Accuri cflow

HCMV HLA-A2 restricted peptide VLEETSVML-specific T cell lines were generated by stimulating PBMC with irradiated (6000 rad) peptide-coated (100 μg/ml for 1 hr at 37°C) autologous fibroblasts in RPMI supplemented with heat-inactivated 10% fetal calf serum, 2% human AB serum (RPMI-AB) and 10 IU/ml recombinant IL-2 (rIL-2). Cultures were re-fed every 3-4 days with an equal volume of RPMI-AB containing 20 IU/ml rIL-2.
CD107 T cell mobilization assays were performed similarly to NK cell mobilization assays. Briefly, autologous fibroblasts were infected with HCMV strain Merlin at an moi of 10 for 72h then coated with VLE peptide (10 μg/ml at 37°C for 1 hr) prior to addition of effectors at an E:T ratio of 10:1. Blocking antibody experiments (anti-CEACAM1, clone ASL-32, Biolegend) were carried out in quadruplicate at 5 μg/ml, either in cultures in excess, or with pre-treatment for 10 min at 37°C before 2 washes with RPMI-10. Anti-CD107a-FITC (BD Biosciences) or isotype control was added with effectors and incubated for 1 hr before addition of BD Golgistop (BD Biosciences). Cultures were then incubated for 4 hr before cell surface staining with anti-CD8-APC (Biolegend). Samples were fixed in 2% PFA before analysis by flow cytometry (BD Biosciences Accuri C Flow).

Annexin V/propidium iodide (PI) double assay was performed with the Annexin V, Alexa Fluor® 488 Conjugate Detection kit (Life Technologies, Paisley, UK). Briefly, 10⁶ cells were resuspended in 500 μl binding buffer and stained with 5 μl Alexa Fluor 488 conjugated annexin V according to the manufacturer's instructions. 1 μl 100 μg/ml PI was added and mixed gently to incubate with cells for 15 min. at room temperature in the dark. After incubation period, samples were subjected to FCM analysis in 1 hr. by using BD Accuri C6 Flow Cytometry (BD Biosciences, San Jose, CA, USA). The data were analysed by using Accuri CFlow^@ and CFlow Plus analysis software (BD Biosciences).

The intracellular free calcium calculation was performed with Fluo-8^® dye (AAT Bioquest, Sunnyvale, USA). To prepare the solution with a final concentration of 5 mM, a total of 0.25 mg of Fluo-8^® dye powder was dissolved into 0.48 mL of DMSO. Before being applied to the cells, the reagent was diluted to 5 μM using PBS or serum-free medium. The cells were incubated in the dark for 20 min after the addition of Fluo-8^® solution, and the fluorescence emission was measured at 520 nm post excitation at 490 nm. Accuri CFlow@ and CFlow Plus tools were used to measure the extent of Fluo-8^® fluorescence (BD Biosciences). The percentage of the fluorescence change was normalized to that of the control group.

ROS increase was measured using aminophenyl fluorescein (APF; Goryo Chemical, Sapporo, Japan). To prepare the solution with a final concentration of 5 mM, 1 mg of APF powder was dissolved in 0.47 mL of N,N-dimethylformamide (DMF). Before being applied to the cells, the reagent was diluted to 5 μM using phosphate-buffered saline (PBS) or serum-free medium. Following the addition of APF, the cells were incubated for 15 min in the dark and the fluorescence emission at 490 nm was measured at 515 nm after excitation. Accuri CFlow@ and CFlow Plus tools were used to measure the extent of APF fluorescence (BD Biosciences) [32 (link)]. The fluorescence shift percentage was normalized to that of the control group.

NK degranulation assays were performed in a similar manner to that described previously [39] , [81] (link). Briefly, PBMC (approved by the Cardiff University School of Medicine Ethics Committee Ref. no: 10/20) and incubated overnight with IFN-α (1000 IU/ml) and IL-15 (15 ng/ml, Milenyi Biotech). PBMC (0.5–1×10⁶) were incubated for 6 h with 0.5–1×10⁵ fibroblast targets per well in a 96 well plate at an effector∶target (E∶T) ratio of 10∶1, with the addition of 3 µl per well FITC-conjugated anti-CD107 antibody (cat. no. 555800, clone H4A3, BD Biosciences) or 3 µl per well FITC-conjugated isotype control (cat. no. 555748, BD Biosciences), adding 1 µl/well BD GolgiStop (BD Biosciences) 1 h after beginning the incubation. (For antibody blocking experiments, targets were pre-incubated with anti-MICA (Clone 159277, mouse IgG2B, MAB1300, R&D Systems) or isotype control (MICB non-blocking antibody, Clone 236511, mouse IgG2B, MAB1599, R&D Systems) antibodies at a concentration of 10 µg/ml for 30 min prior to incubation with PBMC). PBMC were harvested and stained with conjugated antibodies against CD3 (anti-CD3 PE-Cy7, cat. no. 737657, Beckman Coulter, High Wycombe, UK) and CD56 (anti-CD56 PE, cat. no. A07788, Beckman Coulter), and fixed in 2% PFA before analysis by flow cytometry (BD Biosciences Accuri C Flow) (Fig. S10A).