Mab7 b6 1 2

IFNγ ELISPOT was performed using anti-human IFNγ mAb1-D1K(I) and mAb7-B6–1 (II) (Mabtech) in accordance with the manufacturer’s protocol. Effector and target cells (E:T 1:3) were incubated for 24 h with or without pamidronate (10 or 100 μM, Calbiochem) as indicated. Pamidronate was added in all our in vitro experiment in order to enhance TEGs activation as previously reported [10 (link)].

15.000 (Elispot) or 50.000 (ELISA) effector cells and 50.000 target cells were incubated together with or without GAB (different concentrations) for 16 hours at 37°C 5% CO₂. 30 or 100 µM pamidronate (calbiochem, cat no. 109552-15-0) was added to the target cells. For ELISA the supernatant was harvested after 16 hours, and the level of IFNγ was determined using the IFN gamma Human Uncoated ELISA Kit (Invitrogen, cat no. 15541107). For the Elispot assay the co-culture was done in nitrocellulose-bottomed 96-well plates (Millipore, cat no. MSIPN4550) precoated with α-IFNγ antibody (Mabtech, 3420-3-1000, clone 1-D1K 1:200). After 16 hours, the plates were washed with PBS and incubated with mAb7-B6-1 (II; Mabtech, cat no. 3420-6-1000) followed by Streptavadin-HRP (Mabtech, cat no. 3310-9) IFNγ spots were visualized with TMB substrate (Mabtech, cat no. 3651-10) and analyzed using A.EL.VIS ELISPOT Scanner and analysis software (A.EL.VIS).

IFNγ ELISPOT was performed using antihuman IFNγ mAb1-D1K (I) and mAb7-B6–1 (II) (Mabtech) per the manufacturer’s protocol. Then 15,000 TEG cells (TEG011, TEGLM1, TEG011_CD8α, or TEGLM1_CD8α) were co-incubated with 50,000 target cells (E:T ratio 1:3) for 18–24 h in nitrocellulose-bottomed 96-well plates (Millipore). IFNγ spots were visualized with TMB substrate (Sanquin), and subsequently the number of spots was quantified using ELISPOT Analysis Software (Aelvis). Where indicated, blocking of CD8α was performed using 10 μg/ml anti-CD8α antibody clone OKT8 (eBioscience) and blocking of CD8β with 10 μg/ml anti-CD8β clone 2ST8.5H7 (Abcam).