Germinal centers (GCs) are the primary sites of clonal B cell expansion and affinity maturation, directing the production of high-affinity antibodies. This response is a central driver of pathogenesis in autoimmune diseases, such as systemic lupus erythematosus (SLE), but the natural history of autoreactive GCs remains unclear. Here, we present a novel mouse model where the presence of a single autoreactive B cell clone drives the TLR7-dependent activation, expansion, and differentiation of other autoreactive B cells in spontaneous GCs. Once tolerance was broken for one self-antigen, autoreactive GCs generated B cells targeting other self-antigens. GCs became independent of the initial clone and evolved toward dominance of individual clonal lineages, indicating affinity maturation. This process produced serum autoantibodies to a breadth of self-antigens, leading to antibody deposition in the kidneys. Our data provide insight into the maturation of the self-reactive B cell response, contextualizing the epitope spreading observed in autoimmune disease.

During somatic hypermutation (SHM) of immunoglobulin genes, uracils introduced by activation-induced cytidine deaminase are processed by uracil-DNA glycosylase (UNG) and mismatch repair (MMR) pathways to generate mutations at G-C and A-T base pairs, respectively. Paradoxically, the MMR-nicking complex Pms2/Mlh1 is apparently dispensable for A-T mutagenesis. Thus, how detection of U:G mismatches is translated into the single-strand nick required for error-prone synthesis is an open question. One model proposed that UNG could cooperate with MMR by excising a second uracil in the vicinity of the U:G mismatch, but it failed to explain the low impact of UNG inactivation on A-T mutagenesis. In this study, we show that uracils generated in the G1 phase in B cells can generate equal proportions of A-T and G-C mutations, which suggests that UNG and MMR can operate within the same time frame during SHM. Furthermore, we show that Ung-/-Pms2-/- mice display a 50% reduction in mutations at A-T base pairs and that most remaining mutations at A-T bases depend on two additional uracil glycosylases, thymine-DNA glycosylase and SMUG1. These results demonstrate that Pms2/Mlh1 and multiple uracil glycosylases act jointly, each one with a distinct strand bias, to enlarge the immunoglobulin gene mutation spectrum from G-C to A-T bases.

Inborn and acquired deficits of type I interferon (IFN) immunity predispose to life-threatening COVID-19 pneumonia. We longitudinally profiled the B cell response to mRNA vaccination in SARS-CoV-2 naive patients with inherited TLR7, IRF7, or IFNAR1 deficiency, as well as young patients with autoantibodies neutralizing type I IFNs due to autoimmune polyendocrine syndrome type-1 (APS-1) and older individuals with age-associated autoantibodies to type I IFNs. The receptor-binding domain spike protein (RBD)-specific memory B cell response in all patients was quantitatively and qualitatively similar to healthy donors. Sustained germinal center responses led to accumulation of somatic hypermutations in immunoglobulin heavy chain genes. The amplitude and duration of, and viral neutralization by, RBD-specific IgG serological response were also largely unaffected by TLR7, IRF7, or IFNAR1 deficiencies up to 7 mo after vaccination in all patients. These results suggest that induction of type I IFN is not required for efficient generation of a humoral response against SARS-CoV-2 by mRNA vaccines.

ÍSince the introduction of new generation pertussis vaccines, resurgence of pertussis has been observed in many developed countries. Former whole-cell pertussis (wP) vaccines are able to protect against disease and transmission but have been replaced in several industrialized countries because of their reactogenicity and adverse effects. Current acellular pertussis (aP) vaccines, made of purified proteins of Bordetella pertussis, are efficient at preventing disease but fail to induce long-term protection from infection. While the systemic and mucosal T cell immunity induced by the 2 types of vaccines has been well described, much less is known concerning B cell responses. Taking advantage of an inducible activation-induced cytidine deaminase fate-mapping mouse model, we compared effector and memory B cells induced by the 2 classes of vaccines and showed that a stronger and broader memory B cell and plasma cell response was achieved by a wP prime. We also observed that homologous or heterologous vaccine combinations that include at least 1 wP administration, even as a booster dose, were sufficient to induce this broad effector response, thus highlighting its dominant imprint on the B cell profile. Finally, we describe the settlement of memory B cell populations in the lung following subcutaneous wP prime vaccination.

The SARS-CoV-2 Omicron variant can escape neutralization by vaccine-elicited and convalescent antibodies. Memory B cells (MBCs) represent another layer of protection against SARS-CoV-2, as they persist after infection and vaccination and improve their affinity. Whether MBCs elicited by mRNA vaccines can recognize the Omicron variant remains unclear. We assessed the affinity and neutralization potency against the Omicron variant of several hundred naturally expressed MBC-derived monoclonal IgG antibodies from vaccinated COVID-19-recovered and -naive individuals. Compared with other variants of concern, Omicron evaded recognition by a larger proportion of MBC-derived antibodies, with only 30% retaining high affinity against the Omicron RBD, and the reduction in neutralization potency was even more pronounced. Nonetheless, neutralizing MBC clones could be found in all the analyzed individuals. Therefore, despite the strong immune escape potential of the Omicron variant, these results suggest that the MBC repertoire generated by mRNA vaccines still provides some protection against the Omicron variant in vaccinated individuals.

Memory B cells (MBCs) can persist for a lifetime, but the mechanisms that allow their long-term survival remain poorly understood. Here, we isolated and analyzed human splenic smallpox/vaccinia protein B5-specific MBCs in individuals who were vaccinated more than 40 years ago. Only a handful of clones persisted over such an extended period, and they displayed limited intra-clonal diversity with signs of extensive affinity-based selection. These long-lived MBCs appeared enriched in a CD21hiCD20hi IgG+ splenic B cell subset displaying a marginal-zone-like NOTCH/MYC-driven signature, but they did not harbor a unique longevity-associated transcriptional or metabolic profile. Finally, the telomeres of B5-specific, long-lived MBCs were longer than those in patient-paired naive B cells in all the samples analyzed. Overall, these results imply that separate mechanisms such as early telomere elongation, affinity selection during the contraction phase, and access to a specific niche contribute to ensuring the functional longevity of MBCs.

DNA polymerase mu (pol mu) is a template-dependent polymerase closely related to the lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT). We report here the phenotype of pol mu-deficient mice. Such animals display an abnormal B cell differentiation, with a specific alteration in the IgM- to IgM+ transition in bone marrow. In all mice, Ig light chain gene rearrangement is impaired at the level of the Vkappa-Jkappa and Vlambda-Jlambda junctions, which show extensive nibbling of both coding extremities. These alterations lead to a profound defect in the peripheral B cell compartment which, although variable between animals, results in an average 40% reduction in the splenic B cell fraction. Pol mu appears, therefore, as a key element contributing to the relative homogeneity in size of light chain CDR3 and taking part in Ig gene rearrangement at a stage where TdT is no longer expressed.

MBCs (MBCs) generated in T-dependent immune responses can persist for a lifetime and rapidly react upon secondary antigen exposure to differentiate into plasma cells (PCs) and/or to improve the affinity of their BCR through new rounds of hypermutation in germinal centers (GCs). The fate of a MBC in secondary immune reactions appears to depend upon multiple parameters, whose understanding is mandatory for the design of efficient vaccine strategies. We followed the behavior of MBCs in recall responses to SRBCs using an inducible AID fate mapping mouse model in which B cells engaged in a germinal center (GC) response are irreversibly labeled upon simultaneous tamoxifen ingestion and immunization. We used different schemes of mouse immunization and tamoxifen feeding in adoptive-transfer experiments of total splenic B cells into congenic mice that have been pre-immunized or not, to assess the contribution of the different effector subsets in a physiological competitive context. We were able to show that naive B cells can differentiate into GC B cells with kinetics similar to MBCs in the presence of previously activated T follicular helper (TFH) cells and a primed microenvironment. We also showed that MBCs are recruited into secondary GCs, together with naive B cells. In contrast, PC differentiation, which dominated secondary MBC responses, was not dependent upon a previous TFH activation. We observed that the presence of persisting germinal centers and circulating antibody levels are key factors determining the germinal center versus plasma cell fate in a recall response. Notably, disruption of persistent germinal center structures by a lymphotoxin beta-receptor fusion protein or a longer timing between the prime and the boost, which correlated with reduced antigen-specific immunoglobulin levels in serum, were two conditions with an opposite impact, respectively inhibiting or promoting a GC fate for MBCs. Altogether, these studies highlight the complexity of recall responses, whose outcome varies according to immunization contexts.

The mutation pattern of immunoglobulin genes was studied in mice deficient for DNA polymerase eta, a translesional polymerase whose inactivation is responsible for the xeroderma pigmentosum variant (XP-V) syndrome in humans. Mutations show an 85% G/C biased pattern, similar to that reported for XP-V patients. Breeding these mice with animals harboring the stop codon mutation of the 129/Olain background in their DNA polymerase iota gene did not alter this pattern further. Although this G/C biased mutation profile resembles that of mice deficient in the MSH2 or MSH6 components of the mismatch repair complex, the residual A/T mutagenesis of pol eta-deficient mice differs markedly. This suggests that, in the absence of pol eta, the MSH2-MSH6 complex is able to recruit another DNA polymerase that is more accurate at copying A/T bases, possibly pol kappa, to assume its function in hypermutation.

Mouse splenic marginal zone precursors (MZPs) differentiate into marginal zone B (MZB) cells under a signaling pathway involving Notch2 and its ligand, delta-like 1 ligand (Dll1). We report the identification of an MZP subset in the spleen of young children. These MZPs differentiate into MZ-like B cells in vitro in the presence of OP9 cells expressing human DLL1, as demonstrated by the up-regulation of classical MZB cell markers. A set of diagnostic genes discriminating IgM(+)IgD(+)CD27(+) blood and splenic MZB cells from switched B cells was identified (up-regulation of SOX7, down-regulation of TOX, COCH, and HOPX), and their expression during the induction assay mirrored the one of MZB cells. Moreover, Alagille patients with a NOTCH2 haploinsufficiency display a marked reduction of IgM(+)IgD(+)CD27(+) B cells in blood, whereas their switched memory B cells are not affected. Altogether, these results argue in favor of the existence of a rodent-like MZB cell lineage in humans.

Persistent viral infections subvert key elements of adaptive immunity. To compare germinal center (GC) B cell responses in chronic and acute lymphocytic choriomeningitis virus infection, we exploit activation-induced deaminase (AID) fate-reporter mice and perform adoptive B cell transfer experiments. Chronic infection yields GC B cell responses of higher cellularity than acute infections do, higher memory B cell and antibody secreting cell output for longer periods of time, a better representation of the late B cell repertoire in serum immunoglobulin, and higher titers of protective neutralizing antibodies. GC B cells of chronically infected mice are similarly hypermutated as those emerging from acute infection. They efficiently adapt to viral escape variants and even in hypermutation-impaired AID mutant mice, chronic infection selects for GC B cells with hypermutated B cell receptors (BCRs) and neutralizing antibody formation. These findings demonstrate that, unlike for CD8+ T cells, chronic viral infection drives a functional, productive, and protective GC B cell response.

How infection by a viral variant showing antigenic drift impacts a preformed mature human memory B cell (MBC) repertoire remains an open question. Here, we studied the MBC response up to 6 months after SARS-CoV-2 Omicron BA.1 breakthrough infection in individuals previously vaccinated with three doses of the COVID-19 mRNA vaccine. Longitudinal analysis, using single-cell multi-omics and functional analysis of monoclonal antibodies from RBD-specific MBCs, revealed that a BA.1 breakthrough infection mostly recruited pre-existing cross-reactive MBCs with limited de novo response against BA.1-restricted epitopes. Reorganization of clonal hierarchy and new rounds of germinal center reactions, however, combined to maintain diversity and induce progressive maturation of the MBC repertoire against common Hu-1 and BA.1, but not BA.5-restricted, SARS-CoV-2 Spike RBD epitopes. Such remodeling was further associated with a marked improvement in overall neutralizing breadth and potency. These findings have fundamental implications for the design of future vaccination booster strategies.

T cell-dependent immune responses develop soon after birth, whereas it takes 2 yr for humans to develop T cell-independent responses. We used this dissociation to analyze the repertoire diversification of IgM(+)IgD(+)CD27(+) B cells (also known as "IgM memory" B cells), comparing these cells with switched B cells in children <2 yr of age, with the aim of determining whether these two subsets are developmentally related. We show that the repertoire of IgM(+)IgD(+)CD27(+) B cells in the spleen and blood displays no sign of antigen-driven activation and expansion on H-CDR3 spectratyping, despite the many antigenic challenges provided by childhood vaccinations. This repertoire differed markedly from those of switched B cells and splenic germinal center B cells, even at the early stage of differentiation associated with mu heavy chain expression. These data provide evidence for the developmental diversification of IgM(+)IgD(+)CD27(+) B cells, at least in very young children, outside of T cell-dependent and -independent immune responses.

B lymphocytes can suppress immunity through interleukin (IL)-10 production in infectious, autoimmune, and malignant diseases. Here, we have identified a natural plasma cell subset that distinctively expresses the inhibitory receptor LAG-3 and mediates this function in vivo. These plasma cells also express the inhibitory receptors CD200, PD-L1, and PD-L2. They develop from various B cell subsets in a B cell receptor (BCR)-dependent manner independently of microbiota in naive mice. After challenge they upregulate IL-10 expression via a Toll-like receptor-driven mechanism within hours and without proliferating. This function is associated with a unique transcriptome and epigenome, including the lowest amount of DNA methylation at the Il10 locus compared to other B cell subsets. Their augmented accumulation in naive mutant mice with increased BCR signaling correlates with the inhibition of memory T cell formation and vaccine efficacy after challenge. These natural regulatory plasma cells may be of broad relevance for disease intervention.

To what extent immune responses against the gut flora are compartmentalized within mucosal tissues in homeostatic conditions remains a much-debated issue. We describe here, based on an inducible AID fate-mapping mouse model, that systemic memory B cell subsets, including mainly IgM+ B cells in spleen, together with IgA+ plasma cells in spleen and bone marrow, are generated in mice in the absence of deliberate immunization. While the IgA component appears dependent on the gut flora, IgM memory B cells are still generated in germ-free mice, albeit to a reduced extent. Clonal relationships and renewal kinetics after anti-CD20 treatment reveal that this long-lasting splenic population is mainly sustained by output of B cell clones persisting in mucosal germinal centers. IgM-secreting hybridomas established from splenic IgM memory B cells showed reactivity against various bacterial isolates and endogenous retroviruses. Ongoing activation of B cells in gut-associated lymphoid tissues thus generates a diversified systemic compartment showing long-lasting clonal persistence and protective capacity against systemic bacterial infections.

Activation-induced cytidine deaminase (AID) initiates all postrearrangement processes that diversify the immunoglobulin repertoire by specific deamination of cytidines at the immunoglobulin (Ig) locus. As uncontrolled expression of AID is potentially mutagenic, different types of regulation, particularly nucleocytoplasmic shuttling, restrict the likelihood of AID-deoxyribonucleic acid encounters. We studied additional mechanisms of regulation affecting the stability of the AID protein. No modulation of protein accumulation according to the cell cycle was observed in a Burkitt's lymphoma cell line. In contrast, the half-life of AID was markedly reduced in the nucleus, and this destabilization was accompanied by a polyubiquitination that was revealed in the presence of proteasome inhibitors. The same compartment-specific degradation was observed in activated mouse B cells, and also in a non-B cell line. No specific lysine residues could be linked to this degradation, so it remains unclear whether polyubiquitination proceeds through several alternatives sites or through the protein N terminus. The nuclear-restricted form of AID displayed enhanced mutagenicity at both Ig and non-Ig loci, most notably at TP53, suggesting that modulation of nuclear AID content through proteasomal degradation may represent another level of control of AID activity.

Memory B cells play a fundamental role in host defenses against viruses, but to date, their role has been relatively unsettled in the context of SARS-CoV-2. We report here a longitudinal single-cell and repertoire profiling of the B cell response up to 6 months in mild and severe COVID-19 patients. Distinct SARS-CoV-2 spike-specific activated B cell clones fueled an early antibody-secreting cell burst as well as a durable synchronous germinal center response. While highly mutated memory B cells, including pre-existing cross-reactive seasonal Betacoronavirus-specific clones, were recruited early in the response, neutralizing SARS-CoV-2 RBD-specific clones accumulated with time and largely contributed to the late, remarkably stable, memory B cell pool. Highlighting germinal center maturation, these cells displayed clear accumulation of somatic mutations in their variable region genes over time. Overall, these findings demonstrate that an antigen-driven activation persisted and matured up to 6 months after SARS-CoV-2 infection and may provide long-term protection.

In addition to serum immunoglobulins, memory B cell (MBC) generation against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is another layer of immune protection, but the quality of MBC responses in naive and coronavirus disease 2019 (COVID-19)-recovered individuals after vaccination remains ill defined. We studied longitudinal cohorts of naive and disease-recovered individuals for up to 2 months after SARS-CoV-2 mRNA vaccination. We assessed the quality of the memory response by analysis of antibody repertoires, affinity, and neutralization against variants of concern (VOCs) using unbiased cultures of 2,452 MBCs. Upon boosting, the MBC pool of recovered individuals expanded selectively, matured further, and harbored potent neutralizers against VOCs. Although naive individuals had weaker neutralizing serum responses, half of their RBD-specific MBCs displayed high affinity toward multiple VOCs, including delta (B.1.617.2), and one-third retained neutralizing potency against beta (B.1.351). Our data suggest that an additional challenge in naive vaccinees could recall such affinity-matured MBCs and allow them to respond efficiently to VOCs.

The signals controlling marginal zone (MZ) and follicular (FO) B cell development remain incompletely understood. Here, we show that AKT orchestrates MZ B cell formation in mice and humans. Genetic models that increase AKT signaling in B cells or abolish its impact on FoxO transcription factors highlight the AKT-FoxO axis as an on-off switch for MZ B cell formation in mice. In humans, splenic immunoglobulin (Ig) D+CD27+ B cells, proposed as an MZ B cell equivalent, display higher AKT signaling than naive IgD+CD27- and memory IgD-CD27+ B cells and develop in an AKT-dependent manner from their precursors in vitro, underlining the conservation of this developmental pathway. Consistently, CD148 is identified as a receptor indicative of the level of AKT signaling in B cells, expressed at a higher level in MZ B cells than FO B cells in mice as well as humans.

HELLS/LSH (Helicase, Lymphoid Specific) is a SNF2-like chromatin remodelling protein involved in DNA methylation. Its loss-of-function in humans causes humoral immunodeficiency, called ICF4 syndrome (Immunodeficiency, Centromeric Instability, Facial anomalies). Here we show by our newly generated B-cell-specific Hells conditional knockout mouse model that HELLS plays a pivotal role in T-dependent B-cell responses. HELLS deficiency induces accelerated decay of germinal center (GC) B cells and impairs the generation of high affinity memory B cells and circulating antibodies. Mutant GC B cells undergo dramatic DNA hypomethylation and massive de-repression of evolutionary recent retrotransposons, which surprisingly does not directly affect their survival. Instead, they prematurely upregulate either memory B cell markers or the transcription factor ATF4, which is driving an mTORC1-dependent metabolic program typical of plasma cells. Treatment of wild type mice with a DNMT1-specific inhibitor phenocopies the accelerated kinetics, thus pointing towards DNA-methylation maintenance by HELLS being a crucial mechanism to fine-tune the GC transcriptional program and enable long-lasting humoral immunity.