YT () or P-YT (?) cells were incubated with anti-2B4 mAb, C1

YT () or P-YT (?) cells were incubated with anti-2B4 mAb, C1.7, for 45 min prior to 4 hr incubation with target cells. found complete loss of transcriptional activity, including the two-fold increase due to PMA induction of PKC. The present study indicated that PKC may play an important role in 2B4 signalling and activator protein-1 activation. Introduction Natural killer (NK) cells are bone-marrow-derived lymphocytes that function as key players in innate immunity by recognizing viral, bacterial and parasitic infections and neoplastic target cells.1,2 The major effector functions of NK cells are cytotoxicity and cytokine release, including interferon- (IFN-), tumour necrosis factor-, granulocyteCmacrophage colony-stimulating factor as well as matrix metalloproteinases.3C6 NK cell recognition is regulated by specific receptors that, upon interaction with their respective ligands, may send stimulating or inhibitory signals.7C9 An important activating receptor expressed on NK cells is 2B4 (CD244).10 2B4 is a member of the CD2 Prosapogenin CP6 subset of the immunoglobulin superfamily.11,12 2B4 is expressed on NK cells, monocytes, basophils and on subsets of T-cell receptor (TCR) + T cells and CD8+ Itgb8 T cells.13 Ligation Prosapogenin CP6 of 2B4 either by a monoclonal antibody (mAb) or by its natural ligand, CD48, on NK cells results in increased cytotoxicity and secretion of IFN-.6,13C15 Recent findings indicate that 2B4 may function as an inhibitory molecule at early stages of NK cell differentiation.16 Previously, we investigated the possible role of various signalling molecules that may be involved in the activation of NK cells via 2B4. We found through the treatment of YT cells with various specific inhibitors that 2B4-stimulation of YT cells in spontaneous and antibody-dependent cytotoxicity is Ras/Raf dependent and involves multiple mitogen-activated protein kinase (MAPK) signalling pathways [extracellular regulated kinase1/2 (ERK1/2) and p38].17 Inhibition of transcription Prosapogenin CP6 also inhibited 2B4-mediated cytotoxicity, implying that there are transcriptional events critical in regulating NK cell function. When we examined the effect of these inhibitors on 2B4-mediated secretion of IFN-, only inhibitors of transcription and p38 inhibited 2B4-mediated IFN- release. These results indicate that 2B4-mediated activation of NK cell cytolytic function and cytokine production may be regulated by several distinct pathways. Thus, our studies on the signalling of 2B4 revealed that NK cell cytolytic function and cytokine production may be regulated by distinct pathways in activated NK cells.17 Another recent study also indicates that receptor signalling in NK cells can be functionally complex depending on the state of NK cells.18 Rajagopalan gene transcription. Phorbol 12-myristate 13-acetate (PMA) induction resulted in a more than two-fold general increase of 2B4 transcription. However, base substitution mutations of the activator protein-1 (AP-1) binding site at (?106 to ?100) in the promoter resulted in the complete loss of transcriptional activity, including the two-fold increase due to PMA induction of PKC. Materials and methods Cell lines, antibodies and chemicalsYT (human NK cell line), K562 (human erythroleukaemia cell line) and P815 (mouse lymphoma cell line) cells were maintained in complete medium [RPMI-1640 supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 2 mm glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, 10 mm HEPES and 10 mm non-essential amino acids]. Cells were maintained at 37 in a humidified 5% CO2/95% air incubator. Cell culture reagents were obtained from Life Technologies (Gaithersburg, MD) unless otherwise noted. The mAb that specifically recognizes human 2B4 (C1.7),19 was purchased from Coulter (Orlando, FL). All enzymes were purchased from New England.