Distinct Signal Thresholds for the Unique Antigen Receptor–linked Gene Expression Programs in Mature and Immature B Cells By Robert J. Benschop,* Doron Melamed,* David Nemazee,*‡ and John C. Cambier*‡ From the *Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center and the ‡Department of Immunology, University of Colorado Health Sciences Center, Denver, Colorado 80206 Summary Although it is well established that immature B lymphocytes are exquisitely sensitive to toler- ance induction compared with their mature counterparts, the molecular basis for this difference is unknown. We demonstrate that signaling by B cell antigen receptors leads to distinct and mutually exclusive biologic responses in mature and immature B cells: upregulation of CD86, CD69, and MHC class II in mature cells and receptor editing in immature cells. These re- sponses can be induced simply by elevation of intracellular free calcium levels, as occurs after receptor aggregation. Importantly, induction of immature B cell responses requires much smaller increases in intracellular free calcium than does induction of mature B cell responses. These differences in biologic response and sensitivity to intracellular free calcium likely con- tributes to selective elimination at the immature stage of even those B cells that express low af- finity for self-antigens. Key words: signal transduction • B cells • receptor editing • B cell antigen receptor • development A tnoedsseevnetlioapl fperaotuterectiovfetihme mimunmituynetos yfostreemignispiatsthaobgielintys while remaining unresponsive to self-antigens. In the B lymphocyte compartment, self-tolerance can be achieved by antigen stimulation of immature B cells (1–4). Findings that immature B cells are exquisitely sensitive to tolerance induction compared with mature B cells (5–7) suggest that antigen–receptor signaling may be qualitatively or quantita- tively distinct in these populations. Although developmen- tally programmed differences in B cells have been addressed in a number of studies (8–10), underlying differences in sig- nal transduction and transcriptional regulation after antigen stimulation are unknown. Using antireceptor antibodies, it was shown that whereas aggregation of the B cell antigen receptor (BCR)1 induces proliferation of mature B cells (11, 12), immature cells fail to progress beyond the early G1 phase of the cell cycle (13) and in some cases initiate apoptosis (14). Low to absent expression of bcl-2 may pre- dispose immature B cells to the latter response (15). Unlike the situation in their mature counterparts, aggregation of 1Abbreviations used in this paper: BCR, B cell antigen receptor; BM, bone marrow; HEL, hen egg lysozyme; ITAM, immunoreceptor tyrosine- based activation motif; PLC, phospholipase C; RAG, recombinase activa- tor gene; Tg, transgenic. the BCR on immature cells activates receptor editing via induced expression of rag genes (16, 17) but fails to initiate expression of egr-1, c-fos, and c-myc (12, 13). Differential susceptibility to tolerance induction may re- flect differences in proximal events in antigen–receptor sig- nal transduction. In mature and immature B cells, the BCR is composed of membrane (m)Ig noncovalently associated with disulfide-linked heterodimers of immunoreceptor ty- rosine-based activation motif (ITAM)-containing CD79a and CD79b (18–20). BCR aggregation by antigen or anti- receptor antibody mediates clustering of associated Src fam- ily kinase molecules, leading to phosphorylation of ITAM tyrosines. ITAM tyrosines are required for receptor-medi- ated activation of gene expression (21), and their phosphor- ylation leads to the recruitment of additional Src family ki- nases as well as the tyrosine kinase Syk (22–24). Upon recruitment, Syk becomes tyrosyl phosphorylated and acti- vated, which is essential for activation of Btk and the phos- pholipase C (PLC)g pathway (23, 25). Recent studies have defined a B cell–unique linker molecule, BLNK or Slp-65, which serves as a substrate for Syk and is essential for the activation of PLCg2 (26–28). Subsequent tyrosyl phosphor- ylation of CD19 leads to phosphatidylinositol 3 kinase acti- vation and generation of phosphatidylinositol-3,4,5-P3 (PtdIns3,4,5P3) (29, 30). Phosphorylation of Btk and PLCg1 and 2 and translocation of these enzymes to the 749 J. Exp. Med. The Rockefeller University Press • 0022-1007/99/09/749/08 $5.00 Volume 190, Number 6, September 20, 1999 749–756 http://www.jem.org