IntroductionA fundamental hallmark of humoral immunity is the ability to produce class-switched antibodies that have enhanced affinity for antigen. This process, termed affinity maturation, allows clonally selected B cells to refine their response to theoretically target any antigen with high specificity. After activation, B cells mutate the immunoglobulin (Ig) locus in a process known as somatic hypermutation (SHM). 1 Mutated clones that have acquired increased affinity for antigen preferentially expand over their low-affinity counterparts. The selection process occurs in the germinal center (GC), a transient microenvironment that forms in secondary lymphoid tissue shortly after antigen exposure. 2 The GC provides a competitive setting for B cells whereby ineffectual clones are actively cleared from the system via apoptosis, although the processes that govern this selection still remain vague.SHM and class switch recombination (CSR) are initiated by the enzyme activation-induced cytidine deaminase (AID), 3,4 which deaminates cytidines to uridines specifically at the Ig locus. 5-10 AID-generated uridines are then engaged by various DNA repair pathways that either lead to the generation of point mutations in the antibody variable region or recombinogenic events that lead to CSR. AID Ϫ/Ϫ mice lack mutations at their Ig locus and are incapable of producing class-switched antibodies. 4 Interestingly, these mice were previously shown to harbor an abnormally high proportion of splenic GC B cells. 11 This phenotype is also recapitulated in humans with AID deficiencies. 3 Although a link between reduced gut immunity (resulting from an inability to produce mucosal IgA) and peripheral GC formation was hypothesized to account for these abnormalities, this remains to be conclusively proven, and the possibility of a B cell-intrinsic effect that could explain the profound expansion of GC B cells has not been examined.GC B cells represent a unique lymphoid compartment of actively proliferating cells where numerous apoptotic factors must synergize to induce the elimination of nonproductive clones. Factors contributing to the intrinsic pathway of apoptosis, such as Bcl-2, Bcl-x L , and Bim, 12-17 and the extrinsic pathway, such as Fas, [18][19][20][21][22] have been implicated in GC selection. The unique physiology of these cells makes them highly susceptible to disease progression, often serving as etiologic sites for autoimmune and malignant B cells. 13,[23][24][25][26] Recent evidence has implicated AID as a fundamental contributor to the genetic aberrations that lead to these disease phenotypes. 24,[27][28][29] Given the importance of apoptosis as a parameter for both GC B-cell selection and lymphomagenesis, we investigated the relationship between AID-induced DNA mutation and cell death to understand how this enzyme may impinge on survival and death within the GC niche. Here we report that many of the potentially harmful AID-induced genetic alterations may indeed lead to the death of these cells within the GC. Methods Mice...
SummaryIn 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.
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.
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) β 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 LTβ 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.
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.
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