VCAb: a web-tool for structure-guided exploration of antibodies.

Motivation: Effective responses against immune challenges require antibodies of different isotypes performing specific effector functions. Structural information on these isotypes is essential to engineer antibodies with desired physico-chemical features of their antigen-binding properties, and optimal developability as potential therapeutics. mutational scanning profiles on antibody structures would further pinpoint candidate mutations for enhancing antibody stability and function.

sciCSR infers B cell state transition and predicts class-switch recombination dynamics using single-cell transcriptomic data.

Class-switch recombination (CSR) is an integral part of B cell maturation. Here we present sciCSR (pronounced 'scissor', single-cell inference of class-switch recombination), a computational pipeline that analyzes CSR events and dynamics of B cells from single-cell RNA sequencing (scRNA-seq) experiments. Validated on both simulated and real data, sciCSR re-analyzes scRNA-seq alignments to differentiate productive heavy-chain immunoglobulin transcripts from germline 'sterile' transcripts.

Pandemic, Epidemic, Endemic: B Cell Repertoire Analysis Reveals Unique Anti-Viral Responses to SARS-CoV-2, Ebola and Respiratory Syncytial Virus.

Immunoglobulin gene heterogeneity reflects the diversity and focus of the humoral immune response towards different infections, enabling inference of B cell development processes. Detailed compositional and lineage analysis of long read IGH repertoire sequencing, combining examples of pandemic, epidemic and endemic viral infections with control and vaccination samples, demonstrates general responses including increased use of in both Zaire Ebolavirus (EBOV) and COVID-19 patient cohorts. We also show unique characteristics absent in Respiratory Syncytial Virus or yellow fever vaccine samples: EBOV survivors show unprecedented high levels of class switching events while COVID-19 repertoires from acute disease appear underdeveloped.

Tumor-Infiltrating B Lymphocyte Profiling Identifies IgG-Biased, Clonally Expanded Prognostic Phenotypes in Triple-Negative Breast Cancer.

In breast cancer, humoral immune responses may contribute to clinical outcomes, especially in more immunogenic subtypes. Here, we investigated B lymphocyte subsets, immunoglobulin expression, and clonal features in breast tumors, focusing on aggressive triple-negative breast cancers (TNBC). In samples from patients with TNBC and healthy volunteers, circulating and tumor-infiltrating B lymphocytes (TIL-B) were evaluated.

Single-Cell Transcriptomic Analyses Define Distinct Peripheral B Cell Subsets and Discrete Development Pathways.

Separation of B cells into different subsets has been useful to understand their different functions in various immune scenarios. In some instances, the subsets defined by phenotypic FACS separation are relatively homogeneous and so establishing the functions associated with them is straightforward. Other subsets, such as the "Double negative" (DN, CD19+CD27-IgD-) population, are more complex with reports of differing functionality which could indicate a heterogeneous population.

Age-Related Changes in B Cells Relevant to Vaccine Responses.

Older people have reduced immune responses to infection and vaccination. B cell activation is key for the efficacy of the vaccine response, but there are several age-related changes in B cells which may contribute to the loss of vaccine efficacy. Different subpopulations of B cells have different functions and phenotypes.

BRepertoire: a user-friendly web server for analysing antibody repertoire data.

Antibody repertoire analysis by high throughput sequencing is now widely used, but a persisting challenge is enabling immunologists to explore their data to discover discriminating repertoire features for their own particular investigations. Computational methods are necessary for large-scale evaluation of antibody properties. We have developed BRepertoire, a suite of user-friendly web-based software tools for large-scale statistical analyses of repertoire data.

Transitional B Cells in Early Human B Cell Development - Time to Revisit the Paradigm?

The B cell repertoire is generated in the adult bone marrow by an ordered series of gene rearrangement processes that result in massive diversity of immunoglobulin (Ig) genes and consequently an equally large number of potential specificities for antigen. As the process is essentially random, the cells exhibiting excess reactivity with self-antigens are generated and need to be removed from the repertoire before the cells are fully mature. Some of the cells are deleted, and some will undergo receptor editing to see if changing the light chain can rescue an autoreactive antibody.

Significant Differences in Physicochemical Properties of Human Immunoglobulin Kappa and Lambda CDR3 Regions.

Antibody variable regions are composed of a heavy and a light chain, and in humans, there are two light chain isotypes: kappa and lambda. Despite their importance in receptor editing, the light chain is often overlooked in the antibody literature, with the focus being on the heavy chain complementarity-determining region (CDR)-H3 region. In this paper, we set out to investigate the physicochemical and structural differences between human kappa and lambda light chain CDR regions.

A Reassessment of IgM Memory Subsets in Humans.

From paired blood and spleen samples from three adult donors, we performed high-throughput VH sequencing of human B cell subsets defined by IgD and CD27 expression: IgD(+)CD27(+) ("marginal zone [MZ]"), IgD(-)CD27(+) ("memory," including IgM ["IgM-only"], IgG and IgA) and IgD(-)CD27(-) cells ("double-negative," including IgM, IgG, and IgA). A total of 91,294 unique sequences clustered in 42,670 clones, revealing major clonal expansions in each of these subsets. Among these clones, we further analyzed those shared sequences from different subsets or tissues for VH gene mutation, H-CDR3-length, and VH/JH usage, comparing these different characteristics with all sequences from their subset of origin for which these parameters constitute a distinct signature.

Age-related aspects of human IgM(+) B cell heterogeneity.

The CD27(+) IgD(+) B cell population, known as IgM memory, reduces with age. It is thought that this population is responsible for pneumococcal polysaccharide T-independent responses, and that the age-related reduction might be partially responsible for the increased susceptibility of older people to bacterial pathogens. There are other IgM(+) B cell populations that do not express IgD.

Immunoglobulin light chain allelic inclusion in systemic lupus erythematosus.

The principles of allelic exclusion state that each B cell expresses a single light and heavy chain pair. Here, we show that B cells with both kappa and lambda light chains (Igκ and Igλ) are enriched in some patients with the systemic autoimmune disease systemic lupus erythematosus (SLE), but not in the systemic autoimmune disease control granulomatosis with polyangiitis. Detection of dual Igκ and Igλ expression by flow cytometry could not be abolished by acid washing or by DNAse treatment to remove any bound polyclonal antibody or complexes, and was retained after two days in culture.

Monoclonal paraprotein influences baseline B-cell repertoire diversity and perturbates influenza vaccination-induced B-cell response.

Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasma cells in the bone marrow, secreting monoclonal (M) paraprotein. It is associated with increased susceptibility to infections, which may reflect altered B-cell repertoire. To investigate this, we examined the immunoglobulin (Ig) M, IgG, and IgA B-cell repertoire diversity in MGUS at baseline and after influenza vaccination (n = 16) in comparison with healthy controls (HCs; n = 16).

Immunoglobulin kappa variable region gene selection during early human B cell development in health and systemic lupus erythematosus.

The unique specificity of the B cell receptor is generated by an ordered sequence of gene rearrangement events. Once IGH genes have rearranged, rearrangement at the IGK locus is initiated followed by the IGL locus if functional IGK rearrangement is not achieved. Receptor specificity can subsequently be altered by secondary light chain editing based on the features of the heavy and light chain combination.

Immunoglobulin gene repertoire diversification and selection in the stomach - from gastritis to gastric lymphomas.

Chronic gastritis is characterized by gastric mucosal inflammation due to autoimmune responses or infection, frequently with Helicobacter pylori. Gastritis with H. pylori background can cause gastric mucosa-associated lymphoid tissue lymphoma (MALT-L), which sometimes further transforms into diffuse large B-cell lymphoma (DLBCL).

Redemption of autoantibodies on anergic B cells by variable-region glycosylation and mutation away from self-reactivity.

The best-understood mechanisms for achieving antibody self/non-self discrimination discard self-reactive antibodies before they can be tested for binding microbial antigens, potentially creating holes in the repertoire. Here we provide evidence for a complementary mechanism: retaining autoantibodies in the repertoire displayed as low levels of IgM and high IgD on anergic B cells, masking a varying proportion of autoantibody-binding sites with carbohydrates, and removing their self-reactivity by somatic hypermutation and selection in germinal centers (GCs). Analysis of human antibody sequences by deep sequencing of isotype-switched memory B cells or in IgG antibodies elicited against allogeneic RhD+ erythrocytes, vaccinia virus, rotavirus, or tetanus toxoid provides evidence for reactivation of anergic IgM(low) IgD+ IGHV4-34+ B cells and removal of cold agglutinin self-reactivity by hypermutation, often accompanied by mutations that inactivated an N-linked glycosylation sequon in complementarity-determining region 2 (CDR2).

Human lymphocyte repertoires in ageing.

Deterioration of adaptive immunity with ageing may reflect changes in the repertoire of T cells and B cells available to respond to antigenic challenges, due to altered proportions and absolute numbers of lymphocyte subpopulations as well as changes in the repertoire of antigen receptor genes expressed by these cells. High-throughput DNA sequencing (HTS) now facilitates examination of immunoglobulin and T cell receptor gene rearrangements, and initial studies using these methods to study immune system ageing in humans have demonstrated age-related alterations in the receptor populations within lymphocyte subsets, as well as in repertoires responding to vaccination. Accurate measurement of repertoire diversity remains an experimental challenge.

Granulocyte colony stimulating factor exacerbates antineutrophil cytoplasmic antibody vasculitis.

Objectives: Granulocyte colony stimulating factor (GCSF) is important in mobilising neutrophils from the bone marrow but also has a range of proinflammatory effects. We therefore decided to investigate the role of GCSF in antineutrophil cytoplasmic antibody (ANCA) vasculitis.

Methods: We measured GCSF levels in the serum of 38 patients with active ANCA vasculitis compared with 31 age-matched controls, and assessed the effect of GCSF priming on the response of human neutrophils to ANCA.

PCR amplification and high throughput sequencing of immunoglobulin heavy chain genes from formalin-fixed paraffin-embedded human biopsies.

The use of high throughput sequencing (HTS) technologies in biomedicine is expanding in a variety of fields in recent years. The 454 system is an HTS platform that is ideally suited to characterize B cell receptor (BCR) repertoires by sequencing of immunoglobulin (Ig) genes, as it is able to sequence stretches of several hundred nucleotides. Most studies that used this platform for antibody repertoire analyses have started from fresh or frozen tissues or peripheral blood samples, and rely on starting with optimal quality DNA.

Age-Related Changes in Human Peripheral Blood IGH Repertoire Following Vaccination.

Immune protection against pulmonary infections, such as seasonal flu and invasive pneumonia, is severely attenuated with age, and vaccination regimes for the elderly people often fail to elicit effective immune response. We have previously shown that influenza and pneumococcal vaccine responses in the older population are significantly impaired in terms of serum antibody production, and have shown repertoire differences by CDR-H3 spectratype analysis. Here we report a detailed analysis of the B cell repertoire in response to vaccine, including a breakdown of sequences by class and subclass.

B cell immunosenescence: different features of naive and memory B cells in elderly.

Elderly people show a reduced protection against new infections and a decreased response to vaccines as a consequence of impairment of both cellular and humoral immunity. In this paper we have studied memory/naïve B cells in the elderly, evaluating surface immunoglobulin expression, production of the pro- and anti-inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-10, and presence of somatic hypermutation, focusing on the IgG(+)IgD(-)CD27(-) double negative (DN) B cells that are expanded in the elderly. Our results show that naïve B cells from young donors need a sufficiently strong stimulus to be activated "in vitro", while naïve B cells from old subjects are able to produce IL-10 and TNF-α when stimulated "physiologically" (α-CD40/IL-4), suggesting that these cells might play a role in the control of the immuno-inflammatory environment in the elderly.