SSB1/SSB2 Proteins Safeguard B Cell Development by Protecting the Genomes of B Cell Precursors.

Induction of programmed DNA damage and its recognition and repair are fundamental for B cell development. The ssDNA-binding protein SSB1 has been described in human cells as essential for the recognition and repair of DNA damage. To study its relevance for B cells, we recently developed Ssb1 -/- and conditional Ssb1 -/- mice. Although SSB1 loss did not affect B cell development, Ssb1 -/- cells exhibited compensatory expression of its homolog SSB2. We have now generated Ssb2 -/- mice and show in this study that SSB2 is also dispensable for B cell development and DNA damage response activation. In contrast to the single loss of Ssb1 or Ssb2, however, combined SSB1/2 deficiency caused a defect in early B cell development. We relate this to the sensitivity of B cell precursors as mature B cells largely tolerated their loss. Toxicity of combined genetic SSB1/2 loss can be rescued by ectopic expression of either SSB1 or SSB2, mimicked by expression of SSB1 ssDNA-binding mutants, and attenuated by BCL2-mediated suppression of apoptosis. SSB1/2 loss in B cell precursors further caused increased exposure of ssDNA associated with disruption of genome fragile sites, inefficient cell cycle progression, and increased DNA damage if apoptosis is suppressed. As such, our results establish SSB1/2 as safeguards of B cell development and unveil their differential requirement in immature and mature B lymphocytes.

Lineage-Specific Genes Are Prominent DNA Damage Hotspots during Leukemic Transformation of B Cell Precursors.

In human leukemia, lineage-specific genes represent predominant targets of deletion, with lymphoid-specific genes frequently affected in lymphoid leukemia and myeloid-specific genes in myeloid leukemia. To investigate the basis of lineage-specific alterations, we analyzed global DNA damage in primary B cell precursors expressing leukemia-inducing oncogenes by ChIP-seq. We identified more than 1,000 sensitive regions, of which B lineage-specific genes constitute the most prominent targets. Identified hotspots at B lineage genes relate to DNA-DSBs, affect genes that harbor genomic lesions in human leukemia, and associate with ectopic deletion in successfully transformed cells. Furthermore, we show that most identified regions overlap with gene bodies of highly expressed genes and that induction of a myeloid lineage phenotype in transformed B cell precursors promotes de novo DNA damage at myeloid loci. Hence, we demonstrate that lineage-specific transcription predisposes lineage-specific genes in transformed B cell precursors to DNA damage, which is likely to promote the frequent alteration of lineage-specific genes in human leukemia.

A TNF-α-CCL20-CCR6 axis regulates Nod1-induced B cell responses.

Innate immune responses provoke the accumulation of leukocytes at sites of inflammation. In addition to monocytes and granulocytes, B cells also participate in antimicrobial innate immune responses; however, the mechanisms for accumulation of B cells to sites of inflammation are not well understood. To study B cell accumulation following systemic inflammation, we used a model synthetic ligand that stimulates a specific pattern recognition molecule, nucleotide-binding oligomerization domain-containing protein 1 (Nod1). Upon exposure to Nod1 agonists, both B cells and neutrophils rapidly accumulate within the spleen, and dendritic cells migrate into the periarterial lymphoid sheath. Nod1 stimulation led to a marked increase in several chemokines within the spleen, including CXCL13, CCL2, and CCL20. Whereas the lymphotoxin pathway was critical for the induction of the B cell chemoattractant CXCL13 in response to Nod1 agonists, B cell accumulation within the spleen following Nod1-induced systemic inflammation was independent of the lymphotoxin pathway. In contrast, a CCR6/CCL20 chemokine loop instructed rapid increase of B cells in the spleen in response to systemic administration of Nod1 agonists in a TNF-α-dependent manner. Moreover, CCR6 was required to regulate Nod1-mediated B cell responses. These results reveal a novel mechanism of B cells during inflammation and shed light on how B cells participate in innate immune responses to microbial stimulation.

AID and caspase 8 shape the germinal center response through apoptosis.

Germinal centers (GCs) are clusters of activated B cells that form in secondary lymphoid organs during a T-dependent immune response. B cells enter GCs and become rapidly proliferating centroblasts that express the enzyme activation-induced deaminase (AID) to undergo somatic hypermutation and class-switch recombination. Centroblasts then mature into centrocytes to undergo clonal selection. Within the GC, the highest affinity B cell clones are selected to mature into memory or plasma cells while lower affinity clones undergo apoptosis. We reported previously that murine Aicda(-/-) GC B cells have enhanced viability and accumulate in GCs. We now show that murine Aicda(-/-) GC B cells accumulate as centrocytes and inefficiently generate plasma cells. The reduced rate of plasma cell formation was not due to an absence of AID-induced DNA lesions. In addition, we show that the deletion of caspase 8 specifically in murine GC-B cells results in larger GCs and a delay in affinity maturation, demonstrating the importance of apoptosis in GC homeostasis and clonal selection.

AID-expressing germinal center B cells cluster normally within lymph node follicles in the absence of FDC-M1+ CD35+ follicular dendritic cells but dissipate prematurely.

Upon activation with T-dependent Ag, B cells enter germinal centers (GC) and upregulate activation-induced deaminase (AID). AID(+) GC B cells then undergo class-switch recombination and somatic hypermutation. Follicular dendritic cells (FDC) are stromal cells that underpin GC and require constitutive signaling through the lymphotoxin (LT) ß receptor to be maintained in a fully mature, differentiated state. Although it was shown that FDC can be dispensable for the generation of affinity-matured Ab, in the absence of FDC it is unclear where AID expression occurs. In a mouse model that lacks mature FDC, as well as other LT-sensitive cells, we show that clusters of AID(+)PNA(+)GL7(+) Ag-specific GC B cells form within the B cell follicles of draining lymph nodes, suggesting that FDC are not strictly required for GC formation. However, later in the primary response, FDC-less GC dissipated prematurely, correlating with impaired affinity maturation. We examined whether GC dissipation was due to a lack of FDC or other LTß receptor-dependent accessory cells and found that, in response to nonreplicating protein Ag, FDC proved to be more critical for long-term GC maintenance. Our study provides a spatial-temporal analysis of Ag-specific B cell activation and AID expression in the context of a peripheral lymph node that lacks FDC-M1(+) CD35(+) FDC and other LT-sensitive cell types, and reveals that FDC are not strictly required for the induction of AID within an organized GC-like environment.

Secondary B cell receptor diversification is necessary for T cell mediated neuro-inflammation during experimental autoimmune encephalomyelitis.

BACKGROUND: Clinical studies of B cell depletion in Multiple Sclerosis (MS) have revealed that B Lymphocytes are involved in the neuro-inflammatory process, yet it remains unclear how B cells can exert pro- and anti-inflammatory functions during MS. Experimental Autoimmune Encephalomyelitis (EAE) is an animal model of MS whereby myelin-specific T cells become activated and subsequently migrate to the Central Nervous System (CNS) where they perform pro-inflammatory functions such as cytokine secretion. Typically EAE is induced by immunization of mice of a susceptible genetic background with peptide antigen emulsified in Complete Freund's Adjuvant. However, novel roles for B-lymphocytes in EAE may also be explored by immunization with full-length myelin oligodendrocyte glycoprotein (MOG) that contains the B cell conformational epitope. Here we show that full length MOG immunization promotes a chronic disease in mice that depends on antigen-driven secondary diversification of the B cell receptor. METHODS: Activation-Induced Deaminase (AID) is an enzyme that is essential for antigen-driven secondary diversification of the B cell receptor. We immunized AID(-/-) mice with the extracellular domain (amino acids 1-120) of recombinant human MOG protein (rhMOG) and examined the incidence and severity of disease in AID(-/-) versus wild type mice. Corresponding with these clinical measurements, we also evaluated parameters of T cell activation in the periphery and the CNS as well as the generation of anti-MOG antibodies (Ab). CONCLUSIONS: AID(-/-) mice exhibit reduced severity and incidence of EAE. This suggests that the secondary diversification of the B cell receptor is required for B cells to exert their full encephalogenic potential during rhMOG-induced EAE, and possibly also during MS.

Lymphotoxin-sensitive microenvironments in homeostasis and inflammation.

Stromal cell microenvironments within lymphoid tissues are designed to support immune cell homeostasis and to regulate ongoing immune responses to pathogens. Such stromal cell networks have been best characterized within lymphoid tissues including the spleen and peripheral lymph nodes, and systems for classifying stromal cell phenotypes and functions are emerging. In response to inflammation, stromal cell networks within lymphoid tissues change in order to accommodate and regulate lymphocyte activation. Local inflammation in non-lymphoid tissues can also induce de novo formation of lymphoid aggregates, which we term here "follicle-like structures." Of note, the stromal cell networks that underpin such follicles are not as well characterized and may be different depending on the anatomical site. However, one common element that is integral to the maintenance of stromal cell environments, either in lymphoid tissue or in extra-lymphoid sites, is the constitutive regulation of stromal cell phenotype and/or function by the lymphotoxin (LT) pathway. Here we discuss how the LT pathway influences stromal cell environments both in homeostasis and in the context of inflammation in lymphoid and non-lymphoid tissues.

The mismatch repair pathway functions normally at a non-AID target in germinal center B cells.

Deficiency in Msh2, a component of the mismatch repair (MMR) system, leads to an approximately 10-fold increase in the mutation frequency in most tissues. By contrast, Msh2 deficiency in germinal center (GC) B cells decreases the mutation frequency at the IgH V region as a dU:dG mismatch produced by AID initiates modifications by MMR, resulting in mutations at nearby A:T base pairs. This raises the possibility that GC B cells express a factor that converts MMR into a globally mutagenic pathway. To test this notion, we investigated whether MMR corrects mutations in GC B cells at a gene that is not mutated by AID. Strikingly, we found that GC B cells accumulate 5 times more mutations at a reporter gene than during the development of the mouse. Notably, the mutation frequency at this reporter gene was approximately 10 times greater in Msh2(-/-) compared with wild-type GC B cells cells. In contrast to the V region, the increased level of mutations at A:T base pairs in GC B cells was not caused by MMR. These results show that in GC B cells, (1) MMR functions normally at an AID-insensitive gene and (2) the frequency of background mutagenesis is greater in GC B cells than in their precursor follicular B cells.

AID constrains germinal center size by rendering B cells susceptible to apoptosis.

The germinal center (GC) is a transient lymphoid tissue microenvironment that fosters T cell-dependent humoral immunity. Within the GC, the B cell-specific enzyme, activation-induced cytidine deaminase (AID), mutates the immunoglobulin locus, thereby altering binding affinity for antigen. In the absence of AID, larger GC structures are observed in both humans and mice, but the reason for this phenomenon is unclear. Because significant apoptosis occurs within the GC niche to cull cells that have acquired nonproductive mutations, we have examined whether a defect in apoptosis could account for the larger GC structures in the absence of AID. In this report, we reveal significantly reduced death of B cells in AID(-/-) mice as well as in B cells derived from AID(-/-) bone marrow in mixed bone marrow chimeric mice. Furthermore, AID-expressing B cells show decreased proliferation and survival compared with AID(-/-) B cells, indicating an AID-mediated effect on cellular viability. The GC is an etiologic site for B-cell autoimmunity and lymphomagenesis, both of which have been linked to aberrant AID activity. We report a link between AID-induced DNA damage and B-cell apoptosis that has implications for the development of B-cell disorders.

Phylogenetic analysis of Hepatozoon species (Apicomplexa: Adeleorina) infecting frogs of Nova Scotia, Canada, determined by ITS-1 sequences.

Species of Hepatozoon are apicomplexan parasites infecting tetrapod vertebrates and hematophagous arthropods. Two species, Hepatozoon catesbianae and Hepatozoon clamatae, have been described inhabiting the erythrocytes of bullfrogs and green frogs. A number of characteristics typically used to distinguish between members of this genus are shared between these 2 species, prompting speculation as to whether or not these organisms are in fact distinct species. To test the species distinction, bullfrogs and green frogs were captured at various sites across Nova Scotia, blood samples were collected, and DNA was extracted from samples containing parasites. The internal transcribed spacer 1 (ITS-1) from geographically diverse samples of both species was amplified by PCR, sequenced, and analyzed. ITS-1 sequences from the 2 species revealed single-nucleotide polymorphisms at 6 sites. Phylogenetic analysis of these molecular data and cytopathological features place isolates of each species in separate monophyletic groups. Comparison of the ITS-1 sequences between isolates from Nova Scotia and Ontario revealed that ITS-1 sequences of H. catesbianae from a previous study were mischaracterized as being those of H. clamatae. Phylogenetic data based on molecular variation and cytopathological features from this study provide the strongest evidence to date supporting the distinction between these 2 species.