To induce allo-specific CD4+ T cells, BALB/c spleen cells were suspended in RPMI complete medium containing 1 g/ml FITC conjugated anti-CD4 (107 cells/ml) and incubated about snow for 30 min. referrals 1C4). The importance of Fas in B cell apoptosis is definitely supported by B cell build up and autoantibody production in mice with defective Fas or Fas ligand (FasL) genes or in humans with mutated Fas genes (5C8). Although FasCFasL relationships are important for T Famciclovir cell apoptosis after TCR cross-linking, B cell apoptosis induced by cross-linking of the B cell antigen receptor does not involve Fas (9), suggesting that B and T cells differ in Fas-dependent apoptosis. Since FasL manifestation is definitely undetectable in a variety of mouse and human being B cell lines (9, 10), B cells are likely to depend on apoptotic signals through Fas that are generated by relationships with FasL-bearing T cells (11, 12). Fas-mediated signaling in B cells appears to be regulated from the cell activation status. While resting B cells resist Fas cross-linkingCinduced death, mitogen-activated B cells are susceptible to Fas-mediated killing (13). Cross-linking of CD40 prospects to upregulation of Fas manifestation and level of sensitivity to Fas-mediated apoptosis (14, 15). The tasks of adhesion molecules or accessory molecules have not been characterized in CD4+ T cellCmediated B cell lysis from the Fas pathway Famciclovir (16). We examined the tasks of accessory molecules in Fas-mediated B cell lysis. We found that the relationships of LFA-1 on CD4+ T cells and intercellular adhesion molecule-1 and ICAM-2 on B cells are essential for the T cellC mediated B cell lysis. Deficiency in ICAM-1 prospects to decreased B cell apoptosis and the build up of B cells in vivo. MATERIALS AND METHODS Mice. 6C8-wk-old female C57BL/6, C57BL/6-ICAM-1?/?, B10.A, and BALB/c mice were from the (Pub Harbor, ME). Antibodies, Fusion Proteins, and Peptides. AntiCLFA-1 (M17.4), antiCICAM-1 (3E2), antiCICAM-2 (MIC2/4), anti-B7.1 (1G10), anti-B7.2 (GL1), anti-CD2 (RM2-5), anti-CD48 (HM48-1), anti-Fas (Jo2), anti-CD4 (GK1.5), anti-CD8 (53-6.7), antiCThy-1.2 (30-H12), anti-NK1.1 (PK136), anti-CD40 (3/12), anti-FasL, anti-CD16/32, PE conjugated anti-Fas, PE-conjugated antiCICAM-1, PE-conjugated antiCICAM-2, FITC-conjugated antiCLFA-1, FITC- or PE-conjugated anti-CD4, and PE-conjugated anti-CD3 were purchased from (San Diego, CA). FITC-conjugated F(ab)2 goat antiCmouse IgM was from CALTAG (South San Francisco, CA). FasCFc and TNFRCFc fusion proteins were gifts from Dr. D. Lynch (Immunex, Seattle, WA). The pigeon cytochrome C peptide (amino acid residues 88C104: KAERADLIAYLKQATAK) was synthesized from the Peptide Synthesis Facility (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). Purification and Activation of B Cells. Mouse spleen cells were suspended in RPMI 1640 medium comprising 20% FCS (5 106 cells/ml) and 10 ml cells were added to each 100 mm cells tradition dish. After incubation at 37C for 1 h, nonadherent cells were recovered and resuspended in HBSS medium (107/ml) comprising 10 g/ml of antiCThy-1.2, anti-CD4, anti-CD8, and anti-NK1.1 antibodies, and then incubated on snow for 45 min. Next, 1:10 Famciclovir low-tox-M rabbit match (Accurate Chemical and Technology Corp., Westbury, NY) was added and the cells were incubated at 37C for 1 h. Live cells were purified by Ficoll gradient separation and between 95 and 98% of the cells were positive for surface IgM when stained having a PE-conjugated F(ab)2 goat antiCmouse IgM (data not demonstrated). The cells (106 cells/ml) were resuspended in RPMI 1640 medium with 10% FCS, 0.2 mM glutamine, 5 10?4 M -mercaptoethanol (RPMI complete medium) containing 100 g/ml LPS, or 5 g/ml anti-CD40 and cultured at 37C for 2 or 3 3 d before use. Activation of T Cells and 51Cr-release Assay. A.E7 T cells were stimulated with antigen and IL-2 as previously described (17). To induce allo-specific CD4+ T cells, BALB/c spleen cells were suspended in RPMI total medium comprising 1 g/ml FITC conjugated anti-CD4 (107 cells/ml) and incubated on snow for 30 min. CD4+ cells were then purified with magnetic beads conjugated with sheep antifluorescein antibody (PerSeptive Diagnostics, Cambridge, MA). The CD4+ T cells were mixed with 3,000 Rad-irradiated C57BL/6 mouse spleen cells at a percentage of 1 1:5 and suspended in RPMI total medium (106 CD4+ T cells/ ml). After tradition at 37C for 5 d, live cells were purified and used as effector cells to assay B cell lysis. For 51Cr-release assay, B cells were incubated in RPMI medium comprising 5% Famciclovir FCS (5 106/ml) and 200 Ci/ml Na[51Cr]O4 (and and and and mice which gradually develop irregular B cell build up and autoimmunity depending on the background of the mouse strain (26). Although hard to verify experimentally, our result is definitely consistent Famciclovir with Rabbit Polyclonal to OR5AS1 the hypothesis that ICAM-1 is definitely important for B cell apoptosis mediated by T cells in vivo. However, no.
To induce allo-specific CD4+ T cells, BALB/c spleen cells were suspended in RPMI complete medium containing 1 g/ml FITC conjugated anti-CD4 (107 cells/ml) and incubated about snow for 30 min