Age-dependent changes in T follicular helper cells shape the humoral immune response to vaccination.

Vaccination is an excellent strategy to limit the morbidity and mortality associated with infectious disease. Vaccination creates protective, long-lived antibody-mediated immunity by inducing the germinal centre response, an intricate immune reaction that produces memory B cells and long-lived antibody-secreting plasma cells that provide protection against (re)infection. The magnitude and quality of the germinal centre response declines with age, contributing to poor vaccine-induced immunity in older individuals. T follicular helper cells are essential for the formation and function of the germinal centre response. This review will discuss how age-dependent changes in T follicular helper cells influence the germinal centre response, and the evidence that age-dependent changes need not be a barrier to successful vaccination in the later years of life.

Multiscale deep learning framework captures systemic immune features in lymph nodes predictive of triple negative breast cancer outcome in large-scale studies.

The suggestion that the systemic immune response in lymph nodes (LNs) conveys prognostic value for triple-negative breast cancer (TNBC) patients has not previously been investigated in large cohorts. We used a deep learning (DL) framework to quantify morphological features in haematoxylin and eosin-stained LNs on digitised whole slide images. From 345 breast cancer patients, 5,228 axillary LNs, cancer-free and involved, were assessed. Generalisable multiscale DL frameworks were developed to capture and quantify germinal centres (GCs) and sinuses. Cox regression proportional hazard models tested the association between smuLymphNet-captured GC and sinus quantifications and distant metastasis-free survival (DMFS). smuLymphNet achieved a Dice coefficient of 0.86 and 0.74 for capturing GCs and sinuses, respectively, and was comparable to an interpathologist Dice coefficient of 0.66 (GC) and 0.60 (sinus). smuLymphNet-captured sinuses were increased in LNs harbouring GCs (p < 0.001). smuLymphNet-captured GCs retained clinical relevance in LN-positive TNBC patients whose cancer-free LNs had on average ≥2 GCs, had longer DMFS (hazard ratio [HR] = 0.28, p = 0.02) and extended GCs' prognostic value to LN-negative TNBC patients (HR = 0.14, p = 0.002). Enlarged smuLymphNet-captured sinuses in involved LNs were associated with superior DMFS in LN-positive TNBC patients in a cohort from Guy's Hospital (multivariate HR = 0.39, p = 0.039) and with distant recurrence-free survival in 95 LN-positive TNBC patients of the Dutch-N4plus trial (HR = 0.44, p = 0.024). Heuristic scoring of subcapsular sinuses in LNs of LN-positive Tianjin TNBC patients (n = 85) cross-validated the association of enlarged sinuses with shorter DMFS (involved LNs: HR = 0.33, p = 0.029 and cancer-free LNs: HR = 0.21 p = 0.01). Morphological LN features reflective of cancer-associated responses are robustly quantifiable by smuLymphNet. Our findings further strengthen the value of assessment of LN properties beyond the detection of metastatic deposits for prognostication of TNBC patients. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

CD27 on human memory B cells-more than just a surface marker.

Immunological memory protects the human body from re-infection with an earlier recognized pathogen. This memory comprises the durable serum antibody titres provided by long-lived plasma cells and the memory T and B cells with help from other cells. Memory B cells are the main precursor cells for new plasma cells during a secondary infection. Their formation starts very early in life, and they continue to form and undergo refinements throughout our lifetime. While the heterogeneity of the human memory B-cell pool is still poorly understood, specific cellular surface markers define most of the cell subpopulations. CD27 is one of the most commonly used markers to define human memory B cells. In addition, there are molecular markers, such as somatic mutations in the immunoglobulin heavy and light chains and isotype switching to, for example, IgG. Although not every memory B cell undergoes somatic hypermutation or isotype switching, most of them express these molecular traits in adulthood. In this review, I will focus on the most recent knowledge regarding CD27+ human memory B cells in health and disease, and describe how Ig sequencing can be used as a tool to decipher the evolutionary pathways of these cells.

Cervical lymph nodes and ovarian teratomas as germinal centres in NMDA receptor-antibody encephalitis.

Autoantibodies against the extracellular domain of the N-methyl-d-aspartate receptor (NMDAR) NR1 subunit cause a severe and common form of encephalitis. To better understand their generation, we aimed to characterize and identify human germinal centres actively participating in NMDAR-specific autoimmunization by sampling patient blood, CSF, ovarian teratoma tissue and, directly from the putative site of human CNS lymphatic drainage, cervical lymph nodes. From serum, both NR1-IgA and NR1-IgM were detected more frequently in NMDAR-antibody encephalitis patients versus controls (both P < 0.0001). Within patients, ovarian teratoma status was associated with a higher frequency of NR1-IgA positivity in serum (OR = 3.1; P < 0.0001) and CSF (OR = 3.8, P = 0.047), particularly early in disease and before ovarian teratoma resection. Consistent with this immunoglobulin class bias, ovarian teratoma samples showed intratumoral production of both NR1-IgG and NR1-IgA and, by single cell RNA sequencing, contained expanded highly-mutated IgA clones with an ovarian teratoma-restricted B cell population. Multiplex histology suggested tertiary lymphoid architectures in ovarian teratomas with dense B cell foci expressing the germinal centre marker BCL6, CD21+ follicular dendritic cells, and the NR1 subunit, alongside lymphatic vessels and high endothelial vasculature. Cultured teratoma explants and dissociated intratumoral B cells secreted NR1-IgGs in culture. Hence, ovarian teratomas showed structural and functional evidence of NR1-specific germinal centres. On exploring classical secondary lymphoid organs, B cells cultured from cervical lymph nodes of patients with NMDAR-antibody encephalitis produced NR1-IgG in 3/7 cultures, from patients with the highest serum NR1-IgG levels (P < 0.05). By contrast, NR1-IgG secretion was observed neither from cervical lymph nodes in disease controls nor in patients with adequately resected ovarian teratomas. Our multimodal evaluations provide convergent anatomical and functional evidence of NMDAR-autoantibody production from active germinal centres within both intratumoral tertiary lymphoid structures and traditional secondary lymphoid organs, the cervical lymph nodes. Furthermore, we develop a cervical lymph node sampling protocol that can be used to directly explore immune activity in health and disease at this emerging neuroimmune interface.

Physical 'strength' of the multi-protein chain connecting immune cells: Does the weakest link limit antibody affinity maturation?: The weakest link in the multi-protein chain facilitating antigen acquisition by B cells in germinal centres limits antibody affinity maturation.

The affinities of antibodies (Abs) for their target antigens (Ags) gradually increase in vivo following an infection or vaccination, but reach saturation at values well below those realisable in vitro. This 'affinity ceiling' could in many cases restrict our ability to fight infections and compromise vaccines. What determines the affinity ceiling has been an unresolved question for decades. Here, we argue that it arises from the strength of the chain of protein complexes that is pulled by B cells during the process of Ag acquisition. The affinity ceiling is determined by the strength of the weakest link in the chain. We identify the weakest link and show that the resulting affinity ceiling can explain the Ab affinities realized in vivo, providing a conceptual understanding of Ab affinity maturation. We explore plausible evolutionary underpinnings of the affinity ceiling, examine supporting evidence and alternative hypotheses and discuss implications for vaccination strategies.

Critical roles of the E3 ubiquitin ligase FBW7 in B-cell response and the pathogenesis of experimental autoimmune arthritis.

Proper regulation of B-cell function is essential for effective humoral immunity and maintenance of immune tolerance. Here, we found that FBW7 (F-box/WD40 repeat-containing protein 7) is highly expressed in germinal centre B and B1 cells, and confirmed that it has an intrinsic role in maintaining homeostasis of mature B cells and B-1 cells. FBW7 deletion led to an impairment of antibody response, and although germinal centre formation was not affected, antibody class-switch recombination and affinity maturation processes were defective. Likewise, memory immune response was severely impaired. Moreover, FBW7 ablation ameliorated the pathogenesis of an autoimmune disease model, collagen-induced arthritis, by reducing the production of anti-collagen II autoantibodies. Taken together, these data suggest that FBW7 may be an attractive target for developing new therapeutics for the treatment of autoimmune diseases.

Lymphomas complicating primary Sjögren's syndrome: from autoimmunity to lymphoma.

Lymphoma development is the most serious complication of SS and the main factor impacting on mortality rate in patients with this condition. Lymphomas in SS are most commonly extranodal non-Hodgkin B-cell lymphomas of the mucosa-associated lymphoid tissue and frequently arise in salivary glands that are the target of a chronic inflammatory autoimmune process. Extensive work on lymphomagenesis in SS has established that the progression towards B-cell lymphoma is a multistep process related to local chronic antigenic stimulation of B cells. These neoplastic B cells in SS frequently derived from autoreactive clones, most commonly RF-producing B cells, which undergo uncontrolled proliferation and malignant escape. In this review, we highlight the most important recent findings that have enhanced our understanding of lymphoma development in SS, with particular reference to the close link between autoimmunity and lymphomagenesis. We also discuss how the identification of key factors involved in B-cell malignancies may impact on our ability to identify at early stages patients at increased risk of lymphoma with potential significant repercussions for the clinical management of SS patients. Finally, we identified the most promising areas of current and further research with the potential to provide novel basic and translational discoveries in the field. The questions of finding new biomarkers, developing a validated score for predicting lymphoma occurrence and assessing if a better control of disease activity will decrease the risk of lymphoma in primary SS will be the enthralling questions of the next few years.

The role of stroma and epithelial cells in primary Sjögren's syndrome.

Primary SS (pSS) is a chronic autoimmune condition characterized by infiltration of the exocrine glands and systemic B cell hyperactivation. This glandular infiltration is associated with loss of glandular function, with pSS patients primarily presenting with severe dryness of the eyes and mouth. Within the affected glands, the infiltrating lymphocytes are organized in tertiary lymphoid structures. Tertiary lymphoid structures subvert normal tissue architecture and impact on organ function, by promoting the activation and maintenance of autoreactive lymphocytes. This review summarizes the current knowledge about the role of stromal cells (including endothelium, epithelium, nerves and fibroblasts) in the pathogenesis of pSS, in particular the interactions taking place between stromal cells and infiltrating lymphocytes. We will provide evidences pointing towards the driving role of stromal cells in the orchestration of the local inflammatory milieu, thus highlighting the need for therapies aimed at targeting this compartment alongside classical immunosuppression in pSS.

Ubiquitination of IgG1 cytoplasmic tail modulates B-cell signalling and activation.

Upon antigen stimulation, IgG+ B cells rapidly proliferate and differentiate into plasma cells, which has been attributed to the characteristics of membrane-bound IgG (mIgG), but the underlying molecular mechanisms remain elusive. We have found that a part of mouse mIgG1 is ubiquitinated through the two responsible lysine residues (K378 and K386) in its cytoplasmic tail and this ubiquitination is augmented upon antigen stimulation. The ubiquitination of mIgG1 involves its immunoglobulin tail tyrosine (ITT) motif, Syk/Src-family kinases and Cbl proteins. Analysis of a ubiquitination-defective mutant of mIgG1 revealed that ubiquitination of mIgG1 facilitates its ligand-induced endocytosis and intracellular trafficking from early endosome to late endosome, and also prohibits the recycling pathway, thus attenuating the surface expression level of mIgG1. Accordingly, ligation-induced activation of B-cell receptor (BCR) signalling molecules is attenuated by the mIgG1 ubiquitination, except MAP kinase p38 whose activation is up-regulated due to the ubiquitination-mediated prohibition of mIgG1 recycling. Adaptive transfer experiments demonstrated that ubiquitination of mIgG1 facilitates expansion of germinal centre B cells. These results indicate that mIgG1-mediated signalling and cell activation is regulated by ubiquitination of mIgG1, and such regulation may play a role in expansion of germinal centre B cells.

Possibilities for the analysis of peripheral blood B cell subpopulations in a routine immunological laboratory.

B cells play a vital role in the defence of the body against infectious agents. Apart from their ability to present antigen to T cells, B cells are mainly producers of antibodies. These play a crucial role in the effective elimination of infection and are also involved in the regulation of the immune response. The analysis of peripheral blood B cell subpopulations that makes it possible to monitor the development of B cells to the stage of antibody producing plasmablasts provides a valuable laboratory parameter which is important for both the study of the pathogenesis and diagnosis of some diseases. Laboratory analysis of B cell subpopulations is now a routinely available laboratory option thanks to the development of multicolour flow cytometry. This article summarizes the core knowledge which is currently applied to the analysis of B cell subpopulations in immunological laboratories.

Humoral autoimmunity after solid organ transplantation: Germinal ideas may not be natural.

Non-HLA antibody responses following solid organ transplantation have become increasingly emphasised, with several large clinical series suggesting that such responses contribute to late graft failure. Many of the responses described recognise both recipient and donor moieties of the target antigen and thus represent auto-, rather than allo-immunity. Within this rapidly evolving field, many questions remain unanswered: what triggers the response; how innate and adaptive humoral autoimmunity integrate; and most pressingly, how autoimmunity contributes to graft damage and its relationship to other effector mechanisms of graft rejection. This review summarises recent clinical and experimental studies of humoral autoimmunity in transplant rejection, and considers some of the answers to these questions.

Tfh Cells in Health and Immunity: Potential Targets for Systems Biology Approaches to Vaccination.

T follicular helper (Tfh) cells are a specialised subset of CD4+ T cells that play a significant role in the adaptive immune response, providing critical help to B cells within the germinal centres (GC) of secondary lymphoid organs. The B cell receptors of GC B cells undergo multiple rounds of somatic hypermutation and affinity maturation within the GC response, a process dependent on cognate interactions with Tfh cells. B cells that receive sufficient help from Tfh cells form antibody-producing long-lived plasma and memory B cells that provide the basis of decades of effective and efficient protection and are considered the gold standard in correlates of protection post-vaccination. However, the T cell response to vaccination has been understudied, and over the last 10 years, exponential improvements in the technological underpinnings of sampling techniques, experimental and analytical tools have allowed multidisciplinary characterisation of the role of T cells and the immune system as a whole. Of particular interest to the field of vaccinology are GCs and Tfh cells, representing a unique target for improving immunisation strategies. Here, we discuss recent insights into the unique journey of Tfh cells from thymus to lymph node during differentiation and their role in the production of high-quality antibody responses as well as their journey back to the periphery as a population of memory cells. Further, we explore their function in health and disease and the power of next-generation sequencing techniques to uncover their potential as modulators of vaccine-induced immunity.

BAFF, IL-4 and IL-21 separably program germinal center-like phenotype acquisition, BCL6 expression, proliferation and survival of CD40L-activated B cells in vitro.

A B cell culture system using BAFF, IL-4 and IL-21 was recently developed that generates B cells with phenotypic and functional characteristics of in vivo-generated germinal center (GC) B cells. Here, we observe discrete influences of each exogenous signal on the expansion and differentiation of a CD40L-activated B cell pool. IL-4 was expressly necessary, but neither BAFF nor IL-21 was required for B cell acquisition of the GC B cell phenotypes of peanut agglutinin binding and loss of CD38 and IgD expression. Both IL-4 and IL-21 enhanced cell cycle entry upon initial activation dose-dependently, and did so additively. Importantly, while both cytokines acted in concert to increase overall BCL6 expression amounts, IL-21 exposure uniquely caused a small proportion of cells to attain a higher level of BCL6 expression, reminiscent of in vivo GC B cells. In contrast, BAFF supported survival of a fraction of memory-like B cells in extended cultures after removal of surrogate T cell-help signals. Thus, by separably programming proliferation, survival and GC phenotype acquisition, IL-4, BAFF and IL-21 drive distinct components of activated B cell fate.

Toll-like receptor 4 signalling regulates antibody response to adenoviral vector-based vaccines by imprinting germinal centre quality.

Adenoviral vectors (AdV) are considered promising candidates for vaccine applications. A prominent group of Toll-like receptors (TLRs) participate in the adenovirus-induced adaptive immune response, yet there is little information regarding the role of TLR4 in AdV-induced immune responses in recent literature. We investigated the function of TLR4 in both adaptive and innate immune responses to an AdV-based anthrax vaccine. By immunizing wild-type and TLR4 knockout (TLR4-KO) mice, we revealed the requirement of TLR4 in AdV-induced innate responses. We also showed that TLR4 functions are required for germinal centre responses in immunized mice, as expression of the apoptosis-related marker Fas was down-regulated on germinal centre B cells from TLR4-KO mice. Likewise, decreased expression of inducible costimulator on follicular T helper cells was observed in immunized TLR4-KO mice. Moreover, a potent protective antigen-specific humoral immune response was mimicked using an adjuvant system containing the TLR4 agonist monophosphoryl lipid A. Overall, our findings showed that very rapid antigen-specific antibody production is correlated with the TLR4-imprinted germinal centre response to AdV-based vaccine. These results provide additional evidence for the use of the AdV and a TLR agonist to induce humoral responses. Our findings offer new insights into rational vaccine design.

Follicular helper T cells in the pathogenesis of IgG4-related disease.

IgG4-related disease (IgG4-RD) is a recently recognized disease entity characterized by high serum IgG4 concentrations and infiltration of IgG4+ plasma cells with hyperplastic ectopic germinal centres at affected sites. Although the underlying immune mechanism of this disease remains unclear, T cells are abundantly present at affected sites and key players in IgG4-RD pathogenesis. The role of T cell subsets has been investigated thoroughly in this disease. Recent advances in this field have clarified the importance of T follicular helper cells. In this review, we describe the role of T follicular helper cells in the disease process of IgG4-RD, in particular, for IgG4 class-switching, plasmablast and plasma cell differentiation, and germinal centre formation.

BLIMP-1 is insufficient to induce antibody secretion in the absence of IRF4 in DT40 cells.

Differentiation of B cells into antibody-secreting cells (ASCs), plasmablasts and plasma cells is regulated by a network of transcription factors. Within this network, factors including PAX5 and BCL6 prevent ASC differentiation and maintain the B cell phenotype. In contrast, BLIMP-1 and high IRF4 expression promote plasma cell differentiation. BLIMP-1 is thought to induce immunoglobulin secretion, whereas IRF4 is needed for the survival of ASCs. The role of IRF4 in the regulation of antibody secretion has remained controversial. To study the role of IRF4 in the regulation of antibody secretion, we have created a double knockout (DKO) DT40 B cell line deficient in both IRF4 and BCL6. Although BCL6-deficient DT40 B cell line had upregulated BLIMP-1 expression and secreted antibodies, the DKO cell line did not. Even enforced BLIMP-1 expression in DKO cells or IRF4-deficient cells could not induce IgM secretion while in WT DT40 cells, it could. However, enforced IRF4 expression in DKO cells induced strong IgM secretion. Our findings support a model where IRF4 expression in addition to BLIMP-1 expression is required to induce robust antibody secretion.

The elusive role of B lymphocytes and islet autoantibodies in (human) type 1 diabetes.

The role of B lymphocytes in the pathogenesis of type 1 diabetes in humans is not entirely evident. These cells are presumed to be important, but this assumption is largely based on animal models of autoimmune diabetes, where compelling evidence for the contribution of both B lymphocytes and insulin-specific autoantibodies to this disease is in place. For humans, this is much less the case; the exact way in which B lymphocytes and/or autoantibodies may contribute to type 1 diabetes is not yet known but the possibilities include a pathogenic function ('fire'), or they may represent a surrogate of loss of immune tolerance to beta cells ('smoke') or, indeed, they could be a marker of an attempt at immune regulation ('ice water'). In this issue of Diabetologia, a study by Willcox et al (DOI: 10.1007/s00125-017-4221-7 ) adds new information but no greater clarity on the relevance of B lymphocytes in type 1 diabetes, showing a decrease in germinal centre frequencies in donors with recent-onset type 1 diabetes compared with control donors and donors with longstanding type 1 diabetes. These new findings may guide the research community to design experiments to unambiguously define whether B lymphocytes or their products function as fire, smoke or perhaps ice water in the immunopathogenesis of type 1 diabetes.

From dendritic cells to B cells dysfunctions during HIV-1 infection: T follicular helper cells at the crossroads.

T follicular helper (Tfh) cells are essential for B-cell differentiation and the subsequent antibody responses. Their numbers and functions are altered during human and simian immunodeficiency virus (HIV/SIV) infections. In lymphoid tissues, Tfh cells are present in germinal centre, where they are the main source of replicative HIV-1 and represent a major reservoir. Paradoxically, Tfh cell numbers are increased in chronically infected individuals. Understanding the fate of Tfh cells in the course of HIV-1 infection is essential for the design of efficient strategies toward a protective HIV vaccine or a cure. The purpose of this review is to summarize the recent advance in our understanding of Tfh cell dynamics during HIV/SIV infection. In particular, to explore the possible causes of their expansion in lymphoid tissues by discussing the impact of HIV-1 infection on dendritic cells, to identify the molecular players rendering Tfh cells highly susceptible to HIV-1 infection, and to consider the contribution of regulatory follicular T cells in shaping Tfh cell functions.

Ontogeny of human IgE-expressing B cells and plasma cells.

BACKGROUND: IgE-expressing (IgE+ ) plasma cells (PCs) provide a continuous source of allergen-specific IgE that is central to allergic responses. The extreme sparsity of IgE+ cells in vivo has confined their study almost entirely to mouse models. OBJECTIVE: To characterize the development pathway of human IgE+ PCs and to determine the ontogeny of human IgE+ PCs. METHODS: To generate human IgE+ cells, we cultured tonsil B cells with IL-4 and anti-CD40. Using FACS and RT-PCR, we examined the phenotype of generated IgE+ cells, the capacity of tonsil B-cell subsets to generate IgE+ PCs and the class switching pathways involved. RESULTS: We have identified three phenotypic stages of IgE+ PC development pathway, namely (i) IgE+ germinal centre (GC)-like B cells, (ii) IgE+ PC-like 'plasmablasts' and (iii) IgE+ PCs. The same phenotypic stages were also observed for IgG1+ cells. Total tonsil B cells give rise to IgE+ PCs by direct and sequential switching, whereas the isolated GC B-cell fraction, the main source of IgE+ PCs, generates IgE+ PCs by sequential switching. PC differentiation of IgE+ cells is accompanied by the down-regulation of surface expression of the short form of membrane IgE (mIgES ), which is homologous to mouse mIgE, and the up-regulation of the long form of mIgE (mIgEL ), which is associated with an enhanced B-cell survival and expressed in humans, but not in mice. CONCLUSION: Generation of IgE+ PCs from tonsil GC B cells occurs mainly via sequential switching from IgG. The mIgEL /mIgES ratio may be implicated in survival of IgE+ B cells during PC differentiation and allergic disease.

OX40 controls effector CD4+ T-cell expansion, not follicular T helper cell generation in acute Listeria infection.

To investigate the importance of OX40 signals for physiological CD4(+) T-cell responses, an endogenous antigen-specific population of CD4(+) T cells that recognise the 2W1S peptide was assessed and temporal control of OX40 signals was achieved using blocking or agonistic antibodies (Abs) in vivo. Following infection with Listeria monocytogenes expressing 2W1S peptide, OX40 was briefly expressed by the responding 2W1S-specific CD4(+) T cells, but only on a subset that co-expressed effector cell markers. This population was specifically expanded by Ab-ligation of OX40 during priming, which also caused skewing of the memory response towards effector memory cells. Strikingly, this greatly enhanced effector response was accompanied by the loss of T follicular helper (TFH) cells and germinal centres. Mice deficient in OX40 and CD30 showed normal generation of TFH cells but impaired numbers of 2W1S-specific effector cells. OX40 was not expressed by 2W1S-specific memory cells, although it was rapidly up-regulated upon challenge whereupon Ab-ligation of OX40 specifically affected the effector subset. In summary, these data indicate that for CD4(+) T cells, OX40 signals are important for generation of effector T cells rather than TFH cells in this response to acute bacterial infection.