www.nature.com/natureimmunology • october 2002 • volume 3 no 10 • nature immunology Akanksha Chaturvedi, Zaved Siddiqui, Fahri Bayiroglu and Kanury V. S. Rao Published online: 16 September 2002, doi:10.1038/ni839 The induction of a humoral response depends upon efficient cross-linking by antigen of surface immunoglobulin on primary B lymphocytes. We demonstrate here the presence of a glycosylphos- phatidylinositol-linked isoform of membrane IgD (mIgD) receptors on murine resting B cells.This subset was constitutively localized to cell membrane raft microdomains. Its stimulation resulted in the activation of cAMP-dependent signaling pathways, which integrated with signals derived from the transmembrane mIgD receptors. This, in turn, provided a mechanism by which the activation status of the target cells could be variably regulated. Thus, by partitioning receptor activity, preimmune B cells can moderate the extent to which they are activated, depending upon the strength of the antigenic stimulus. Immunology Group, International Centre for Genetic Engineering and Biotechnology,Aruna Asaf Ali Marg, New Delhi 110067, India. Correspondence should be addressed to K.V. S. R. (kanury@icgeb.res.in). A GPI-linked isoform of the IgD recep- tor regulates resting B cell activation Both the development and physiological function of B lymphocytes is tightly regulated by signals generated from the B cell antigen receptor complex (BCR) 1 . Whereas their maintenance in the periphery depends on low-intensity constitutive signaling—by an as yet unknown mecha- nism—through the BCR 2 , signaling induced upon cross-linking of the BCR by antigen stimulates a cellular response that can either be apop- totic or proliferative, depending on the maturation status of the cell 1 . The BCR is a complex hetero-oligomeric structure in which the lig- and binding and signal transduction properties are compartmentalized into distinct receptor subunits 3,4 . The membrane immunoglobulin (mIg) unit binds ligand, whereas the associated disulfide-bonded heterodimer of CD79a (Ιgα) and CD79b (Igβ) transduces signals. Cross-linking of mIg molecules induces phosphorylation of the immunoreceptor tyro- sine-based activation motif (ITAM) sequences within CD79a and CD79b by the Src family of protein tyrosine kinases (PTKs) 3,4 . This results in the recruitment of at least four major intracellular signaling pathways, including those that are dependent upon phospholipase C-γ (PLC-γ), the Rho family of GTPases, the Ras GTPase and the phos- pholipid kinase phosphatidylinositol-3 kinase (PI3K) 3,4 . Each of these pathways plays a critical role in defining B cell responsiveness to both activating and differentiating stimuli. Although information on BCR-dependent signaling pathways and their regulation continues to accumulate 1–5 , the mechanisms by which these biochemical processes interact to modulate B cell fate and func- tion are not clear. Indeed, how plasticity in BCR signaling is achieved with a limited number of downstream pathways remains a key con- founding issue in B cell signaling. We demonstrate here the presence of a glycosylphosphatidylinositol (GPI)-linked isoform of IgD (mIgD) receptors on murine resting B cells. Activation of GPI-linked mIgD induced intracellular cAMP accumulation, which triggered pathways that engaged in cross-talk with the known BCR-dependent signaling pathways. The extent of this cross-talk defined the spectrum of cell surface activation markers that were induced. Our results suggest that it is this segregation of receptor activities that facilitates the graded responsiveness of preimmune B cells, depending on the strength of the antigenic stimulus. Results Activation of resting B lymphocytes Immunization with a T cell–dependent antigen induces B cells to fol- low two compartmentalized and distinct pathways of differentiation. After activation of B cells in the T cell–rich extrafollicular sites, foci of antigen-specific antibody-forming cells appear in the periphery of the periarteriolar lymphoid sheath. In a second pathway, some of these cells within the next few days move to the germinal center (GC) in the primary B cell follicles, where the processes of somatic mutation and differentiation of antigen-activated B cells are initiated 6,7 . To examine phenotypic alterations that characterize the above two pathways in resting B lymphocytes, we used the F(ab´)2 fragment of a monoclonal anti-IgD as a surrogate antigen (referred to hereafter as anti-IgD). Purified resting B cells were cocultured with anti-IgD and monitored for cell-surface modulation of specific proteins associated with activation and GC B cells. We found that mIgD expression was down-regulated, whereas surface amounts of CD86 and major histo- compatibility complex (MHC) class II molecules were enhanced 8–10 . Several additional markers—CD80, peanut agglutinin receptor (PNA- R), CD24 and GL7—that characterize murine GC B cells were also up-regulated (Web Fig. 1a online), which confirmed published data 11–19 . Thus, stimulation of murine resting B cells with anti-IgD induced phenotypic changes that characterized both the activated and the GC B cell. In addition to phenotypic changes, optimal stimulation of resting B cells with anti-IgD also conferred upon these cells the ability to popu- late GCs in a T cell–dependent manner. Thus, stimulated BALB/c IgH a B cells, loaded with a synthetic T cell epitope peptide (peptide CT3) 20 reconstituted GCs when transferred into CT3-primed BALB/c IgH b mice (Web Fig. 1b online). In contrast, peptide-loaded unstimulated B cells or stimulated B cells loaded with an irrelevant peptide were unable ARTICLES 951 © 2002 Nature Publishing Group http://www.nature.com/natureimmunology