Figure 1. Generation of immature B cells in BM cultures containing IL-7. B cells isolated from BM from 3-83 md Tg and non-Tg littermate mice (B10.D2) were cultured for 7 d in the presence of IL-7. Cells, as well as mature splenic B cells, were analyzed by flow cytometry. Negative control his- tograms are shown as dashed lines. Shown in the upper right corner of each panel is the percentage of cells positive for the respective marker along with the relative mean fluorescence intensity (MFI). pared with mature B cells), whereas they were negative for CD21/35 and CD23, which are only expressed on mature B cells (38–40). Equivalent phenotypes were seen when BM cells from a-HEL Tg animals were grown in IL-7 (data not shown). These data confirm a previous report (34) that the presence of mIgM transgenes is sufficient to accelerate B cell maturation through the pro- and pre-B cell stages to an immature phenotype. Although it is diffi- cult to exclude the possibility that the cells grown in this system are developmentally pre-B cells that simply express a Tg BCR on their surfaces, these cells express all known markers associated with the immature B cell stage (41, 42). We then compared early biologic responses of the re- spective B cell populations to antigen, analyzing induction of rag gene expression, indicative of clonal elimination by receptor editing (3), and CD69, CD86, and MHC class II expression, indicative of BCR signal transduction leading to initiation of an immune response. Immature B cells from BM cultures and mature splenic B cells from 3-83 md or a-HEL Tg animals were incubated with antigen, and the ex- pression of activation markers was assessed (Fig. 2 A). Ma- ture B cells upregulated CD69, CD86, and MHC class II molecules within 12 h of stimulation, as previously shown (43–45). In immature 3-83 md Tg cells, however, only slight upregulation of CD69 and CD86 was detected, and this was seen only at 12 h after stimulation. HEL stimula- tion of immature, a-HEL Tg cells also led to upregulation of these markers at 12 h. The responses were more pro- 751 Benschop et al. nounced than those of immature 3-83 md cells but much less than those of mature a-HEL Tg B cells (Fig. 2 A). No antigen induction of MHC class II was observed in imma- ture cells. The difference in the degree of upregulation be- tween the 3-83 md and a-HEL Tg cells is probably due to the affinity of the transgene-encoded receptor for its ligand (z2 3 1025 M for 3-83 md [reference 46] and 2 3 1029 M for a-HEL [reference 2]). These results demonstrate that immature B cells are unable to effectively upregulate mole- cules essential for T–B cell collaboration; this probably pre- vents their participation in T cell–dependent immune re- sponses. Recent studies demonstrate that binding of antigen to mIgM on immature B cells induces recombinase gene ex- pression and L chain receptor rearrangements (16, 17), a process that results in alteration of receptor specificity, termed receptor editing (3). This process leads to clonal elimination of immature cells but may also contribute to increasing diversity in a germinal center reaction (47). To compare the ability of immature and mature B cells to un- dergo a receptor editing response to antigen, we tested for antigen-induced reactivation of rag genes using RT-PCR. These analyses demonstrated a greater than twofold (nor- malized to CD19 mRNA levels) increase in RAG-2 ex- pression upon antigen stimulation in immature cells (Fig. 2 B), resembling responses observed in vivo (3). No RAG-2 mRNA was detectable in mature B cells before or after 18-h antigen stimulation. Finally, no antigen-induced cell death