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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.Significance:Tumor-infiltrating B lymphocytes assemble in clusters, undergoing B-cell receptor–driven activation, proliferation, and isotype switching. Clonally expanded, IgG isotype-biased humoral immunity associates with favorable prognosis primarily in triple-negative breast cancers.

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Supplementary materials and methods and Supplementary figure legends revised

Class-switch recombination (CSR) is an integral part of B cell maturation. Steady-state analyses of isotype distribution (e.g. B cell receptor [BCR] repertoire analysis of snapshots during an immune response) do not directly measure CSR dynamics, which is crucial in understanding how B cell maturation is regulated across time. We present sciCSR (pronounced ‘scissor’, single-cell inference of class switch recombination), a computational pipeline which analyses CSR events and dynamics of B cells from single-cell RNA-sequencing (scRNA-seq) experiments. sciCSR re-analyses transcriptomic sequence 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 by the pipeline to infer the dynamics and direction of CSR. Applying sciCSR on SARS-CoV-2 vaccination time-course scRNA-seq data, we observe that sciCSR predicts, using data from an earlier timepoint in the collected time-course, the isotype distribution of BCR repertoires of subsequent timepoints with high accuracy (cosine similarity ∼ 0.9). sciCSR also recapitulates CSR patterns in mouse models where B cell maturation was perturbed using gene knockouts. sciCSR infers cell state transitions using processes specific to B cells, identifies transitions which are often missed by conventional RNA velocity analyses, and can reveal insights into the regulation of CSR and the dynamics of B cell maturation during an immune response.

BackgroundThe COVID-19 pandemic is likely to represent an ongoing global health issue given the potential for vaccine escape and the low likelihood of eliminating all reservoirs of the disease. Whilst diagnostic testing has progressed at pace, there is an unmet clinical need to develop tests that are prognostic, to triage the high volumes of patients arriving in hospital settings. Recent research has shown that serum metabolomics has potential for prognosis of disease progression. 1 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. We demonstrate here for the first time that saliva metabolomics can reveal COVID-19 severity.Methods88 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. COVID severity was classified using clinical descriptors first proposed by SR Knight et al. Metabolites were extracted from saliva samples and analysed using liquid chromatography mass spectrometry.ResultsIn this work, positive percent agreement of 1.00 between a PLS-DA metabolomics model and the clinical diagnosis of COVID severity was achieved. The negative percent agreement with the clinical severity diagnosis was also 1.00, for overall percent agreement of 1.00.ConclusionsThis research demonstrates that liquid chromatography-mass spectrometry can identify salivary biomarkers capable of separating high severity COVID-19 patients from low severity COVID-19 patients in a small cohort study.

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., Graphical Abstract Graphical Abstract

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. Significance: Tumor-infiltrating B lymphocytes assemble in clusters, undergoing B-cell receptor–driven activation, proliferation, and isotype switching. Clonally expanded, IgG isotype-biased humoral immunity associates with favorable prognosis primarily in triple-negative breast cancers.

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.

In this short report we present a preliminary assessment of the serum 25-hydroxyvitamin D status (25(OH)D), body mass index (BMI), ethnicity and other lifestyle factors in the first-reported UK Biobank COVID-19 positive cases (n 580) compared with negative controls (n 723). The COVID-19 cases include those who have been treated as a hospital inpatient as well as those who have not, and are from England only. Mean (SD) for age was 57.5 (8.7) in positive cases and 57.9 (8.7) in negative controls.

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.

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

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 is unclear but it is suggested to provide host protection against a variety of noxious environmental substances and parasitic infections at epithelial barrier surfaces. Here we show that skin inflammation enhances levels of IgE with natural specificities and with a similar repertoire, VDJ rearrangements and CDRH3 characteristics as in healthy tissue. IgE-bearing basophils are recruited to inflamed skin via CXCL12 and 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 H1R and H4R. Furthermore, this natural IgE response strongly drives tumour outgrowth of epithelial cells harbouring oncogenic mutation. These findings indicate that natural IgE support skin barrier defences however during chronic tissue inflammation this may be subverted to promote tumour growth.

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 H1R and H4R. 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.

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

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 FceRI, provided protection against epithelial carcinogenesis, and expression of the gene encoding FceRI 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.

Purpose To assess the impact of a masked Endpoint Committee on estimates of the incidence of primary open-angle glaucoma (POAG) treatment efficacy and statistical power of the Ocular Hypertension Treatment Study-Phase 1, 1994-2002 (OHTS-1). Design Retrospective interrater reliability analysis of endpoint attribution by the Endpoint Committee. Methods After study closeout, we recalculated estimates of endpoint incidence, treatment efficacy, and statistical power using all-cause endpoints and POAG endpoints. To avoid bias, only the first endpoint per participant is included in this report. Results The Endpoint Committee reviewed 267 first endpoints from 1636 participants. The Endpoint Committee attributed 58% (155 of 267) of the endpoints to POAG. The incidence of all-cause endpoints vs POAG endpoints was 19.5% and 13.2%, respectively, in the observation group and 13.1% and 5.8%, respectively, in the medication group. Treatment effect for all-cause endpoints was a 33% reduction in risk (relative risk = 0.67, 95% confidence interval [CI] of 0.54-0.84) and a 56% reduction in risk for POAG endpoints (relative risk = 0.44, 95% CI of 0.31-0.61). Post hoc statistical power for detecting treatment effect was 0.94 for all-cause endpoints and 0.99 for POAG endpoints. Conclusion Endpoint Committee adjudication of endpoints improved POAG incidence estimates, increased statistical power, and increased calculated treatment effect by 23%. An Endpoint Committee should be considered in therapeutic trials when common ocular and systemic comorbidities, other than the target condition, could compromise study results.

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.

Summary 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.

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.

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

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.

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.

The B cell repertoire is generated in the adult bone marrow by an ordered series of gene rearrangement processes that result in massive diversity of immunoglobulin (Ig) genes and consequently an equally large number of potential specificities for antigen. As the process is essentially random, the cells exhibiting excess reactivity with self-antigens are generated and need to be removed from the repertoire before the cells are fully mature. Some of the cells are deleted, and some will undergo receptor editing to see if changing the light chain can rescue an autoreactive antibody. 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 CDRH3 characteristics. Age-related changes are small, and only the size of the CDRH3 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.

Antibody variable regions are composed of a heavy and a light chain, and in humans,\ud there are two light chain isotypes: kappa and lambda. Despite their importance in\ud receptor editing, the light chain is often overlooked in the antibody literature, with the\ud focus being on the heavy chain complementarity-determining region (CDR)-H3 region.\ud In this paper, we set out to investigate the physicochemical and structural differences\ud between human kappa and lambda light chain CDR regions. We constructed a dataset\ud containing over 29,000 light chain variable region sequences from IgM-transcribing,\ud newly formed B cells isolated from human bone marrow and peripheral blood. We also\ud used a published human naïve dataset to investigate the CDR-H3 properties of heavy\ud chains paired with kappa and lambda light chains and probed the Protein Data Bank to\ud investigate the structural differences between kappa and lambda antibody CDR regions.\ud We found that kappa and lambda light chains have very different CDR physicochemical\ud and structural properties, whereas the heavy chains with which they are paired do not\ud differ significantly. We also observed that the mean CDR3 N nucleotide addition in the\ud kappa, lambda, and heavy chain gene rearrangements are correlated within donors but\ud can differ between donors. This indicates that terminal deoxynucleotidyl transferase may\ud work with differing efficiencies between different people but the same efficiency in the\ud different classes of immunoglobulin chain within one person. We have observed large\ud differences in the physicochemical and structural properties of kappa and lambda light\ud chain CDR regions. This may reflect different roles in the humoral immune response.

Many diversification mechanisms occurr during central tolerance leading to a wide array of antibodies (Abs) with a range of binding specificities, some of which lead to polyreactivity. The repertoire of preB cells (before central tolerance), immature B cells (during central tolerance) and naive B cells in the peripheral blood (after central tolerance) has been interrogated by high throughput sequencing of heavy and light chain Ig genes. It has been shown that older people display a less diverse repertoire of antibody-producing cells, and that this loss of diversity is associated with poor health. Within a population of responding cells there are favoured characteristics of a the Ab gene CDRH3 loop that is crucial in binding the foreign antigen. We hypothesise that favoured physico-chemical CDRH3 characteristics make a structure capable of binding to multiple foreign antigens (polyspecific).The ability to predict this promiscuous behaviour with respect to binding specificity would greatly improve the efficiency of the therapeutic antibody discovery process. From these data we want to extract the molecular determinants playing a role in polyreactivity by exploiting structural and dynamical information. We use statistical analyses to classify CDRH3 physico-chemical properties and Molecular Dynamics (MD) simulations at the atomistic and/or coarse grained levels to characterise flexibility requirements for binding multiplicity.Martin V, Wu YC, Kipling D, Dunn-Walters DK. Ann N Y Acad Sci. 2015 http://dx.doi.org/10.1111/nyas.12823.Fornili A, Pandini A, Lu HC, Fraternali F. J Chem Theory Comput. 2013 9:5127-5147.Marcatili P, et al. J Immunol. 2013 190:5771-8.

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 18–49 and 65–89. 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.

The Association to Advance Collegiate Schools of Business (AACSB) describes their accreditation as the hallmark of business education. According to information at BestBizSchools.com (n.d.), AACSB accreditation represents the highest standard of achievement for business schools worldwide. Being AACSB accredited means a business school is able to continuously pass a strict set of standards that ensure quality. As of December 2010, only 5%, or 607, of the academic business programs globally were accredited by AACSB. This number represents schools in 38 countries where the majority of programs incorporate both undergraduate and graduate education covering business, accounting, or both. An institution must be a member of AACSB in order to apply for accreditation. It is important to note, however, that membership does not imply that the program is accredited (The Association to Advance Collegiate Schools of Business, n.d.-a). Recent emphasis demanding external validation on the quality of Business Schools has resulted in the promotion of AACSB accreditation as the de facto quality standard. Earning this quality seal of approval, business programs can verify they have met the 21 AACSB standards that cover strategic, participant, and assurance of learning achievements and processes. Programs with AACSB accreditation are encouraged to promote the standard using it to externally validate their quality and to market their programs to external groups including students, employers, and contributors (The Association to Advance Collegiate Schools of Business, n.d.-b). Despite established standards, no single approach to meeting standards for accreditation is suggested by AACSB. Rather, varying approaches to meeting standards should be developed to fit individual programs of institutions (Bryant & Scherer, 2009). This position by AACSB underscores its recognition of the diversity across accredited programs and allows educators wide latitude in developing and implementing approaches to excellence. Small programs are not disadvantaged so long as their students, faculty, graduates, and the employers who hire them receive the quality outputs that help them meet the external competitive requirements (Olian, 2007). In recognition of member institutions diversity, the AACSB has established the Affinity Group program where school administrators from schools sharing similar characteristics can interact, exchange ideas, and present views on a wide range of issues (Olian, 2007). This allows AACSB member schools, who have varying missions and constituents, to find and link with other programs of a similar nature where creativity and synergy can more easily occur. The AACSB wants the accreditation process to help facilitate creativity in designing business school strategies rather than being viewed as an impediment to a programs push to quality (Romero, 2008).

SummaryAgeing 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 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 Sjögren'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.

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.

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.

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.

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.

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.

Transitional (TR) B cells are immature B cells that have migrated from the bone marrow to peripheral lymphoid organs, but can still undergo selection against autoreactivity. TR cells that survive selection eventually develop into mature naïve B cells (CD27-IgD+, NA). Upon exposure to antigen, NA cells may become IgM memory (CD27+IgD+, MM) or "classical", classswitched memory (CD27+IgD-, SM) B cells. Although MM immunoglobulin (Ig) genes do not undergo class switching, they do undergo somatic hypermutation, albeit with lower frequency than SM. It has been postulated that MM B cells originate from T-independent immune responses, while SM cells originate from T-dependent responses. Alternatively, MM cells may be early emigrants from T-dependent germinal centers. Double negative B cells (CD27-IgD-, DN) have been said to be exhausted memory cells, but their precise origin is unclear. Therefore, a definitive elucidation of lineage relationships between these different B cell subsets is needed.

There is very little change in the quantity of antibodies people produce, of any isotype, with age. However, there is a change in the quality of the antibody response. Older people produce fewer antibodies that are specific for the activating pathogen or vaccine. At the same time, the number of nonspecific antibodies increases. Quite often these antibodies have self-reactivity (e.g., anti-dsDNA). The appearance of these antibodies is not associated with pathogenic autoimmune disease, although it is true that the incidence of some autoimmune diseases increases with age. The authors postulate that the process of antibody affinity maturation is compromised in old age. No evidence was found that the process of hypermutation is compromised with age. However, using graph theory to study the dynamics of a germinal center selection process, a decrease in the extent of selection occurring in the germinal centers of mucosal tissue was observed with age. This is a tissue-specific phenomenon because the decrease was not seen in the germinal centers of spleen. Because selection of highly specific cells in the germinal center depends on a number of factors (number and quality of founder cells, help from T cells, and follicular dendritic cells) these need to be investigated further to determine what is needed to improve the affinity mutation process.