Professor Deborah Dunn-Walters FRSB

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 IGHV4-39 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. Despite the high levels of clonal expansion in COVID-19 IgG1 repertoires there is a striking lack of evidence of germinal centre mutation and selection. Given the differences in COVID-19 morbidity and mortality with age, it is also pertinent that we find significant differences in repertoire characteristics between young and old patients. Our data supports the hypothesis that a primary viral challenge can result in a strong but immature humoral response where failures in selection of the repertoire risk off-target effects.

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. Recent advances in single-cell techniques enable an alternative route to characterize cells based on their transcriptome. To maximize immunological insight, we need to match prior data from phenotype-based studies with the finer granularity of the single-cell transcriptomic signatures. We also need to be able to define meaningful B cell subsets from single cell analyses performed on PBMCs, where the relative paucity of a B cell signature means that defining B cell subsets within the whole is challenging. Here we provide a reference single-cell dataset based on phenotypically sorted B cells and an unbiased procedure to better classify functional B cell subsets in the peripheral blood, particularly useful in establishing a baseline cellular landscape and in extracting significant changes with respect to this baseline from single-cell datasets. We find 10 different clusters of B cells and applied a novel, geometry-inspired, method to RNA velocity estimates in order to evaluate the dynamic transitions between B cell clusters. This indicated the presence of two main developmental branches of memory B cells. A T-independent branch that involves IgM memory cells and two DN subpopulations, culminating in a population thought to be associated with Age related B cells and the extrafollicular response. The other, T-dependent, branch involves a third DN cluster which appears to be a precursor of classical memory cells. In addition, we identify a novel DN4 population, which is IgE rich and closely linked to the classical/precursor memory branch suggesting an IgE specific T-dependent cell population.

The COVID-19 pandemic has led to an unprecedented demand for testing - for diagnosis and prognosis - as well as for investigation into the impact of the disease on the host metabolism. Sebum sampling has the potential to support both needs by looking at what the virus does to us, rather than looking for the virus itself. In this pilot study, sebum samples were collected from 67 hospitalised patients (30 COVID-19 positive and 37 COVID-19 negative) by gauze swab. Lipidomics analysis was carried out using liquid chromatography mass spectrometry, identifying 998 reproducible features. Univariate and multivariate statistical analyses were applied to the resulting feature set. Lipid levels were depressed in COVID-19 positive participants, indicative of dyslipidemia; p-values of 0·022 and 0·015 were obtained for triglycerides and ceramides respectively, with effect sizes of 0·44 and 0·57. Partial Least Squares-Discriminant Analysis showed separation of COVID-19 positive and negative participants with sensitivity of 57% and specificity of 68%, improving to 79% and 83% respectively when controlled for confounding comorbidities. COVID-19 dysregulates many areas of metabolism; in this work we show that the skin lipidome can be added to the list. Given that samples can be provided quickly and painlessly, we conclude that sebum is worthy of future consideration for clinical sampling. The authors acknowledge funding from the EPSRC Impact Acceleration Account for sample collection and processing, as well as EPSRC Fellowship Funding EP/R031118/1, the University of Surrey and BBSRC BB/T002212/1. Mass Spectrometry was funded under EP/P001440/1.

Description This dataset of participant, field blank and quality control liquid-chromatography-mass spectrometry .raw files supports the following article: Changes to the sebum lipidome upon COVID-19 infection observed via rapid sampling from the skin - EClinicalMedicine (thelancet.com) Background The COVID-19 pandemic has led to an unprecedented demand for testing - for diagnosis and prognosis - as well as for investigation into the impact of the disease on the host metabolism. Sebum sampling has the potential to support both needs by looking at what the virus does to us, rather than looking for the virus itself. Methods and attached dataset description In this pilot study, sebum samples were collected from 67 hospitalised patients (30 COVID-19 positive and 37 COVID-19 negative) by gauze swab. Lipidomics analysis was carried out using liquid chromatography mass spectrometry, identifying 998 reproducible features. Univariate and multivariate statistical analyses were applied to the resulting feature set. The dataset uploaded here represents .raw liquid chromatography-mass spectrometry files for participants (triplicate injections), field blanks and pooled quality control standards, as well as the output peak:area matrix. Findings Lipid levels were depressed in COVID-19 positive participants, indicative of dyslipidemia; p-values of 0·022 and 0·015 were obtained for triglycerides and ceramides respectively, with effect sizes of 0·44 and 0·57. Partial Least Squares-Discriminant Analysis showed separation of COVID-19 positive and negative participants with sensitivity of 57% and specificity of 68%, improving to 79% and 83% respectively when controlled for confounding comorbidities. Interpretation COVID-19 dysregulates many areas of metabolism; in this work we show that the skin lipidome can be added to the list. Given that samples can be provided quickly and painlessly, we conclude that sebum is worthy of future consideration for clinical sampling.

Treatments for COVID-19 infections have improved dramatically since the beginning of the pandemic, and glucocorticoids have been a key tool in improving mortality rates. The UK’s National Institute for Health and Care Excellence guidance is for treatment to be targeted only at those requiring oxygen supplementation, however, and the interactions between glucocorticoids and COVID-19 are not completely understood. In this work, a multi-omic analysis of 98 inpatient-recruited participants was performed by quantitative metabolomics (using targeted liquid chromatography-mass spectrometry) and data-independent acquisition proteomics. Both ‘omics datasets were analysed for statistically significant features and pathways differentiating participants whose treatment regimens did or did not include glucocorticoids. Metabolomic differences in glucocorticoid-treated patients included the modulation of cortisol and bile acid concentrations in serum, but no alleviation of serum dyslipidemia or increased amino acid concentrations (including tyrosine and arginine) in the glucocorticoid-treated cohort relative to the untreated cohort. Proteomic pathway analysis indicated neutrophil and platelet degranulation as influenced by glucocorticoid treatment. These results are in keeping with the key role of platelet-associated pathways and neutrophils in COVID-19 pathogenesis and provide opportunity for further understanding of glucocorticoid action. The findings also, however, highlight that glucocorticoids are not fully effective across the wide range of ‘omics dysregulation caused by COVID-19 infections.

Abstract The majority of metabolomics studies to date have utilised blood serum or plasma, biofluids that do not necessarily address the full range of patient pathologies. Here, correlations between serum metabolites, salivary metabolites and sebum lipids are studied for the first time. 83 COVID-19 positive and negative hospitalised participants provided blood serum alongside saliva and sebum samples for analysis by liquid chromatography mass spectrometry. Widespread alterations to serum-sebum lipid relationships were observed in COVID-19 positive participants versus negative controls. There was also a marked correlation between sebum lipids and the immunostimulatory hormone dehydroepiandrosterone sulphate in the COVID-19 positive cohort. The biofluids analysed herein were also compared in terms of their ability to differentiate COVID-19 positive participants from controls; serum performed best by multivariate analysis (sensitivity and specificity of 0.97), with the dominant changes in triglyceride and bile acid levels, concordant with other studies identifying dyslipidemia as a hallmark of COVID-19 infection. Sebum performed well (sensitivity 0.92; specificity 0.84), with saliva performing worst (sensitivity 0.78; specificity 0.83). These findings show that alterations to skin lipid profiles coincide with dyslipidaemia in serum. The work also signposts the potential for integrated biofluid analyses to provide insight into the whole-body atlas of pathophysiological conditions.

The effect of COVID-19 infection on the human metabolome has been widely reported, but to date all such studies have focused on a single wave of infection. COVID-19 has generated numerous waves of disease with different clinical presentations, and therefore it is pertinent to explore whether metabolic disturbance changes accordingly, to gain a better understanding of its impact on host metabolism and enable better treatments. This work used a targeted metabolomics platform (Biocrates Life Sciences) to analyze the serum of 164 hospitalized patients, 123 with confirmed positive COVID-19 RT-PCR tests and 41 providing negative tests, across two waves of infection. Seven COVID-19-positive patients also provided longitudinal samples 2–7 months after infection. Changes to metabolites and lipids between positive and negative patients were found to be dependent on collection wave. A machine learning model identified six metabolites that were robust in diagnosing positive patients across both waves of infection: TG (22:1_32:5), TG (18:0_36:3), glutamic acid (Glu), glycolithocholic acid (GLCA), aspartic acid (Asp) and methionine sulfoxide (Met-SO), with an accuracy of 91%. Although some metabolites (TG (18:0_36:3) and Asp) returned to normal after infection, glutamic acid was still dysregulated in the longitudinal samples. This work demonstrates, for the first time, that metabolic dysregulation has partially changed over the course of the pandemic, reflecting changes in variants, clinical presentation and treatment regimes. It also shows that some metabolic changes are robust across waves, and these can differentiate COVID-19-positive individuals from controls in a hospital setting. This research also supports the hypothesis that some metabolic pathways are disrupted several months after COVID-19 infection.

Around 80 % of immunoglobulin (Ig)‐producing cells in man are located in the gut, with a preponderance of IgA‐ and IgM‐producing cells that express heavily mutated IgVH genes. Here we describe the characteristics of Ig light chain genes isolated from human ileal and colonic lamina propria plasma cells. We focused on the properties of the two most commonly used light chain families, Vκ1 and Vλ2. Out‐of‐frame λ rearrangements were very rare, suggesting that these λ light chains may have undergone sequential rearrangements until successful conformation was achieved. This has not been observed in the human peripheral B cell population. The in‐frame λ gene rearrangements were highly mutated, with a frequency of mutation that was indistinguishable from that observed in many groups of heavy chain variable regions used by intestinal plasma cells. The in‐frame κ chain rearrangements were also highly mutated, but contained a subgroup of genes (27.3 %) that showed over 98 % homology with the germ‐line gene. The majority of unused κ chain genes were unmutated. A strong tendency for preferential mutation of G over C nucleotides was observed. Detailed analysis of the sequences in which the biases were observed suggested that this was likely to be due to selection, rather than a characteristic of the mechanism introducing the mutations.

Immunoglobulin V region genes acquire point mutations during affinity maturation of the T-cell-dependent B-cell response. It has been proposed that both selection by antigen and characteristics of the DNA sequence are involved in determining the distribution of mutations along the genes. There is a tendency for replacement mutations to occur in the complementarity-determining regions and for silent mutations to accumulate in the framework regions of used genes. By analysing a group of highly mutated human IgVH4-34 (VH4. 21) and family 5 genes derived from human gut-associated lymphoid tissues, which were out-of-frame between VH and JH (and therefore not used) we have investigated the distribution of mutations acquired in the absence of selection. We observed that these genes may show the statistical hallmarks of selected genes, suggesting that intrinsic biases alone may be enough to give the appearance of selection. These data suggest that analysis of the distribution of mutations in IgVH genes cannot be used reliably to state whether antigenic selection of the B-cell carrying the genes occurred. In-frame genes had more silent mutations than the out-of-frame genes and lacked stop codons. These characteristics were considered to be indicative of selection in the in-frame genes derived from human gut-associated lymphoid tissue.

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. Current antibody structure databases lack consistent annotations of isotypes and structural coverage of 3D antibody structures, as well as computed deep mutation profiles. The and region bearing nti ody (VCAb) web-tool is established to clarify these annotations and provides an accessible resource to facilitate antibody engineering and design. VCAb currently provides data on 7,166 experimentally determined antibody structures including both V and C regions from different species. Additionally, VCAb provides annotations of species and isotypes with numbering schemes applied. These information can be interactively queried or downloaded in batch. VCAb is implemented as a R shiny application to enable interactive data interrogation. The online application is freely accessible https://fraternalilab.cs.ucl.ac.uk/VCAb/. The source code to generate the database and the online application is available open-source at https://github.com/Fraternalilab/VCAb.

Abstract Somatic hypermutation introduces mutations into IgV genes during affinity maturation of the B cell response. Mutations are introduced nonrandomly, and are generally targeted to the complementarity determining regions (CDRs). Subsequent selection against mutations that result in lower affinity or nonfunctional Ig increases the relative number of mutations in the CDRs. Investigation of somatic hypermutation is hampered by the effects of selection. We have avoided this by studying out-of-frame human IgVH4.21 and 251 genes, which, being unused alleles, are unselected. By comparison of the frequency of A, C, G, and T nucleotides at positions −3 to +3 around mutated or unmutated A, C, and G nucleotides, we have identified flanking sequences that most commonly surround mutated bases. Distinct trends in flanking sequences that were unique for each base were observed. Statistically significant trends that were common to both IgVH4.21 and 251 were used to deduce motifs that bias somatic hypermutation. The motifs deduced from this data, with targeted bases in regular type, are AANB, WDCH, and DGHD (where W = A/T, B = C/G/T, D = A/G/T, H = A/C/T, and N = any base). Mutations from C and G in two further groups of out-of-frame human IgVH genes, not used in the deduction of the motifs, occurred significantly within the motifs for C and G. The proposed target sequence for G is within the reverse complement of the target sequence for C, suggesting that the hypermutation mechanism may target only G or C. The mutation in the complementary base would appear on the other strand following replication.

Abstract The origin of human ileal lamina propria (LP) plasma cells has been investigated using a technique based on microdissection of cells from immunohistochemically stained tissue sections. We have sequenced rearranged IgV H 4.21, IgV H 5 family and IgV H 6 genes from Peyer's patch germinal center (GC) cells and plasma cells from the ileal LP. Clonally related cells were identified by comparison of their complementarity determining region (CDR) 3 sequences and patterns of somatic mutation. We observed Ig genes from B cells in the GC of Peyer's patches which were related to Ig genes from plasma cells in the ileal LP, demonstrating that clonally related cells span these sites. In addition, groups of clonally related, but diversified plasma cells were common in the lamina propria. All Ig genes isolated from LP plasma cells were heavily mutated. The distribution of mutations in the CDR of the Ig heavy chain variable region (V H ) genes which were considered to be the expressed alleles in LP plasma cells was consistent with an affinity‐matured response. These observations provide compelling evidence for the origin of human ileal plasma cells from the Peyer's patches.

The elderly produce increased levels of antibodies to autologous antigens and are less able to make high-affinity antibodies to foreign antigens. Ig gene hypermutation is integral to the affinity maturation process but previous studies of hypermutation with age have yielded conflicting results. The cells studied have represented post-germinal center (GC) populations and, therefore, the results may be complicated by possible differences in activation history. We studied Ig genes from GC B cells to elucidate which factors in the affinity maturation process change with age. Age-related changes in the pattern of hypermutation were seen, although the analysis of variable region heavy chain (VH) genes and their lineage trees shows that an alteration in the mechanism of somatic hypermutation is unlikely. The changes are due to founder cell effects and/or the process of selection. Striking tissue-specific differences were seen. All measurements indicated that selection of Ig genes may decrease in Peyer's patch GC but increase in splenic GC with age. These tissue-specific differences highlight the importance of considering the activation and effector sites when studying immune senescence.

Abstract Somatic hypermutation affects Ig genes during T-dependent B cell responses and is characterized by a high frequency of single base substitutions. Hypermutation is not a completely random process; a study of mutations in different systems has revealed the presence of sequence motifs that target mutation. In a recent analysis of the sequences surrounding individual mutated bases in out-of-frame human IgVH genes, we found that the target motifs around mutated G’s and C’s are reverse complements of each other. This finding suggests that hypermutation acts on both strands of DNA, which contradicts evidence of a strand-dependent mechanism as suggested by an observed bias in A and T mutations and the involvement of transcriptional machinery. We have now extended our database of out-of-frame genes and determined the sequence motifs flanking mutated A and T nucleotides. In addition, we have analyzed the flanking sequences for different types of nucleotide substitutions separately. Our results confirm the relationship between the motifs for G and C mutations and show that the motifs surrounding mutated A’s and T’s are weaker and do not have the same relationship. Taken together with our observation of A/T strand bias in out-of-frame genes, this observation suggests that there is a semitargeted G/C mutator that is strand-independent and a separate A/T mutator that is strand-dependent and is less reliant on the local target sequence.

BACKGROUND Ulcerative colitis is an inflammatory disease of the colonic and rectal mucosa. Autoantibodies have been observed in ulcerative colitis which may have a role in the pathogenesis of the disease. Evidence also suggests that there is an hereditary predisposition towards the disease, although no individual genes have been identified. AIMS This is a pilot study of immunoglobulin heavy chain genes (IgH) in ulcerative colitis to determine whether they have any particular genetic characteristics which may lead to a better understanding of the disease aetiology. SUBJECTS Colonic or rectal tissue was obtained from five children with ulcerative colitis. Tissue was also obtained from five children with Crohn’s disease and five children who did not have inflammatory bowel disease as controls. METHODS B cells and IgD+ B cells were identified by immunohistochemistry on frozen sections. Areas of lamina propria containing plasma cells, and areas of IgD+ B cells were microdissected. The immunoglobulin genes were PCR amplified, cloned, and sequenced. Sequences were analysed for content of somatic mutations and composition of heavy chain. RESULTS An increase in the use of JH6 and DXP′1, and a decrease in the use of JH4, gene segments in immunoglobulin genes from lamina propria plasma cells, and from virgin IgD+ B cells, was found in patients with ulcerative colitis. These biases were not present in the control groups. CONCLUSIONS There is a fundamental difference in the immunoglobulin genes from patients with ulcerative colitis. Whether this is caused by a difference in content of immunoglobulin gene segments in the germline or a difference in the recombination mechanism is not known.

The Peyer's patches of the terminal ileum are a source of IgA plasma cells in the intestinal lamina propria of experimental animals. They are also thought to harbour IgA memory cells. However, the microanatomical location of Peyer's patch memory cells, and whether they are also present in man is not known. Human Peyer's patches have a pronounced marginal zone (MGZ) of sIgD-negative B cells. In this study we analysed the sequence of polymerase chain reaction-amplified, rearranged IgVH genes from microdissected MGZ B cells, to determine whether this is a site of B-cell memory in Peyer's patches. We observed that the majority of Peyer's patch MGZ B cells contain heavily mutated IgVH genes and are therefore clearly memory B cells. Sequences of rearranged mutated genes in the MGZ have a pattern of replacement and silent mutations expected of selected products of the affinity maturation process. Related clones, with identical CDR3 but different patterns of mutation, are seen. This suggests that either these memory cells are formed as the germinal centre selection process proceeds, or a memory cell has re-entered the germinal centre for further rounds of mutation. Interestingly, in one patient, the MGZ in the Peyer's patches also contains a proportion of B cells with unmutated IgVH 4.21 genes.

The organised gut associated lymphoid tissue (GALT) exists adjacent to an extensive and diverse luminal flora. The follicle associated epithelium and associated dendritic cells and lymphocytes form a tightly fortified gateway between the flora and the host that permits connectivity between them and chronic activation of the lymphoid compartment. As a consequence, plasma cell precursors are generated continuously, and in abundance, in GALT by clonal proliferation. Clonal proliferation alone on this scale would reduce the spectrum of B cell specificity. To compensate, GALT also houses molecular machinery that diversifies the receptor repertoire by somatic hypermutation, class switch recombination and receptor revision. These three processes of enhancing the diversity of mature B cells ensure that although clonally related plasma cells may secrete immunoglobulin side by side in the mucosa they rarely have identical antigen binding sites. (C) 2009 Elsevier Ltd. All rights reserved.

Immunoglobulins are known to be variably glycosylated. Although most carbohydrate is likely to be associated with the constant region, the variable region may also be glycosylated. Variable region glycosylation is known to be associated with antigen binding; in one model, the presence of carbohydrate on the external surface of CDR2 was shown to increase the affinity by up to ten-fold. In this study we have studied the effect of somatic hypermutation on potential sites of N-glycosylation in IgV H genes used by mucosal plasma cells secreting IgM, IgA and IgG in adults and children. Mucosal plasma cells secreting IgM, IgA and IgG are all heavily mutated from childhood. We have observed a tendency to lose the germline encoded N-glycosylation sites in all populations studied (a range of 43–67% of genes showing loss of the site). The tendency to lose the site was associated with a higher frequency of somatic hypermutation. We have also analysed the tendency to create potential N-glycosylation sites, and observed that this was greater in IgA and IgG than IgM and was again associated with the high frequency of somatic hypermutation. We observed no evidence of selection for either loss or gain of potential N-glycosylation sites. The changes in potential glycosylation status associated with a high frequency of somatic hypermutation is likely to increase the diversity of mucosal immunoglobulins, but is not as likely to affect the peripheral immune system where the frequency of somatic hypermutation is generally lower.

Autoimmune diseases show high diversity in the affected organs, clinical manifestations and disease dynamics. Yet they all share common features, such as the ectopic germinal centers found in many affected tissues. Lineage trees depict the diversification, via somatic hypermutation (SHM), of immunoglobulin variable-region (IGV) genes. We previously developed an algorithm for quantifying the graphical properties of IGV gene lineage trees, allowing evaluation of the dynamical interplay between SHM and antigen-driven selection in different lymphoid tissues, species, and disease situations. Here, we apply this method to ectopic GC B cell clones from patients with Myasthenia Gravis, Rheumatoid Arthritis, and Sjögren’s Syndrome, using data scaling to minimize the effects of the large variability due to methodological differences between groups. Autoimmune trees were found to be significantly larger relative to normal controls. In contrast, comparison of the measurements for tree branching indicated that similar selection pressure operates on autoimmune and normal control clones.

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. In this paper we demonstrate that BCR repertoire analysis can be done using DNA from formalin-fixed paraffin-embedded (FFPE) human tissue samples. The heterogeneity of BCR repertoires we obtained confirms the plausibility of HTS of DNA from FFPE specimens. The establishment of experimental protocols and computational tools that enable sequence data analysis from the low quality DNA of FFPE tissues is important for enabling research, as it would enable the use of the rich source of preserved samples in clinical biobanks and biopsy archives. (C) 2012 Elsevier Inc. All rights reserved.

IgA contributes to homeostatic balance between host and intestinal microbiota. Mechanisms that initiate the IgA response are unclear and likely to differ between humans and animal models. We used multiple experimental approaches to investigate the origin of human intestinal plasma cells that produce IgA in the gastrointestinal tract. Complexity of IgA-producing plasma cell populations in human gastrointestinal mucosa and bone marrow and the specific response to oral cholera vaccine were compared by analysis of immunoglobulin genes. Flow cytometry, gene expression analysis, and immunohistochemistry were used to analyze signaling pathways induced by B-cell receptor engagement in human gut-associated lymphoid tissue (GALT) and involvement of innate immunity in B-cell activation in GALT compared with nonintestinal sites. Human intestinal IgA-producing plasma cells appeared to be of germinal center origin; there was no evidence for the population complexity that accompanies multiple pathways of derivation observed in bone marrow. In germinal center B cells of human GALT, Btk and Erk are phosphorylated, CD22 is down-regulated, Lyn is translocated to the cell membrane, and Fos and Jun are up-regulated; these features indicate B-cell receptor ligation during germinal center evolution. No differences in innate activation of B cells were observed in GALT, compared with peripheral immune compartments. IgA-producing plasma cells appear to be derived from GALT germinal centers in humans. B-cell receptor engagement promotes formation of germinal centers of GALT, with no more evidence for innate immune receptor activation in the mucosa than nonintestinal immune compartments. Germinal centers in GALT should be targets of mucosal vaccinations because they are the source of human intestinal IgA response.

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 (HIS) 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. Studies of larger numbers of human subjects, analysis of defined lymphocyte subpopulations including antigen-specific populations, and controlling for factors such as chronic viral infections, will be important for gaining additional understanding of the impact of ageing on human lymphocyte populations.

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/na < ve B cells in the elderly, evaluating surface immunoglobulin expression, production of the pro- and anti-inflammatory cytokines, tumor necrosis factor (TNF)-alpha 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 na < ve B cells from young donors need a sufficiently strong stimulus to be activated "in vitro", while na < ve B cells from old subjects are able to produce IL-10 and TNF-alpha when stimulated "physiologically" (alpha-CD40/IL-4), suggesting that these cells might play a role in the control of the immuno-inflammatory environment in the elderly. In addition, in the elderly there is an accumulation of DN B cells with a reduced rate of somatic hypermutation. Thus, DN B lymphocytes may be exhausted cells that are expanded and accumulate as a by-product of persistent stimulation or impaired germinal center formation.

The diversity of the antibody response is achieved, in part, by rearrangement of different immunoglobulin (Ig) genes. The Ig heavy chain is made up of a variable region (IGHV), a diversity region (IGHD) and a joining region (IGHJ). Human germline IGHV genes have been grouped into seven multigene subgroups. Size and usage of these subgroups is not equal, the IGHV3 subgroup is the most commonly used (36%), followed by IGHV1/7 (26%), then IGHV4, IGHV5, IGHV2, IGHV6 (15%, 12%, 4%, 3% respectively). The rhesus macaque (Macaca mulatta) is a useful non-human primate model for studies of infection and the database of germline Ig genes for the macaque is gradually growing to become a useful tool in the study of B-cell responses. The proportions of IGHV subgroup usage in the macaque are similar to those in man. Representatives from IGHV3 and IGHV4 subgroups for the macaque have been published, as have germline sequences of the IGHD and IGHJ genes. However, to date there have been no sequences published from the second largest IGHV subgroup, IGHV1. We report the isolation and sequencing of a genomic fragment containing an IGHV1 gene from the macaque. Polymerase chain reaction (PCR) primers designed from this sequence enabled us to amplify and sequence 25 new IGHV1 germline genes. We also isolated two IGHV7 genes, using the same primers, and two IGHV5 genes, using human IGHV5 primers.

Complementarity-determining region 3 (CDR3) is the most hyper-variable region in B cell receptor (BCR) and T cell receptor (TCR) genes, and the most critical structure in antigen recognition and thereby in determining the fates of developing and responding lymphocytes. There are millions of different TCR Vβ chain or BCR heavy chain CDR3 sequences in human blood. Even now, when high-throughput sequencing becomes widely used, CDR3 length distributions (also called spectratypes) are still a much quicker and cheaper method of assessing repertoire diversity. However, distribution complexity and the large amount of information per sample (e.g. 32 distributions of the TCRα chain, and 24 of TCRβ) calls for the use of machine learning tools for full exploration. We have examined the ability of supervised machine learning, which uses computational models to find hidden patterns in predefined biological groups, to analyze CDR3 length distributions from various sources, and distinguish between experimental groups. We found that (a) splenic BCR CDR3 length distributions are characterized by low standard deviations and few local maxima, compared to peripheral blood distributions; (b) healthy elderly people's BCR CDR3 length distributions can be distinguished from those of the young; and (c) a machine learning model based on TCR CDR3 distribution features can detect myelodysplastic syndrome with approximately 93% accuracy. Overall, we demonstrate that using supervised machine learning methods can contribute to our understanding of lymphocyte repertoire diversity.

Revision of Ab L chains by secondary rearrangement in mature B cells has the potential to change the specific target of the immune response. In this study, we show for the first time that L chain revision is normal and widespread in the largest Ab producing population in man: intestinal IgA plasma cells (PC). Biases in the productive and non-productive repertoire of lambda L chains, identification of the circular products of rearrangement that have the characteristic biases of revision, and identification of RAG genes and protein all reflect revision during normal intestinal IgA PC development. We saw no evidence of IgH revision, probably due to inappropriately orientated recombination signal sequences, and little evidence of kappa-chain revision, probably due to locus inactivation by the kappa-deleting element. We propose that the lambda L chain locus is available and a principal modifier and diversifier of Ab specificity in intestinal IgA PCs.

B cells have been found to play a critical role in the pathogenesis of several autoimmune (AI) diseases A common feature amongst many AI diseases is the formation of ectopic germinal centers (GC) within the afflicted tissue or organ in which activated B cells expand and undergo somatic hypermutation (SHM) and antigen-driven selection on their immunoglobulin variable region (IgV) genes However it is not yet clear whether these processes occurring in ectopic GCs are identical to those in normal GCs The analysis of IgV mutations has aided in revealing many aspects concerning B cell expansion mutation and selection in GC reactions We have applied several mutation analysis methods based on lineage tree construction to a large set of data containing IgV productive and non-productive heavy and light chain sequences from several different tissues to examine three of the most profoundly studied AI diseases - Rheumatoid Arthritis (RA) Multiple Sclerosis (MS) and Sjogren s Syndrome (SS) We have found that RA and MS sequences exhibited normal mutation spectra and targeting motifs but a stricter selection compared to normal controls which was more apparent in RA SS sequence analysis results deviated from normal controls in both mutation spectra and indications of selection also showing differences between light and heavy chain IgV and between different tissues The differences revealed between AI diseases and normal control mutation patterns may result from the different microenvironmental influences to which ectopic GCs are exposed relative to those in normal secondary lymphoid tissues (c) 2010 Elsevier Ltd All rights reserved

On April 7,8, 2009 a Symposium entitled "Pathophysiology of Successful and Unsuccessful Ageing" took place in Palermo, Italy. Here, the lectures of G. Pawelec, D. Dunn-Walters and. G. Colonna-Romano on T and B immunosenescence are summarized. In the elderly, many alterations of both innate and acquired immunity have been described. Alterations to the immune system in the older person are generally viewed as a deterioration of immunity, leading to the use of the catch-all term immunosenescence. Indeed, many immunological parameters are often markedly different in elderly compared to young people, and some, mostly circumstantial, evidence suggests that retained function of both innate and acquired immunity in the elderly is correlated with health status. What is often not clear from studies is how far immune dysfunction is a cause or an effect. A better understanding of immunosenescence and mechanisms responsible for proven deleterious changes is needed to maintain a healthy state in later life and to design possible therapeutic interventions.

Class switch recombination (CSR) and somatic hypermutation (SHM) are mechanistically related processes initiated by activation-induced cytidine deaminase. Here, we have studied the role of ataxia telangiectasia and Rad3-related protein (ATR) in CSR by analyzing the recombinational junctions, resulting from in vivo switching, in cells from patients with mutations in the ATR gene. The proportion of cells that have switched to immunoglobulin (Ig)A and IgG in the peripheral blood seems to be normal in ATR-deficient (ATRD) patients and the recombined S regions show a normal “blunt end-joining,” but impaired end joining with partially complementary (1–3 bp) DNA ends. There was also an increased usage of microhomology at the μ-α switch junctions, but only up to 9 bp, suggesting that the end-joining pathway requiring longer microhomologies (≥10 bp) may be ATR dependent. The SHM pattern in the Ig variable heavy chain genes is altered, with fewer mutations occurring at A and more mutations at T residues and thus a loss of strand bias in targeting A/T pairs within certain hotspots. These data suggest that the role of ATR is partially overlapping with that of ataxia telangiectasia–mutated protein, but that the former is also endowed with unique functional properties in the repair processes during CSR and SHM.

Age related changes in the structure and function of the immune system, collectively termed immunosenescence, result in poor responses to infections, increased susceptibility to cancers and increased incidence of autoimmune diseases. The humoral immune response, maintained by the B cell compartment, has a key role in an effective immune system-not only in producing high affinity antibodies that are crucial for vaccination strategies, but in assisting other components of the immune system in their function. Hence an understanding of B cell immunosenescence in particular is vital in designing strategies to combat the effects of age on immune function. Numerous studies have been undertaken using small animal models in order to understand immunosenescence, and these have contributed greatly to our understanding of the events that underpin impaired immune responses. However, there are key differences between the human and the mouse and a clear understanding of these differences is required when extrapolating from one species to the other. In this article we present an overview of B cell development and summarise current data on age-related B cell changes, at both the population level and at the individual mechanistic level. Areas of similarity and difference between human and mouse models are highlighted.

Many preclinical experiments have attested to the critical role of CD4+ T cell help in CD8+ cytotoxic T lymphocyte (CTL)-mediated immunity. Recent clinical trials have demonstrated that reinfusion of CD4+ T cells can induce responses in infectious diseases and cancer. However, few standardized and versatile systems exist to expand antigen-specific CD4+ Th for clinical use. K562 is a human erythroleukemic cell line, which lacks expression of HLA class I and class II, invariant chain and HLA-DM but expresses adhesion molecules such as intercellular adhesion molecule-1 and leukocyte function-associated antigen-3. With this unique immunologic phenotype, K562 has been tested in clinical trials of cancer immunotherapy. Previously, we created a K562-based artificial antigen-presenting cell (aAPC) that generates ex vivo long-lived HLA-A2-restricted CD8+ CTL with a central/effector memory phenotype armed with potent effector function. We successfully generated a clinical version of this aAPC and conducted a clinical trial where large numbers of anti-tumor CTL are reinfused to cancer patients. In this article, we shifted focus to CD4+ T cells and developed a panel of novel K562-derived aAPC, where each expresses a different single HLA-DR allele, invariant chain, HLA-DM, CD80, CD83 and CD64; takes up soluble protein by endocytosis and processes and presents CD4+ T-cell peptides. Using this aAPC, we were able to determine novel DR-restricted CD4+ T-cell epitopes and expand long-lived CD4+ T-cells specific for multiple antigens without growing bystander Foxp3+ regulatory T cells. Our results suggest that K562-based aAPC may serve as a translatable platform to generate both antigen-specific CD8+ CTL and CD4+ Th.

CD27 expression has been used to distinguish between memory and naive B cells in humans. However, low levels of mutated and isotype-switched CD27−IgD− cells are seen in healthy adults, and these are increased in some autoimmune diseases and in the elderly. Thus CD27 is not a universal marker of memory B cells in humans. Various hypotheses have been put forward as to the function of the CD27− memory population. Since we have previously found high-throughput IGHV repertoire analysis useful to distinguish “innate-like” memory B cells (CD27+IgD+), we have employed similar analyses to elucidate the relationship between CD27− and CD27+ memory B cells. IgM+IgD− memory cells in both the CD27+ and CD27− compartments share the unique characteristics of the “innate-like” IgM+IgD+CD27+ cells. The switched CD27+ and CD27− memory cells share a similar IGHV repertoire, having more in common with each other than with “innate-like” memory cells, although it is interesting that IgG2 and IgA2 subclasses of antibody in both switched memory populations have a more “innate-like” repertoire. Clonality analysis shows evidence of a close clonal relationship between the two populations in that both CD27− and CD27+ switched memory cells can be found in the same genealogical tree. The expression of CD27 does not appear to occur in a linear developmental fashion, since we see CD27− cells as precursors of CD27+ cells and vice versa. Despite the similarities, the CDR-H3 repertoire of the CD27− cells is significantly different from both the CD27+IgD+ and CD27+IgD− populations, indicating that perhaps the lack of CD27 might be related to binding properties of the Ig CDR-H3 region.

Hypermutation is thought to be a two-phase process. The first phase is via the action of activation-induced cytidine deaminase (AID), which deaminates C nucleotides in WRC motifs. This results in the RGYW/WRCY hot spot motifs for mutation from G and C observed in vivo. The resemblance between the hot spot for C mutations and the reverse complement of that for G mutations implies a process acting equally on both strands of DNA. The second phase of hypermutation generates mutations from A and T and exhibits strand bias, with more mutations from A than T. Although this does not concur with the idea of one mechanism acting equally on both strands, it has been suggested that the AT mutator also has a reversible motif; WA/TW. We show here that the motifs surrounding the different substitutions from A vary significantly; there is no single targeting motif for all A mutations. Sequence preferences associated with mutations from A more likely reflect an influence of adjacent nucleotides over what the A mutates "to." This influence tends toward "like" replacements: Purines (A or G) in the 5' position bias toward replacement by another purine (G), whereas replacement with pyrimidines (C or T) is more likely if the preceding base is also a pyrimidine. There is no reverse complementarity in these observations, in that similar influences of nucleotides adjacent to T are not seen. Hence, WA and TW should not be considered as reverse complement hot spot motifs for A and T mutations.

B cells are present in human fetal intestine from approximately 14 weeks of gestation. Here we show that this population includes mature, dividing B cells. These are large cells with dendritic processes, resembling human thymic B cells. In addition, we observed IgM+, light chain-, and CD20- cells and local expression of V pre-B, demonstrating that the human fetal intestine is a site of B cell development. Ig V(H)DJ(H) gene sequencing can confirm clonal identity of B cells. Identification of the same IgV(H)4-34 sequence in serial sections in two fetuses confirmed local accumulation of related cells in each case. IgV(H)4-34 was also amplified from an additional two samples, and the D and J repertoire compared with a unique database of unselected V(H)4-34 genes from postnatal gut. Distinguishing characteristics of Ig lambda genes in postnatal gut were also studied in the fetus. According to these parameters, fetal and postnatal B cells are unrelated.

Ageing is associated with multiple changes in many different components of the immune system. A healthy immune system exists in a state of balance between efficient effector responses against pathogens and tolerance to self antigens. This balance is changed with age; functions such as antigen recognition, phagocytosis, antigen presentation, chemotaxis, cytokine secretion and killing ability are all compromised. Aberrant cellular responses lead to an altered cytokine network with increases in inflammatory cytokines and decreases in anti-inflammatory cytokines leading to a pro-inflammatory state. Consequently older patients require extra care in diagnosis of infections as symptoms may be perturbed, resulting in unusual presentations of common conditions. The defects in immunity due to immunosenescence also mean that older patients require more care and screening than other patients in the same disease cohort. Though it is generally understood by clinicians that older patients are more at risk from multiple infections, the wider clinical effects of immunosenescence are less understood. The immune system is involved in several neurodegenerative conditions and the inflammatory conditions of immunosenescence may be a key factor in pathogenesis. Similarly, there is reason to believe that immunosenescence might be a key factor explaining the increased incidence of cancer in older age. With increasing understanding of the immune system's involvement in many of these pathological processes, and the contribution that immunosenescence makes to these, more efficient vaccines and novel therapies may be developed to prevent/treat these conditions.

B-cell receptor (BCR) diversity is achieved centrally by rearrangement of Variable, Diversity, and Joining genes, and peripherally by somatic hypermutation and class-switching of the rearranged genes. Peripheral B-cell populations are subject to both negative and positive selection events in the course of their development that have the potential to shape the BCR repertoire. The origin of IgM + IgD + CD27 + (IgM memory) cells is controversial. It has been suggested that they may be a prediversified, antigen-independent, population of cells or that they are a population of cells that develop in response to T-independent antigens. Most recently, it was suggested that the majority of IgM memory cells are directly related to switched memory cells and are early emigrants from the germinal center reaction. Advances in sequencing technology have enabled us to undertake large scale IGH repertoire analysis of transitional, naive, IgM memory and switched memory B-cell populations. We find that the memory B-cell repertoires differ from the transitional and naive repertoires, and that the IgM memory repertoire is distinct from that of class-switched memory. Thus we conclude that a large proportion of IgM memory cells develop in response to different stimuli than for class-switched memory cell development.

In studies of inflammatory bowel diseases (IBD), research has so far focused mainly on the role of T cells. Despite evidence suggesting that B cells and the production of auto-antibodies may play a significant role in IBD pathogenesis, the role of B cells in gut inflammation has not yet been thoroughly investigated. In the present study we used the new approach of lineage tree analysis for studying immunoglobulin variable region gene diversification in B cells found in the inflamed intestinal tissue of two ulcerative colitis patients as well as B cells from mucosa-associated lymph nodes (LN) in the same patients. Healthy intestinal tissue of three patients with carcinoma of the colon was used as normal control. Lineage tree shapes revealed active immune clonal diversification processes occurring in ulcerative colitis patients, which were quantitatively similar to those in healthy controls. B cells from intestinal tissues and the associated LN are shown here to be clonally related, thus supplying the first direct evidence supporting B-cell trafficking between gut and associated LN in IBD and control tissues.

Graves’ disease is characterized by production of agonist antibodies to the thyroid-stimulating hormone receptor (TSHR), but knowledge of the genetic and somatic events leading to their aberrant production is limited. We describe the genetic analysis of two monoclonal antibodies (mAbs) with thyroid-stimulating activity (TSAb) obtained from a single mouse with experimental Graves’ disease. The mAbs were class switched, but used the same rearrangement of immunoglobulin heavy chain, variable region (IGHV) and immunoglobulin light chain, variable region (IGLV) germline genes, implying a clonal relationship and derivation from a single precursor B-cell clone. The IGHV-region genes of the two mAbs underwent high degrees of somatic hypermutation by sharing numerous mutations before diverging, while the IGLV genes evolved separately. Interestingly, the mutations were present in both the complementarity-determining regions (CDRs) and the framework regions. The cloned IGHV and IGLV genes were confirmed to have TSAb properties in experiments in which they were expressed as recombinant Fabs (rFabs). In other experiments, we swapped the IGLV genes with IGHV genes by constructing chimeric rFabs and showed that the chimeras retained TSAb activities, confirming the close functional relatedness of the V-region genes. Importantly, the IGLV genes in chimeric rFabs had a dominant stimulatory effect at low concentrations, while the IGHV genes had a dominant effect at higher concentrations. Our findings demonstrate that, in experimentally immunized mice, multiple pathogenic antibodies to TSHR can arise from a single clone by a series of somatic mutations in the V-region genes and may give an insight into how such antibodies develop spontaneously in autoimmune Graves’ disease.

Older people suffer from a decline in immune system, which affects their ability to respond to infections and to raise efficient responses to vaccines. Effective and specific antibodies in responses from older individuals are decreased in favour of non-specific antibody production. We investigated the B-cell repertoire in DNA samples from peripheral blood of individuals aged 86–94 years, and a control group aged 19–54 years, using spectratype analysis of the IGHV complementarity determining region (CDR)3. We found that a proportion of older individuals had a dramatic collapse in their B-cell repertoire diversity. Sequencing of polymerase chain reaction products from a selection of samples indicated that this loss of diversity was characterized by clonal expansions of B cells in vivo. Statistical analysis of the spectratypes enabled objective comparisons and showed that loss of diversity correlated very strongly with the general health status of the individuals; a distorted spectratype can be used to predict frailty. Correlations with survival and vitamin B12 status were also seen. We conclude that B-cell diversity can decrease dramatically with age and may have important implications for the immune health of older people. B-cell immune frailty is also a marker of general frailty.

Recent advances allow aging-associated changes in B-cell function to be approached at a mechanistic level. Reduced expression of genes crucial to lineage commitment and differentiation yield diminished B-cell production. Moreover, intrinsic differences in the repertoire generated by B-cell precursors in aged individuals, coupled with failing B-cell generation rates and life-long homeostatic competition, result in narrowed clonotypic diversity. Similarly, reductions in gene products crucial for immunoglobulin class switch recombination and somatic hypermutation impact the efficacy of humoral immune responses. Together, these findings set the stage for integrated analyses of how age-related changes at the molecular, cellular and population levels interact to yield the overall aging phenotype.

The splenic marginal zone (MGZ), which surrounds the mantle zone (MTZ) in human splenic white pulp, contains a phenotypically and morphologically distinct population of B cells. The origin of MGZ B cells is uncertain. Whereas some experiments in rodents have suggested that they are a distinct cell lineage responsible for the immune response to T-independent type 2 antigens, others have suggested that they are memory B cells derived from a germinal center (GC) response. The progeny of a GC reaction is expected to have rearranged immunoglobulin (Ig) genes that are mutated. The distribution of mutations would be expected to reflect the selection of Ig by its affinity for antigen. We have analyzed rearranged Ig heavy chain variable region (VH) 6 and VH 4.21 genes in MGZ and MTZ B cells microdissected from frozen sections of human spleen to determine whether these genes have the properties of an affinity-selected memory B cell population. MTZ B cells contained germline Ig VH genes, confirming previous reports and providing an internal control for mutational analysis. MGZ B cells contained Ig VH genes that were mutated, showing that these cells had been subjected to a mutational mechanism characteristically active in the GC. The rearranged VH 6 genes showed patterns of mutation indicative of an antigen selection process, whereas the distribution of mutations in VH 4.21 genes was not characteristic of gene selection by conventional T-dependent antigen. Our studies provide the first evidence that the human splenic MGZ is a reservoir of memory B cells.

Immunosenescence contributes to the decreased ability of the elderly to control infectious diseases, which is also reflected in their generally poor response to new antigens and vaccination. It is known that the T cell branch of the immune system is impaired in the elderly mainly due to expansion of memory/effector cells that renders the immune system less able to respond to new antigens. B lymphocytes are also impaired in the elderly in terms of their response to new antigens. In this paper we review recent work on B cell immunosenescence focusing our attention on memory B cells and a subset of memory B cells (namely IgG(+)IgD(-)CD27(-)) that we have demonstrated is increased in healthy elderly. (C) 2010 Elsevier B.V. All rights reserved.

Activation-induced cytidine deaminase (AID) is believed to initiate somatic hypermutation (SHM) by deamination of deoxycytidines to deoxyuridines within the immunoglobulin variable regions genes. The deaminated bases can subsequently be replicated over, processed by base excision repair or mismatch repair, leading to introduction of different types of point mutations (G/C transitions, G/C transversions and A/T mutations). It is evident that the base excision repair pathway is largely dependent on uracil-DNA glycosylase (UNG) through its uracil excision activity. It is not known, however, which endonuclease acts in the step immediately downstream of UNG, i.e. that cleaves at the abasic sites generated by the latter. Two candidates have been proposed, an apurinic/apyrimidinic endonuclease (APE) and the Mre11-Rad50-NBS1 complex. The latter is intriguing as this might explain how the mutagenic pathway is primed during SHM. We have investigated the latter possibility by studying the in vivo SHM pattern in B cells from ataxia-telangiectasia-like disorder (Mre11 deficient) and Nijmegen breakage syndrome (NBS1 deficient) patients. Our results show that, although the pattern of mutations in the variable heavy chain (V-H) genes was altered in NBS1 deficient patients, with a significantly increased number of G (but not C) transversions occurring in the SHM and/or AID targeting hotspots, the general pattern of mutations in the V-H genes in Mre11 deficient patients was only slightly altered, with an increased frequency of A to C transversions. The Mre11-Rad50-NBS1 complex is thus unlikely to be the major nuclease involved in cleavage of the abasic sites during SHM, whereas NBS1 might have a specific role in regulating the strand-biased repair during phase Ib mutagenesis.

Somatic hypermutation (SH) of V(D)J rearrangements at the IGH and IGL loci diversifies the IG repertoire during the germinal center response. SH is absolutely dependent on the enzyme activation induced cytidine deaminase (AID) that initiates the SH process by deaminating C nucleotides in ssDNA. Mutations from G and C are thought to occur as a result of strand symmetrical deamination of C by AID on the coding and non-coding strands respectively. Mutations from A and Tare introduced in a strand biased way during error prone repair of the AID induced lesion. SH is linked to transcription and it has been proposed that bidirectional transcription across V(D)J rearrangements occurs in activated and quiescent B cells and that it is co-opted to facilitate the accessibility of the two DNA strands by regulating accessibility of single stranded DNA to AID. We have developed a quantitative method to study directional transcription. Our method controls for differences in efficiency and specificity of reverse transcription that are known to be able to generate false positive data. This method does not detect antisense transcripts in exonic or intronic regions within the hypermutation domain of the spontaneously hypermutating cell line Ramos, or in human blood B cells or tonsil cells, providing convincing evidence that antisense transcripts are rare or absent in human B cells. (C) 2009 Elsevier Ltd. All rights reserved.

Follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL) and primary central nervous system lymphoma are B cell malignancies. FL and DLBCL have a germinal center origin. We have applied mutational analyses and a novel algorithm for quantifying shape properties of mutational lineage trees to investigate the nature of the diversification, somatic hypermutation and selection processes that affect B cell clones in these malignancies and reveal whether they differ from normal responses. Lineage tree analysis demonstrated higher diversification and mutations per cell in the lymphoma clones. This was caused solely by the longer diversification times of the malignant clones, as their recent diversification processes were similar to those of normal responses, implying similar mutation frequencies. Since previous analyses of antigen-driven selection were shown to yield false positives, we performed a corrected analysis of replacement and silent mutation patterns, which revealed selection against replacement mutations in the framework regions, responsible for the structural integrity of the B cell receptor, but not for positive selection for replacements in the complementary determining regions. Most replacements, however, were neutral or conservative, suggesting that if at all selection operates in these malignancies it is for structural B cell receptor integrity but not for antigen binding.

Plasma cells secreting immunoglobulin M (IgM) and IgA in human:intestinal mucosa are the largest antibody-producing population in the human body. Despite this there have been relatively few studies of the characteristics and maturation of the genes which encode the mucosal immunoglobulins. We have previously demonstrated that intestinal plasma cells use highly mutated IgV(H) genes, likely to reflect germinal centre origin. Here we show that IgV(H) genes used by intestinal lamina propria plasma cells secreting IgM and IgA are highly mutated from childhood, with no change in the frequency of mutation through to adulthood, though IgV(H) genes used. by IgM are significantly less mutated than those used by IgA. There was no difference between the IgA subclasses in either the frequency or distribution of mutations. The Frequency of mutation in IgV(H)4-34 genes used by IgG was also studied in the adult biopsies, and was found to be of the same order as that observed in IgA and was significantly higher than that observed in IgM. We have identified IgM and IgA sequences which share identical CDR3 and distribution of mutations. Isotype switching may therefore occur after extensive mutation of IgM sequences; and IgM- and IgA-secreting plasma cells with the same specificity may occur within the same microenvironment. IgM should therefore be considered to be a component of secondary immune responses in the gut.

The combined processes of immunoglobulin (IG) gene rearrangement and somatic hypermutation allow for the creation of an extremely diverse antibody repertoire. Knowledge of the germline sequence of the IG genes is required so that hypermutation and the affinity matured humoral response can be properly studied. Variable region genes can be arranged into subgroups; in humans, there are 11 IGLV subgroups and six IGKV subgroups. The rhesus macaque (Macaca mulatta) is a relevant non-human primate model for human immunological systems. A number of macaque IGHV, IGHD and IGHJ genes have already been reported, but only one light-chain germline gene has been published so far. Here we report the isolation of new macaque IGKV genes by polymerase chain reaction (PCR) amplification from macaque genomic DNA using primers based on the human sequences. Twenty-eight IGKV1, 22 IGKV2 and 12 IGKV3 germline genes for the macaque were found, the open reading frames of which exhibit high homology to their human counterparts (>96, >99 and >96%, respectively).[PUBLICATION ABSTRACT]

Splenic marginal zone lymphoma (SMZL) is a low-grade primary splenic B cell lymphoma, originally thought to be related to splenic marginal zone B cells. Later studies showed that SMZL sometimes may be accompanied by villous lymphocytes in the peripheral blood, a condition previously characterized as splenic lymphoma with villous lymphocytes (SLVL). The relationship between SMZL and splenic marginal zone B cells has recently been called into question. We report four further cases of SMZL, two of which were associated with villous lymphocytes in the peripheral blood. In addition to immunophenotypical analysis, we have studied the IgV H genes in each case, because the extent and patterns of their mutation can indicate the normal B cell counterpart of lymphomas. The IgV H genes in the four cases of SMZL studied are mutated, which is consistent with their origin from postfollicular marginal zone B cells. Evidence of ongoing mutation was also observed. This contrasts with a study showing that blood-borne tumor cells in SLVL show no sign of ongoing mutation. It is possible that the ongoing mutations in the cases studied here are acquired in a splenic microenvironment, such as that found in the follicle center.

In this work we have microdissected lamina propria plasma cells and used polymerase chain reaction and sequencing to investigate immunoglobulin (Ig) gene rearrangements and mutations in human intestine. In addition, specific primers were designed for individual Ig gene rearrangements to analyze the distribution of related B cell and plasma cell clones at different sites along the bowel. Confirming our earlier work, intestinal IgVH genes were highly mutated in plasma cells from older individuals (> 30 years). IgVH genes were significantly less mutated in samples taken from patients aged 11–30 years, and there were fewer mutations again in samples from young children (< 11 years). In age‐matched specimens the number of mutations was equivalent in the duodenum and colon. Using complementarity‐determining region 3 primers to amplify specific Ig gene rearrangements, evidence was also found for the existence of related lamina propria plasma cells along the small bowel and colon, although these were quite scarse. In addition, analysis of the numbers of related clones in a random sampling from discrete areas of lamina propria indicates that the local population is diverse. These results suggest that the highly mutated IgVH genes in adult intestinal plasma cells are a consequence of chronic antigen exposure with age. Duodenal plasma cells are as highly mutated as colonic plasma cells, despite the fact that the upper bowel has no indigenous microbial flora (the stimulus for intestinal plasma cells). They also show that the plasma cell population is diverse and can be widely disseminated along the bowel.

During the B-cell response to T-cell-dependent antigens, the B cells undergo a rapid proliferative phase in the germinal centre. This is accompanied by the introduction of mutations into the immunoglobulin (Ig) variable region (V) genes. The B cells are then selected according to the affinity of the encoded immunoglobulin for antigen, resulting in affinity maturation of the response. Analysis of mutations in IgV genes has given insight into the history of individual B cells and their malignancies. In most cases, analysis of mutations confirms classifications of B-cell lineage designated by studies of cellular morphology and surface antigen expression. However, of particular interest is the subdivision of groups of malignancies by analysis of somatic hypermutation. It is now apparent that there are two subsets of chronic lymphocytic leukaemia (CLL), one with a low load of mutations and poor prognosis, and one with a heavy load of mutations with a much more favourable prognosis. In addition, in Burkitt's lymphoma, sporadic and endemic subtypes are now considered possibly to have a different pathogenesis, reflected in differences in the numbers of mutations. Hodgkin's disease, which was a mystery for many years, has now been shown to be a B-cell tumour. Although in many cases the Ig genes are crippled by somatic hypermutation, it is thought that failure to express Ig is more likely to be associated with problems of transcription. It has been proposed that the distribution of mutations in a B-cell lymphoma can be used to determine whether a lymphoma is selected. We have investigated the load and distribution of mutations in one group of lymphomas-marginal zone B-cell lymphomas of mucosa-associated lymphoid tissues (MALT-type lymphoma), which are dependent on Helicobacter pylori for disease progression, to investigate the limits of information that can be derived from such studies. Comparison of the load of mutations demonstrates that these tumours have approximately the same load of mutations as normal mucosal marginal zone B cells from the Peyer's patches and mucosal plasma cells. This is consistent with the origin of these cells from mucosal marginal zone B cells with plasma cell differentiation. To investigate selection in MALT lymphomas we compared a region of the framework region three in ten MALT lymphomas which use the VH4 family, with the same codons in groups of VH4 genes that are out of frame between V and J. The latter accumulate mutations but are not used and are not selected. A group of VH4 genes are in-frame between V and J were also included for comparison. There were no obvious differences in the distribution of mutations between the groups of genes; the same hot spots and cold spots were apparent in each. In the MALT lymphomas, selection was apparent in the framework regions only and the tendency was to conserve. We therefore feel that there is selection to conserve antibody structure and that this does not reflect selection for antigen. We do not believe that antigen selection can be deduced reliably from sequence information alone. It is possible that somatic hypermutation could be a cause of malignancy since it has been shown that the process may generate DNA strand breaks and is known to be able to generate insertions and deletions. Such events may mediate the translocation of genes-a process that is pivotal in the evolution of many lymphomas.

Thymic B cells are a proliferating B cell population concentrated in normal thymic medulla. They are large cells, some with dendritic morphology, and are not associated with any organized follicular structure. Previous work in this laboratory has shown that most of these B cells are surrounded by tightly adherent thymocytes. The literature on human thymic B cells contains many inconsistencies. There is no consensus on whether they express CD5. Even the existence of thymic B cells has been questioned. In this study we have undertaken the first analysis of rearranged immunoglobulin (Ig) genes, looking in particular for evidence of affinity maturation. The Ig VH genes of human thymic B cells in this study are those of the fetal repertoire, though the resemblence to fetal Ig genes is limited in other respects. They are mostly unmutated, but the presence of mutated sequences suggests that this is not a uniform population, as has been previously indicated by phenotypic studies.

The combined processes of immunoglobulin (IG) gene rearrangement and somatic hypermutation allow for the creation of an extremely diverse antibody repertoire. Knowledge of the germline sequence of the IG genes is required so that hypermutation and the affinity matured humoral response can be properly studied. Variable region genes can be arranged into subgroups; in humans, there are 11 IGLV subgroups and 6 IGKV subgroups. The rhesus macaque (Macaca mulatta) is a relevant non-human primate model for human immunological systems. A number of macaque IGHV, IGHD and IGHJ genes have already been reported. We have also previously reported a number of macaque IGKV genes. Here we report the isolation of new macaque IGLV genes by polymerase chain reaction amplification from macaque genomic DNA using primers based on the human sequences. Nine IGLV1, 10 IGLV2, 21 IGLV3, 5 IGLV4 and 7 IGLV5 germline genes for the macaque were found, the open-reading frames of which exhibit high homology to their human counterparts (>89.3, >88.6, >89.0, >94.7 and >87.1%, respectively).[PUBLICATION ABSTRACT]

The elderly are more susceptible to infectious diseases. Mortality and morbidity from infections increase sharply over the age of 65 years. At the same time, the efficacy of vaccinations in the elderly is decreased. The elderly also have an increased incidence of cancer and inflammatory diseases. All the above indicate an age-related dysregulation of the immune system. Evidence suggests that the change in the humoral immune response with age is a qualitative rather than a quantitative one, i.e. it is the affinity and specificity of the antibody that changes, rather than the quantity of antibody produced. There are a number of possible causes of this failure, one of which is a defect in the mechanism of hypermutation of immunoglobulin genes. We have studied individual clonal responses within germinal centres of spleen and Peyer's patches in young and old patient groups. Our results indicate that there is no difference in the actual mechanism of hypermutation with age. There are, however, differences that are due either to a change in selection processes or to a change in the founder cells available for activation.

A diverse B cell repertoire is essential for an effective immune response. Not only to provide a variety of antibodies to recognise the Multiplicity of likely pathogen challenge, but also because B cells are important regulators of the immune response. In addition to their excellent capabilities as antigen presenting and activating cells, recent work shows that some subpopulations of B cells can have suppressive functions. The diversity of the B cell population as a whole decreases with age, and is associated with ill health. Whether decreased diversity is a feature of all B cells or a reflection of altered subpopulations is not clear, since different subsets of B cells have different functions and their repertoires are shaped by different selection pressures.

Young patients with myasthenia gravis (MG) frequently have ectopic GC in their thymus. We investigated these ectopic GC by microdissection of GC B cells and analysis of their Ig gene characteristics, in comparison to normal GC. CDR3 length distribution, a measure of clonal variability, and Ig gene family usage were similar in MG and normal tonsil samples. Lineage tree analysis demonstrated similar diversification and mutations per cell compared with normal control trees. Mutations were observed in the framework regions, responsible for the structural integrity of the BCR; however, these mutations were mostly conservative or neutral, confirming that a functional BCR is conserved in MG. In the CDR, responsible for Ag binding, selection against replacement mutations was revealed. This may indicate that the MG clones analyzed are already highly Ag-specific, and therefore potential affinity-reducing replacement mutations in the CDR3 are not propagated, due to Ag-driven selection. Somatic hypermutation (SHM) targeting motifs and aa substitution preferences in MG were similar to those of normal controls. Overall, these results suggest that B cells in the ectopic GC in MG appear to undergo normal diversification and selection, in spite of the chronic nature and different environment of the response.

Germinal center (GC) T cells are a poorly investigated population with a unique helper‐inducer memory phenotype. Murine GC T cells have been demonstrated to be oligoclonal and antigen specific. The aim of this study was to investigate the diversity of human tonsillar GC T cells. Immunohistochemistry with a panel of different TCRVβ family antibodies and sequencing of TCRγ gene rearrangements obtained from individual microdissected GC demonstrated local diversity of human tonsillar GC T cells. No evidence of local clonal dominance or of a local migratory pathway from the T cell zone to adjacent GC was seen. Primers specific to the junctional region of the TCRγ gene of individual GC T cell clones were designed, and used to trace the dissemination of the clones. One was found to be concentrated locally. Both clones studied were present in both of the patient's tonsils, suggesting widespread dissemination. In addition, no evidence of somatic hypermutation was found in the TCRγ gene sequences, or in expressed TCRα and β gene sequences obtained by reverse transcriptase‐PCR from isolated tonsillar GC.

Lineage trees have frequently been drawn to illustrate diversification, via somatic hypermutation (SHM), of immunoglobulin variable-region ( IGV) genes. In order to extract more information from IGV sequences, we developed a novel mathematical method for analyzing the graphical properties of IgV gene lineage trees, allowing quantification of the differences between the dynamics of SHM and antigen-driven selection in different lymphoid tissues, species, and disease situations. Here, we investigated trees generated from published IGV sequence data from B cell clones participating in autoimmune responses in patients with Myasthenia Gravis (MG), Rheumatoid Arthritis (RA), and Sjögren's Syndrome (SS). At present, as no standards exist for cell sampling and sequence extraction methods, data obtained by different research groups from two studies of the same disease often vary considerably. Nevertheless, based on comparisons of data groups within individual studies, we show here that lineage trees from different individual patients are often similar and can be grouped together, as can trees from two different tissues in the same patient, and even from IgG- and IgA-expressing B cell clones. Additionally, lineage trees from most studies reflect the chronic character of autoimmune diseases.

The term Richter's syndrome is used to describe the transformation of chronic lymphatic leukaemia (CLL) into a high-grade systemic lymphoma and is associated with a poor prognosis. We have undertaken detailed molecular studies in two patients with cutaneous B-cell lymphoma (CBCL) and CLL. Patient 1 exhibited a low-grade CBCL with different immunoglobulin gene rearrangements in blood and skin. By contrast, patient 2 showed identical gene rearrangements, confirmed by gene sequencing, and died within 4 months of presentation. The latter patient fulfilled the criteria for a diagnosis of cutaneous Richter's syndrome, whereas the former patient demonstrated the coincidence of CLL with a primary CBCL. Our results highlight the importance of gene rearrangement studies with sequencing for the accurate diagnosis of cutaneous Richter's syndrome.

It is well known that older people are more susceptible to morbidity and mortality from infectious diseases, particularly from pulmonary diseases such as pneumococcal pneumonia where vaccines do not provide efficient protection as in younger populations. We have previously shown that the B-cell repertoire in the old is reduced and hypothesise that this may contribute to the impaired humoral responses of the elderly. Here, we investigated the repertoire and antibody responses to winter vaccination in two age groups, aged 1849 and 6589. We found that the serum IgM and IgA pneumococcal responses were significantly impaired in the older group, with no difference in IgG levels. IGHM spectratype analysis seems to be the most promising in terms of its predictive ability for vaccine responses. Spectratypes showed a clear change in the repertoire at day 7 after vaccination, with a return to the baseline levels at day 28. The changes at day 7 reflected expansion of IGH sequences that have smaller, more hydrophilic, CDR3 regions, and these changes were attenuated in the older group. The older group was more likely to have spectratypes indicative of a reduced diversity at day 0 and day 28. On average, the baseline repertoire in the older group was comprised of larger CDR3 regions than in the younger group. In conclusion, IgA and IgM responses are significantly impaired in the elderly pneumococcal response and are likely key mediators of protection. Hydrophilicity and/or small size of the IGH CDR3 appear to be important in these responses.

Abstract Immunologically, the parotid salivary gland is an effector site that secretes large quantities of polyspecific Abs into the saliva, mainly of the IgA isotype. It is considered to be part of the common mucosal immune system but the inductive site for the Ab-producing cells of the salivary gland has not yet been clearly identified. The origin and diversity of cells of B lineage can be investigated by analyzing their Ig heavy chain genes (IgH). We have obtained sequences of IgM and IgA VH4–34 genes from plasma cells in human salivary gland, duodenal lamina propria, and splenic red pulp. Related sequences were found in different areas sampled within each tissue studied, indicating that the plasma cells carrying these genes are widespread with limited diversity. Examples of related IgH genes that are isotype switched were also seen in the salivary gland. The genes from plasma cells of the salivary gland were highly mutated, as were duodenal plasma cell sequences. The level of mutation was significantly higher than that seen in splenic plasma cell sequences. Analysis of CDR3 regions showed that the sequences from salivary gland had significantly smaller CDR3 regions than sequences from spleen, due to differences in number and type of DH regions used. Sequences from duodenum also had smaller CDR3 regions. Therefore, plasma cells from human duodenum and salivary gland showed characteristics that differed from those of human splenic plasma cells.

Graves' disease results from thyroid-stimulating Abs (TSAbs) activating the thyrotropin receptor (TSHR). How TSAbs arise from early precursor B cells has not been established. Genetic and environmental factors may contribute to pathogenesis, including the bacterium Yersinia enterocolitica. We developed two pathogenic monoclonal TSAbs from a single experimental mouse undergoing Graves' disease, which shared the same H and L chain germline gene rearrangements and then diversified by numerous somatic hypermutations. To address the Ag specificity of the shared germline precursor of the monoclonal TSAbs, we prepared rFab germline, which showed negligible binding to TSHR, indicating importance of somatic hypermutation in acquiring TSAb activity. Using rFab chimeras, we demonstrate the dominant role of the H chain V region in TSHR recognition. The role of microbial Ags was tested with Y. enterocolitica proteins. The monoclonal TSAbs recognize 37-kDa envelope proteins, also recognized by rFab germline. MALDI-TOF identified the proteins as outer membrane porin (Omp) A and OmpC. Using recombinant OmpA, OmpC, and related OmpF, we demonstrate cross-reactivity of monoclonal TSAbs with the heterogeneous porins. Importantly, rFab germline binds recombinant OmpA, OmpC, and OmpF confirming reactivity with Y. enterocolitica. A human monoclonal TSAb, M22 with similar properties to murine TSAbs, also binds recombinant porins, showing cross-reactivity of a spontaneously arising pathogenic Ab with Y. enterocolitica. The data provide a mechanistic framework for molecular mimicry in Graves' disease, where early precursor B cells are expanded by Y. enterocolitica porins to undergo somatic hypermutation to acquire a cross-reactive pathogenic response to TSHR.

Marginal-zone B cells are likely to be the first B cells in lymphoid tissue to encounter antigen. Here, Jo Spencer and colleagues review their distribution, properties and malignancies, and suggest that they are designated `marginal-zone B cells' because of their phenotype and location, and that this does not reflect a specific function, since the population appears to be functionally heterogeneous.

The immune system learns from its encounters with pathogens and memorizes its experiences. One of the mechanisms it uses for this purpose is the intra-individual evolution of antigen receptors on B lymphocytes, achieved via hypermutation and selection of antigen receptor variable region genes during an immune response. We have developed a novel method for analyzing the graphical properties of phylogenetic trees of receptor genes which have been mutated and selected during an immune response. In the study presented here, we address the artifacts introduced by experimental methods of cell collection for DNA analysis, the meaning of each parameter measured on the tree graphs, and the differences between the dynamics of the humoral immune response in different lymphoid tissues.

Affinity maturation is a process by which low-affinity antibodies are transformed into highly specific antibodies in germinal centres. This process occurs by hypermutation of immunoglobulin heavy chain variable (IgH V) region genes followed by selection for high-affinity variants. It has been proposed that statistical tests can identify affinity maturation and antigen selection by analysing the frequency of replacement and silent mutations in the complementarity determining regions (CDRs) that contact antigen and the framework regions (FRs) that encode structural integrity. In this study three different methods that have been proposed for detecting selection: the binomial test, the multinomial test and the focused binomial test, have been assessed for their reliability and ability to detect selection in human IgH V genes. We observe first that no statistical test is able to identify selection in the CDR antigen-binding sites, second that tests can reliably detect selection in the FR and third that antibodies from nasal biopsies from patients with Wegener's granulomatosis and pathogenic antibodies from systemic lupus erythematosus do not appear to be as stringently selected for structural integrity as other groups of functional sequences. Lupus (2010) 19, 1161-1170.

It is thought that senescence of the immune system is responsible, at least in part, for many health problems associated with ageing. Previous studies on changes in lymphocyte composition have used flow cytometry to study peripheral blood lymphocytes (PBL’s), or cells isolated from rodent tissue, and have yielded conflicting results. We have used immunohistochemistry to determine whether the B and T cells in human tissue from spleen and gut are affected by age. Areas of germinal centre, mantle zone and marginal zone of B cell follicles were measured. In addition, CD4 and CD8 T cells in T cell areas and in B cell follicles were counted. We observed a striking age-related decrease in the proportion of CD8+ T cells in the T cell zones of the spleen. This decrease was not apparent in the T cell population that occupies splenic B cell areas, or in GALT. Further differences, in CD4+ cells, were seen between T cell populations in the T cell zones and those in B cell areas. These findings highlight differences between lymphocyte populations in different lymphoid tissues, and different compartments within each tissue, which may be of importance in future studies of the ageing immune system.

Background: Ulcerative colitis (UC) is a chronic inflammatory bowel disease in which the colonic mucosa is infiltrated with plasma cells producing IgG autoantibodies. It is not known whether this represents a local mucosal response which has switched to IgG or a peripheral response which may have been initiated by peripheral antigen which homed to the colonic mucosa. The clonal distribution of IgG secreting cells and isotype switched variants in UC is not known. Aims: To investigate the clonal distribution of mucosal IgG in UC and to search for related IgG and IgA secreting cells in normal and diseased mucosa and blood in UC. To investigate characteristics which may discriminate between the mucosal and peripheral repertoire in the normal mucosa and in UC. Patients: Blood and normal and diseased mucosa from two patients with UC were studied. Methods: Immunoglobulin gene analysis and clone specific polymerase chain reaction were used to study the clonal distribution and characteristics of IgG and related IgA in the mucosa and blood of patients with UC. Results: The IgG response in the mucosa of UC patients included widespread clones of cells that were present in both the diseased mucosa and blood but that were scarce in normal mucosa. Clonally related IgA class switch variants, all IgA1, were detected but also only in the diseased mucosa and blood. This suggests that these clones home preferentially to the diseased mucosa. We showed that JH1 usage was characteristic of the peripheral repertoire, and that examples of JH1 usage were observed in mucosal IgG in UC. Conclusions: Overall, these data are consistent with a model of UC in which a peripheral response is expressed and expanded in the colonic mucosa.

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. We also examined the effect of exogenous GCSF administration in a murine model of antimyeloperoxidase (anti-MPO) vasculitis, and the effect of GCSF on murine neutrophil activation. Results The serum levels of GCSF in patients with active ANCA vasculitis were significantly higher than those of age matched healthy controls (mean 38.04 vs 18.35 pg/ml, p

Intraepithelial lymphocytes have been shown to be oligoclonal and to be disseminated widely along the human intestine. However, studies using monoclonal antibodies have suggested that superimposed on the widespread clones, there is local variability in the mucosal T cell population. We have investigated the possibility that local dominant clones of T cells are present in the colonic mucosa by polymerase chain reaction (PCR) amplification of T cell receptor β chain junctional regions using DNA extracted from microdissected fragments of tissue sections. Colon from two right hemicolectomy specimens was sampled at 7‐cm intervals. Adjacent areas of mucosa were microdissected from sections from each colon sample. When the PCR products were separated according to size on polyacrylamide gels, bands of identical size were often observed when DNA extracted from adjacent fragments of mucosa had been used. Different bands were present when the different samples of colon had been studied. Sequencing of the PCR products confirmed that clonally related T cells were present in adjacent areas of mucosa, whereas different clones dominated at distant sites. DNA extracted from cells microdissected from the T cell zone of Peyer's patch was treated identically. The sequences obtained from the Peyer's patch, as expected, were diverse. However, one of the sequences identified was identical to that of one of the clones in the colon, implying that this clone was either trafficking through the Peyer's patch or possibly originated from the Peyer's patch. In this study, we also identified the Peyer's patches as a site of proliferation of CD4+ T cells. No T cell division was observed in the lamina propria. The molecular and immunohistochemical observations together support the hypothesis that the Peyer's patches are a source of mucosal T cells.

It has been well established that the efficiency of the immune system declines with increasing age. Immunosenescence causes increased susceptibility to infectious diseases, and infection is, in fact, the third leading cause of mortality in people aged 65 and over [1]. As is clearly apparent from the other chapters of this book, there are many components of the immune system that can change with age, and are crucial to maintaining an effective immune system. The humoral immune system interacts with the other components, both as part of its own development and via its effector mechanisms. The most important function of B-cells is to produce antibodies, the indispensable soluble effectors of many functions. There are a number of different stages of development for B-cells and their antibodies (Fig. 1).

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 CDB-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. Clustering analysis of high throughput sequencing data enables us to visualize the response in terms of expansions of clonotypes, changes in CDB-H3 characteristics, and somatic hypermutation as well as identifying the commonly used IGH genes. We have highlighted a number of significant age-related changes in the B cell repertoire. Interestingly, in light of the fact that IgG is the most prevalent serum antibody and the most widely used as a correlate of protection, the most striking age related differences are in the IgA response, with defects also seen in the IgM repertoire. In addition there is a skewing toward IgG2 in the IgG sequences of the older samples at all time points. This analysis illustrates the importance of antibody classes other than IgG and has highlighted a number of areas for future consideration in vaccine studies of the elderly

We have developed a rigorous graph-theoretical algorithm for quantifying the shape properties of mutational lineage trees. We show that information about the dynamics of hypermutation and antigen-driven clonal selection during the humoral immune response is contained in the shape of mutational lineage trees deduced from the responding clones. Age and tissue related differences in the selection process can be studied using this method. Thus, tree shape analysis can be used as a means of elucidating humoral immune response dynamics in various situations.

•Groups of immunoglobulin kappa light chain gene (IGK) rearrangements were compared.•Positive and negative selection of IGK gene segments during repertoire development was identified.•Productive rearrangements of IGKs can be rejected in favour of rearrangement of IGL.•Although IGKV4.1 may not be selected against during B cell development, expression may be suppressed.•Suppression of expression of IGKV4.1 appears to be defective in SLE. 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. The final profile of expressed genes is not random and biases in this profile are associated with several autoimmune diseases. However, how and when biases are created is not known. To increase our understanding of the processes of selection and editing of IGK rearrangements, we compared four groups of rearrangements of IGK acquired by next generation sequencing. First, expressed rearrangements of IGK from cDNA of IGK expressing B cells. Second, productive rearrangements of IGK from DNA of the same kappa expressing B cells. Third, non-productive rearrangements of IGK from DNA of IGK and IGL expressing B cells, and fourth productively rearranged IGK from DNA of IGL expressing B cells. The latter group would have been rejected during B cell development in favour of rearrangement at the IGL locus and are therefore selected against. We saw evidence that rearranged IGK segments can be selected at a checkpoint where the decision to rearrange the IGL locus is made. In addition, our data suggest that mechanisms regulating the expression or not of IGK rearrangements may also contribute to repertoire development and also that this latter component of the selection process is defective in SLE.

Aim—To determine whether the immunoglobulin genes used by three gastric mucosa associated lymphoid tissue type lymphomas with known autoreactivity are mutated from germline as mutation from germline is an indicator of exposure to a mutational mechanism which characteristically acts on B cells as they undergo a follicle centre response. Methods—Cell lines established from two cases of MALT type lymphoma secrete autoantibodies recognising follicular dendritic cells (one case) and basement membrane (one case). The immunoglobulin heavy chain variable region genes (IgVH) and light chain variable region genes (IgVL) used by these cell lines, and the IgVH genes from a third case recognising human IgG were sequenced. Results—All three cases studied had mutated IgVH genes, while the IgVL genes were unmutated. Conclusion—The presence of mutations in IgVH genes is consistent with the origin of gastric MALT type lymphomas from B cells which have traversed the lymphoid follicle.

B cells have a number of different roles in the immune response. Their excellent antigen presentation potential can contribute to the activation of other cells of the immune system, and evidence is emerging that specialized subsets of these cells, that may be increased with age, can influence the cell-mediated immune system in antitumor responses. They can also regulate immune responses, to avoid autoreactivity and excessive inflammation. Deficiencies in regulatory B cells may be beneficial in cancer but will only exacerbate the inflammatory environment that is a hallmark of aging. The B cell role as antibody producers is particularly important, since antibodies perform numerous different functions in different environments. Although studying tissue responses in humans is not as easy as in mice, we do know that certain classes of antibodies are more suited to protecting the mucosal tissues (IgA) or responding to T-independent bacterial polysaccharide antigens (IgG2) so we can make some inference with respect to tissue-specific immunity from a study of peripheral blood. We can also make inferences about changes in B cell development with age by looking at the repertoire of different B cell populations to see how age affects the selection events that would normally occur to avoid autoreactivity, or increase specificity, to antigen.

Mononuclear Phagocytes defend tissues, present antigens and mediate recovery and healing. To date we lack a marker to unify mononuclear phagocytes in humans or that informs us about their origin. Here, we reassess Mononuclear Phagocyte ontogeny in human blood through the lineage receptor CSF1R, in the steady state and in COVID-19. We define CSF1R as the first sensitive and reproducible pan-phagocyte lineage marker, to identify and enumerate all conventional monocytes, and the myeloid dendritic cells. In the steady state CSF1R is sufficient for sorting and immuno-magnetic isolation. In pathology, changes in CSF1R are more sensitive than CD14 and CD16. In COVID-19, a significant drop in membrane CSF1R is useful for stratifying patients, beyond the power of cell categories published thus far, which fail to capture COVID-19 specific events. Importantly, CSF1R defines cells which are neither conventional monocytes nor DCs, which are missed in published analysis. CSF1R decrease can be linked ex vivo to high CSF1 levels. Blood assessment of CSF1R+ cells opens a developmental window to the Mononuclear Phagocyte System in transit from bone marrow to tissues, supports isolation and phenotypic characterisation, identifies novel cell types, and singles out CSF1R inhibition as therapeutic target in COVID-19 and other diseases.

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. We compared the different IgM populations using high-throughput sequencing of the immunoglobulin (Ig) gene repertoire and multidimensional cell phenotyping and found that the different populations of IgM cells, defined by CD27 and IgD expression, have repertoire differences. Some of these differences are likely indicative of different selection pressures in an immune response, although the older individuals were found to have a changed repertoire in naive B cells, which may contribute to some of the changes seen in memory cells. In addition, even within the CD27+IgD+ IgM memory population there are multiple cell types. We show that the level of IgM expression varies substantially and hypothesize that this distinguishes between T-dependent and T-independent types of IgM memory cells. Significant age-related changes in the relative proportions of these populations may exacerbate the reduction in T-independent responders in old age.

Abstract Sustainable modern poultry production depends on effective protection against infectious diseases and a diverse range of antibodies is key for an effective immune response. In the domestic chicken, somatic gene conversion is the dominant process in which the antibody immunoglobulin genes are diversified. Affinity maturation by somatic hypermutation (SHM) also occurs, but the relative contribution of gene conversion versus somatic hypermutation to immunoglobulin (Ig) gene diversity is poorly understood. In this study, we use high throughput long-read sequencing to study immunoglobulin diversity in multiple immune-associated tissues in Rhode Island Red chickens. To better understand the impact of genetic diversification in the chicken, a novel gene conversion identification software was developed (BrepConvert). In this study, BrepConvert enabled the identification of over 1 million gene conversion events. Mapping the occurrence of putative somatic gene conversion (SGC) events throughout the variable gene region revealed repetitive and highly restricted patterns of genetic insertions in both the antibody heavy and light chains. These patterns coincided with the locations of genetic variability in available pseudogenes and align with antigen binding sites, predominately the complementary determining regions (CDRs). We found biased usage of pseudogenes during gene conversion, as well as immunoglobulin heavy chain diversity gene (IGHD) preferences during V(D)J gene rearrangement, suggesting that antibody diversification in chickens is more focused than the genetic potential for diversity would suggest.

Background The COVID-19 pandemic is likely to represent an ongoing global health issue given the potential for new variants, vaccine escape and the low likelihood of eliminating all reservoirs of the disease. Whilst diagnostic testing has progressed at a fast pace, the metabolic drivers of outcomes-and whether markers can be found in different biofluids-are not well understood. Recent research has shown that serum metabolomics has potential for prognosis of disease progression. In a hospital setting, collection of saliva samples is more convenient for both staff and patients, and therefore offers an alternative sampling matrix to serum. Methods Saliva samples were collected from hospitalised patients with clinical suspicion of COVID-19, alongside clinical metadata. COVID-19 diagnosis was confirmed using RT-PCR testing, and COVID-19 severity was classified using clinical descriptors (respiratory rate, peripheral oxygen saturation score and C-reactive protein levels). Metabolites were extracted and analysed using high resolution liquid chromatography-mass spectrometry, and the resulting peak area matrix was analysed using multivariate techniques. Results Positive percent agreement of 1.00 between a partial least squares-discriminant analysis metabolomics model employing a panel of 6 features (5 of which were amino acids, one that could be identified by formula only) and the clinical diagnosis of COVID-19 severity was achieved. The negative percent agreement with the clinical severity diagnosis was also 1.00, leading to an area under receiver operating characteristics curve of 1.00 for the panel of features identified. Conclusions In this exploratory work, we found that saliva metabolomics and in particular amino acids can be capable of separating high severity COVID-19 patients from low severity COVID-19 patients. This expands the atlas of COVID-19 metabolic dysregulation and could in future offer the basis of a quick and non-invasive means of sampling patients, intended to supplement existing clinical tests, with the goal of offering timely treatment to patients with potentially poor outcomes.

Both antigenic drive and genetic change play a critical role in the development of MALT lymphoma, but neither alone is sufficient for malignant transformation, and lymphoma development critically depends on their cooperation. However, which of these different events concur and how they cooperate in MALT lymphomagenesis is totally unknown. To explore this, we investigated somatic mutations of 17 genes and IGHV usage in 179 MALT lymphomas from various sites. We showed that: 1) there was a significant association between the biased usage of IGHV4-34 (binds to the carbohydrate I/i antigens) and inactivating mutation of TNFAIP3 (encoding a global negative regulator of the canonical NF-B pathway) in ocular adnexal MALT lymphoma; 2) IGHV1-69 was significantly overrepresented (54%) in MALT lymphoma of salivary gland, but not associated with mutation in any of the 17 genes investigated; and 3) MALT lymphoma lacked mutations frequently seen in other B-cell lymphomas characterised by constitutive NF-B activities, including CD79B, CARD11, MYD88, TNFRSF11A and TRAF3. Our findings show for the first time a significant association between biased usage of autoreactive IGHV and somatic mutation of NF-B regulators in MALT lymphoma, arguing for their cooperation in sustaining chronic BCR signalling and driving oncogenesis in lymphoma development.

The B cell receptor (BCR) repertoire is highly diverse. Repertoire diversity is achieved centrally by somatic recombination of immunoglobulin (Ig) genes and peripherally by somatic hypermutation and Ig heavy chain class-switching. Throughout these processes, there is selection for functional gene rearrangements, selection against gene combinations resulting in self-reactive BCRs, and selection for BCRs with high affinity for exogenous antigens after challenge. Hence, investigation of BCR repertoires from different groups of B cells can provide information on stages of B cell development and shed light on the etiology of B cell pathologies. In most instances, the third complementarity determining region of the Ig heavy chain (CDR-H3) contributes the majority of amino acids to the antibody/antigen binding interface. Although CDR-H3 spectratype analysis provides information on the overall diversity of BCR repertoires, this fairly simple technique analyzes the relative quantities of CDR-H3 regions of each size, within a range of approximately 10–80 bp, without sequence detail and thus is limited in scope. High-throughput sequencing (HTS) techniques on the Roche 454 GS FLX Titanium system, however, can generate a wide coverage of Ig sequences to provide more qualitative data such as V, D, and J usage as well as detailed CDR3 sequence information. Here we present protocols in detail for CDR-H3 spectratype analysis and HTS of human BCR repertoires.

The elderly immune system is characterized by reduced responses to infections and vaccines, and an increase in the incidence of autoimmune diseases and cancer. Age-related deficits in the immune system may be caused by peripheral homeostatic pressures that limit bone marrow B-cell production or migration to the peripheral lymphoid tissues. Studies of peripheral blood B-cell receptor spectratypes have shown that those of the elderly are characterized by reduced diversity, which is correlated with poor health status. In the present study, we performed for the first time high-throughput sequencing of immunoglobulin genes from archived biopsy samples of primary and secondary lymphoid tissues in old (74 ± 7 years old, range 61—89) versus young (24 ± 5 years old, range 18–45) individuals, analyzed repertoire diversities and compared these to results in peripheral blood. We found reduced repertoire diversity in peripheral blood and lymph node repertoires from old people, while in the old spleen samples the diversity was larger than in the young. There were no differences in somatic hypermutation characteristics between age groups. These results support the hypothesis that age-related immune frailty stems from altered B-cell homeostasis leading to narrower memory B-cell repertoires, rather than changes in somatic hypermutation mechanisms.

The best-understood mechanisms for achieving antibody self/nonself 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 IgMlow 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). In a Hy10 antibody transgenic model where anergic B cells respond to a biophysically defined lysozyme epitope displayed on both foreign and self-antigens, cell transfers revealed that anergic IgMlow IgD+ B cells form twice as many GC progeny as naïve IgMhi IgD+ counterparts. Their GC progeny were rapidly selected for CDR2 mutations that blocked 72% of antigen-binding sites with N-linked glycan, decreased affinity 100-fold, and then cleared the binding sites of blocking glycan. These results provide evidence for a mechanism to acquire self/non-self discrimination by somatic mutation away from self-reactivity, and reveal how varying the efficiency of N-glycosylation provides a mechanism to modulate antibody avidity.

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. From a snapshot of B cell scRNA-seq data, a Markov state model is built to infer the dynamics and direction of CSR. Applying sciCSR on severe acute respiratory syndrome coronavirus 2 vaccination time-course scRNA-seq data, we observe that sciCSR predicts, using data from an earlier time point in the collected time-course, the isotype distribution of B cell receptor repertoires of subsequent time points with high accuracy (cosine similarity ~0.9). Using processes specific to B cells, sciCSR identifies transitions that are often missed by conventional RNA velocity analyses and can reveal insights into the dynamics of B cell CSR during immune response.

Metabolic programming is important for B cell fate, but the bioenergetic requirement for regulatory B (Breg) cell differentiation and function is unknown. Here we show that Breg cell differentiation, unlike non-Breg cells, relies on mitochondrial electron transport and homeostatic levels of reactive oxygen species (ROS). Single-cell RNA sequencing analysis revealed that TXN, encoding the metabolic redox protein thioredoxin (Trx), is highly expressed by Breg cells, unlike Trx inhibitor TXNIP which was downregulated. Pharmacological inhibition or gene silencing of TXN resulted in mitochondrial membrane depolarization and increased ROS levels, selectively suppressing Breg cell differentiation and function while favoring pro-inflammatory B cell differentiation. Patients with systemic lupus erythematosus (SLE), characterized by Breg cell deficiencies, present with B cell mitochondrial membrane depolarization, elevated ROS and fewer Trx⁺ B cells. Exogenous Trx stimulation restored Breg cells and mitochondrial membrane polarization in SLE B cells to healthy B cell levels, indicating Trx insufficiency underlies Breg cell impairment in patients with SLE.

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. The IgM-only repertoire profile differed notably from that of MZ B cells by a higher mutation frequency and lower VH4 and higher JH6 gene usage. Strikingly, IgM sequences from clones shared between the MZ and the memory IgG/IgA compartments showed a mutation and repertoire profile of IgM-only and not of MZ B cells. Similarly, all IgM clonal relationships (among MZ, IgM-only, and double-negative compartments) involved sequences with the characteristics of IgM-only B cells. Finally, clonal relationships between tissues suggested distinct recirculation characteristics between MZ and switched B cells. The “IgM-only” subset (including cells with its repertoire signature but higher IgD or lower CD27 expression levels) thus appear as the only subset showing precursor–product relationships with CD27+ switched memory B cells, indicating that they represent germinal center–derived IgM memory B cells and that IgM memory and MZ B cells constitute two distinct entities.

The British Society for Immunology is the leading UK membership organisation working with scientists andclinicians from academia and industry to forward immunology research and application around the world. Ourfriendly, accessible community consists of more than 4,200 immunologists, giving us a powerful voice to advocate for immunological science and health for the benefit of society.As this has been a rapid review, it is a summary of the research at time of writing; it is not an exhaustive literature review. It is the considered input of the advisory group and does not necessarily represent the position of the British Society for Immunology, its members or the individual members of the advisory group.All web references were accessed in January 2021.© British Society for Immunology

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. Both surface and intracytoplasmic dual light chain expression was evident by flow cytometry and confocal microscopy. We observed reduced frequency of rearrangements of the kappa-deleting element (KDE) in SLE and an inverse correlation between the frequency of KDE rearrangement and the frequency of dual light chain expressing B cells. We propose that dual expression of Igκ and Igλ by a single B cell may occur in some patients with SLE when this may be a consequence of reduced activity of the KDE.

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, then 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. As a consequence, the binding properties of the B cell receptor are changed as development progresses through pre- B>>immature>>transitional>>naïve phenotypes. Using long-read, high-throughput, sequencing we have produced a unique set of sequences from these four cell types in human bone marrow and matched peripheral blood and our results describe the effects of tolerance selection on the B cell repertoire at the Ig gene level. Most strong effects of selection are seen within the heavy chain repertoire, and can be seen both in gene usage and in CDR-H3 characteristics. Age-related changes are small and only the size of the CDR-H3 shows constant and significant change in these data. The paucity of significant changes in either kappa or lambda light chain repertoires implies that either the heavy chain has more influence over autoreactivity than light chain and/or that switching between kappa and lambda light chains, as opposed to switching within the light chain loci, may effect a more successful autoreactive rescue by receptor editing. Our results show that the transitional cell population contains cells other than those that are part of the pre-B>>immature>>transitional>>naïve development pathway, since the population often shows a repertoire that is outside the trajectory of gene loss/gain between pre-B and naïve stages.

The immune system defends against infection, but older people paradoxically suffer not only from failing immunity resulting in increased susceptibility to infections and decreased responsiveness to vaccination, but at the same time increased inflammation and immunopathology accompanying immune responses. Interventions to reduce such deleterious effects while enhancing protective immunity are challenging but need to be confronted if we are to deal successfully with the increasing numbers of elderly and frail people in modern societies. To do this, we need to understand the mechanisms responsible for age-associated increased susceptibility to infections and immune-influenced chronic degenerative diseases of ageing. Defining relevant age-associated alterations and identifying reliable biomarkers for monitoring clinically-relevant immune status in the elderly population is crucial to overcoming these problems. Here, we briefly outline age-associated changes to immunity collectively termed ‘immunosenescence’

IgE is an ancient and conserved immunoglobulin isotype with potent immunological function. Nevertheless, the regulation of IgE responses remains an enigma, and evidence of a role for IgE in host defense is limited. Here we report that topical exposure to a common environmental DNA-damaging xenobiotic initiated stress surveillance by γδTCR+ intraepithelial lymphocytes that resulted in class switching to IgE in B cells and the accumulation of autoreactive IgE. High-throughput antibody sequencing revealed that γδ T cells shaped the IgE repertoire by supporting specific variable-diversity-joining (VDJ) rearrangements with unique characteristics of the complementarity-determining region CDRH3. This endogenous IgE response, via the IgE receptor FcεRI, provided protection against epithelial carcinogenesis, and expression of the gene encoding FcεRI in human squamous-cell carcinoma correlated with good disease prognosis. These data indicate a joint role for immunosurveillance by T cells and by B cells in epithelial tissues and suggest that IgE is part of the host defense against epithelial damage and tumor development.

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. The software is able to use data preprocessed by IMGT, and performs statistical and comparative analyses with versatile plotting options. BRepertoire has been designed to operate in various modes, for example analysing sequence-specific V(D)J gene usage, discerning physico-chemical properties of the CDR regions and clustering of clonotypes. Those analyses are performed on the fly by a number of R packages and are deployed by a shiny web platform. The user can download the analysed data in different table formats and save the generated plots as image files ready for publication.We believe BRepertoire to be a versatile analytical tool that complements experimental studies of immune repertoires. To illustrate the server’s functionality, we show use cases including differential gene usage in a vaccination dataset and analysis of CDR3H properties in old and young individuals. The server is accessible under http://mabra.biomed.kcl.ac.uk/BRepertoire.

B cells rely on a broad receptor repertoire to provide protection against a wide range of pathogens. This is in part achieved through V(D)J recombination, which, by assembling various combinations of variable (V), diversity (D), and joining (J) genes, creates different IgV regions.1 The recombination processes is initiated by recombination-activating gene (RAG) 1/RAG2 enzymes and requires a functional nonhomologous end-joining (NHEJ) machinery. B cells can further diversify their IgV regions through somatic hypermutation (SHM) to improve affinity between the antibody and antigen and switch the isotype of antibody produced by class-switch recombination (CSR).

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). However, gastric DLBCL can also be initiated de novo. The mechanisms underlying transformation into DLBCL are not completely understood.We analyzed immunoglobulin repertoires and clonal trees to investigate whether and how immunoglobulin gene repertoires, clonal diversification, and selection in gastritis, gastric MALT-L, and DLBCL differ from each other and from normal responses. The two gastritis types (positive or negative for H. pylori) had similarly diverse repertoires. MALT-L dominant clones (defined as the largest clones in each sample) presented higher diversification and longer mutational histories compared with all other conditions. DLBCL dominant clones displayed lower clonal diversification, suggesting the transforming events are triggered by similar responses in different patients. These results are surprising, as we expected to find similarities between the dominant clones of gastritis and MALT-L and between those of MALT-L and DLBCL.

B cells undergo a number of different developmental stages, from initial formation of their B cell receptor (BCR) genes to differentiation into antibody-secreting plasma cells. Because the BCR is vital in these differentiation steps, autoreactive and exogenous antigen binding to the BCR exert critical selection pressures to shape the B cell repertoire. Older people are more prone to infectious disease, less able to respond well to vaccination and more likely to have autoreactive antibodies. Here we review evidence of changes in B cell repertoires in older people, which may be a reflection of age-related changes in B cell selection processes.

Executive summary Understanding immunity to COVID-19, induced by both natural infection and through vaccination, is key to our ability to exit the current pandemic. In this report, you will find the answers to key questions around what we know and don’t know about immunity to the virus SARS-CoV-2, which causes the disease, COVID-19. This includes the effectiveness of the immune response, how to measure and track immunity, the benefits of vaccine-mediated immunity, and the longevity of any immunity conferred. We have sought to make the subject matter relevant to both the public policy discussions that are ongoing and those that will arise as the pandemic situation begins to change over the next few months. It is important to say that there are differing degrees of immunity. Different individuals will create different immune responses to invasive pathogens, and the case of the SARS-CoV-2 virus is no exception. Some people create a very effective immune response, so they will not get sick again from SARS-CoV-2 and will not pass the virus to anyone else (so-called ‘sterilising’ immunity), while others will make antibodies* and be protected from the disease COVID-19, but may still be infected with the SARS-CoV-2 virus and transmit it to others (‘protective’ immunity). Immunity can be difficult to measure. The best marker currently is neutralising antibodies*, which have been shown to persist in some individuals up to 8 months after original infection. While immunity can also be measured by looking at memory immune cells, methods for doing this at scale are not currently available. Immunity can wane over time and this can lead to the small chance of reinfection. Exactly how long immunity following COVID-19 lasts will need a longer time to determine. Vaccine-mediated immunity is preferable and safer than naturally acquired immunity. While clinical trials recorded the ability of the vaccines to protect from COVID-19 disease, questions remain around whether the vaccines being administered currently will prevent people from being able to carry and transmit the SARS-CoV-2 virus. Currently not enough time has elapsed between the vaccines first being administered in humans and the present time for durability of vaccine-induced immunity to be determined, but this is the subject of ongoing phase 3 vaccine studies. The answers to all these questions will have a profound impact on the policy decisions that the Government makes. Questions over how immunity can be measured, how long immunity lasts and the reliability of such tests can undermine the usefulness of ‘vaccine passports’, and whether the vaccine stops the spread of the SARS-CoV-2 virus to others or simply stops the person who has been vaccinated from contracting the disease, COVID-19, will be vital to finding our way out of this pandemic. The longevity of immunity conferred by a vaccine will determine whether there will be need for an annual COVID-19 vaccination programme, like that currently carried out for flu. With so many key policy issues resting on issues of COVID-19 immunity, it is integral to the country’s future that we immediately implement a robust and widespread immune monitoring programme to understand in detail the immunity conferred through vaccination in different individuals. It is also crucial that we ensure proper surveillance of viral variance at a global scale and through this the ability of any variants to escape vaccine-mediated immunity. With the UK being an international leader in the rollout of COVID-19 vaccines, we can lead the world in immune monitoring protocols that will allow us to emerge from this pandemic more safely and quickly. This is an opportunity that we should seize with both hands.

The human immunoglobulin repertoire is a hugely diverse set of sequences that are formed by processes of gene rearrangement, heavy and light chain gene assortment, class switching and somatic hypermutation. Early B cell development produces diverse IgM and IgD B cell receptors on the B cell surface, resulting in a repertoire that can bind many foreign antigens but which has had self-reactive B cells removed. Later antigen-dependent development processes adjust the antigen affinity of the receptor by somatic hypermutation. The effector mechanism of the antibody is also adjusted, by switching the class of the antibody from IgM to one of seven other classes depending on the required function. There are many instances in human biology where positive and negative selection forces can act to shape the immunoglobulin repertoire and therefore repertoire analysis can provide useful information on infection control, vaccination efficacy, autoimmune diseases and cancer. It can also be used to identify antigen-specific sequences that may be of use in therapeutics. The juxtaposition of lymphocyte development and numerical evaluation of immune repertoires has resulted in the growth of a new sub-speciality in immunology where immunologists and computer scientists/physicists collaborate to assess immune repertoires and develop models of immune action.

Human B cells produce antibodies, which bind to their cognate antigen based on distinct molecular properties of the antibody CDR loop. We have analysed a set of 10 antibodies showing a clear difference in their binding properties to a panel of antigens, resulting in two subsets of antibodies with a distinct binding phenotype. We call the observed binding multiplicity ‘promiscuous’ and selected physico-chemical CDRH3 characteristics and conformational preferences may characterise these promiscuous antibodies. To classify CDRH3 physico-chemical properties playing a role in their binding properties, we used statistical analyses of the sequences annotated by Kidera factors. To characterise structure-function requirements for antigen binding multiplicity we employed Molecular Modelling and Monte Carlo based coarse-grained simulations. The ability to predict the molecular causes of promiscuous, multi-binding behaviour would greatly improve the efficiency of the therapeutic antibody discovery process.

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 contain have different functions and phenotypes. These populations can change as we age; older people have been shown to have fewer “IgM memory” cells, regulatory B cells and plasma cells and more IgD-CD27- “double negative” and “Age-related B cells”. While the overall quantity of antibody in the blood does not change, the quality of the B cell response changes; producing less specific antibodies upon challenge and more autoreactive antibodies. This could be due to changes in selection pressures, as has been demonstrated by repertoire sequencing of different subsets of B cells at different ages. Other changes in antibody repertoire are seen, including: greater levels of IgG2 in older people, and altered IgG1 IGHV gene usage. Since B cells rely on their environment for efficient responses, some of these changes may be due to age-related changes in accessory cells/signals. Other changes appear to be intrinsic to older/aged B cells themselves, such as their tendency to produce greater levels of inflammatory cytokines.

Intestinal homeostasis relies on a continuous dialogue between the commensal bacteria and the immune system. Natural killer T (NKT) cells, which recognize CD1d-restricted microbial lipids and self-lipids, contribute to the regulation of mucosal immunity, yet the mechanisms underlying their functions remain poorly understood. Here, we demonstrate that NKT cells respond to intestinal lipids and CD11c+ cells (including dendritic cells (DCs) and macrophages) are essential to mediate lipid presentation within the gut ultimately controlling intestinal NKT cell homeostasis and activation. Conversely, CD1d and NKT cells participate in the control of the intestinal bacteria composition and compartmentalization, in the regulation of the IgA repertoire and in the induction of regulatory T cells within the gut. These changes in intestinal homeostasis require CD1d expression on DC/macrophage populations as mice with conditional deletion of CD1d on CD11c+ cells exhibit dysbiosis and altered immune homeostasis. These results unveil the importance of CD11c+ cells in controlling lipid-dependent immunity in the intestinal compartment and reveal an NKT cell–DC crosstalk as a key mechanism for the regulation of gut homeostasis.

B cells are known to contribute to the anti-tumor immune response, especially in immunogenic tumors such as melanoma, yet humoral immunity has not been characterized in these cancers to detail. Here we show comprehensive phenotyping in samples of circulating and tumor-resident B cells as well as serum antibodies in melanoma patients. Memory B cells are enriched in tumors compared to blood in paired samples and feature distinct antibody repertoires, linked to specific isotypes. Tumor-associated B cells undergo clonal expansion, class switch recombination, somatic hypermutation and receptor revision. Compared with blood, tumor-associated B cells produce antibodies with proportionally higher levels of unproductive sequences and distinct complementarity determining region 3 properties. The observed features are signs of affinity maturation and polyreactivity and suggest an active and aberrant autoimmune-like reaction in the tumor microenvironment. Consistent with this, tumor-derived antibodies are polyreactive and characterized by autoantigen recognition. Serum antibodies show reactivity to antigens attributed to autoimmune diseases and cancer, and their levels are higher in patients with active disease compared to post-resection state. Our findings thus reveal B cell lineage dysregulation with distinct antibody repertoire and specificity, alongside clonally-expanded tumor-infiltrating B cells with autoimmune-like features, shaping the humoral immune response in melanoma.

Older people are more susceptible to infection, less responsive to vaccination and have a more inflammatory immune environment. Using spectratype analysis, we have previously shown that the B-cell repertoire of older people shows evidence of inappropriate clonal expansions in the absence of challenge, and that this loss of B-cell diversity correlates with poor health. Studies on response to vaccination, using both spectratyping and high-throughput sequencing of the repertoire, indicate that older responses to challenge are lacking in magnitude and/or delayed significantly. Also that some of the biologically significant differences may be in different classes of antibody. We have also previously shown that normal young B-cell repertoires can vary between different phenotypic subsets of B cells. In this paper, we present an analysis of immunoglobulin repertoire in different subclasses of antibody in five different populations of B cell, and show how the repertoire in these different groups changes with age. Although some age-related repertoire differences occur in naive cells, before exogenous antigen exposure, we see indications that there is a general dysregulation of the selective forces that shape memory B-cell populations in older people.

IgE is the least abundant circulating antibody class but is constitutively present in healthy tissues bound to resident cells via its high-affinity receptor, FcεRI. The physiological role of endogenous IgE antibodies is unclear but it has been suggested that they provide host protection against a variety of noxious environmental substances and parasitic infections at epithelial barrier surfaces. Here we show, in mice, that skin inflammation enhances levels of IgE antibodies that have natural specificities and a repertoire, VDJ rearrangements and CDRH3 characteristics similar to those of IgE antibodies in healthy tissue. IgE-bearing basophils are recruited to inflamed skin via CXCL12 and thymic stromal lymphopoietin (TSLP)/IL-3-dependent upregulation of CXCR4. In the inflamed skin, IgE/FcεRI-signalling in basophils promotes epithelial cell growth and differentiation, partly through histamine engagement of H 1 R and H 4 R. Furthermore, this IgE response strongly drives tumour outgrowth of epithelial cells harbouring oncogenic mutation. These findings indicate that natural IgE antibodies support skin barrier defences, but that during chronic tissue inflammation this role may be subverted to promote tumour growth.

The mechanisms of B-cell diversification differ greatly between aves and mammals, but both produce B cells and antibodies capable of supporting an effective immune response. To see how differences in the generation of diversity might affect overall repertoire diversity, we have compared the diversity characteristics of immunoglobulin genes from domestic chickens to those from humans. Both use V(D)J gene rearrangement and somatic hypermutation, but only chickens use somatic gene conversion. A range of diversity analysis tools were used to investigate multiple aspects of amino acid diversity at both the germline and repertoire levels. The effect of differing amino acid usages on antibody characteristics was assessed. At both the germline and repertoire levels, chickens exhibited lower amino acid diversity in comparison to the human immunoglobulin genes, especially outside of the complementarity-determining region (CDR). Chickens were also found to possess much larger and more hydrophilic CDR3s with a higher predicted protein binding potential, suggesting that the antigen-binding site in chicken antibodies is more flexible and more polyreactive than that seen in human antibodies.

B cells have a number of different roles in the immune response. Their excellent antigen presentation potential can contribute to the activation of other cells of the immune system, and evidence is emerging that specialized subsets of these cells, that may be increased with age, can influence the cell-mediated immune system in antitumor responses. They can also regulate immune responses, to avoid autoreactivity and excessive inflammation. Deficiencies in regulatory B cells may be beneficial in cancer but will only exacerbate the inflammatory environment that is a hallmark of aging. The B cell role as antibody producers is particularly important, since antibodies perform numerous different functions in different environments. Although studying tissue responses in humans is not as easy as in mice, we do know that certain classes of antibodies are more suited to protecting the mucosal tissues (IgA) or responding to T-independent bacterial polysaccharide antigens (IgG2) so we can make some inference with respect to tissue-specific immunity from a study of peripheral blood. We can also make inferences about changes in B cell development with age by looking at the repertoire of different B cell populations to see how age affects the selection events that would normally occur to avoid autoreactivity, or increase specificity, to antigen.

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. CD20⁺CD27⁺IgD⁻ isotype-switched B lymphocytes were increased in tumors, compared with matched blood. TIL-B frequently formed stromal clusters with T lymphocytes and engaged in bidirectional functional cross-talk, consistent with gene signatures associated with lymphoid assembly, costimulation, cytokine–cytokine receptor interactions, cytotoxic T-cell activation, and T-cell–dependent B-cell activation. TIL-B–upregulated B-cell receptor (BCR) pathway molecules FOS and JUN, germinal center chemokine regulator RGS1, activation marker CD69, and TNFα signal transduction via NFκB, suggesting BCR–immune complex formation. Expression of genes associated with B lymphocyte recruitment and lymphoid assembly, including CXCL13, CXCR4, and DC-LAMP, was elevated in TNBC compared with other subtypes and normal breast. TIL-B–rich tumors showed expansion of IgG but not IgA isotypes, and IgG isotype switching positively associated with survival outcomes in TNBC. Clonal expansion was biased toward IgG, showing expansive clonal families with specific variable region gene combinations and narrow repertoires. Stronger positive selection pressure was present in the complementarity determining regions of IgG compared with their clonally related IgA in tumor samples. Overall, class-switched B lymphocyte lineage traits were conspicuous in TNBC, associated with improved clinical outcomes, and conferred IgG-biased, clonally expanded, and likely antigen-driven humoral responses.

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. Recent advances in single-cell techniques enable an alternative route to characterize cells based on their transcriptome. To maximize immunological insight, we need to match prior data from phenotype-based studies with the finer granularity of the single-cell transcriptomic signatures. We also need to be able to define meaningful B cell subsets from single cell analyses performed on PBMCs, where the relative paucity of a B cell signature means that defining B cell subsets within the whole is challenging. Here we provide a reference single-cell dataset based on phenotypically sorted B cells and an unbiased procedure to better classify functional B cell subsets in the peripheral blood, particularly useful in establishing a baseline cellular landscape and in extracting significant changes with respect to this baseline from single-cell datasets. We find 10 different clusters of B cells and applied a novel, geometry-inspired, method to RNA velocity estimates in order to evaluate the dynamic transitions between B cell clusters. This indicated the presence of two main developmental branches of memory B cells. A T-independent branch that involves IgM memory cells and two DN subpopulations, culminating in a population thought to be associated with Age related B cells and the extrafollicular response. The other, T-dependent, branch involves a third DN cluster which appears to be a precursor of classical memory cells. In addition, we identify a novel DN4 population, which is IgE rich and closely linked to the classical/precursor memory branch suggesting an IgE specific T-dependent cell population.

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 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. We constructed a dataset containing over 29,000 - light chain variable region sequences from IgM-transcribing, newly formed B cells isolated from human bone marrow and peripheral blood. We also used a published human naïve dataset to investigate the CDR-H3 properties of heavy chains paired with kappa and lambda light chains, and probed the Protein Data Bank (PDB) to investigate the structural differences between kappa and lambda antibody CDR regions. We found that kappa and lambda light chains have very different CDR physicochemical and structural properties, whereas the heavy chains with which they are paired do not differ significantly. We also observed that the mean CDR3 N nucleotide addition in the kappa, lambda and heavy chain gene rearrangements are correlated within donors, but can differ between donors. This indicates that TdT may work with differing efficiencies between different people, but the same efficiency in the different classes of immunoglobulin chain within one person. We have observed large differences in the physicochemical and structural properties of kappa and lambda light chain CDR regions. This may reflect different roles in the humoral immune response.