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• 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]

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]

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]

Infection with encapsulated bacteria can be prevented by vaccination with capsular polysaccharides, either plain or conjugated to a protein carrier. However, results concerning these vaccinations raise several paradoxes. Polysaccharides from encapsulated bacteria are generally considered to be T cell-independent antigens which are unable to trigger a T cell-dependent germinal center reaction, but, strikingly, anti-polysaccharide antibodies are often mutated in humans. Polysaccharide–protein conjugate vaccines are able to induce a true T cell-dependent memory response with a rise in antibody titers and a switch to high-affinity IgG antibodies in children below two years of age, but neither the plain nor the conjugated vaccine can induce memory in older infants and adults. We propose some explanations for these paradoxes based on our recent observation that humans display a circulating splenic marginal zone B-cell population with a pre-diversified immunoglobulin repertoire in charge of the immune response to T cell-independent antigens. [Copyright &y& Elsevier]

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]

A very unusual molecular mechanism is involved in generating the preimmune repetoire in the chicken bursa of Fabricius. A unique rearranged V gene is diversified through a progam of segmental gene conversion with a pool of noncoding pseudogenes being used as donors. A specifically committed progenitor that originates in the embryonic bursa is responsible for long-term maintenance of the B cell population. Both these properties and the characteristics of the peripheral B cell compartment are discussed in terms of the evolution of the T and B immune systems. [ABSTRACT FROM AUTHOR]

We show in this review that there is a continuum between the chicken B-cell system classified as the first GALT model described and the human B-cell system. We propose that humans have conserved for one B-cell subpopulation, the marginal zone B-cell subset in charge of T-independent responses, the strategies of diversification used by GALT species to generate their pre-immune repertoire. [Copyright &y& Elsevier]

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]

Previous data have suggested that B-cell-depletion therapy may induce the settlement of autoreactive long-lived plasma cells (LLPCs) in the spleen of patients with autoimmune cytopenia. To investigate this process, we used the AID-CreERT2-EYFP mouse model to follow plasma cells (PCs) engaged in an immune response. Multiplex polymerase chain reaction at the single-cell level revealed that only a small fraction of splenic PCs had a long-lived signature, whereas PCs present after anti-CD20 antibody treatment appeared more mature, similar to bone marrow PCs. This observation suggested that, in addition to a process of selection, a maturation induced on B-cell depletion drove PCs toward a long-lived program. We showed that B-cell activating factor (BAFF) and CD4+ T cells play a major role in the PC survival niche, because combining anti-CD20 with anti-BAFF or anti-CD4 antibody greatly reduce the number of splenic PCs. Similar results were obtained in the lupus-prone NZB/W model. These different contributions of soluble and cellular components of the PC niche in the spleen demonstrate that the LLPC expression profile is not cell intrinsic but largely depends on signals provided by the splenic microenvironment, implying that interfering with these components at the time of B-cell depletion might improve the response rate in autoimmune cytopenia. [ABSTRACT FROM AUTHOR]

The article presents a study for identifying Kelch-like (Klhl)6 deficiency leading to 2-fold reduction in mature B cells. Topics include interaction between the B cell–activating factor (BAFF) cytokine and its specific receptor; broad complex involved in protein–protein interaction and protein self-oligomerization; and chronic lymphocytic leukemias (CLLs) with mutated genes.

Rev3, the catalytic subunit of yeast DNA polymerase ζ, is required for UV resistance and UV-induced mutagenesis, while its mammalian ortholog, REV3L, plays further vital roles in cell proliferation and embryonic development. To assess the contribution of REV3L catalytic activity to its in vivo function, we generated mutant mouse strains in which one or two Ala residues were substituted to the Asp of the invariant catalytic YGDTDS motif. The simultaneous mutation of both Asp (ATA) phenocopies the Rev3l knockout, which proves that the catalytic activity is mandatory for the vital functions of Rev3L, as reported recently. Surprisingly, although the mutation of the first Asp severely impairs the enzymatic activity of other B-family DNA polymerases, the corresponding mutation of Rev3 (ATD) is hypomorphic in yeast and mouse, as it does not affect viability and proliferation and moderately impacts UVC-induced cell death and mutagenesis. Interestingly, Rev3l hypomorphic mutant mice display a distinct, albeit modest, alteration of the immunoglobulin gene mutation spectrum at G-C base pairs, further documenting its role in this process. [ABSTRACT FROM AUTHOR]

Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with nonimmunodominant specificities must be elicited, as is the case for HIV-1 and influenza. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Splenic vaccinia B5-specific MBCs can be isolated decades after last vaccination • Long-lived B5-specific MBCs display signs of extensive affinity-based selection • Long-lived B5-specific MBCs are enriched in a splenic CD21hiCD20hi IgG+ B cell subset • Long-lived B5-specific MBCs harbor long-lasting elongated telomeres Immune memory in humans has been shown to extend well beyond decades. Chappert et al. provide an extensive functional characterization of human splenic smallpox/vaccinia protein B5-specific MBCs, generated more than four decades ago, to decipher the distinct selection and survival mechanisms associated with MBCs longevity. [ABSTRACT FROM AUTHOR]

From paired blood and spleen samples from three adult donors, we performed high-throughput VH sequencing of human B cell subsets defined by IgD and CD27 expression: IgD+CD27+ ("marginal zone [MZ]"), IgD-CD27+ ("memory," including IgM ["IgM-only"], IgG and IgA) and IgD-CD27- cells ("double-negative," including IgM, IgG, and IgA). A total of 91,294 unique sequences clustered in 42,670 clones, revealing major clonal expansions in each of these subsets. Among these clones, we further analyzed those shared sequences from different subsets or tissues for VH gene mutation, H-CDR3-length, and Vh/Jh usage, comparing these different characteristics with all sequences from their subset of origin for which these parameters constitute a distinct signature. The IgM-only repertoire profile differed notably from that of MZ B cells by a higher mutation frequency and lower VH4 and higher JH6 gene usage. Strikingly, IgM sequences from clones shared between the MZ and the memory IgG/IgA compartments showed a mutation and repertoire profile of IgM-only and not of MZ B cells. Similarly, all IgM clonal relationships (among MZ, IgM-only, and double-negative compartments) involved sequences with the characteristics of IgM-only B cells. Finally, clonal relationships between tissues suggested distinct recirculation characteristics between MZ and switched B cells. The "IgM-only" subset (including cells with its repertoire signature but higher IgD or lower CD27 expression levels) thus appear as the only subset showing precursor-product relationships with CD27+ switched memory B cells, indicating that they represent germinal centerderived IgM memory B cells and that IgM memory and MZ B cells constitute two distinct entities. [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]

Primary warm autoimmune hemolytic anemia (wAIHA) is a rare autoimmune disease in which red blood cells are eliminated by IgG autoantibodies. We analyzed the antibody-secreting cells in the spleen and the peripheral blood of wAIHA patients in various contexts of treatment. Plasmablasts were observed in peripheral blood of newly diagnosed wAIHA patients and, accordingly, active germinal center reactions were present in the spleen of patients receiving short-term corticosteroid therapy. Long-term corticosteroid regimens markedly reduced this response while splenic plasma cells were able to persist, a fraction of them secreting anti-red blood cell IgG in vitro . In wAIHA patients treated by rituximab and who underwent splenectomy because of treatment failure, plasma cells were still present in the spleen, some of them being autoreactive. By using a set of diagnostic genes that allowed us to assess the plasma cell maturation stage, we observed that these cells displayed a long-lived program, differing from the one of plasma cells from healthy donors or from wAIHA patients with various immunosuppressant treatments, and more similar to the one of normal long-lived bone-marrow plasma cells. Interestingly, an increased level of B-cell activating factor (BAFF) was observed in the supernatant of spleen cell cultures from such rituximab-treated wAIHA patients. These results suggest, in line with our previous report on primary immune thrombocytopenia, that the B-cell depletion induced by rituximab promoted a suitable environment for the maturation and survival of auto-immune long-lived plasma cells in the spleen. [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]

Summary: Segmented filamentous bacterium (SFB) is a symbiont that drives postnatal maturation of gut adaptive immune responses. In contrast to nonpathogenic E. coli, SFB stimulated vigorous development of Peyer’s patches germinal centers but paradoxically induced only a low frequency of specific immunoglobulin A (IgA)-secreting cells with delayed accumulation of somatic mutations. Moreover, blocking Peyer’s patch development abolished IgA responses to E. coli, but not to SFB. Indeed, SFB stimulated the postnatal development of isolated lymphoid follicles and tertiary lymphoid tissue, which substituted for Peyer’s patches as inductive sites for intestinal IgA and SFB-specific T helper 17 (Th17) cell responses. Strikingly, in mice depleted of gut organized lymphoid tissue, SFB still induced a substantial but nonspecific intestinal Th17 cell response. These results demonstrate that SFB has the remarkable capacity to induce and stimulate multiple types of intestinal lymphoid tissues that cooperate to generate potent IgA and Th17 cell responses displaying only limited target specificity. [Copyright &y& Elsevier]

Highlights: [•] Multiple memory B-cell subsets are generated by the immune response. [•] IgM+ and IgG+ memory B cells fullfill different effector functions. [•] Early memory B cells emerge as germinal center-independent subpopulations. [•] Persisting germinal centers modify the outcome of the recall response. [Copyright &y& Elsevier]

Primary immune thrombocytopenia (ITP) is a disorder caused by autoantibody-mediated platelet destruction and decreased platelet production. Rituximab, a B cell-depleting agent, has become the first-line treatment for ITP; however, patients with refractory disease usually require splenectomy. We identified antibody-secreting cells as the major splenic B cell population that is resistant to rituximab. The phenotype, antibody specificity, and gene expression profile of these cells were characterized and compared to those of antibody-secreting cells from untreated ITP spleens and from healthy tissues. Antiplatelet-specific plasma cells (PC) were detected in the spleens of patients with ITP up to 6 months after rituximab treatment, and the PC population displayed a long-lived program similar to the one of bone marrow PC, thus explaining for most of these patients the absence of response to rituximab and the response to splenectomy. When analyzed by multiplex PCR at the single-cell level, normal splenic PC showed a markedly different gene expression profile, with an intermediate signature, including genes characteristic of both long-lived PC and proliferating plasmablasts. Surprisingly, long-lived PC were not detected in untreated ITP spleens. These results suggest that the milieu generated by B cell depletion promotes the differentiation and settlement of long-lived PC in the spleen. [ABSTRACT FROM AUTHOR]

Abstract: The generation of high affinity antibodies in B cells critically depends on translesion synthesis (TLS) polymerases that introduce mutations into immunoglobulin genes during somatic hypermutation (SHM). The majority of mutations at A/T base pairs during SHM require ubiquitination of PCNA at lysine 164 (PCNA-Ub), which activates TLS polymerases. By comparing the mutation spectra in B cells of WT, TLS polymerase η (Polη)-deficient, PCNAK164R-mutant, and PCNAK164R;Polη double-mutant mice, we now find that most PCNA-Ub-independent A/T mutagenesis during SHM is mediated by Polη. In addition, upon exposure to various DNA damaging agents, PCNAK164R mutant cells display strongly impaired recruitment of TLS polymerases, reduced daughter strand maturation and hypersensitivity. Interestingly, compared to the single mutants, PCNAK164R;Polη double-mutant cells are dramatically delayed in S phase progression and far more prone to cell death following UV exposure. Taken together, these data support the existence of PCNA ubiquitination-dependent and -independent activation pathways of Polη during SHM and DNA damage tolerance. [Copyright &y& Elsevier]

Post-rearrangement diversification of the antibody repertoire relies on a DNA editing factor, the cytidine deaminase AID. How B lymphocytes avoid generalized mutagenesis while expressing high levels of AID remained a long-standing question. Genome-wide studies of AID targeting combined to the discovery of several co-factors controlling its recruitment and its local activity shed new light on this enigma. [Copyright &y& Elsevier]

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]

The presence of intratumoral tertiary lymphoid structures (TLS) is associated with positive clinical outcomes and responses to immunotherapy in cancer. Here, we used spatial transcriptomics to examine the nature of B cell responses within TLS in renal cell carcinoma (RCC). B cells were enriched in TLS, and therein, we could identify all B cell maturation stages toward plasma cell (PC) formation. B cell repertoire analysis revealed clonal diversification, selection, expansion in TLS, and the presence of fully mature clonotypes at distance. In TLS+ tumors, IgG- and IgA-producing PCs disseminated into the tumor beds along fibroblastic tracks. TLS+ tumors exhibited high frequencies of IgG-producing PCs and IgG-stained and apoptotic malignant cells, suggestive of anti-tumor effector activity. Therapeutic responses and progression-free survival correlated with IgG-stained tumor cells in RCC patients treated with immune checkpoint inhibitors. Thus, intratumoral TLS sustains B cell maturation and antibody production that is associated with response to immunotherapy, potentially via direct anti-tumor effects. [Display omitted] • Tertiary lymphoid structures are sites of in situ B cell maturation toward plasma cells • IgG+ and IgA+ plasma cells disseminate into the tumor tissue along fibroblastic tracks • Tumor cells are labeled by locally produced IgG • Patients with IgG-labeled tumor cells have high response rate to ICI and prolonged PFS Meylan et al. show that tertiary lymphoid structures found in tumors are sites of generation of fully mature B cell immunity. Plasma cells disseminate into tumor beds, producing antibodies that bind to tumor cells and initiate their apoptosis, providing a mechanism to support cancer immunotherapies that modulate the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Abstract: Genes coding for DNA polymerases η, ι and ζ, or for both Pol η and Pol ι have been inactivated by homologous recombination in the Burkitt''s lymphoma BL2 cell line, thus providing for the first time the total suppression of these enzymes in a human context. The UV sensitivities and UV-induced mutagenesis on an irradiated shuttle vector have been analyzed for these deficient cell lines. The double Pol η/ι deficient cell line was more UV sensitive than the Pol η-deficient cell line and mutation hotspots specific to the Pol η-deficient context appeared to require the presence of Pol ι, thus strengthening the view that Pol ι is involved in UV damage translesion synthesis and UV-induced mutagenesis. A role for Pol ζ in a damage repair process at late replicative stages is reported, which may explain the drastic UV-sensitivity phenotype observed when this polymerase is absent. A specific mutation pattern was observed for the UV-irradiated shuttle vector transfected in Pol ζ-deficient cell lines, which, in contrast to mutagenesis at the HPRT locus previously reported, strikingly resembled mutations observed in UV-induced skin cancers in humans. Finally, a Pol η PIP-box mutant (without its PCNA binding domain) could completely restore the UV resistance in a Pol η deficient cell line, in the absence of UV-induced foci, suggesting, as observed for Pol ι in a Pol η-deficient background, that TLS may occur without the accumulation of microscopically visible repair factories. [Copyright &y& Elsevier]

Summary: DNA polymerases mu (pol μ), lambda (pol λ), and terminal deoxynucleotidyltransferase (TdT) are enzymes of the pol X family that share homology in sequence and functional domain organization. We showed previously that pol μ participates in light chain but surprisingly not heavy chain gene rearrangement. We show here that immunoglobulin heavy chain junctions from pol λ-deficient animals have shorter length with normal N-additions, thus indicating that pol λ is recruited during heavy chain rearrangement at a step that precedes the action of TdT. In contrast to previous in vitro studies, analysis of animals with combined inactivation of these enzymes revealed no overlapping or compensatory activities for V(D)J recombination between pol μ, pol λ, and TdT. This complex usage of polymerases with distinct catalytic specificities may correspond to the specific function that the third hypervariable region assumes for each immunoglobulin chain, with pol λ maintaining a large heavy chain junctional heterogeneity and pol μ ensuring a restricted light chain junctional variability. [Copyright &y& Elsevier]

DNA polymerase mu (pol μ) is a template-dependent polymerase closely related to the lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT). We report here the phenotype of pol μ-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 Vκ-Jκ and Vλ-Jλ 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 μ 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. [Copyright &y& Elsevier]

Ig gene hypermutation was originally described as the molecular process underlying B cell affinity maturation following a T-dependent immune response. Somatic hypermutation is also used in some species such as sheep, to generate diversity during formation of the primary antibody repertoire. In sheep, B cells mutate their Ig receptor during antigen-independent development in the lymphoid follicles of ileal Peyer’s patches, but this process is arrested when these same B cells are cultured in vitro. We have used these differences between in vivo and in vitro B cell development to perform a cDNA subtraction between these two cell populations, in order to search for genes that might be involved in the hypermutation process. We describe in this paper the characterization of two genes, highly expressed in sheep ileal Peyer’s patch B cells and also in centroblasts of human tonsils: deltex (Drosophila) homolog 1 (DTX1), which is related to the Notch pathway and a new Kelch-like protein, KLHL6. The putative role of these proteins, which are more likely involved in the germinal center B cell differentiation pathway than in the hypermutation mechanism per se, is discussed. [Copyright &y& Elsevier]

Somatic hypermutation of immunoglobulin genes is a unique, targeted, adaptive process. While B cells are engaged in germinal centres in T-dependent responses, single base substitutions are introduced in the expressed V[sub H]/V[sub L] genes to allow the selection of mutants with a higher affinity for the immunizing antigen. Almost every possible DNA transaction has been proposed to explain this process, but each of these models includes an errorprone DNA synthesis step that introduces the mutations. The Y family of DNA polymerases—pol η, pol ι, pol κ and rev1—are specialized for copying DNA lesions and have high rates of error when copying a normal DNA template. By performing gene inactivation in a Burkitt's lymphoma cell line inducible for hypermutation, we show here that somatic hypermutation is dependent on DNA polymerase iota. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]