Immunoglobulin (Ig) isotype switching is a recombination event that changes the constant domain of antibody genes and is catalyzed by activation-induced cytidine deaminase (AID). Upon recruitment to Ig genes, AID deaminates cytidines at switch (S) recombination sites, leading to the formation of DNA breaks. In addition to their role in isotype switching, AIDinduced lesions promote Igh-cMyc chromosomal translocations and tumor development. However, cMyc translocations are also present in lymphocytes from healthy humans and mice, and thus, it remains unclear whether AID directly contributes to the dynamics of B cell transformation. Using a plasmacytoma mouse model, we show that AID +/ ؊ mice have reduced AID expression levels and display haploinsuffi ciency both in the context of isotype switching and plasmacytomagenesis. At the Ig loci, AID +/ ؊ lymphocytes show impaired intra-and interswitch recombination, and a substantial decrease in the frequency of S mutations and chromosomal breaks. In AID +/ ؊ mice, these defects correlate with a marked decrease in the accumulation of B cell clones carrying Igh-cMyc translocations during tumor latency. These results thus provide a causality link between the extent of AID enzymatic activity, the number of emerging Igh-cMyc -translocated cells, and the incidence of B cell transformation.

Previous studies using cytochalasins and latrunculin B, inhibitors of actin polymerization, showed that filamentous (F)-actin had a negative regulatory role in Fc 4 receptor I (Fc 4 RI) signaling. How F-actin is involved in regulating the activation of mast cells is unknown. In this study we investigated the role of F-actin in mast cell activation induced by aggregation of the glycosylphosphatidylinositol (GPI)-anchored proteins Thy-1 and TEC-21, and compared it to activation via Fc 4 RI. Pretreatment of rat basophilic leukemia cells with latrunculin B inhibited the Thy-1-induced actin polymerization and elevated the Thy-1-mediated secretory and calcium responses. Inhibition of actin polymerization followed by Thy-1 aggregation resulted in an increased tyrosine phosphorylation of Syk, phospholipase C + (PLC + ), Gab2 and linker for activation of T cells (LAT) adapters, and some other signaling molecules. Enzymatic activities of phosphatidylinositol 3-kinase, PLC + , and phosphatase SHP-2 were also upregulated, but tyrosine phosphorylation of ezrin was inhibited. Similar changes were observed in Fc 4 RI-activated cells. Significant changes in intracellular distribution, tyrosine phosphorylation, and/or enzymatic activities of signaling molecules occurred in latrunculinpretreated cells before cell triggering. The combined data suggest that actin polymerization is critical for setting the thresholds for mast cell signaling via aggregation of both Fc 4 RI and GPI-anchored proteins.

Gangliosides released from tumor cells, as well as administered exogenously, suppress the immune responses by largely unknown mechanisms. We show here that a pretreatment of rat basophilic leukemia cells with isolated brain gangliosides inhibited the release of preformed secretory mediators from cells activated via FcεRI but not Thy-1 glycoprotein. Exogenously administered gangliosides also affected the cell-substrate adhesion and the levels of polymeric filamentous actin in Ag-activated cells. Although the production of phosphoinositides was also decreased, enzymatic activity of phosphatidylinositol 3-kinase was not inhibited. Gangliosides had no or only marginal effect on the association of aggregated FcεRI with glycosphingolipid-enriched membranes and on tyrosine phosphorylation of FcεRI and the linker for activation of T cells. Though pretreatment with gangliosides did not inhibit the association of linker for activation of T cells with phospholipase C (PLC)γ1 and PLCγ2, tyrosine phosphorylation of these enzymes, as well as their enzymatic activities and association with detergent-insoluble signaling assemblies were reduced. This resulted in a decreased production of inositol 1,4,5-trisphosphate and an inhibition of Ca2+ mobilization. The combined data support the concept that exogenously administered gangliosides interfere with those properties of glycosphingolipid-enriched membranes that are important for the formation of plasma membrane-associated signaling assemblies containing PLCγ but not for initial tyrosine phosphorylation of FcεRI subunits.