plasma membrane, where they bind PtdIns3,4,5P3, leads to PLCg1/2-mediated hydrolysis of phosphoinositides, gen- eration of inositol-1,4,5-triphosphate (Ins1,4,5P3), and mo- bilization of calcium (31–33). Comparative analysis of signal transduction has revealed that immature B cells mobilize intracellular calcium in response to BCR aggregation by anti- receptor antibodies but fail to produce levels of Ins1,4,5P3 found in mature B cells (12). These findings suggest that (a) certain intermediary events required for antigen receptor– mediated phosphoinositide hydrolysis are impaired in im- mature cells and (b) immature B cell calcium stores are more sensitive to Ins1,4,5P3. Neither the ability of cognate antigen to induce distinct signal transduction responses in these cells nor the role of these responses in determining the unique biologic outcome of BCR signaling in imma- ture cells has been assessed. Here we report a comparative analysis of events after an- tigen-induced signal transduction in mature and immature B cells. Results indicate that antigen stimulates distinct and mutually exclusive biologic responses in immature and ma- ture B cells, receptor editing and upregulation of activation markers (CD86, CD69, and MHC class II), respectively. Both responses can be attributed to receptor-mediated ele- vation of intracellular free calcium levels ([Ca21] ), as eleva- i tion of [Ca21]i, using agents such as ionomycin, is sufficient to trigger them. Importantly, induction of receptor editing in immature B cells requires much lower increases in [Ca21]i than does the induction of activation marker ex- pression in mature B cells. These results demonstrate differ- ential activation of gene expression in immature and ma- ture B cells after BCR aggregation and that immature B cells display increased sensitivity in the induction of these genetic programs. Materials and Methods Animals and Tissue Culture. B10.D2nSn/J and two Ig trans- genic (Tg) mouse models were used in the experiments: the 3-83 md Ig Tg mice express IgM and IgD specific for H-2Kk (1), and Ig Tg anti–hen egg lysozyme (a-HEL) mice express IgM and IgD specific for HEL (2). Bone marrow (BM) was obtained from 3–4-mo-old mice; a single-cell suspension was prepared by flush- ing femurs with IMDM to dislodge cells, followed by gentle de- aggregation using a 5-ml syringe. BM was depleted of erythro- cytes using Gey’s solution, washed twice in IMDM, and cultured at 5 3 105 cells/ml per 10-cm petri dish (7% CO2, 378C) in IMDM containing 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mg/ml gentamycin, 2 mM l-glutamine, 1 mM sodium pyru- vate, 50 mM 2-ME, 10% FCS (Hyclone), and 50–100 U IL-7 (34) (derived from culture supernatant of J558L cells transfected with mIL-7 cDNA, a gift from Dr. A. Rolink, Basel Institute for Immunology, Switzerland). Typically, cells were harvested after 6–7 d in culture, washed twice with IMDM, and used in subse- quent experiments. Resting splenic B cells (r . 1.066) were ob- tained from 3–4-mo-old adult mice and prepared as previously described (35). Cell viability was assessed using trypan blue dye exclusion. Reagents. Antigens: an H-2Kk mimetic peptide (CSGFGG- FQHLCCGAAGA) that binds specifically to the 3-83 receptor (35, 36) was synthesized and multimerized by coupling to N-eth- ylmaleamide–activated dextran (a gift from CorTech, Inc.) at a 100:1 peptide/dextran molar ratio; HEL was purchased from Sigma Chemical Co. Antibodies directed against the following molecules were used: MHC class II (Iab/d; D3.137); CD69 (H1.2F3, PharMingen); CD86 (GL-1, PharMingen); CD21/35 (CR2/CR1; 7G6, PharMingen); CD19 (1D3, PharMingen); IgM (b-7-6); IgD (JA12.5); CD22 (CY34.1.2); CD23 (IgE Fc receptor; B3B4, PharMingen); CD45 (I3/12.5); and CD45R (anti-B220; RA3-3A1 and RA3-6B2). Ionomycin was purchased from Calbiochem Corp.; propidium iodide was from Sigma Chemical Co. Phenotypic Analysis. Cells were washed once, resuspended in PBS containing 1% BSA and 0.1% sodium azide, and incubated with an optimal amount of biotinylated or directly fluorescinated antibody. Cells were incubated for 30 min at 48C and washed twice in PBS/BSA/azide. In the case of biotinylated antibodies, cells were incubated as before with avidin–FITC or avidin–PE (Becton Dickinson). After washing, cells were analyzed on a flow cytometer. Histograms were constructed based on analysis of 10,000 cells. Calcium Mobilization. For measurements of [Ca21]i, cells were loaded with Indo-1AM (Molecular Probes, Inc.), suspended at 106 cells/ml in IMDM, and stimulated with either antigen or anti-IgM antibody (b-7-6). Mean [Ca21]i was evaluated over time using a flow cytometer (Model 50H; Ortho Diagnostic Sys- tems Inc.) with appended data acquisition system and MultiTime software (Phoenix Flow Systems) as previously described (35). Analysis for Recombinase Activator Gene 2 Expression. Levels of recombinase activator gene (RAG)-2 and CD19 mRNA were determined by RT-PCR assay as described in detail elsewhere (34). To obtain a semiquantitative estimate of gene expression, the signal for RAG-2 was normalized to the CD19 signal. Results and Discussion Analysis of molecular mechanisms underlying the re- sponses of immature and mature B cells to antigen required generation of homogeneous populations of antigen-specific cells. To generate antigen-specific immature B cells, cells were isolated from BM of 3-83 md (specific for H-2Kk) Ig Tg (1) or a-HEL Ig Tg (2) mice and cultured for 6–7 d in IL-7–containing medium. Over this period, the total number of cells per dish increased from 5 3106 to 30–40 3 106 (data not shown). The percentage of B cells (CD45R1) increased from 12–18% at the onset of the culture to .95% after 5 d, demonstrating the selective outgrowth of IL-7– responsive B cell precursors (34, 37). Phenotypic analysis further indicated that few cells from non-Tg BM grown in IL-7 progressed to the IgM1IgD2 immature B cell stage compared with the Ig Tg animals (Fig 1). Cells from Tg BM grown under these conditions were .90% IgMlow IgD2; upon removal of IL-7, cells progressed through IgMhighIgD2, the immature stage, to IgMhighIgDlow, the transitional stage, and finally to mature B cells (data not shown and reference 34). Relative expression of mIgM and mIgD transgenes as well as other markers was consistent with development of a homogeneous population of imma- ture cells in these cultures (Fig. 1): cells were CD45low (also CD45Rlow, not shown), CD22low, and CD19low (all com- 750 BCR Signaling in Immature B Cells