Within inflammatory environments, B cells encountering foreign or self-Ag can develop tertiary lymphoid tissue expressing activation-induced cytosine deaminase (AID). Recently, this DNA-modifying enzyme was detected in nonlymphoid cells within several inflamed tissues and strongly implicated in malignant transformation. This study examines whether a cyclooxygenase 2 (COX-2) pathway, often linked to inflammation, influences AID expression in activated B lymphocytes. In this paper, we report that dividing human B cells responding to surrogate C3d-coated Ag, IL-4, and BAFF express AID, as well as COX-2. A progressive increase in AID with each division was paralleled by a division-related increase in a COX-2–linked enzyme, microsomal PGE2 synthase-1, and the PGE2R, EP2. Cells with the greatest expression of AID expressed the highest levels of EP2. Although COX-2 inhibitors diminished both AID expression and IgG class switching, exogenous PGE2 and butaprost, a selective EP2 agonist, augmented AID mRNA/protein and increased the numbers of IgG+ progeny. Despite the latter, the proportion of IgG+ cells within viable progeny generally declined with PGE2 supplementation. This was not due to PGE2-promoted differentiation to plasma cells or to greater downstream switching. Rather, because phosphorylated ataxia telangiectasia mutated levels were increased in progeny of PGE2-supplemented cultures, it appears more likely that PGE2 facilitates AID-dependent DNA double-strand breaks that block B cell cycle progression or promote activation-induced cell death, or both. Taken together, the results suggest that a PGE2 feed-forward mechanism for augmenting COX-2 pathway proteins promotes progressively increased levels of AID mRNA, protein, and function.

Resting mature human B cells undergo a dynamic process of clonal expansion, followed by clonal contraction, during an in vitro response to surrogate C3d-coated antigen and innate immune system cytokines, IL-4 and BAFF. We here explore the mechanism for clonal contraction through following the time- and division-influenced expression of several pro- and anti-apoptotic proteins within CFSE-labeled cultures. Several findings, involving both human and mouse B cells, show that a mitochondria-dependent apoptotic pathway involving p53 contributes to the high AICD susceptibility of replicating blasts. Activated B cell clones exhibit elevated p53 protein and elevated mRNA/protein of pro-apoptotic molecules known to be under direct p53 transcriptional control, Bax, Bad, Puma, Bid, and pro-caspase 6, accompanied by reduced anti-apoptotic Bcl-2. Under these conditions, Bim levels were not increased. Findings that full length Bid protein significantly declines in AICD-susceptible replicating blasts, while Bid mRNA does not, suggests that Bid is actively cleaved to short-lived, pro-apoptotic tBid. AICD was diminished, albeit not eliminated, by p53 siRNA transfection, genetic deletion of p53, or Bcl-2 overexpression. DNA damage is a likely trigger for p53-dependent AICD since susceptible lymphoblasts expressed significantly elevated levels of both phospho-ATMser1980 and phospho-H2AXser139. Deficiency in activation-induced cytosine deaminase (AID) diminishes but does not ablate murine B cell AICD, indicating that AID-induced DNA damage is only in part responsible. Evidence for p53-influenced AICD during this route of TI clonal expansion raises the possibility that progeny bearing p53 mutations might undergo positive selection in peripherally inflamed tissues with elevated levels of IL-4 and BAFF.

Since its discovery in follicular lymphoma cells at the breakpoint t(14;18), Bcl-2 has been studied extensively in many basic and clinical science settings. Bcl-2 can locate as an integral mitochondrial membrane component, where its primary role is to block apoptosis by maintaining membrane integrity. Here we show that Bcl-2 also can position on the outer cell surface membrane of B cells from patients with chronic lymphocytic leukemia (B-CLL) and certain other leukemias that do not classically possess the chromosomal breakpoint t(14;18). Although low levels of Bcl-2 can be detected on the surface membrane of apparently healthy leukemic and normal B cells, expression of Bcl-2 correlates best with spontaneous or induced apoptosis. Notably, upon induction of apoptosis, B-CLL cells were much more efficient in upregulating surface Bcl-2 than normal B cells. It is not clear if this surface membrane expression is a passive consequence of the apoptotic process or an active attempt by the B cell to abort cell death by stabilizing the plasma membrane.

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