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Secondary immunoglobulin diversification by somatic hypermutation and class switch recombination in B cells is instrumental for an adequate adaptive humoral immune response. These genetic events may, however, also introduce aberrations into other cellular genes and thereby cause B cell malignancies. While the basic mechanism of somatic hypermutation and class switch recombination is now well understood, their regulation and in particular the mechanism of their specific targeting to immunoglobulin genes is still rather mysterious. In this review, we summarize the current knowledge on the mechanism and regulation of secondary immunoglobulin diversification and discuss known mechanisms of physiological targeting to immunoglobulin genes and mistargeting to other cellular genes. We summarize open questions in the field and provide an outlook on future research.

Candida albicans is usually a benign member of the human gut microbiota, but can become pathogenic under certain circumstances, for example in an immunocompromised host. The innate immune system, in particular neutrophils and macrophages, constitutes a crucial first line of defense against fungal invasion, however adaptive immunity may provide long term protection and thus allow vaccination of at risk patients. While T H 1 and T H 17 cells are important for antifungal responses, the role of B cells and antibodies in protection from C. albicans infection is less well defined. In this study, we show that C. albicans hyphae but not yeast, as well as fungal cell wall components, directly activate B cells via MyD88 signaling triggered by Toll- like receptor 2, leading to increased IgG1 production. While Dectin-1 signals and specific recognition by the B cell receptor are dispensable for B cell activation in this system, TLR2/MyD88 signals cooperate with CD40 signals in promoting B cell activation. Importantly, recognition of C. albicans via MyD88 signaling is also essential for induction of IL-6 secretion by B cells, which promotes T H 17 polarization in T-B cell coculture experiments. B cells may thus be activated directly by C. albicans in its invasive form, leading to production of antibodies and T cell help for fungal clearance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ferreira-Gomes, Wich, Böde, Hube, Jacobsen and Jungnickel.)

Checkpoint signaling in the context of a functional DNA damage response is crucial for the prevention of oncogenic transformation of cells. Our immune system, though, takes the risk of attenuated checkpoint responses during immunoglobulin diversification. B cells undergo continuous DNA damage and error-prone repair of their immunoglobulin genes during the process of somatic hypermutation. An accompanying attenuation of the DNA damage response via the ATR-Chk1 axis in B cells is believed to allow for a better DNA damage tolerance and for evasion of apoptosis, so as to ensure mutations to be passed on. We sought to determine whether the downregulation of Chk1 could also directly influence the process of hypermutation in vivo by altering the relative activity of error-prone DNA repair pathways. We analyzed the humoral response and the hypermutation process in mice whose B cells express reduced levels of the Chk1 protein. We found that Chk1 heterozygosity limits the accumulation of mutations in the immunoglobulin loci, likely by impacting on the survival of B cells as they accumulate DNA damage. Nevertheless, we unveiled an unanticipated role for Chk1 downregulation in favoring A/T mutagenesis at the antibody-variable regions during hypermutation. Even though immunoglobulin mutagenesis was found to be reduced, Chk1 signaling attenuation allows for sustained mutagenesis outside the immunoglobulin loci. Our study thus reveals that a proper Chk1 dosage is crucial for adequate somatic hypermutation in B cells., (© 2021 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Members of the RAF family of serine-threonine kinases are intermediates in the mitogen-activated protein kinase and extracellular signal-regulated kinase (MAPK-ERK) signaling pathway, which controls key differentiation processes in B cells. By analyzing mice with B cell-specific deletion of Raf1 , Braf , or both, we showed that Raf-1 and B-Raf acted together in mediating the positive selection of pre-B and transitional B cells as well as in initiating plasma cell differentiation. However, genetic or chemical inactivation of RAFs led to increased ERK phosphorylation in mature B cells. ERK activation in the absence of Raf-1 and B-Raf was mediated by multiple RAF-independent pathways, with phosphoinositide 3-kinase (PI3K) playing an important role. Furthermore, we found that ERK phosphorylation strongly increased during the transition from activated B cells to pre-plasmablasts. This increase in ERK phosphorylation did not occur in B cells lacking both Raf-1 and B-Raf, which most likely explains the partial block of plasma cell differentiation in mice lacking both RAFs. Collectively, our data indicate that B-Raf and Raf-1 are not necessary to mediate ERK phosphorylation in naïve or activated B cells but are essential for mediating the marked increase in ERK phosphorylation during the transition from activated B cells to pre-plasmablasts., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

During secondary immunoglobulin (Ig) diversification in vertebrates, the sequence of the variable region of Ig genes may be altered by templated or non-templated mechanisms. In both cases, cytidine deamination by activation-induced cytidine deaminase (AID) in the transcribed Ig loci leads to DNA lesions, which are repaired by conservative homologous recombination (HR) during Ig gene conversion, or by non-templated mutagenesis during somatic hypermutation. The molecular basis for the differential use of these two pathways in different species is unclear. While experimental ablation of HR in avian cells performing Ig gene conversion may promote a switch to somatic hypermutation, the activity of HR processes in intrinsically hypermutating mammalian cells has not been measured to date. Employing a functional HR assay in human germinal centre like B cell lines, we detect elevated HR activity that can be enhanced by transcription and AID. Products of such recombination events mostly arise through non-conservative HR pathways, while the activity of conservative HR is low to absent. Our results identify non-conservative HR as a novel DNA transaction pathway promoted by AID and suggest that somatic hypermutation in germinal centre B cells may be based on a physiological suppression of conservative HR.

Somatic hypermutation and class-switch recombination in germinal centers critically depend on activation-induced cytidine deaminase (AID). Deregulation of AID may lead to the aberrant activation or persistence of both genetic processes, thus contributing to the pathogenesis of B-cell lymphomas by mistargeted mutagenesis or recombination. The Epstein-Barr virus (EBV) establishes an asymptomatic latent infection in more than 90% of the human population, but it has also been linked to lymphomagenesis. A cooperative relationship of EBV and the germinal center reaction during the establishment of viral persistence has been postulated, but the contribution of EBV latent genes to the respective genetic events remains to be investigated in detail. In the present study, we show that activation of the EBV growth program has a clear inhibitory effect on AID expression, due to a negative effect of the master transcription factor of this program, EBNA2. This mechanism may counterbalance AID induction by the LMP1 protein, in order to prevent deleterious genetic changes during EBV-induced B-cell growth. EBNA2-mediated AID inhibition also provides a molecular explanation for the previously observed differences in somatic hypermutation activity in EBV-associated lymphoproliferative diseases, thus pointing to a crucial mechanism of EBV-mediated regulation of genomic integrity.

Epstein-Barr virus (EBV) is associated with B-cell lymphomas such as Hodgkin lymphoma, Burkitt lymphoma, and post-transplantation lymphoma, which originate from clonal germinal center (GC) B cells. During the process of somatic hypermutation, GC B cells can acquire deleterious or nonsense mutations in the heavy and light immunoglobulin genes. Such mutations abrogate the cell surface expression of the B-cell receptor (BCR), which results in the elimination of these nonfunctional B cells by immediate apoptosis. EBV encodes several latent genes, among them latent membrane protein 1 (LMP1) and LMP2A, which are regularly expressed in EBV-positive Hodgkin lymphoma and posttransplantation lymphomas. Since LMP1 and LMP2A mimic the function of 2 key receptors on B cells, CD40 and BCR, respectively, we wanted to learn whether EBV infection can rescue proapoptotic GC B cells with crippling mutations in the heavy chain immunoglobulin locus from apoptosis. We show here that BCR-negative GC B cells readily enter the cell cycle upon infection with EBV in vitro and yield clonal lymphoblastoid cell lines that are incapable of expressing a functional BCR because the rearranged and formerly functional heavy chain immunoglobulin alleles carry deleterious mutations. Our findings imply an important role for EBV in the process of lymphomagenesis in certain cases of Hodgkin lymphoma and posttransplantation lymphomas.

It has been speculated that somatic hypermutation of rearranged immunoglobulin variable (V) region genes does not only take place in the germinal center (GC) microenvironment, but also in the marginal zone (MZ) of the spleen, and that human peripheral blood IgM-positive B cells with somatically mutated V region genes may derive from mutating MZ B cells. As somatic hypermutation is strictly dependent on the enzyme activation-induced cytidine deaminase (AID), we used an AID-specific monoclonal antibody that is suitable for immunohistochemical staining to analyze human splenic MZ cells for AID expression. Analysis of tissue sections from 29 spleens revealed only very rare MZ cells (approx. 0.05%) showing AID staining, whereas in 25 of the spleen samples strong AID staining of GC B cells was observed. Thus, there are virtually no AID-expressing MZ B cells, indicating that somatic hypermutation does not take place at a significant level in the MZ. Consequently, it appears unlikely that the somatically mutated IgM B cells are generated in the splenic MZ. Moreover, the lack of AID-positive MZ B cells questions the recent speculation that B cell chronic lymphocytic leukemias with mutated V genes are derived from mutating MZ B cells.

Hodgkin and Reed-Sternberg (HRS) cells represent the malignant cells in classical Hodgkin lymphoma (HL). Because their immunophenotype cannot be attributed to any normal cell of the hematopoietic lineage, the origin of HRS cells has been controversially discussed, but molecular studies established their derivation from germinal center B cells. In this study, gene expression profiles generated by serial analysis of gene expression (SAGE) and DNA chip microarrays from HL cell lines were compared with those of normal B-cell subsets, focusing here on the expression of B-lineage markers. This analysis revealed decreased mRNA levels for nearly all established B-lineage-specific genes. For 9 of these genes, lack of protein expression was histochemically confirmed. Down-regulation of genes affected multiple components of signaling pathways active in B cells, including B-cell receptor (BCR) signaling. Because several genes down-regulated in HRS cells are positively regulated by the transcriptional activator Pax-5, which is expressed in most HRS cells, we studied HL cell lines for mutations in the Pax-5 gene. However, no mutations were found. We propose that the lost B-lineage identity in HRS cells may explain their survival without BCR expression and reflect a fundamental defect in maintaining the B-cell differentiation state in HRS cells, which is likely caused by a novel, yet unknown, pathogenic mechanism.

Somatic hypermutation specifically modifies rearranged immunoglobulin (Ig) genes in germinal center (GC) B cells. However, the bcl-6 gene can also acquire somatic mutations during the GC reaction, indicating that certain non-Ig genes can be targeted by the somatic hypermutation machinery. The CD95 gene, implicated in negative selection of B lymphocytes in GCs, is specifically expressed by GC B cells and was recently identified as a tumor suppressor gene being frequently mutated in (post) GC B cell lymphomas. In this study, the 5' region (5'R) and/or the last exon coding for the death domain (DD) of the CD95 gene were investigated in naive, GC, and memory B cells from seven healthy donors. About 15% of GC and memory, but not naive, B cells carried mutations within the 5'R (mutation frequency 2.5 x 10(-4) per basepair). Mutations within the DD were very rare but could be efficiently selected by inducing CD95-mediated apoptosis: in 22 apoptosis-resistant cells, 12 DD mutations were found. These results indicate that human B cells can acquire somatic mutations of the CD95 gene during the GC reaction, which potentially confers apoptosis resistance and may counteract negative selection through the CD95 pathway.