These cells continuously went through the cell cycle in the following 11 h. HeLa cells was preferentially found in the early S phase. Furthermore, in CDK2 hypomorphic cells there was reduced nuclear AID accumulation. Thus, our data are compatible with the idea that division-linked Ig class switching is in part due to CDK2-regulated AID nuclear access at the G1/S border. Introduction Activated B cells can switch their Ig expression from IgM and IgD to IgG, IgE, or IgA through class switch recombination (CSR). The main regulator of CSR is activation-induced cytidine deaminase (AID) (1, 2), which deaminates cytosine to uracil in switch (S) region DNA (3, 4). This leads to recruitment of factors involved in DNA repair and double-strand breaks (DSBs) are created. A mechanism similar to classical nonhomologous end joining (C-NHEJ) is employed to join donor S region to a downstream acceptor S region, with looping out the intervening DNA sequence. In the absence of key factors in C-NHEJ, an alternative end joining (A-EJ) pathway is suggested to mediate the SCS joining with increased use of microhomology in the SCS junctions (5). In this way, the V(D)J unit is joined with close proximity to a downstream C region. As a result, B cells are able to maintain the Ag specificity while changing Ab effector function. Little is known about how Ig class switching is coordinated with cell cycle control, although cell proliferation is required for Ig class switching (6). It was shown that two to three rounds of cell division was required before switching to IgG and IgA and five to six rounds for IgE (7, 8). This requirement is partly because the AID expression level is upregulated after two cell divisions. Additionally, AID expression levels increase with Tedizolid Phosphate successive divisions, providing a possible explanation to proliferation-dependent class switching (9). Although Tedizolid Phosphate there are some early studies suggesting that CSR may occur in the S phase of the cell cycle (10, 11), there is evidence suggesting that AID-dependent DSBs in the IgH locus occur mainly in the G1 phase (12, 13). However, AID is present all through the cell cycle in activated B cells. Because of the existence of the G1/S checkpoint, it would appear unlikely that B cells can pass through the cell cycle checkpoint before CSR is achieved and all the breaks are repaired. Therefore, CSR was postulated to occur in the G1 phase. However, other studies indicate that the G1/S checkpoint is not fully functional in activated B cells and that AID-dependent DSBs can leak into S phase (14C16). This raises the question whether Ig class switching itself is subjected to cell cycle regulation, for example by cyclin-dependent kinases (CDKs). CDKs are the central players in regulating cell cycle progression. Several CDKs have been identified in mammalian cells with functional redundancy and tissue specificity (17). Recent studies suggest that CDKs may also be involved in the DNA damage response and apoptosis. For example, mammalian CDK2 plays an important role in DNA repair by enhancing the NHEJ pathway (18). So far, it is still unclear how CDKs are involved in these processes. Similar to exogenous DNA damage reagents, class switching also induces a DNA damage response and triggers the same set of repair proteins. Instead of faithful repair, these proteins promote a deletional recombination event in switching cells. However, to our knowledge there is no information whether CDKs are also involved in regulating Ig class switching. In the present study, we examined the early kinetics of Tedizolid Phosphate Ig class switching in mouse splenic B cells in vitro. We give evidence that Ig class switching ends in the early S phase. Experiments are presented that CDK2 can control access of AID to the S region. Our data thus provide an explanation for proliferation-dependent switching. Materials and Methods Tedizolid Phosphate Mice C57BL/6 mice were purchased from Scanbur and bred Tedizolid Phosphate in pathogen-free conditions at the animal facility of the Department of Molecular Biosciences, Wenner-Gren Institute, Stockholm University. All animal experiments were approved by the Stockholm North Animal Ethics Committee. B cell isolation and cell culture Enriched spleen B cells were cultured by treatment with Abs to CD4, CD8, CD90.2, and CD11b (BD Biosciences or eBioscience) and low-toxin rabbit complement (Cedarlane) followed by Percoll-gradient separation. Rabbit Polyclonal to RPS20 Cells were cultured at 2C4 105 cells/ml. Monoclonal rat anti-mouse CD40 (1C10) was purified as described (19) and was used at 10C20 g/ml. IL-4 (PeproTech) was used at 8 ng/ml. LPS O55:B5 (Sigma-Aldrich) was used at 10 g/ml. RPMI 1640 culture medium was supplemented with sodium pyruvate, penicillin-streptomycin, l-glutamine, 2-ME, and 10%.