Mab1599

The following primary antibodies were used for flow cytometry: anti-MICA (R&D, MAB1300), anti-MICB (R&D, MAB1599), anti-ULBP1 (R&D, MAB1380), anti-ULBP2 (R&D, MAB1298, with cross reactivity to ULBP5 and ULBP6), anti-ULBP3 (R&D, MAB1517), anti-GLUT-1 (abcam, Ab150299), anti-IFNƔ (R&D, MAB285), anti-CD56 (BioLegend, 36503), anti-CD3 (ThermoFisher Scientific, 56-0038-82), anti-σ1 (Sigma-Aldrich, MAB994-I-25UG), anti-CD4 (BioLegend, 344604), anti-CRT (Invitrogen, 902A), anti-ERp57 (BioLegend, 937301) and anti-PVR (BioLegend, 337602). Additionally, anti-KIRL2D3, murine anti-NKG2D, and anti-NKP46 antibodies were purified from hybridomas as previously described (48 (link)). These primary antibodies were stained with AF448-coupled donkey anti-mouse IgG (Jackson ImmunoResearch, 715-545-151) or APC-coupled goat anti-mouse IgG (Thermo Fisher Scientific, 31981). Murine NKG2D-Ig and human NKG2D-Ig fusion protein (R&D, 1299-NK) were followed by secondary staining with PE-coupled F(ab’)₂ fragment goat anti-human IgG (Jackson ImmunoResearch 109-116-088). Additionally, a Fixable Viability Dye eFluor™ 780 (FVD) (Thermo Fisher Scientific, 65-0865-14) was included in the flow cytometry staining to exclude the dead population.

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).