Phenotype analysis and function of NK receptors in LGL patient. All experiments were performed in vitro using cells collected before treatment with tipifarnib. (A) The phenotype was determined by flow cytometry and dot plots are shown on CD56⁺/CD16⁺/CD3⁻ NK-cells (R2) from the NK-LGL patient (a) and healthy control (d). The reactivity of NK cells to anti-CD158a (KIR2DS1/KIR2DL1) and anti-CD158b (KIR2DS2/KIR2DL2/KIR2DL3) antibody in the patient (b and c, respectively) compared with a healthy donor (e and f, respectively). (B) RT-PCR of this patient (P) and a healthy control (N) for KIR2DS1 and KIR2DL1. Control (Bl, blank) is shown containing no template. (C) NK-cells (effector cells) from the LGL patient (■) and healthy donor () were used for reverse ADCC experiments. Effector cells were exposed to medium (negative control), isotype antibody control, anti-NKp46 antibody, anti-CD158a antibody, anti-CD158b antibody, and anti-CD16 antibody before interaction with the NK-resistant (FcR)–expressing P815 multiple myeloma cell line at an effector-to-target cell (E:T) ratio of 50:1. (D) NK cells from a healthy control or the LGL patient were used at 6:1, 12:1, 25:1, and 50:1 E:T ratios using both K562 (NK sensitive target, —) and CRL-2598 (endothelial cell line, ----) cells as the target. (E) NK cells were cultured in medium (no stimulation), admixed with CRL-2598 at an E:T cell ratio of 1:1, or cultured with staphylococcal enterotoxin B (SEB) as a positive control for IFNγ and TNFα release. Intracellular cytokine flow cytometry was used to detect these cytokines in the NK cells from the LGL patient (■) and a healthy donor (). (F) dnDAP10 and dnDAP12 viruses were engineered to carry a single Y→A mutation at the location of Y75A or a double mutation at both Y64A and Y75A within the YxxM activating ITAM motif; shown previously to be an effective inhibitor of endogenous ITAM signaling by other T-cell receptors and NK receptors.^16-19  NK cells from the LGL leukemia patient were treated under mock conditions, infected with recombinant CD56 vaccinia virus (CD56, control), dnDAP10 alone, dnDAP12 alone, or a combination of dnDAP10 plus dnDAP12 recombinant vaccinia viruses. These groups of effector cells were admixed at 6:1, 12:1, 25:1, and 50:1 E:T ratios with the CRL-2598 target cells. Cells were examined for cytotoxicity in 5-hour ⁵¹-Cr release assays (E:T ratio, 1:1).