Immunization with a peptide mimicking lipoteichoic acid induces memory B cells in BALB/c mice.

BACKGROUND: There is an urgent clinical need for developing novel immunoprophylaxis and immunotherapy strategies against Staphylococcus aureus (S. aureus). In our previous work, immunization with a tetra-branched multiple antigenic peptide, named MAP2-3 that mimics lipoteichoic acid, a cell wall component of S. aureus, successfully induced a humoral immune response and protected BALB/c mice against S. aureus systemic infection. In this study, we further investigated whether vaccination with MAP2-3 can elicit immunologic memory. METHODS: BALB/c mice were immunized with MAP2-3 five times. After one month of the last vaccination, mice were challenged with heat-killed S. aureus via intraperitoneal injection. After a 7-day inoculation, the percentage of plasma cells, memory B cells, effector memory T cells, and follicular helper T cells were detected by flow cytometry. The levels of IL-6, IL-21, IL-2, and IFN-γ were measured by real-time PCR and ELISA. Flow cytometry results were compared by using one-way ANOVA or Mann-Whitney test, real-time PCR results were compared by using one-way ANOVA, and ELISA results were compared by using one-way ANOVA or student's t-test. RESULTS: The percentage of plasma cells and memory B cells in the spleen and bone marrow from the MAP2-3 immunized mice was significantly higher than that from the control mice. The percentage of effector memory T cells in spleens and lymphoid nodes as well as follicular helper T cells in spleens from the MAP2-3 immunized mice were also higher. Moreover, the levels of IL-6 and IL-21, two critical cytokines for the development of memory B cells, were significantly higher in the isolated splenocytes from immunized mice after lipoteichoic acid stimulation. CONCLUSIONS: Immunization with MAP2-3 can efficiently induce memory B cells and memory T cells.

AS03 adjuvant enhances the magnitude, persistence, and clonal breadth of memory B cell responses to a plant-based COVID-19 vaccine in humans.

Vaccine adjuvants increase the breadth of serum antibody responses, but whether this is due to the generation of antigen-specific B cell clones with distinct specificities or the maturation of memory B cell clones that produce broadly cross-reactive antibodies is unknown. Here, we longitudinally analyzed immune responses in healthy adults after two-dose vaccination with either a virus-like particle COVID-19 vaccine (CoVLP), CoVLP adjuvanted with AS03 (CoVLP+AS03), or a messenger RNA vaccination (mRNA-1273). CoVLP+AS03 enhanced the magnitude and durability of circulating antibodies and antigen-specific CD4+ T cell and memory B cell responses. Antigen-specific CD4+ T cells in the CoVLP+AS03 group at day 42 correlated with antigen-specific memory B cells at 6 months. CoVLP+AS03 induced memory B cell responses, which accumulated somatic hypermutations over 6 months, resulting in enhanced neutralization breadth of monoclonal antibodies. Furthermore, the fraction of broadly neutralizing antibodies encoded by memory B cells increased between day 42 and 6 months. These results indicate that AS03 enhances the antigenic breadth of B cell memory at the clonal level and induces progressive maturation of the B cell response.

CD4 T Cell-Derived IL-21 Is Critical for Sustaining Plasmodium Infection-Induced Germinal Center Responses and Promoting the Selection of Memory B Cells with Recall Potential.

Development of Plasmodium-specific humoral immunity is critically dependent on CD4 Th cell responses and germinal center (GC) reactions during blood-stage Plasmodium infection. IL-21, a cytokine primarily produced by CD4 T cells, is an essential regulator of affinity maturation, isotype class-switching, B cell differentiation, and maintenance of GC reactions in response to many infection and immunization models. In models of experimental malaria, mice deficient in IL-21 or its receptor IL-21R fail to develop memory B cell populations and are not protected against secondary infection. However, whether sustained IL-21 signaling in ongoing GCs is required for maintaining GC magnitude, organization, and output is unclear. In this study, we report that CD4+ Th cells maintain IL-21 expression after resolution of primary Plasmodium yoelii infection. We generated an inducible knockout mouse model that enabled cell type-specific and timed deletion of IL-21 in peripheral, mature CD4 T cells. We found that persistence of IL-21 signaling in active GCs had no impact on the magnitude of GC reactions or their capacity to produce memory B cell populations. However, the memory B cells generated in the absence of IL-21 exhibited reduced recall function upon challenge. Our data support that IL-21 prevents premature cellular dissolution within the GC and promotes stringency of selective pressures during B cell fate determination required to produce high-quality Plasmodium-specific memory B cells. These data are additionally consistent with a temporal requirement for IL-21 in fine-tuning humoral immune memory responses during experimental malaria.

ARID1A orchestrates SWI/SNF-mediated sequential binding of transcription factors with ARID1A loss driving pre-memory B cell fate and lymphomagenesis.

ARID1A, a subunit of the canonical BAF nucleosome remodeling complex, is commonly mutated in lymphomas. We show that ARID1A orchestrates B cell fate during the germinal center (GC) response, facilitating cooperative and sequential binding of PU.1 and NF-kB at crucial genes for cytokine and CD40 signaling. The absence of ARID1A tilts GC cell fate toward immature IgM+CD80-PD-L2- memory B cells, known for their potential to re-enter new GCs. When combined with BCL2 oncogene, ARID1A haploinsufficiency hastens the progression of aggressive follicular lymphomas (FLs) in mice. Patients with FL with ARID1A-inactivating mutations preferentially display an immature memory B cell-like state with increased transformation risk to aggressive disease. These observations offer mechanistic understanding into the emergence of both indolent and aggressive ARID1A-mutant lymphomas through the formation of immature memory-like clonal precursors. Lastly, we demonstrate that ARID1A mutation induces synthetic lethality to SMARCA2/4 inhibition, paving the way for potential precision therapy for high-risk patients.

Frequency-potency analysis of IgG+ memory B cells delineates neutralizing antibody responses at single-cell resolution.

Identifying individual functional B cell receptors (BCRs) is common, but two-dimensional analysis of B cell frequency versus BCR potency would delineate both quantity and quality of antigen-specific memory B cells. We efficiently determine quantitative BCR neutralizing activities using a single-cell-derived antibody supernatant analysis (SCAN) workflow and develop a frequency-potency algorithm to estimate B cell frequencies at various neutralizing activity or binding affinity cutoffs. In an HIV-1 fusion peptide (FP) immunization study, frequency-potency curves elucidate the quantity and quality of FP-specific immunoglobulin G (IgG)+ memory B cells for different animals, time points, and antibody lineages at single-cell resolution. The BCR neutralizing activities are mainly determined by their affinities to soluble envelope trimer. Frequency analysis definitively demonstrates dominant neutralizing antibody lineages. These findings establish SCAN and frequency-potency analyses as promising approaches for general B cell analysis and monoclonal antibody (mAb) discovery. They also provide specific rationales for HIV-1 FP-directed vaccine optimization.

Frequencies of SARS-CoV-2 Spike Protein-Specific Memory B Cells in Human PBMCs, Quantified by ELISPOT Assay.

Vaccination against SARS-CoV-2 with coronavirus vaccines that elicit protective immune responses is critical to the prevention of severe disease and mortality associated with SARS-CoV-2 infection. Understanding the adaptive immune responses to SARS-CoV-2 infection and/or vaccination will continue to aid in the development of next-generation vaccines. Studies have shown the important role of SARS-CoV-2-specific antibodies for both disease resolution and prevention of COVID-19 serious sequelae following vaccination. However, antibody responses are short-lived, highlighting the importance of studying antigen-specific B-cell responses to better understand durable immunity and immunologic memory. Since the spike protein is the main target of antibody-producing B cells, we developed a SARS-CoV-2 memory B cell ELISPOT assay to measure the frequencies of spike-specific B cells after COVID-19 infection and/or vaccination. Here, we describe in detail the methodology for using this ELISPOT assay to quantify SARS-CoV-2 spike-specific memory B cells produced by infection and/or vaccination in human PBMC samples. Application of this assay may help better understand and predict SARS-CoV-2 recall immune responses and to develop potential B cell correlates of protection at the methodological level.

Monitoring Memory B Cells by Next-Generation ImmunoSpot® Provides Insights into Humoral Immunity that Measurements of Circulating Antibodies Do Not Reveal.

Memory B cells (Bmem) provide the second wall of adaptive humoral host defense upon specific antigen rechallenge when the first wall, consisting of preformed antibodies originating from a preceding antibody response, fails. This is the case, as recently experienced with SARS-CoV-2 infections and previously with seasonal influenza, when levels of neutralizing antibodies decline or when variant viruses arise that evade such. While in these instances, reinfection can occur, in both scenarios, the rapid engagement of preexisting Bmem into the recall response can still confer immune protection. Bmem are known to play a critical role in host defense, yet their assessment has not become part of the standard immune monitoring repertoire. Here we describe a new generation of B cell ELISPOT/FluoroSpot (collectively ImmunoSpot®) approaches suited to dissect, at single-cell resolution, the Bmem repertoire ex vivo, revealing its immunoglobulin class/subclass utilization, and its affinity distribution for the original, and for variant viruses/antigens. Because such comprehensive B cell ImmunoSpot® tests can be performed with minimal cell material, are scalable, and robust, they promise to be well-suited for routine immune monitoring.

Tracking Circulating HLA-Specific IgG-Producing Memory B Cells with the B-Cell ImmunoSpot Assay.

Donor-specific antibodies (DSA) against human leukocyte antigen (HLA) molecules are a major risk factor for rejection of transplanted organs (in antibody-mediated rejection [ABMR]), particularly in patients who have prior sensitization or receive insufficient immunosuppression through minimization or noncompliance. These DSA are measured routinely in the serum of patients prior to transplantation mainly using bead-based technologies or cell-based assays. However, the absence of detectable serum DSA does not always reflect the absence of sensitization or histologically defined ABMR, and so it has been proposed that the detection and measurement of memory B cells capable of secreting antibodies against donor HLA antigens could be carried out using B-cell ImmunoSpot, to better inform the degree of immune sensitization of transplant patients prior to as well as after transplantation. Such an assay is described here.

B cells on the brink.

Type 2-polarized memory B cells sustain food allergy and allergic rhinitis by rapidly differentiating into pathogenic IgE-producing plasma cells.

Interleukin-4 downregulates transcription factor BCL6 to promote memory B cell selection in germinal centers.

Germinal center (GC)-derived memory B cells (MBCs) are critical for humoral immunity as they differentiate into protective antibody-secreting cells during re-infection. GC formation and cellular interactions within the GC have been studied in detail, yet the exact signals that allow for the selection and exit of MBCs are not understood. Here, we showed that IL-4 cytokine signaling in GC B cells directly downregulated the transcription factor BCL6 via negative autoregulation to release cells from the GC program and to promote MBC formation. This selection event required additional survival cues and could therefore result in either GC exit or death. We demonstrate that both increasing IL-4 bioavailability or limiting IL-4 signaling disrupted MBC selection stringency. In this way, IL-4 control of BCL6 expression serves as a tunable switch within the GC to tightly regulate MBC selection and affinity maturation.

Twin study dissects CXCR3+ memory B cells as non-heritable feature in multiple sclerosis.

BACKGROUND: In multiple sclerosis (MS), B cells are considered main triggers of the disease, likely as the result of complex interaction between genetic and environmental risk factors. Studies on monozygotic twins discordant for MS offer a unique way to reduce this complexity and reveal discrepant subsets. METHODS: In this study, we analyzed B cell subsets in blood samples of monozygotic twins with and without MS using publicly available data. We verified functional characteristics by exploring the role of therapy and performed separate analyses in unrelated individuals. FINDINGS: The frequencies of CXCR3+ memory B cells were reduced in the blood of genetically identical twins with MS compared to their unaffected twin siblings. Natalizumab (anti-VLA-4 antibody) was the only treatment regimen under which these frequencies were reversed. The CNS-homing features of CXCR3+ memory B cells were supported by elevated CXCL10 levels in MS cerebrospinal fluid and their in vitro propensity to develop into antibody-secreting cells. CONCLUSIONS: Circulating CXCR3+ memory B cells are affected by non-heritable cues in people who develop MS. This underlines the requirement of environmental risk factors such as Epstein-Barr virus in triggering these B cells. We propose that after CXCL10-mediated entry into the CNS, CXCR3+ memory B cells mature into antibody-secreting cells to drive MS. FUNDING: This work was supported by Nationaal MS Fonds (OZ2021-016), Stichting MS Research (19-1057 MS, 20-490f MS, and 21-1142 MS), the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program grant agreement no. 882424, and the Swiss National Science Foundation (733 310030_170320, 310030_188450, and CRSII5_183478).

Migration Inhibition Factor Secreted by Peripheral Blood Memory B Cells Binding to CD74-CD44 Receptor Complex Drives Macrophage Behavior in Alzheimer's Disease.

Dysregulation of the peripheral immune system is be involved in the neuroinflammation in Alzheimer disease (AD) and accelerate the disease progression. The contribution of immune cells, particularly B cells, to AD pathogenesis has gained attention in recent research. In this study, we investigated the role of Peripheral Blood Memory B cells (PBMBs) and their secreted Migration Inhibition Factor (MIF) in driving macrophage behavior in AD based on the scRNA-seq technique, immunofluorescence and flow cytometry. We discovered that MIF binds to the CD74-CD44 receptor complex on macrophages, influencing their behavior. The dysregulated macrophage response hampers the clearance of amyloid-beta (Aß) plaques, exacerbating AD pathology. Targeting the MIF-CD74-CD44 signal pathway may hold therapeutic potential in modulating macrophage activity and mitigating neuroinflammation in AD. This study provides a further understanding of peripheral immune cells dysregulated in AD.

Antiviral memory B cells exhibit enhanced innate immune response facilitated by epigenetic memory.

The long-lasting humoral immunity induced by viral infections or vaccinations depends on memory B cells with greatly increased affinity to viral antigens, which are evolved from germinal center (GC) responses. However, it is unclear whether antiviral memory B cells represent a distinct subset among the highly heterogeneous memory B cell population. Here, we examined memory B cells induced by a virus-mimicking antigen at both transcriptome and epigenetic levels and found unexpectedly that antiviral memory B cells exhibit an enhanced innate immune response, which appeared to be facilitated by the epigenetic memory that is established through the memory B cell development. In addition, T-bet is associated with the altered chromatin architecture and is required for the formation of the antiviral memory B cells. Thus, antiviral memory B cells are distinct from other GC-derived memory B cells in both physiological functions and epigenetic landmarks.

Isotype switching in human memory B cells sets intrinsic antigen-affinity thresholds that dictate antigen-driven fates.

Memory B cells (MBCs) play a critical role in protection against homologous and variant pathogen challenge by either differentiating to plasma cells (PCs) or to germinal center (GC) B cells. The human MBC compartment contains both switched IgG+ and unswitched IgM+ MBCs; however, whether these MBC subpopulations are equivalent in their response to B cell receptor cross-linking and their resulting fates is incompletely understood. Here, we show that IgG+ and IgM+ MBCs can be distinguished based on their response to κ-specific monoclonal antibodies of differing affinities. IgG+ MBCs responded only to high-affinity anti-κ and differentiated almost exclusively toward PC fates. In contrast, IgM+ MBCs were eliminated by apoptosis by high-affinity anti-κ but responded to low-affinity anti-κ by differentiating toward GC B cell fates. These results suggest that IgG+ and IgM+ MBCs may play distinct yet complementary roles in response to pathogen challenge ensuring the immediate production of high-affinity antibodies to homologous and closely related challenges and the generation of variant-specific MBCs through GC reactions.

Atypical memory B cell frequency correlates with antibody breadth and function in malaria immune adults.

Clinical immunity to malaria develops slowly after repeated episodes of infection and antibodies are essential in naturally acquired immunity against malaria. However, chronic exposure to malaria has been linked to perturbation in B-cell homeostasis with the accumulation of atypical memory B cells. It is unclear how perturbations in B cell subsets influence antibody breadth, avidity, and function in individuals naturally exposed to malaria. We show that individuals living in high malaria transmission regions in Ghana have higher Plasmodium falciparum merozoite antigen-specific antibodies and an increased antibody breadth score but lower antibody avidities relative to low transmission regions. The frequency of circulating atypical memory B cells is positively associated with an individual's antibody breadth. In vitro growth inhibition is independent of the ability to bind to free merozoites but associated with the breadth of antibody reactivity in an individual. Taken together, our data shows that repeated malaria episodes hamper the development of high avid antibodies which is compensated for by an increase in antibody breadth. Our results provide evidence to reinforce the idea that in regions with high malaria prevalence, repeated malaria infections lead to the broadening of antibody diversity and the continued presence of atypical memory B cell populations.

Type 2-polarized memory B cells hold allergen-specific IgE memory.

Allergen-specific immunoglobulin E (IgE) antibodies mediate pathology in diseases such as allergic rhinitis and food allergy. Memory B cells (MBCs) contribute to circulating IgE by regenerating IgE-producing plasma cells upon allergen encounter. Here, we report a population of type 2-polarized MBCs defined as CD23hi, IL-4Rαhi, and CD32low at both the transcriptional and surface protein levels. These MBC2s are enriched in IgG1- and IgG4-expressing cells while constitutively expressing germline transcripts for IgE. Allergen-specific B cells from patients with allergic rhinitis and food allergy were enriched in MBC2s. Furthermore, MBC2s generated allergen-specific IgE during sublingual immunotherapy, thereby identifying these cells as a major reservoir for IgE. The identification of MBC2s provides insights into the maintenance of IgE memory, which is detrimental in allergic diseases but could be beneficial in protection against venoms and helminths.

CD23+IgG1+ memory B cells are poised to switch to pathogenic IgE production in food allergy.

Food allergy is caused by allergen-specific immunoglobulin E (IgE) antibodies, but little is known about the B cell memory of persistent IgE responses. Here, we describe, in human pediatric peanut allergy, a population of CD23+IgG1+ memory B cells arising in type 2 immune responses that contain high-affinity peanut-specific clones and generate IgE-producing cells upon activation. The frequency of CD23+IgG1+ memory B cells correlated with circulating concentrations of IgE in children with peanut allergy. A corresponding population of "type 2-marked" IgG1+ memory B cells was identified in single-cell RNA sequencing experiments. These cells differentially expressed interleukin-4 (IL-4)- and IL-13-regulated genes, such as FCER2/CD23+, IL4R, and germline IGHE, and carried highly mutated B cell receptors (BCRs). In children with high concentrations of serum peanut-specific IgE, high-affinity B cells that bind the main peanut allergen Ara h 2 mapped to the population of "type 2-marked" IgG1+ memory B cells and included clones with convergent BCRs across different individuals. Our findings indicate that CD23+IgG1+ memory B cells transcribing germline IGHE are a unique memory population containing precursors of high-affinity pathogenic IgE-producing cells that are likely to be involved in the long-term persistence of peanut allergy.

SARS-CoV-2 spike-reactive naïve B cells and pre-existing memory B cells contribute to antibody responses in unexposed individuals after vaccination.

Introduction: Since December 2019, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) has presented considerable public health challenges. Multiple vaccines have been used to induce neutralizing antibodies (nAbs) and memory B-cell responses against the viral spike (S) glycoprotein, and many essential epitopes have been defined. Previous reports have identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-reactive naïve B cells and preexisting memory B cells in unexposed individuals. However, the role of these spike-reactive B cells in vaccine-induced immunity remains unknown. Methods: To elucidate the characteristics of preexisting SARS-CoV-2 S-reactive B cells as well as their maturation after antigen encounter, we assessed the relationship of spike-reactive B cells before and after vaccination in unexposed human individuals. We further characterized the sequence identity, targeting domain, broad-spectrum binding activity and neutralizing activity of these SARS-CoV-2 S-reactive B cells by isolating monoclonal antibodies (mAbs) from these B cells. Results: The frequencies of both spike-reactive naïve B cells and preexisting memory B cells before vaccination correlated with the frequencies of spike-reactive memory B cells after vaccination. Isolated mAbs from spike-reactive naïve B cells before vaccination had fewer somatic hypermutations (SHMs) than mAbs isolated from spike-reactive memory B cells before and after vaccination, but bound SARS-CoV-2 spike in vitro. Intriguingly, these germline-like mAbs possessed broad binding profiles for SARS-CoV-2 and its variants, although with low or no neutralizing capacity. According to tracking of the evolution of IGHV4-4/IGKV3-20 lineage antibodies from a single donor, the lineage underwent SHMs and developed increased binding activity after vaccination. Discussion: Our findings suggest that spike-reactive naïve B cells can be expanded and matured by vaccination and cocontribute to vaccine-elicited antibody responses with preexisting memory B cells. Selectively and precisely targeting spike-reactive B cells by rational antigen design may provide a novel strategy for next-generation SARS-CoV-2 vaccine development.

Aberrant B cell receptor signaling in circulating naïve and IgA+ memory B cells from newly-diagnosed autoantibody-positive rheumatoid arthritis patients.

OBJECTIVE: Altered B cell receptor (BCR) signaling has been implicated in the pathogenesis of rheumatoid arthritis (RA). Here we aimed to identify signaling aberrations in autoantibody-positive and autoantibody-negative RA patients by performing a comprehensive analysis of the BCR signaling cascade in different B cell subsets. METHODS: We first optimized phosphoflow cytometry for an in-depth analysis of BCR signaling across immunoglobulin isotypes in healthy donors. Subsequently, we compared BCR signaling in circulating B cell subsets from treatment-naïve, newly-diagnosed autoantibody-positive RA and autoantibody-negative RA patients and healthy controls (HCs). RESULTS: We observed subset-specific phosphorylation patterns of the BCR signalosome in circulating B cells from healthy donors. Compared with HCs, autoantibody-positive RA patients displayed enhanced responses to BCR stimulation for multiple signaling proteins, specifically in naïve and IgA+ memory B cells. Whereas in unstimulated healthy donor B cells, the phosphorylation status of individual signaling proteins showed only limited correlation, BCR stimulation enhanced the interconnectivity in phosphorylation within the BCR signalosome. However, this strong interconnectivity within the BCR signalosome in stimulated B cells from HCs was lost in RA, especially in autoantibody-positive RA patients. Finally, we observed strong correlations between SYK and BTK protein expression, and IgA and IgG anti-citrullinated protein antibody concentrations in serum from autoantibody-positive RA patients. CONCLUSION: Collectively, the isotype-specific analysis of multiple key components of the BCR signalosome identified aberrant BCR signaling responses in treatment-naïve autoantibody-positive RA patients, particularly in naïve B cells and IgA+ memory B cells. Our findings support differential involvement of dysregulated BCR signaling in the pathogenesis of autoantibody-positive and autoantibody-negative RA.

Memory B cell subsets have divergent developmental origins that are coupled to distinct imprinted epigenetic states.

Memory B cells (MBCs) are phenotypically and functionally diverse, but their developmental origins remain undefined. Murine MBCs can be divided into subsets by expression of CD80 and PD-L2. Upon re-immunization, CD80/PD-L2 double-negative (DN) MBCs spawn germinal center B cells (GCBCs), whereas CD80/PD-L2 double-positive (DP) MBCs generate plasmablasts but not GCBCs. Using multiple approaches, including generation of an inducible GCBC-lineage reporter mouse, we demonstrate in a T cell-dependent response that DN cells formed independently of the germinal center (GC), whereas DP cells exhibited either extrafollicular (DPEX) or GCBC (DPGC) origins. Chromatin and transcriptional profiling revealed similarity of DN cells with an early memory precursor. Reciprocally, GCBC-derived DP cells shared distinct genomic features with GCBCs, while DPEX cells had hybrid features. Upon restimulation, DPEX cells were more prone to divide, while DPGC cells differentiated toward IgG1+ plasmablasts. Thus, MBC functional diversity is generated through distinct developmental histories, which imprint characteristic epigenetic patterns onto their progeny, thereby programming them for divergent functional responses.