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BCR editing in the bone marrow contributes to B cell tolerance by orchestrating secondary Ig rearrangements in self-reactive B cells. We have recently shown that loss of the BCR or a pharmacologic blockade of BCR proximal signaling pathways results in a global "back-differentiation" response in which immature B cells down-regulate genes important for the mature B cell program and up-regulate genes characteristic of earlier stages of B cell development. These observations led us to test the hypothesis that self-Ag-induced down-regulation of the BCR, and not self-Ag-induced positive signals, lead to Rag induction and hence receptor editing. Supporting this hypothesis, we found that immature B cells from xid (x-linked immunodeficiency) mice induce re-expression of a Rag2-GFP bacterial artificial chromosome reporter as well as wild-type immature B cells following Ag incubation. Incubation of immature B cells with self-Ag leads to a striking reversal in differentiation to the pro-/pre-B stage of development, consistent with the idea that back-differentiation results in the reinduction of genes required for L chain rearrangement and receptor editing. Importantly, Rag induction, the back-differentiation response to Ag, and editing in immature and pre-B cells are inhibited by a combination of phorbol ester and calcium ionophore, agents that bypass proximal signaling pathways and mimic BCR signaling. Thus, mimicking positive BCR signals actually inhibits receptor editing. These findings support a model whereby Ag-induced receptor editing is inhibited by BCR basal signaling on developing B cells; BCR down-regulation removes this basal signal, thereby initiating receptor editing.

Receptor editing is an important mechanism to modify the Ag specificity of newly developing B cells that are reactive with self-Ags. Previous studies have suggested that late immature B cells, bearing high levels of IgM on their cell surface, are unable to initiate receptor editing and instead are deleted by apoptosis. Using the hen egg lysozyme transgenic system, we show that IgM(high) late-immature B cells are fully capable of receptor editing to soluble self-Ag. This was demonstrated by the induction of new endogenous light chain locus rearrangements and by a single-cell flow cytometric assay using a recombination-activating gene 2-green fluorescence protein reporter transgene. These studies suggest that the developmental window available for immature B cells to edit their Ig receptors, at least in response to certain soluble Ags, extends through the IgM(high) late immature B cell stage.

Receptor editing in the bone marrow (BM) serves to modify the Ag receptor specificity of immature self-reactive B cells, while anergy functionally silences self-reactive clones. Here, we demonstrate that anergic B cells in hen egg lysozyme Ig (HEL-Ig)/soluble HEL double transgenic mice show evidence of having undergone receptor editing in vivo, as demonstrated by the presence of elevated levels of endogenous kappa light chain rearrangements in the BM and spleen. In an in vitro IL-7-driven BM culture system, HEL-Ig BM B cells grown in the presence of soluble HEL down-regulated surface IgM expression and also showed induction of new endogenous kappa light chain rearrangements. Using a panel of soluble protein ligands with reduced affinity for the HEL-Ig receptor, the editing response was shown to correlate in a dose-dependent fashion with the strength of signaling through the B cell receptor. The finding that the level of B cell receptor cross-linking sufficient to induce anergy in B cells is also capable of engaging the machinery required for receptor editing suggests an intimate relationship between these two mechanisms in maintaining B cell tolerance.

Ligation of the B cell antigen receptor (BCR) complex initiates tyrosine phosphorylation of the receptor's transducer components, Ig-alpha and Ig-beta and tyrosine kinase-dependent accumulation of GTP-bound, activated p21ras. The mechanism of receptor coupling to p21ras activation and the roles of Ig-alpha and Ig-beta are unknown. The results reported here indicate that the resting, nonphosphorylated BCR associates with the Grb-2/Sos-linker SHC via the Ig-alpha immunoreceptor-based tyrosine activation motif (ITAM). Ig-alpha specificity of this interaction is determined by the sequence DCSM found in Ig-alpha, but not Ig-beta. Tyrosine phosphorylation of Ig-alpha and Ig-beta ITAM allows recruitment of SHC, which now binds directly to both Ig-alpha and Ig-beta via a phosphotyrosine/SH2 interaction. In confirmation of recent studies by Saxton et al. (J. Immunol. 1994. 153: 623) receptor ligation leads to tyrosine phosphorylation of SHC and to the formation of a phospho-SHC/Grb2/Sos complex. In view of previous studies which demonstrated p21ras co-capping with ligated BCR, the data presented here suggest that Ig-alpha/beta- and SHC tyrosine phosphorylation-dependent recruitment of the Grb2/Sos complex to the receptor can occur and may provide a mechanism by which the nucleotide exchange activity of Sos could mediate activation of BCR-localized p21ras.

Coligation of the Fc receptor on B cells, Fc gamma RIIB1, with the B cell antigen receptor (BCR) leads to abortive BCR signaling. Here it was shown that the Fc gamma RIIB1 recruits the phosphotyrosine phosphatase PTP1C after BCR coligation. This association is mediated by the binding of a 13-amino acid tyrosine-phosphorylated sequence to the carboxyl-terminal Src homology 2 domain of PTP1C and activates PTP1C. Inhibitory signaling and PTP1C recruitment are dependent on the presence of the tyrosine within the 13-amino acid sequence. Inhibitory signaling mediated by Fc gamma RIIB1 is deficient in motheaten mice which do not express functional PTP1C. Thus, PTP1C is an effector of BCR-Fc gamma RIIB1 negative signal cooperativity.