Ly49D Receptor Expressed on Immature B Cells Regulates Their IFN- Secretion, Actin Polymerization, and Homing 1 Gili Hart, Liat Flaishon, Shirly Becker-Herman, and Idit Shachar 2 Low levels of IFN- secreted by immature B cells prevent their own migration and homing to the lymph nodes and premature encounter with Ag. In this study we followed the mechanism regulating IFN- secretion by immature B cells. We show that the MHC class I receptor, Ly49D, is expressed on immature B cells and is down-regulated during maturation. Activation of this receptor leads to increase in IFN- transcription and translation and results in the altered ability of B cells to polymerize actin in response to chemokine stimulation. Moreover, we show that H2-D blockage inhibits the ability of immature B cells to transcribe the IFN- gene and results in rescue of cytoskeletal rearrangement. Thus, Ly49D that is expressed on immature B cells recognizes MHC class I on the peripheral tissues, inducing the secretion of low levels of IFN- and thereby down-regulating immature B cell homing to the lymph nodes or to sites of inflammation. The Journal of Immunology, 2003, 171: 4630 – 4638. T he differentiation pathway from stem cell to mature B lymphocyte can be divided into several stages, character- ized by differentiation processes, proliferation phases, and control steps. Precursor B cells differentiate into immature B lym- phocytes after successfully expressing a surface Ig receptor (IgM). These immature B cells emerge from the bone marrow to the pe- riphery and migrate into the spleen for their final maturation step. This migration proceeds through the terminal branches of central arterioles to blood sinusoids of the marginal zone (1, 2). Before arriving at the spleen, immature B cells are excluded from non- splenic secondary lymphoid organs, which are specialized tissues for collecting Ags (3), and from sites of infection and inflamma- tion. In these secondary lymphoid organs, in which differentiation to a mature phenotype does not occur, Ag encounter could lead to effective clonal elimination because of a negative selection process. Previously we demonstrated that immature B cells can down- regulate their own integrin-mediated adhesion to the extracellular matrix and thereby suppress their migration into nonsplenic sites (4). This inhibition is mediated by IFN-, which is transcribed and secreted at low levels by immature B cells. The inhibitory signal of IFN- is transmitted through the IFN- receptor, whose engage- ment leads to inhibition of cytoskeleton rearrangement, which is required for promoting integrin-mediated adhesion and migration of B cells (5, 6). The NK-activating receptor, Ly49D, is a type II transmembrane glycoprotein of the C-type lectin superfamily that recognizes MHC class I proteins. Ly49D does not contain the cytoplasmic immune receptor tyrosine-based inhibitory motifs (ITIMs) 3 that are phos- phorylated upon stimulation, confirming that it is not an inhibitory receptor (7–9). This receptor was recently shown to be a potent inducer of IFN- expression. Furthermore, stimulation with mAb Ly49D on NK cells induces IFN- gene transcription and higher levels of IFN- protein following receptor ligation (10), although the function of this induction was not analyzed. The expression of Ly49 family proteins was described mostly on NK cells; however, a member of this family, Ly49A/D, was recently found on the peritoneal B1 subpopulation of B cells (11). In this study we in- vestigated whether Ly49D may be expressed on immature B cells and may control B cell homing. Our results show that immature B cells express Ly49D, which controls their IFN- secretion and consequently their actin polymerization and migration. Materials and Methods Mice C57BL/6, Ii -/- (12), IFN- -/- (Jackson), K b-/- and D b-/- (13) mice were used at 6 – 8 wk of age. The Animal Research Committee at the Weizmann Institute approved all animal procedures. Cells and Separation of B cells The murine pre-B cell-like lymphoma line, 70Z/3 (14) was grown in sus- pension culture at 37°C in RPMI 1640 medium containing 10% (v/v) FCS and 200 M 2-ME in the presence or absence of LPS (5 g/ml). Spleen cells were obtained as previously described (15). Control IgD - , IgD + CD21 + , and CD21-B were separated as previously described (16). CD19 + cells were separated using anti-CD19 magnetic beads (Miltenyi Biotec, Auburn, CA). Bone marrow cells were obtained from the femurs of control and Ii -/- mice. The marrow was rinsed with PBS, and the RBC were lysed. Pre-pro- and pro-B cells were separated by FACS sorting using anti-B220, CD19, Bp1, and IgM Abs (BD PharMingen, San Diego, CA). Bone mar- row immature B cells were purified by incubating bone marrow cells with anti-IgD-PE Ab (BD PharMingen) and separation with anti-PE magnetic beads using the MACS system (Miltenyi Biotec). After depletion of the mature IgD + B population, cells were incubated with anti-IgM-FITC Ab (BD PharMingen), and the positive population was separated with anti- FITC beads using the MACS system (Miltenyi Biotec). NK cells were purified using the anti-Dex5 magnetic beads (Miltenyi Biotec). 493 + cells were separated using anti-493-PE Abs (BD PharMingen), followed by sep- aration with anti-PE magnetic beads (Miltenyi Biotec). Department of Immunology, Weizmann Institute of Science, Rehovot, Israel Received for publication February 7, 2003. Accepted for publication August 28, 2003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Israel Science Foundation founded by the Academy of Sciences and Humanities and by the Minerva Foundation. I.S. is the incumbent of the Alvin and Gertrude Levine Career Development Chair of Cancer Research. 2 Address correspondence and reprint requests to Dr. Idit Shachar, Department of Immunology, Weizmann Institute of Science, 76100 Rehovot, Israel. E-mail address: idit.shachar@weizmann.ac.il 3 Abbreviations used in this paper: ITIM, immune receptor tyrosine-based inhibitory motif; HPRT, hypoxanthine phosphoribosyltransferase; LN, lymph nodes; SDF-1, stromal cell-derived factor 1. The Journal of Immunology Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00