Rituximab® was used in the concentration 0·1 μg/ 0·2 × 106 target cells. After counting and centrifugation

(200 g, 10 min) the target cells were adjusted to 2 × 106 cells/ml AIM-V medium. Ten μl antibodies were added to 0·2 × 106 target cells (0·1 ml) and incubated for 15 min at room temperature. The effector cells were counted and resuspended in AIM-V to a final concentration of 2 × 106 cells/ml; 0·2 × 106 of these cells were added to the antibody-coated target cells and after centrifugation (30 g for 3 min) the cells were incubated in a humidified incubator with 5% CO2 at 37°C for 2 h. After one wash in phosphate-buffered saline (PBS) the cells were ready for staining with the monoclonal antibodies given below and subsequent flow cytometry. Samples were labelled with monoclonal antibodies for 30 min in the dark at 4°C, washed once in PBS (pH 7·4) and finally resuspended ERK inhibitor in PBS. The following monoclonal mouse antibodies and other markers were used: anti-CD3 fluorescein isothiocyanate (FITC) (clone UCHT1, IgG1, F0818; Dako, Glostrup, Denmark), anti-CD56 phycoerythrin (PE) [clone c5·9, immunoglobulin (Ig)G2b, R7251; Dako], anti-CD107a Alexa 647 (clone eBio H4A3, IgG1, #51-1079; eBioscience, San Diego, CA, USA), anti-CD8 PC7 (clone SFCI21Thy2D3, IgG1, #737661; Beckman Coulter, Indianapolis, IN, USA), CD2/CD2R (CD2 clone L303·1,CD2R clone L304·1; #340366; BD Pharmingen, San Jose, CA, USA), AlexaFluor 647 mouse IgG1k isotype

control (clone MOPC-21, #557714; BD Pharmingen) and 7-aminoactinomycin D (7-AAD) (# 555816; BD Via Probe, BD Pharmingen). Flow cytometric analyses were performed using a Cytomics FC500 five-colour flow cytometer Sirolimus (Beckman Coulter) equipped with two lasers, an argon laser (488 nm) and a HeNe laser (633 nm). FlowJo software version 9·3 (Tree Star, Inc., Ashland, OR, USA) was used for data analysis. A total of 20 000 events were collected for further analysis. NK cells were defined as CD3−/CD56+ lymphocytes. Effector cells alone were used to define the initial CD107a level of positive NK cells or CD8+ cells. In Fig. 1,

we present examples of spontaneous up-regulation of CD107a on effector cells, as well as FMO (fluorescence-minus one), an isotype antibody control for CD107a and 7AAD viability staining. Using the CD2/CD2R system, we also performed positive effector cell control experiments, confirming the PRKACG activation potential of the effector cells (data not shown). In 51Cr cytotoxicity assays results are given normally as percentages of cell killing, with the maximum killing as a basic value. In assays measuring granularity by CD107a this is not meaningful, as a maximum value is difficult, if not impossible, to give. The results are therefore given as increments, where either the NK value or the value with preimmune serum is subtracted from the value with immune serum. The increase can also be given as a ratio between, for example, immune sera and preimmune sera.