Regulation of B-cell function and expression of CD11c, T-bet, and FcRL5 in response to different activation signals.

CD11c, FcRL5, or T-bet are commonly expressed by B cells expanding during inflammation, where they can make up >30% of mature B cells. However, the association between the proteins and differentiation and function in the host response remains largely unclear. We have assessed the co-expression of CD11c, T-bet, and FcRL5 in an in vitro B-cell culture system to determine how stimulation via the BCR, toll-like receptor 9 (TLR9), and different cytokines influence CD11c, T-bet, and FcRL5 expression. We observed different expression dynamics for all markers, but a largely overlapping regulation of CD11c and FcRL5 in response to BCR and TLR9 activation, while T-bet was strongly dependent on IFN-γ signaling. Investigating plasma cell differentiation and APC functions, there was no association between marker expression and antibody secretion or T-cell help. Rather the functions were associated with TLR9-signalling and B-cell-derived IL-6 production, respectively. These results suggest that the expression of CD11c, FcRL5, and T-bet and plasma cell differentiation and improved APC functions occur in parallel and are regulated by similar activation signals, but they are not interdependent.

Unravelling B cell heterogeneity: insights into flow cytometry-gated B cells from single-cell multi-omics data.

Introduction: B cells play a pivotal role in adaptive immunity which has been extensively characterised primarily via flow cytometry-based gating strategies. This study addresses the discrepancies between flow cytometry-defined B cell subsets and their high-confidence molecular signatures using single-cell multi-omics approaches. Methods: By analysing multi-omics single-cell data from healthy individuals and patients across diseases, we characterised the level and nature of cellular contamination within standard flow cytometric-based gating, resolved some of the ambiguities in the literature surrounding unconventional B cell subsets, and demonstrated the variable effects of flow cytometric-based gating cellular heterogeneity across diseases. Results: We showed that flow cytometric-defined B cell populations are heterogenous, and the composition varies significantly between disease states thus affecting the implications of functional studies performed on these populations. Importantly, this paper draws caution on findings about B cell selection and function of flow cytometric-sorted populations, and their roles in disease. As a solution, we developed a simple tool to identify additional markers that can be used to increase the purity of flow-cytometric gated immune cell populations based on multi-omics data (AlliGateR). Here, we demonstrate that additional non-linear CD20, CD21 and CD24 gating can increase the purity of both naïve and memory populations. Discussion: These findings underscore the need to reconsider B cell subset definitions within the literature and propose leveraging single-cell multi-omics data for refined characterisation. We show that single-cell multi-omics technologies represent a powerful tool to bridge the gap between surface marker-based annotations and the intricate molecular characteristics of B cell subsets.

Emerging insights into atypical B cells in pediatric chronic infectious diseases and immune system disorders: T(o)-bet on control of B-cell immune activation.

Repetitive or persistent cellular stimulation in vivo has been associated with the development of a heterogeneous B-cell population that exhibits a distinctive phenotype and, in addition to classical B-cell markers, often expresses the transcription factor T-bet and myeloid marker CD11c. Research suggests that this atypical population consists of B cells with distinct B-cell receptor specificities capable of binding the antigens responsible for their development. The expansion of this population occurs in the presence of chronic inflammatory conditions and autoimmune diseases where different nomenclatures have been used to describe them. However, as a result of the diverse contexts in which they have been investigated, these cells have remained largely enigmatic, with much ambiguity remaining regarding their phenotype and function in humoral immune response as well as their role in autoimmunity. Atypical B cells have garnered considerable interest because of their ability to produce specific antibodies and/or autoantibodies and because of their association with key disease manifestations. Although they have been widely described in the context of adults, little information is present for children. Therefore, the aim of this narrative review is to describe the characteristics of this population, suggest their function in pediatric immune-related diseases and chronic infections, and explore their potential therapeutic avenues.

Atypical B cells (CD21-CD27-IgD-) correlate with lack of response to checkpoint inhibitor therapy in NSCLC.

INTRODUCTION: Checkpoint inhibitor (CI) therapy has revolutionized treatment for non-small cell lung cancer (NSCLC). However, a proportion of patients do not respond to CI therapy for unknown reasons. Although the current paradigm in anti-tumor immunity evolves around T cells, the presence of tertiary lymphoid structures and memory B cells has been positively correlated with response to CI therapy in NSCLC. In addition, double negative (DN) (CD27- IgD-) B cells have been shown to be abundant in NSCLC compared to healthy lung tissue and inversely correlate with the intratumoral presence of memory B cells. Nonetheless, no study has correlated DN B cells to survival in NSCLC. METHODS: In this study, we evaluated the presence and phenotype of B cells in peripheral blood with flow cytometry of patients with NSCLC and mesothelioma before receiving CI therapy and correlated these with clinical outcome. RESULTS: Non-responding patients showed decreased frequencies of B cells, yet increased frequencies of antigen-experienced CD21- DN (Atypical) B cells compared to responding patients and HC, which was confirmed in patients with mesothelioma treated with CI therapy. CONCLUSIONS: These data show that the frequency of CD21- DN B cells correlates with lack of response to CI therapy in thoracic malignancies. The mechanism by which CD21- DN B cells hamper CI therapy remains unknown. Our findings support the hypothesis that CD21- DN B cells resemble phenotypically identical exhausted B cells that are seen in chronic infection or function as antigen presenting cells that induce regulatory T cells.

Acquisition of complement fixing antibodies targeting Plasmodium falciparum merozoites in infants and their mothers in Uganda.

Background: Antibody-mediated complement fixation has previously been associated with protection against malaria in naturally acquired immunity. However, the process of early-life development of complement-fixing antibodies in infants, both in comparison to their respective mothers and to other immune parameters, remains less clear. Results: We measured complement-fixing antibodies in newborns and their mothers in a malaria endemic area over 5 years follow-up and found that infants' complement-fixing antibody levels were highest at birth, decreased until six months, then increased progressively until they were similar to birth at five years. Infants with high levels at birth experienced a faster decay of complement-fixing antibodies but showed similar levels to the low response group of newborns thereafter. No difference was observed in antibody levels between infant cord blood and mothers at delivery. The same result was found when categorized into high and low response groups, indicating placental transfer of antibodies. Complement-fixing antibodies were positively correlated with total schizont-specific IgG and IgM levels in mothers and infants at several time points. At nine months, complement-fixing antibodies were negatively correlated with total B cell frequency and osteopontin concentrations in the infants, while positively correlated with atypical memory B cells and P. falciparum-positive atypical memory B cells. Conclusion: This study indicates that complement-fixing antibodies against P. falciparum merozoites are produced in the mothers and placentally-transferred, and they are acquired in infants over time during the first years of life. Understanding early life immune responses is crucial for developing a functional, long lasting malaria vaccine.

BAFF system expression in double negative 2, activated naïve and activated memory B cells in systemic lupus erythematosus.

Introduction: B cell activating factor (BAFF) has an important role in normal B cell development. The aberrant expression of BAFF is related with the autoimmune diseases development like Systemic Lupus Erythematosus (SLE) for promoting self-reactive B cells survival. BAFF functions are exerted through its receptors BAFF-R (BR3), transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA) that are reported to have differential expression on B cells in SLE. Recently, atypical B cells that express CD11c have been associated with SLE because they are prone to develop into antibody-secreting cells, however the relationship with BAFF remains unclear. This study aims to analyze the BAFF system expression on CXCR5- CD11c+ atypical B cell subsets double negative 2 (DN2), activated naïve (aNAV), switched memory (SWM) and unswitched memory (USM) B cells. Methods: Forty-five SLE patients and 15 healthy subjects (HS) were included. Flow cytometry was used to evaluate the expression of the receptors in the B cell subpopulations. Enzyme-linked immunosorbent assay (ELISA) was performed to quantify the soluble levels of BAFF (sBAFF) and IL-21. Results: We found increased frequency of CXCR5- CD11c+ atypical B cell subpopulations DN2, aNAV, SWM and USM B cells in SLE patients compared to HS. SLE patients had increased expression of membrane BAFF (mBAFF) and BCMA receptor in classic B cell subsets (DN, NAV, SWM and USM). Also, the CXCR5+ CD11c- DN1, resting naïve (rNAV), SWM and USM B cell subsets showed higher mBAFF expression in SLE. CXCR5- CD11c+ atypical B cell subpopulations DN2, SWM and USM B cells showed strong correlations with the expression of BAFF receptors. The atypical B cells DN2 in SLE showed significant decreased expression of TACI, which correlated with higher IL-21 levels. Also, lower expression of TACI in atypical B cell DN2 was associated with high disease activity. Discussion: These results suggest a participation of the BAFF system in CXCR5- CD11c+ atypical B cell subsets in SLE patients. Decreased TACI expression on atypical B cells DN2 correlated with high disease activity in SLE patients supporting the immunoregulatory role of TACI in autoimmunity.

A sex-biased imbalance between Tfr, Tph, and atypical B cells determines antibody responses in COVID-19 patients.

Sex-biased humoral immune responses to COVID-19 patients have been observed, but the cellular basis for this is not understood. Using single-cell proteomics by mass cytometry, we find disrupted regulation of humoral immunity in COVID-19 patients, with a sex-biased loss of circulating follicular regulatory T cells (cTfr) at a significantly greater rate in male patients. In addition, a male sex-associated cellular network of T-peripheral helper, plasma blasts, proliferating and extrafollicular/atypical CD11c+ memory B cells was strongly positively correlated with neutralizing antibody concentrations and negatively correlated with cTfr frequency. These results suggest that sex-specific differences to the balance of cTfr and a network of extrafollicular antibody production-associated cell types may be a key factor in the altered humoral immune responses between male and female COVID-19 patients.

Autoantibodies during infectious diseases: Lessons from malaria applied to COVID-19 and other infections.

Autoimmunity is a common phenomenon reported in many globally relevant infections, including malaria and COVID-19. These and other highly inflammatory diseases have been associated with the presence of autoantibodies. The role that these autoantibodies play during infection has been an emerging topic of interest. The vast numbers of studies reporting a range of autoantibodies targeting cellular antigens, such as dsDNA and lipids, but also immune molecules, such as cytokines, during malaria, COVID-19 and other infections, underscore the importance that autoimmunity can play during infection. During both malaria and COVID-19, the presence of autoantibodies has been correlated with associated pathologies such as malarial anemia and severe COVID-19. Additionally, high levels of Atypical/Autoimmune B cells (ABCs and atypical B cells) have been observed in both diseases. The growing literature of autoimmune B cells, age-associated B cells and atypical B cells in Systemic Lupus erythematosus (SLE) and other autoimmune disorders has identified recent mechanistic and cellular targets that could explain the development of autoantibodies during infection. These new findings establish a link between immune responses during infection and autoimmune disorders, highlighting shared mechanistic insights. In this review, we focus on the recent evidence of autoantibody generation during malaria and other infectious diseases and their potential pathological role, exploring possible mechanisms that may explain the development of autoimmunity during infections.

CD21- CD27- Atypical B Cells in a Pediatric Cohort Study: An Extensive Single Center Flow Cytometric Analysis.

Atypical B cells (atBCs) are a distinct B-cell population and represent approximately 5% of B cells in peripheral blood (PB) of healthy adult individuals. However, in adults these cells are expanded in conditions of chronic infections, inflammation, primary immunodeficiencies, autoimmune diseases, and aging. Their immunophenotype is characterized by the lack of CD21 expression and the hallmark human memory B-cell marker CD27. In this study, we investigated the immunophenotype of atBCs in different pediatric pathological conditions and correlated their expansion with the children's clinical diagnosis. We were able to retrospectively evaluate 1,571 consecutive PB samples, corresponding to 1,180 pediatric patients, by using a 9-color flow-cytometric panel. The results, compared with a pediatric healthy cohort, confirmed an expansion of atBCs in patient samples with percentages greater than 5% of total B cells. Four subpopulations with different expressions of IgM and IgD were discriminated: IgM+IgD+, IgM+-only, IgD+-only, and IgM-IgD-. IgG+ atBCs were predominant in the IgM- IgD- subpopulation. Moreover, the study highlighted some features of atBCs, such as a low CD38 expression, a heterogeneity of CD24, a high expression of CD19 and a large cell size. We also demonstrated that an increase of atBCs in a pediatric cohort is correlated with immunodeficiencies, autoimmune, inflammatory, and hematological disorders, consistent with previous studies mainly performed in adults. Furthermore, our flow cytometric clustering analysis corroborated the recent hypothesis of an alternative B origin for atBCs.

Generation of plasma cells and CD27-IgD- B cells during hantavirus infection is associated with distinct pathological findings.

OBJECTIVE: Human hantavirus infections can cause haemorrhagic fever with renal syndrome (HFRS). The pathogenic mechanisms are not fully understood, nor if they affect the humoral immune system. The objective of this study was to investigate humoral immune responses to hantavirus infection and to correlate them to the typical features of HFRS: thrombocytopenia and transient kidney dysfunction. METHODS: We performed a comprehensive characterisation of longitudinal antiviral B-cell responses of 26 hantavirus patients and combined this with paired clinical data. In addition, we measured extracellular adenosine triphosphate (ATP) and its breakdown products in circulation and performed in vitro stimulations to address its effect on B cells. RESULTS: We found that thrombocytopenia was correlated to an elevated frequency of plasmablasts in circulation. In contrast, kidney dysfunction was indicative of an accumulation of CD27-IgD- B cells and CD27-/low plasmablasts. Finally, we provide evidence that high levels of extracellular ATP and matrix metalloproteinase 8 can contribute to shedding of CD27 during human hantavirus infection. CONCLUSION: Our findings demonstrate that thrombocytopenia and kidney dysfunction associate with distinctly different effects on the humoral immune system. Moreover, hantavirus-infected individuals have significantly elevated levels of extracellular ATP in circulation.

Deep Phenotyping of CD11c+ B Cells in Systemic Autoimmunity and Controls.

Circulating CD11c+ B cells are a key phenomenon in certain types of autoimmunity but have also been described in the context of regular immune responses (i.e., infections, vaccination). Using mass cytometry to profile 46 different markers on individual immune cells, we systematically initially confirmed the presence of increased CD11c+ B cells in the blood of systemic lupus erythematosus (SLE) patients. Notably, significant differences in the expression of CD21, CD27, and CD38 became apparent between CD11c- and CD11c+ B cells. We observed direct correlation of the frequency of CD21-CD27- B cells and CD21-CD38- B cells with CD11c+ B cells, which were most pronounced in SLE compared to primary Sjögren's syndrome patients (pSS) and healthy donors (HD). Thus, CD11c+ B cells resided mainly within memory subsets and were enriched in CD27-IgD-, CD21-CD27-, and CD21-CD38- B cell phenotypes. CD11c+ B cells from all donor groups (SLE, pSS, and HD) showed enhanced CD69, Ki-67, CD45RO, CD45RA, and CD19 expression, whereas the membrane expression of CXCR5 and CD21 were diminished. Notably, SLE CD11c+ B cells showed enhanced expression of the checkpoint molecules CD86, PD1, PDL1, CD137, VISTA, and CTLA-4 compared to HD. The substantial increase of CD11c+ B cells with a CD21- phenotype co-expressing distinct activation and checkpoint markers, points to a quantitative increased alternate (extrafollicular) B cell activation route possibly related to abnormal immune regulation as seen under the striking inflammatory conditions of SLE which shows a characteristic PD-1/PD-L1 upregulation.

Atypical B cells are part of an alternative lineage of B cells that participates in responses to vaccination and infection in humans.

The diversity of circulating human B cells is unknown. We use single-cell RNA sequencing (RNA-seq) to examine the diversity of both antigen-specific and total B cells in healthy subjects and malaria-exposed individuals. This reveals two B cell lineages: a classical lineage of activated and resting memory B cells and an alternative lineage, which includes previously described atypical B cells. Although atypical B cells have previously been associated with disease states, the alternative lineage is common in healthy controls, as well as malaria-exposed individuals. We further track Plasmodium-specific B cells after malaria vaccination in naive volunteers. We find that alternative lineage cells are primed after the initial immunization and respond to booster doses. However, alternative lineage cells develop an atypical phenotype with repeated boosts. The data highlight that atypical cells are part of a wider alternative lineage of B cells that are a normal component of healthy immune responses.

Autoimmune Anemia in Malaria.

Severe anemia is a major cause of death by malaria. The loss of uninfected erythrocytes is an important contributor to malarial anemia; however, the mechanisms underlying this pathology are not well understood. Malaria-induced anemia is related to autoimmune antibodies against the membrane lipid phosphatidylserine (PS). In mice, these antibodies induce the clearance of uninfected erythrocytes after binding to PS exposed in their membrane. In human malaria patients there is a strong correlation between anemia and anti-PS antibodies. During malaria, anti-PS antibodies are produced by atypical B cells, whose levels correlate with the development of anemia in patients. Autoimmune responses, which are documented frequently in different infections, contribute to the pathogenesis of malaria by inducing the clearance of uninfected erythrocytes.

Challenges and Opportunities for Consistent Classification of Human B Cell and Plasma Cell Populations.

The increasingly recognized role of different types of B cells and plasma cells in protective and pathogenic immune responses combined with technological advances have generated a plethora of information regarding the heterogeneity of this human immune compartment. Unfortunately, the lack of a consistent classification of human B cells also creates significant imprecision on the adjudication of different phenotypes to well-defined populations. Additional confusion in the field stems from: the use of non-discriminatory, overlapping markers to define some populations, the extrapolation of mouse concepts to humans, and the assignation of functional significance to populations often defined by insufficient surface markers. In this review, we shall discuss the current understanding of human B cell heterogeneity and define major parental populations and associated subsets while discussing their functional significance. We shall also identify current challenges and opportunities. It stands to reason that a unified approach will not only permit comparison of separate studies but also improve our ability to define deviations from normative values and to create a clean framework for the identification, functional significance, and disease association with new populations.