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Marginal zone (MZ) B cells are one of the main actors of T-independent (TI) responses in mice. To identify the B cell subset(s) involved in such responses in humans, we vaccinated healthy individuals with Pneumovax, a model TI vaccine. By high-throughput repertoire sequencing of plasma cells (PCs) isolated 7 days after vaccination and of different B cell subpopulations before and after vaccination, we show that the PC response mobilizes large clones systematically, including an immunoglobulin M component, whose diversification and amplification predated the pneumococcal vaccination. These clones could be mainly traced back to MZ B cells, together with clonally related IgA+ and, to a lesser extent, IgG+CD27+ B cells. Recombinant monoclonal antibodies isolated from large PC clones recognized a wide array of bacterial species from the gut flora, indicating that TI responses in humans largely mobilize MZ and switched B cells that most likely prediversified during mucosal immune responses against bacterial antigens and acquired pneumococcal cross-reactivity through somatic hypermutation. Tracing the origin of T-independent responses: T-independent (TI) B cell responses develop without T cell help and are mostly directed against repetitive structures such as surface polysaccharides derived from encapsulated bacteria. Using high throughput BCR repertoire sequencing, Weller et al. studied the B cell subsets contributing to human TI responses in healthy individuals vaccinated with the pneumococcal polysaccharide vaccine Pneumovax. The most expanded plasma cells were clonally related to previously mutated peripheral blood B cell precursors, which included marginal zone B cells and remained stable without acquiring further mutations up to 2 months after vaccination. Antibodies specific to bacterial capsular polysaccharides isolated from vaccine-elicited plasma cells cross-reacted with gut bacterial antigens, supporting a model in which human TI responses mobilize marginal zone B cells that are pre-diversified in gut-associated lymphoid tissues. —CO [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: This study was undertaken to characterize kidney and urine antibody‐secreting cells (ASCs) from patients with active lupus nephritis, before and after induction therapy. Methods: We included patients with biopsy‐proven active lupus nephritis and performed anti‐CD138 staining of kidney biopsy samples to visualize ASCs. We performed single‐cell gene expression profiling on sorted ASCs from fresh biopsy samples using multiplex reverse transcriptase–polymerase chain reaction. We used a gene set that allowed for the study of ASC maturation from plasmablasts to long‐lived plasma cells. We quantified urine ASCs from untreated patients with lupus nephritis at diagnosis and after 6 months of prospective follow‐up during induction therapy. Results: The number of kidney CD138+ ASCs in 46 untreated patients with lupus nephritis was correlated with a low estimated glomerular filtration rate and with tubulointerstitial damage. Most kidney ASCs from 3 untreated patients had a plasmablast molecular signature; in contrast, in 4 patients with refractory lupus nephritis, the kidney ASCs were mainly long‐lived plasma cells, representing an ASC transcriptional profile similar to that in the bone marrow of 2 healthy donors. Some urine ASCs with a plasmablast signature were detected in patients with untreated active lupus nephritis. The presence of urine ASCs at 6 months was associated with treatment failure. Conclusion: Our results suggest potential for ASC‐directed therapy in refractory lupus nephritis. [ABSTRACT FROM AUTHOR]

B cells behaving badly: Patients diagnosed with B cell–mediated autoimmune diseases, such as immune thrombocytopenia (ITP), can benefit from treatment with the B cell–depleting antibody, rituximab. Unfortunately, a large proportion of patients relapse after treatment. To understand why, Crickx et al. evaluated B cell phenotypes and specificity, finding two populations of B cells that may contribute to such relapses. The first consisted of a population of rituximab-resistant memory B cells, whereas the second consisted of a new population of autoreactive B cells never exposed to rituximab. Rituximab-resistant B cell populations maintained expression of the B cell marker CD19, suggesting that CD19-targeting therapies could provide another line of treatment for patients who do not respond to rituximab. Rituximab (RTX), an antibody targeting CD20, is widely used as a first-line therapeutic strategy in B cell–mediated autoimmune diseases. However, a large proportion of patients either do not respond to the treatment or relapse during B cell reconstitution. Here, we characterize the cellular basis responsible for disease relapse in secondary lymphoid organs in humans, taking advantage of the opportunity offered by therapeutic splenectomy in patients with relapsing immune thrombocytopenia. By analyzing the B and plasma cell immunoglobulin gene repertoire at bulk and antigen-specific single-cell level, we demonstrate that relapses are associated with two responses coexisting in germinal centers and involving preexisting mutated memory B cells that survived RTX treatment and naive B cells generated upon reconstitution of the B cell compartment. To identify distinctive characteristics of the memory B cells that escaped RTX-mediated depletion, we analyzed RTX refractory patients who did not respond to treatment at the time of B cell depletion. We identified, by single-cell RNA sequencing (scRNA-seq) analysis, a population of quiescent splenic memory B cells that present a unique, yet reversible, RTX-shaped phenotype characterized by down-modulation of B cell–specific factors and expression of prosurvival genes. Our results clearly demonstrate that these RTX-resistant autoreactive memory B cells reactivate as RTX is cleared and give rise to plasma cells and further germinal center reactions. Their continued surface expression of CD19 makes them efficient targets for current anti-CD19 therapies. This study thus identifies a pathogenic contributor to autoimmune diseases that can be targeted by available therapeutic agents. [ABSTRACT FROM AUTHOR]

• IgM memory B cells can achieve multiple fates upon recall adaptive responses in the mouse. • IgM memory B cells with lower mutation load show broader reactivity against variant pathogens. • Innate-like IgM B cells educated in the gut can provide broad systemic protection against cross-reactive glycan epitopes. • Human marginal zone B cells are clonally related to cells diversifying in gut germinal centers. Antigen-experienced IgM+ B cells with mutated V genes have emerged as important effectors of both adaptive and innate-like immune responses. While their precise role in recall responses appear to differ according to the nature of the immunogen or the infectious agent, they are able to achieve rapid plasma cell differentiation, germinal center re-initiation, as well as IgM and IgG memory pool replenishment, which establishes them as multi-lineage precursors of the various functional memory subsets. For innate-like responses, recent data have shown that activation by gut commensals is able to generate, both in mice and humans, a systemic IgM+ population with specificity against glycan epitopes, which displays broad cross-reactivity towards multiple micro-organisms, and ensures a first line of defense against systemic infections. [ABSTRACT FROM AUTHOR]

Mice derived from the 129 strain have a nonsense codon mutation in exon 2 of the polymerase iota (Polι) gene and are therefore considered Polι deficient. When we amplified Polι mRNA from 129/SvJ or 129/Ola testes, only a small fraction of the full-length cDNA contained the nonsense mutation; the major fraction corresponded to a variant Polι isoform lacking exon 2. Polι mRNA lacking exon 2 contains an open reading frame, and the corresponding protein was detected using a polyclonal antibody raised against the C terminus of the murine Polι protein. The identity of the corresponding protein was further confirmed by mass spectrometry. Although the variant protein was expressed at only 5 to 10% of the level of wild-type Polι, it retained de novo DNA synthesis activity, the capacity to form replication foci following UV irradiation, and the ability to rescue UV light sensitivity in Polι-/- embryonic fibroblasts derived from a new, fully deficient Polι knockout (KO) mouse line. Furthermore, in vivo treatment of 129-derived male mice with Velcade, a drug that inhibits proteasome function, stabilized and restored a substantial amount of the variant Polι in these animals, indicating that its turnover is controlled by the proteasome. An analysis of two xeroderma pigmentosum-variant (XPV) cases corresponding to missense mutants of Polι, a related translesion synthesis (TLS) polymerase in the same family, similarly showed a destabilization of the catalytically active mutant protein by the proteasome. Collectively, these data challenge the prevailing hypothesis that 129-derived strains of mice are completely deficient in Polι activity. The data also document, both for 129-derived mouse strains and for some XPV patients, new cases of genetic defects corresponding to the destabilization of an otherwise functional protein, the phenotype of which is reversible by proteasome inhibition. [ABSTRACT FROM AUTHOR]

A/T mutations at immunoglobulin loci are introduced by DNA polymerase η (Polη) during an Msh2/6-dependent repair process which results in A's being mutated 2-fold more often than T's. This patch synthesis is initiated by a DNA incision event whose origin is still obscure. We report here the analysis of A/T oligonucleotide mutation substrates inserted at the heavy chain locus, including or not including internal C's or G's. Surprisingly, the template composed of only A's and T's was highly mutated over its entire 90-bp length, with a 2-fold decrease in mutation from the 5' to the 3' end and a constant A/T ratio of 4. These results imply that Polη synthesis was initiated from a break in the 5'-flanking region of the substrate and proceeded over its entire length. The A/T bias was strikingly altered in an Ung-/- background, which provides the first experimental evidence supporting a concerted action of Ung and Msh2/6 pathways to generate mutations at A/T bases. New analysis of Pms2-/- animals provided a complementary picture, revealing an A/T mutation ratio of 4. We therefore propose that Ung and Pms2 may exert a mutual backup function for the DNA incision that promotes synthesis by Polη, each with a distinct strand bias. [ABSTRACT FROM AUTHOR]

A large number of auto-immune diseases are treated with rituximab, an antibody against CD20 that depletes most of the B-cells in the organism. The response to this treatment depends largely on the disease and the type of lymphoid cells involved in the auto-immune process. We recently reported that B-cell depletion in immune thrombocytopenia induced the appearance of pathogenic long-lived plasma cells in the spleen, which were not present before treatment or in non-auto-immune conditions. The spleen of treated patients produced an excess of the cytokine B-cell activating factor, which in in vitro-cultured splenic cells, could increase the longevity of plasma cells. Our results suggested that, paradoxically, the B-cell depletion itself, by altering the splenic milieu, promoted the differentiation of short-lived auto-immune plasma cells into long-lived ones. We describe the cellular and cytokinic components of the splenic plasma cell niche, notably CD4+ T cells and discuss possible survival factors that could be targeted simultaneously with rituximab-mediated B-cell depletion to interfere with plasma cell persistence. [ABSTRACT FROM AUTHOR]

Humoral memory is maintained by two types of persistent cells, memory B cells and plasma cells, which have different phenotypes and functions. Long-lived plasma cells can survive for a lifespan within a complex niche in the bone marrow and provide continuous protective serum antibody levels. Memory B cells reside in secondary lymphoid organs, where they can be rapidly mobilized upon a new antigenic encounter. Surface IgG has long been taken as a surrogate marker for memory in the mouse. Recently, however, we have brought evidence for a long-lived IgM memory B cell population in the mouse, while we have also argued that, in humans, these same cells are not classical memory B cells but marginal zone (MZ) B cells which, as opposed to their mouse MZ counterpart, recirculate and carry a mutated B cell receptor. In this review, we will discuss these apparently paradoxical results. [ABSTRACT FROM AUTHOR]

Memory B cells are at the center of longstanding controversies regarding the presence of antigen for their survival and their re-engagement in germinal centers after secondary challenge. Using a new mouse model of memory B cell labeling dependent on the cytidine deaminase AID, we show that after immunization with a particulate antigen, B cell memory appeared in several subsets, comprising clusters of immunoglobulin M–positive (IgM+) and IgG1+ B cells in germinal center–like structures that persisted up to 8 months after immunization, as well as IgM+ and IgG1+ B cells with a memory phenotype outside of B cell follicles. After challenge, the IgG subset differentiated into plasmocytes, whereas the IgM subset reinitiated a germinal center reaction. This model, in which B cell memory appears in several layers with different functions, reconciles previous conflicting propositions. [ABSTRACT FROM AUTHOR]

To cope with an unpredictable variety of potential pathogenic insults, the immune system must generate an enormous diversity of recognition structures, and it does so by making stepwise modifications at key genetic loci in each lymphoid cell. These modifications proceed through the action of lymphoid-specific proteins acting together with the general DNA-repair machinery of the cell. Strikingly, these general mechanisms are usually diverted from their normal functions, being used in rather atypical ways in order to privilege diversity over accuracy. In this Review, we focus on the contribution of a set of DNA polymerases discovered in the past decade to these unique DNA transactions. [ABSTRACT FROM AUTHOR]

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The mutation pattern of immunoglobulin genes was studied in mice deficient for DNA polymerase η, 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 L 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 polη-deficient mice differs markedly. This suggests that, in the absence of polη, the MSH2-MSH6 complex is able to recruit another DNA polymerase that is more accurate at copying A/T bases, possibly polκ, to assume its function in hypermutation. [ABSTRACT FROM AUTHOR]

Central and peripheral tolerance checkpoints are in place to remove autoreactive B cell populations and prevent the development of autoimmunity. In this issue of the JCI, Pala and colleagues reveal that individuals with the X-linked immunodeficiency Wiskott-Aldrich syndrome (WAS) have opposite alterations at central and peripheral B cell checkpoints: a more stringent selection for central tolerance, resulting in reduced numbers of autoreactive cells at the emergent immature B cell stage, and a relaxed selection for peripheral tolerance, resulting in an increased frequency of autoreactive cells in the mature naive B cell compartment. Moreover, reinstatement of the WAS gene in these patients restored both B cell tolerance checkpoints. These results suggest that, in a normal situation, mature naive B cells undergo a positive selection step driven by self-antigens, kept in control by Tregs. [ABSTRACT FROM AUTHOR]