Ataxia Telangiectasia Mutated and MSH2 Control Blunt DNA End Joining in Ig Class Switch Recombination.

Class-switch recombination (CSR) produces secondary Ig isotypes and requires activation-induced cytidine deaminase (AID)-dependent DNA deamination of intronic switch regions within the IgH (Igh) gene locus. Noncanonical repair of deaminated DNA by mismatch repair (MMR) or base excision repair (BER) creates DNA breaks that permit recombination between distal switch regions. Ataxia telangiectasia mutated (ATM)-dependent phosphorylation of AID at serine 38 (pS38-AID) promotes its interaction with apurinic/apyrimidinic endonuclease 1 (APE1), a BER protein, suggesting that ATM regulates CSR through BER. However, pS38-AID may also function in MMR during CSR, although the mechanism remains unknown. To examine whether ATM modulates BER- and/or MMR-dependent CSR, Atm-/- mice were bred to mice deficient for the MMR gene mutS homolog 2 (Msh2). Surprisingly, the predicted Mendelian frequencies of Atm-/-Msh2-/- adult mice were not obtained. To generate ATM and MSH2-deficient B cells, Atm was conditionally deleted on an Msh2-/- background using a floxed ATM allele (Atmf) and B cell-specific Cre recombinase expression (CD23-cre) to produce a deleted ATM allele (AtmD). As compared with AtmD/D and Msh2-/- mice and B cells, AtmD/DMsh2-/- mice and B cells display a reduced CSR phenotype. Interestingly, Sµ-Sγ1 junctions from AtmD/DMsh2-/- B cells that were induced to switch to IgG1 in vitro showed a significant loss of blunt end joins and an increase in insertions as compared with wild-type, AtmD/D, or Msh2-/- B cells. These data indicate that the absence of both ATM and MSH2 blocks nonhomologous end joining, leading to inefficient CSR. We propose a model whereby ATM and MSH2 function cooperatively to regulate end joining during CSR through pS38-AID.

TGF-ß Promotes the Postselection Thymic Development and Peripheral Function of IFN-γ-Producing Invariant NKT cells.

IFN-γ-producing invariant NKT (iNKT)1 cells are lipid-reactive innate-like lymphocytes that are resident in the thymus and peripheral tissues where they protect against pathogenic infection. The thymic functions of iNKT1 cells are not fully elucidated, but subsets of thymic iNKT cells modulate CD8 T cell, dendritic cell, B cell, and thymic epithelial cell numbers or function. In this study, we show that a subset of murine thymic iNKT1 cells required TGF-ß-induced signals for their postselection development, to maintain hallmark TGF-ß-induced genes, and for expression of the adhesion receptors CD49a and CD103. However, the residency-associated receptor CD69 was not TGF-ß signaling-dependent. Recently described CD244+ c2 thymic iNKT1 cells, which produce IFN-γ without exogenous stimulation and have NK-like characteristics, reside in this TGF-ß-responsive population. Liver and spleen iNKT1 cells do not share this TGF-ß gene signature, but nonetheless TGF-ß impacts liver iNKT1 cell phenotype and function. Our findings provide insight into the heterogeneity of mechanisms guiding iNKT1 cell development in different tissues and suggest a close association between a subset of iNKT1 cells and TGF-ß-producing cells in the thymus that support their development.

BRWD1 establishes epigenetic states for germinal center initiation, maintenance, and function.

Germinal center (GC) B cells segregate into three subsets that compartmentalize the antagonistic molecular programs of selection, proliferation, and somatic hypermutation. In bone marrow, the epigenetic reader BRWD1 orchestrates and insulates the sequential stages of cell proliferation and Igk recombination. We hypothesized BRWD1 might play similar insulative roles in the periphery. In Brwd1 -/- follicular B cells, GC initiation and class switch recombination following immunization were inhibited. In contrast, in Brwd1 -/- GC B cells there was admixing of chromatin accessibility across GC subsets and transcriptional dysregulation including induction of inflammatory pathways. This global molecular GC dysregulation was associated with specific defects in proliferation, affinity maturation, and tolerance. These data suggest that GC subset identity is required for some but not all GC-attributed functions. Furthermore, these data demonstrate a central role for BRWD1 in orchestrating epigenetic transitions at multiple steps along B cell developmental and activation pathways.

Regulatory Non-Coding RNAs Modulate Transcriptional Activation During B Cell Development.

B cells play a significant role in the adaptive immune response by secreting immunoglobulins that can recognize and neutralize foreign antigens. They develop from hematopoietic stem cells, which also give rise to other types of blood cells, such as monocytes, neutrophils, and T cells, wherein specific transcriptional programs define the commitment and subsequent development of these different cell lineages. A number of transcription factors, such as PU.1, E2A, Pax5, and FOXO1, drive B cell development. Mounting evidence demonstrates that non-coding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), modulate the expression of these transcription factors directly by binding to the mRNA coding for the transcription factor or indirectly by modifying cellular pathways that promote expression of the transcription factor. Conversely, these transcription factors upregulate expression of some miRNAs and lncRNAs to determine cell fate decisions. These studies underscore the complex gene regulatory networks that control B cell development during hematopoiesis and identify new regulatory RNAs that require additional investigation. In this review, we highlight miRNAs and lncRNAs that modulate the expression and activity of transcriptional regulators of B lymphopoiesis and how they mediate this regulation.

BCR Affinity Influences T-B Interactions and B Cell Development in Secondary Lymphoid Organs.

B cells produce high-affinity immunoglobulins (Igs), or antibodies, to eliminate foreign pathogens. Mature, naïve B cells expressing an antigen-specific cell surface Ig, or B cell receptor (BCR), are directed toward either an extrafollicular (EF) or germinal center (GC) response upon antigen binding. B cell interactions with CD4+ pre-T follicular helper (pre-Tfh) cells at the T-B border and effector Tfh cells in the B cell follicle and GC control B cell development in response to antigen. Here, we review recent studies demonstrating the role of B cell receptor (BCR) affinity in modulating T-B interactions and the subsequent differentiation of B cells in the EF and GC response. Overall, these studies demonstrate that B cells expressing high affinity BCRs preferentially differentiate into antibody secreting cells (ASCs) while those expressing low affinity BCRs undergo further affinity maturation or differentiate into memory B cells (MBCs).