Figure 2. Antigen induces qualitatively distinct biologic responses in immature and mature B cells. (A) Expression of CD69, CD86, and MHC class II was analyzed in immature and mature B cells from 3-83 md and a-HEL Tg animals after stimulation with peptidic 3-83 antigen–dextran conjugates (500 ng/106 cells) or HEL (1,000 ng/106 cells), respectively for 0 (dotted lines), 12 (dashed lines), or 24 h (bold lines); surface expression of markers was monitored by immunofluorescence with analysis by flow cytometry. (B) RT-PCR analysis of RAG-2 and CD19 mRNA in im- mature and mature B cells from 3-83 md and a-HEL Tg animals before and after 18-h stimulation with their respective antigens. was observed over the 18-h period in mature or immature cells (data not shown). Thus, as previously shown (16), an- tigen-induced cross-linking of the BCR in immature cells results in the induction of receptor editing, and this re- sponse is not seen in mature, naive B cells. Together, the data demonstrate two mutually exclusive response patterns in mature and immature cells. These distinct patterns may ensure that autoreactive, immature B cells do not mature into autoreactive, mature B cells and that only B cells that have been properly vetted to eliminate those that are au- toreactive can effectively upregulate ligands for CD28 (CD86) and T cell antigen receptor (MHC class II) and thus participate in a productive immune response. The observed differences in the biologic responses of mature and immature cells may result from differences in BCR coupling to proximal signal transduction pathways. Alternatively and/or additionally, these B cells may be pro- grammed to respond differently to the same constellation of “second” messengers. Calcium mobilization plays an im- portant role in triggering a wide array of cellular processes (48), including RAG induction (49), and could drive the BCR-mediated expression for CD69, MHC class II, and CD86. To determine if calcium mobilization is sufficient to initiate the biologic responses of immature and mature B cells, ionomycin was used to induce graded rises in the in- tracellular free Ca21 concentration ([Ca21] ). Ionomycin i induced comparable increases in [Ca21]i in immature and mature B cells at each concentration used (Fig. 3 A). Anal- ysis of marker induction after 18 h of ionomycin stimula- tion demonstrated profound upregulation of CD86 in ma- ture B cells but only at doses of ionomycin that induced increases of .600 nM [Ca21]i; small increases were ob- served at doses that raised [Ca21]i by 80–120 nM, and no upregulation was seen when [Ca21]i was raised by 50 nM (Fig. 3 B). Consistent with the effects of antigen (Fig. 2), ionomycin did not induce CD86 in immature B cells. Sim- ilar observations were made for MHC class II and CD69 (data not shown). This demonstrates the sufficiency of cal- cium mobilization in antigen activation of CD86 and MHC class II expression in mature B cells. As previously reported (49), elevation of [Ca21]i induced RAG-2 expression in immature B cells. However, as shown in Fig. 3, this response followed much smaller in- creases in [Ca21]i than required to induce CD86 in mature cells: RAG-2 expression was increased 1.7–2.3-fold after 80–120-nM rises in [Ca21]i and 3.1–5-fold after a 50-nM rise in [Ca21]i (Fig. 3 C). High doses of ionomycin (induc- ing .600 nM increase in [Ca21]i) only raised RAG-2 mar- ginally and were accompanied by an increased apoptotic response (two- to threefold increase over unstimulated control), whereas little to no apoptosis was induced at 100 and 10 nM ionomycin (data not shown and reference 49). This suggests that extreme increases in [Ca21]i in immature B cells (possibly reflecting a signal through a high-avidity interaction with self-antigen) leads to an apoptotic rather than a receptor editing response. No RAG-2 induction was detected in mature B cells. These data demonstrate that increases in [Ca21]i can mediate the unique, antigen-induced changes in CD86 and RAG-2 seen in mature and imma- ture B cells, respectively. These distinct biologic responses could be generated via two mechanisms. First, BCR stimu- lation could lead to activation of different transcription fac- tors that are developmental stage specific in their expres- sion. Second, different genetic loci could be more accessible in immature and mature B cells by regulated demethylation and chromatin remodeling; it has been demonstrated that these processes are involved in differential induction of egr-1 in immature versus mature B cells (9), allelic exclusion dur- ing B cell development (10), and regulation of cytokine gene expression in Th1 and Th2 cells (50). According to this model, BCR stimulation would activate calcium-depen- dent transcription factors (e.g., NFAT [nuclear factor of activated T lymphocytes]), leading to rapid transcription of the accessible loci in each cell type; these would include rag in immature cells and CD86 and MHC class II in mature cells. The ability of HEL to induce transient upregulation 752 BCR Signaling in Immature B Cells