The role of apoptosis in affinity maturation was investigated by determining

The role of apoptosis in affinity maturation was investigated by determining the affinity of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific antibody-forming cells (AFCs) and serum antibody in transgenic mice that overexpress a suppressor of apoptosis, Bcl-xL, in the B cell compartment. CD40 extends the survival of cultured GC cells and upregulates Bcl-2 14. Reciprocally, a positive regulator of apoptotic cell death, Fas (CD95), is highly expressed in GC B cells 1819, and GC B cells are susceptible to Fas-mediated apoptosis in vitro 2021. Despite these in vitro models, studies of genetically modified mice do not support major roles for Bcl-2 or Fas in affinity maturation. Neither the overexpression of Bcl-2 nor the lack of Fas has detectable effects on the affinity maturation of serum antibodies 1922. These findings raise the possibility that affinity maturation is achieved solely by positive selection, or that other apoptosis-regulatory molecules are involved in the negative selection process. A homologue of transgenic mice, which support higher numbers and longer-lived splenic AFCs 22. Frequencies and kinetics of specific BM AFCs were indistinguishable between transgenic and control mice (Fig. 3 C). The expanded CS-088 splenic AFC pool in transgenic mice resulted in a minor increase in serum antibody titers on day 12, but later levels of antibody did not differ significantly between transgenic and control mice. In both groups, antibody concentrations were at maximal levels on day 12 and then slowly declined to about one third of this peak by day 69 (Fig. 3 D). Thus, overexpression of Bcl-xL modestly expands recruitment into the splenic AFC pool but does not change cellular recruitment into GCs, entry into the BM AFC pool, or maintenance of long-lasting serum antibody. bcl-xL Transgenic Mice Have Fewer Apoptotic Cells in GCs. GCs contain more apoptotic lymphocytes as determined by TUNEL than other regions of spleen 17. These TUNEL+ cells are thought to represent lymphocytes that have been negatively selected during the GC response. We performed TUNEL assays on spleen sections from transgenic and control mice to determine if the small addition of transgenic Bcl-xL expressed in GC B cells was sufficient to reduce programmed cell death. TUNEL+ cells in GCs from both groups were counted by microscopic examination, and the frequency of TUNEL+ cells per unit area was calculated. These frequencies were subdivided into 12 categories, and the distribution histogram for CS-088 each category was plotted (Fig. 4). GCs from < 0.01) than those from control mice (Fig. 4). The most common apoptotic index in wild-type animals was 2.0C2.5 TUNEL+ cells/unit area but only 1 1.0C1.5 in the transgenics. Perhaps more significantly, >20% of GCs in control mice contained >3 TUNEL+ cells/unit area, whereas only 5% of GCs in > 0.05) in the ratios of replacement versus silent mutations (R/S ratios) in CDRs (Table ). Other characteristics indicative of high-affinity, NP-specific B cells, e.g., the fraction of rearrangements containing DFL16.1 and the YYGS CDR3 motif, were also similar in both groups. Thus, cellular recruitment, V(D)J hypermutation, and positive selection in GCs are unaffected by the = 5) and transgenic (= 5) mice by cell sorting 11. Typically, at day 69 after immunization >50% of sorted cells from both groups of mice secreted IgG1 antibody specific for NP. Enriched BM AFC populations were subjected to a reverse Influenza B virus Nucleoprotein antibody transcription PCR that preferentially amplifies cDNA representing rearrangements of the V186.2 and V3 subfamilies of VH gene segments joined to C1 11. Amplified VDJ rearrangements were cloned and sequenced to identify the VH and D gene segments used and any mutations present. Table summarizes this work and shows that only half (11/21) of the VDJ sequences recovered from and transgenes also CS-088 act differently during negative selection in immature B cells, as CS-088 transgenic Bcl-xL has the ability to block negative selection and promote developmental maturation, whereas autoreactive cells transgenic for remain arrested in development 4243. Given the similar reciprocal expression of and in GC B cells and CS-088 pre-B cells, may have a.