacity of the immune system to affinity-mature large numbers of Env-specific B cell lineages simultaneously, supporting the use of regimens consisting of repeated boosts to improve each Ab, even those belonging to less expanded lineages.

PB1868 B‐CELL PROLYMPHOCYTIC LEUKEMIA (B‐PLL) AND PROLYMPHOCYTOID MANTLE CELL LYMPHOMA (PMCL) (MORE THAN 55% OF PROLYMPHOCYTES) ARE CLOSED BUT DISTINCT ENTITIES. ON BEHALF GFCH AND FILO GROUPS

Background:B‐PLL is a rare entity that was debated. It is now recognized in the WHO classification and defined by the presence of prolymphocytes in peripheral blood (PB) exceeding 55% of lymphoid cells, different from transformed chronic lymphocytic leukemia (CLL) and cases with translocation t(11;14) that are classified as MCL. We previously showed that B‐PLL have a high frequency of MYC activation by translocation [t(MYC)] or gain, and frequent del17p, patients with both abnormalities (abs) having the worst prognosis. It has been suggested that B‐PLL should be considered as a subgroup of MCL.Aims:In order to evaluate the relationship between B‐PLL and MCL, we compared cytogenetics and molecular data.Methods:We studied 35 B‐PLL and 4 pMCL (≥55% of prolymphocytes in PB) (multicentric study by the Groupe Francophone de Cytogénétique Hématologique and the FILO group), 24 MCL and 16 CLL as control, using karyotype (K), FISH, whole‐exome sequencing (WES), IGHV and RNA‐sequencing. All MCL had a t(11;14), while the presence of a CCND1, CCND2 or CCND3 rearrangement was excluded in B‐PLL cases.Results:A morphological review, performed by 3 independent experts, did not show any distinctive features between B‐PLL and pMCL. In B‐PLL, we found a complex K (CK) (≥3 abs) in 71%, and a highly complex K (HCK ≥ 5) in 44%. The most frequent chromosomal abs were: MYC abs (n = 27, 77%): t(MYC) (n = 22, 63%) or MYC gain (n = 5, 14%) and del17p (n = 13, 37%). Among the 19/24 MCL with an informative K, we found CK in 13 (68%), including 11 HCK (58%) and del11q (ATM) in 3 (16%). By FISH, we found 9 (38%) del17p and 7 (29%) MYC abs (6 gains and 1 t(MYC)). All 4 pMCL had a CK, including 3 HCK, 2 had a t(MYC), 1 a MYC gain, 2 a del17p and 2 a del11q (Figure A). Using WES on 23 B‐PLL, the most frequent mutated genes were TP53 (n = 12, 52%), MYC, BCOR (n = 5, 22% each), SETD2, MYD88 (n = 4, 17% each), SF3B1 and CHD2 (n = 3, 13% each). WES performed on 3 pMCL showed 2 cases ATMmut, and 1 CCND1mut/TP53mut (Figure B). Overall, ATM inactivation was present in 1/35 (2.9%) B‐PLL vs 3/4 (75%) pMCL (p = .001). The majority of B‐PLL used a VH3 or VH4 gene (17/19, 89%), but none of the 4 pMCL (VH1, n = 3, VH2, n = 1). The level of somatic hypermutation was higher in B‐PLL: 16/19 (84%) had ≤98% of homology vs 1/4 pMCL (p = .04). Principal component analysis of gene expression data in 12 B‐PLL, 3 pMCL and 16 CLL analyzed by RNA‐Seq showed that B‐PLL and pMCL clustered together, distinctively from CLL (Figure C). However, 386 genes were differently expressed between B‐PLL and pMCL, with enrichment in cell cycle genes in pMCL, including CCND1 and CCND2. The level of SOX11 expression was significantly lower in B‐PLL than in pMCL (p = .02). Comparing B‐PLL and CLL, GSEA analysis revealed enrichment in MYC signaling pathway, and cell metabolism signatures in B‐PLL.Summary/Conclusion:In conclusion, our results show that B‐PLL have a genomic profile distinct from MCL and CLL, with frequent t(MYC), TP53, MYC and BCOR mutations. However, B‐PLL have similarities with the rare subgroup of pMCL: morphology, high frequency of MYC abs and gene expression profiles related to cell cycle and proliferation. Nevertheless, we highlighted distinctive features in addition to t(11;14): IGHV repertoire and mutation level, SOX11 expression and frequency of ATM abs. Our data advocate that pMCL could be an aggressive form of MCL, which have to be definitely distinguished from B‐PLL with a routine test, the karyotype.image

High-Quality Library Preparation for NGS-Based Immunoglobulin Germline Gene Inference and Repertoire Expression Analysis.

Next generation sequencing (NGS) of immunoglobulin (Ig) repertoires (Rep-seq) enables examination of the adaptive immune system at an unprecedented level. Applications include studies of expressed repertoires, gene usage, somatic hypermutation levels, Ig lineage tracing and identification of genetic variation within the Ig loci through inference methods. All these applications require starting libraries that allow the generation of sequence data with low error rate and optimal representation of the expressed repertoire. Here, we provide detailed protocols for the production of libraries suitable for human Ig germline gene inference and Ig repertoire studies. Various parameters used in the process were tested in order to demonstrate factors that are critical to obtain high quality libraries. We demonstrate an improved 5′RACE technique that reduces the length constraints of Illumina MiSeq based Rep-seq analysis but allows for the acquisition of sequences upstream of Ig V genes, useful for primer design. We then describe a 5′ multiplex method for library preparation, which yields full length V(D)J sequences suitable for genotype identification and novel gene inference. We provide comprehensive sets of primers targeting IGHV, IGKV, and IGLV genes. Using the optimized protocol, we produced IgM, IgG, IgK, and IgL libraries and analyzed them using the germline inference tool IgDiscover to identify expressed germline V alleles. This process additionally uncovered three IGHV, one IGKV, and six IGLV novel alleles in a single individual, which are absent from the IMGT reference database, highlighting the need for further study of Ig genetic variation. The library generation protocols presented here enable a robust means of analyzing expressed Ig repertoires, identifying novel alleles and producing individualized germline gene databases from humans.

Autoreactive B-lymphocytes in SLE and RA patients: Isolation and characterisation using extractable nuclear and citrullinated antigens bound to immunobeads.

Systemic lupus erythematosus and rheumatoid arthritis are autoimmune diseases characterised by B-cell hyperactivation and production of autoantibodies (AutoAbs) against various self-antigens, including extractable nuclear antigens and citrullinated peptides. Therefore, B lymphocytes and antibody-secreting cells are considered relevant targets for therapies. However, isolation and characterisation of auto-reactive specific B lymphocytes are limited, primarily due to technical issues. In this work, we purified extractable nuclear antigen-specific and citrullinated peptide-specific auto-reactive B lymphocytes by magnetic selection with ENA- and citrullinated peptide-bound immunobeads. We obtained blood auto-reactive B lymphocytes from most patients. Their nature was primarily naïve B cells, some of them in an active status, with low levels of somatic hypermutations in the immunoglobulin heavy-chain variable regions. Their presence correlated with serum levels of autoAb. Auto-reactive B lymphocytes were able to differentiate into auto-reactive antibody-secreting cells under conditions of stimulation. In addition, based on the presence of circulating auto-reactive B cells and/or antibody-secreting cells, four different profiles were described in lupus patients. Thus, tracking auto-reactive B cells and/or antibody-secreting cells in patient blood could represent a biomarker for deciding whether to use therapies blocking either B cells, plasma cells or both, as well as a new tool for monitoring minimal residual autoimmune disease in patients.

Longitudinal Analysis Reveals Early Development of Three MPER-Directed Neutralizing Antibody Lineages from an HIV-1-Infected Individual

Lineage-based vaccine design is an attractive approach for eliciting broadly neutralizing antibodies (bNAbs) against HIV-1. However, most bNAb lineages studied to date have features indicative of unusual recombination and/or development. From an individual in the prospective RV217 cohort, we identified three lineages of bNAbs targeting the membrane-proximal external region (MPER) of the HIV-1 envelope. Antibodies RV217-VRC42.01, -VRC43.01, and -VRC46.01 used distinct modes of recognition and neutralized 96%, 62%, and 30%, respectively, of a 208-strain virus panel. All three lineages had modest levels of somatic hypermutation and normal antibody-loop lengths and were initiated by the founder virusMPER. The broadest lineage, VRC42, was similar to the known bNAb 4E10. A multimeric immunogen based on the founder MPER activatedB cells bearing the unmutated common ancestor of VRC42, with modest maturation of early VRC42 intermediates imparting neutralization breadth. These features suggest that VRC42 may be a promising template for lineage-based vaccine design.

VDJH Gene Repertoire Analysis in Multiple Myeloma (MM) Patients: Correlation with Clinical Data

IntroductionVDJH usage description in B-cell hematological malignancies has brought new insights into the clonal differentiation and clinical implications for certain pathologies, improving patients' care, although no correlation has been made in MM.We present the largest-to-date VDJH gene repertoire analysis in MM, consisting in biological and clinical data from 413 patients. This database was used to comprehensively investigate the characteristics of VDJH rearrangements: gene segment usage, somatic hypermutation (SHM), CDR3 characterization, and immunoglobulin stereotyped clusters assessment. We also investigated the potential relationship of these molecular features with the outcome.MethodsNewly diagnosed MM patients were included in the study. All were managed according to the recommendations of the GEM-PETHEMA Spanish MM group, including the inclusion of most patients in the GEM2000 and GEM2005 schemes. Monoclonal assessment was carried out with FR1 VH-JH amplification, following the BIOMED-2 methods. Moreover, 113 cases were also analyzed by NGS using the LymphoTrack IGH FR1 assay (Invivoscribe Tech, San Diego, CA).VDJH usage and mutational status were analyzed with IMGT-V-Quest. ClustalX2.0 was used to perform clustering analysis with CDR3 regions from our cohort and 1117 additional sequences obtained from IMGT/LIGM-DB corresponding to unique rearrangements from both normal and tumor human B cells. Molecular and clinical data were used to perform survival studies.ResultsThe overall VDJH detection rate was 92.5% (382/413), including 376 patients with only one detectable rearrangement, five with a biallelic rearrangement, and one with a biclonal rearrangement, also detectable by flow cytometry. Thus, 388 rearrangements where detected: 362 were productive, and 26 unproductive.Gene segments were identified in productive rearrangements. VH repertoire in MM reflected its normal counterpart, with IGHV3 being the predominant selected family, followed by IGHV4, IGHV1, IGHV2 and IGHV5. IGHD and IGHJ segment usage showed a skewed distribution, with IGHD2 and IGHD3 families accounting for 55.5% of cases, and IGHJ4 and IGHJ6 accounting for 70.8%.SHM level could be identified in 349/362 productive sequences (mean: 9.16%, SD 3.95) with statistically significant differences between certain IGHV families, namely IGHV2 was less hypermutated than IGHV1 or IGHV4. We could also collect other rearrangement features: nucleotide addition by TdT appeared in 92.5% for N1 and 88.6% for N2, with evidence of exonuclease activity in all cases. CDR3 mean length was 16±4 aminoacids (median 15, range 6-29), with preference for Gly, Ala, Asp, Tyr (~10% each one). The R/S mutation ratio was 2.39 for CDRs versus 1.74 for FWRs, showing the high influence of antigen selection over the former. No stereotyped clusters were found in our cohort.Relationship between these finding and clinical evolution was done through univariate and multivariate analyses. A shorter Progression-Free Survival (PFS) related to well-known clinical factors: presence of plasmacytomas, poor performance status or ISS stages, response to therapy, and poor-risk cytogenetics (p<0.05), as well as with gene segment selection (IGHV5, IGHD4, non IGHD2 and IGHD3), and SHM below 7%, among others (Table 1). Similar clinical data were observed for Overall Survival (OS), where the association between a short overall survival and low SHM rate (<7%) was remarkable (Figure 1).ConclusionsVDJH usage somatic hypermutation levels and CDR3 composition in multiple myeloma resembles the normal mature B cell repertoire. In contrast to Chronic Lymphocytic Leukemia (CLL), Mantle Cell Lymphoma or Marginal Zone Lymphoma, stereotyped receptors were absent in MM, which shows no evidence of autoantigen-driven selection.Cases with SHM levels below 7% were associated with aggressive outcome that, in the same line of CLL, would reflect the expansion of more immature clones in these cases.More studies are needed to deepen in the clinical meaning of these new findings. [Display omitted] DisclosuresPuig:Celgene: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria. Ocio:Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy; Pharmamar: Consultancy; AbbVie: Consultancy; Novartis: Consultancy, Honoraria; Sanofi: Research Funding; BMS: Consultancy; Amgen: Consultancy, Honoraria, Research Funding; Mundipharma: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Array Pharmaceuticals: Research Funding. Mateos:Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Lahuerta:Amgen: Honoraria; Janssen: Honoraria; Celgene: Honoraria. San-Miguel:Celgene: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; MSD: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Brystol-Myers Squibb: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees. García-Sanz:Incyte: Consultancy; Amgen Inc.: Research Funding; Gilead: Research Funding; Spanish Government: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; BMS: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses.

Structural Survey of Broadly Neutralizing Antibodies Targeting the HIV-1 Env Trimer Delineates Epitope Categories and Characteristics of Recognition

Over the past decade, structures have been determined for broadly neutralizing antibodies that recognize all major exposed surfaces of the prefusion-closed HIV-1-envelope (Env) trimer. To understand this recognition and its implications, we analyzed 206 antibody-HIV-1 Env structures from the Protein Data Bank with resolution suitable to define interaction chemistries and measured antibody neutralization on a 208-strain panel. Those with >25% breadth segregated into almost two dozen classes based on ontogeny and recognition and into six epitope categories based on recognized Env residues. For paratope, the number of protruding loops and level of somatic hypermutation were significantly higher for broad HIV-1 neutralizing antibodies than for a comparison set of non-HIV-1 antibodies (p< 0.0001). For epitope, the number of independent sequence segments was higher (p< 0.0001), as well as the glycan component surface area (p= 0.0005). The unusual characteristics of epitope and paratope delineated here are likely to reflect respectively virus-immune evasion and antibody-recognition solutions that allow effective neutralization of HIV-1.

Absence of Surface IgD Does Not Impair Naive B Cell Homeostasis or Memory B Cell Formation in IGHD Haploinsufficient Humans.

Surface IgD is coexpressed with IgM on naive mature B cells. Still, the role of surface IgD remains enigmatic even 50 y after its initial discovery. In this study, we examined the in vivo role of surface IgD in human B cell homeostasis and Ab responses in four individuals with heterozygous nonsense mutations in IGHD All IGHD heterozygous individuals had normal numbers of B cells and serum Igs and did not show signs of immunodeficiency or immune dysregulation. IgD+ and IgD- naive mature B cells were present in equal numbers and showed similar immunophenotypes, except for decreased expression of CD79b in the IgD- subset. Furthermore, both IgD+ and IgD- naive mature B cells had normal replication histories and similar capacities to differentiate into plasma cells upon in vitro stimulation, and Ig class-switched memory B cells showed similar levels of somatic hypermutations. Thus, human B cells lacking IgD expression develop normally and generate immunological memory in vivo, suggesting that surface IgD might function more restrictedly in regulating of B cell activation to specific antigenic structures.

On the origin of rheumatoid factors: Insights from analyses of variable region sequences

ObjectivesRheumatoid factors (RFs) are thought to play an important role in rheumatoid arthritis (RA), but are also found in healthy donors (HDs). Previous studies examined variable region sequences of these autoantibodies at a time when knowledge of the human germline repertoire was incomplete. Here we collected and analyzed RF sequence data from the literature to elucidate how RFs develop and whether their characteristics differ between RA patients and HDs. MethodsA database was built containing nucleotide sequences of RF heavy and light chain variable domains and characteristics including affinity, isotype and specificity, all collected from published papers. Gene usage and mutation frequencies were analyzed using IMGT/HiV-QUEST. Selection strength was assessed with the BASELINe tool. ResultsSequences were retrieved for 183 RF clones (87 RA; 67 HDs; 29 other). No biased gene usage was observed for RA and HDs. However, there does appear to be skewed gene usage in RFs from patients with mixed cryoglobulinemia. Mutation frequency varies considerably between RFs, and isotype-switched clones have significantly more mutations. Monospecific RFs carry more mutations than polyspecific RFs; no difference was found for RA- versus HD-derived RFs. Overall, reported affinity is low (median 1 µM), with a non-significant trend toward higher affinity of RA-derived RFs. Mutation frequency and affinity did not appear to be correlated. BASELINe analysis suggests an overall lack of positive selection and less negative selection strength in RA-derived RFs. ConclusionsRFs derived from RA patients have similar properties as those derived from HDs. The RF response can be characterized as a moderately matured autoantibody response, with variable levels of somatic hypermutation, but low affinity.

Harnessing T Follicular Helper Cell Responses for HIV Vaccine Development.

Passive administration of broadly neutralizing antibodies (bNAbs) capable of recognizing a broad range of viral strains to non-human primates has led to protection from infection with chimeric SIV/HIV virus (SHIV). This data suggests that generating protective antibody responses could be an effective strategy for an HIV vaccine. However, classic vaccine approaches have failed so far to induce such protective antibodies in HIV vaccine trials. HIV-specific bNAbs identified in natural infection show high levels of somatic hypermutations, demonstrating that they underwent extensive affinity maturation. It is likely that to gain ability to recognize diverse viral strains, vaccine-induced humoral responses will also require complex, iterative maturation. T follicular helper cells (Tfh) are a specialized CD4+ T cell subset that provides help to B cells in the germinal center for the generation of high-affinity and long-lasting humoral responses. It is therefore probable that the quality and quantity of Tfh responses upon vaccination will impact development of bNAbs. Here, we review studies that advanced our understanding of Tfh differentiation, function and regulation. We discuss correlates of Tfh responses and bNAb development in natural HIV infection. Finally, we highlight recent strategies to optimize Tfh responses upon vaccination and their impact on prophylactic HIV vaccine research.

The reference epigenome and regulatory chromatin landscape of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a frequent hematological neoplasm in which underlying epigenetic alterations are only partially understood. Here, we analyze the reference epigenome of seven primary CLLs and the regulatory chromatin landscape of 107 primary cases in the context of normal B cell differentiation. We identify that the CLL chromatin landscape is largely influenced by distinct dynamics during normal B cell maturation. Beyond this, we define extensive catalogues of regulatory elements de novo reprogrammed in CLL as a whole and in its major clinico-biological subtypes classified by IGHV somatic hypermutation levels. We uncover that IGHV-unmutated CLLs harbor more active and open chromatin than IGHV-mutated cases. Furthermore, we show that de novo active regions in CLL are enriched for NFAT, FOX and TCF/LEF transcription factor family binding sites. Although most genetic alterations are not associated with consistent epigenetic profiles, CLLs with MYD88 mutations and trisomy 12 show distinct chromatin configurations. Furthermore, we observe that non-coding mutations in IGHV-mutated CLLs are enriched in H3K27ac-associated regulatory elements outside accessible chromatin. Overall, this study provides an integrative portrait of the CLL epigenome, identifies extensive networks of altered regulatory elements and sheds light on the relationship between the genetic and epigenetic architecture of the disease.