Figure 3. Calcium elevation induces RAG-2 expression in immature B cells and CD86 in mature B cells, and these responses exhibit different sensi- tivities to [Ca21]i. (A) [Ca21]i was measured in immature and mature B cells from 3-83 md Tg animals after stimulation with ionomycin. (B) Immature (filled bars) and mature (open bars) B cells from 3-83 md and a-HEL Tg animals were stimulated with varying doses of ionomycin (10–1,000 nM) or their respective antigens (500 ng peptidic 3-83 antigen–dextran/106 cells and 1,000 ng/ml HEL) for 18 h; induction of CD86 is expressed as fold increase in mean fluorescence intensity over unstimulated control. (C) RT-PCR analysis of mRNA expression of RAG-2 was normalized to that of CD19; in- duction of RAG-2 is expressed as fold increase over unstimulated control. Asterisks indicate absence of data because RAG-2 could not be detected in mature B cells before or after ionomycin. Results are representative of three experiments. of CD86 in immature cells appears inconsistent with both of these hypotheses. The high frequency of cells displaying this response excludes the possibility that it is a consequence of contaminating mature cells. It is possible that the accessibil- ity of the loci is not absolute. Perhaps a sufficiently strong antigen receptor signal can lead to some expression of a rel- atively inaccessible locus. More studies are required to re- solve this question. The observations presented here are consistent with the behavior of the 3-83 md Ig Tg model of tolerance induc- tion wherein immature B cells encounter autoantigen in the BM and are induced to edit their receptors (1, 3). However, they appear inconsistent with the HEL/a-HEL model. Specifically, findings that HEL induces RAG in im- mature B cells from a-HEL mice predict that B cells in HEL/a-HEL double-Tg mice should undergo editing in vivo. Evidence for receptor editing is observed when HEL is expressed in a membrane-associated form (51) but is less obvious when HEL is present as a secreted protein (2). In the latter double-Tg animals, however, a decline in the number and frequency of HEL-specific B cells is observed, suggesting that a portion of B cells may have been deleted (e.g., the IgMhigh cells) (2). It is possible that soluble HEL may not reach sufficient concentrations in the BM milieu to generate a strong enough signal to induce RAG in most developing B cells. Upon exiting the BM, B cells would 753 Benschop et al. encounter concentrations of soluble HEL in the periphery sufficient to render them anergic. Considering that the sol- uble HEL Tg is under the control of a metallothionein promoter, highest secretion of soluble HEL would be ex- pected in the liver, kidney, or pancreas (52, 53). Thus, it is conceivable that immature B cells would only encounter low concentrations of soluble HEL in the BM. In addition, it could be hypothesized that only a limited number of B cells (e.g., only IgMhigh B cells) receive a signal strong enough to induce receptor editing; these cells are then ei- ther deleted or successfully edited away from their specific- ity for HEL but are not seen in vivo because of the size of the anergic B cell population. Mice expressing Ig Tg recep- tors for another soluble but multivalent ligand, DNA, dem- onstrate that B cells are rendered anergic when recognizing single-stranded DNA (low affinity), whereas B cells bind- ing double-stranded DNA (high affinity) are deleted (4, 54). This supports the hypothesis that antigen valency in combination with receptor affinity determines the mecha- nism of tolerance induction in immature B cells. Resolu- tion of these and other alternatives awaits further study. The data presented also demonstrate a clear difference in sensitivity of the responses of mature and immature B cell to rises in intracellular calcium. Differences in these re- sponses may be linked to the observed difference in toler- ance sensitivity (5–7), where low signals in immature B