ELISA

Serum from mice transplanted with PBS or PT-3 ALL cells was obtained by centrifugation of 500 μL of blood (10,000 × g for 10 minutes) on day 35. Serum samples were stored at −80°C and thawed prior to use. Mouse TGF-β was measured with the Legend Max Mouse Latent TGFβ ELISA Kit (BioLegend) and 1:50 serum dilution in manufacturer’s buffer was performed as specified by the manufacturer’s instructions. Per condition, 4 μL of sample was diluted with 196 μL of Assay Buffer provided by manufacturer, after which 50 μL was added to the well in triplicates. The Human/Mouse TGF-beta 1 Uncoated Elisa Kit (Invitrogen) was also used to measure TGFβ and serum was diluted 1:5 in PBS, following specific instructions from manufacturer. Per condition, 20 μL of serum diluted in 80 μL of PBS (1:5). Samples were activated with acid: 20 μL of 1N HCL (Fisher Scientific) was added to 100 μL of serum diluted in 80 μL of PBS, incubated at room temperature for 10 minutes followed by addition of 20 μL of 1N NaOH, after which 100 μL was added to each well in triplicates. Correction to the dilution factor of 1.4 was made for final calculations. For granzyme B, TNFα, and IFNγ ELISAs, indicated NK cells (1×10⁶) were cocultured with (2×10^⁵) PT-3 ALL cells that had been pre-treated with or without VAY736 (20 μg/mL for 2 hours) for 4 hours. Legend Max Human Granzyme B ELISA Kit was obtained from Biolegend. TNFα, and IFNγ ELISAs were obtained from Invitrogen. Per condition, 50 μL (granzyme B ELISA) and 100 μL (TNFα, and IFNγ ELISAs) of non-diluted sample was added to each well in triplicates. Binding of TGFβ, granzyme B, TNFα, and IFNγ was detected using secondary antibody, streptavidin-HRP, and TMB Substrate solution (provided with specified ELISA kit). Substrate conversion was stopped after 20 minutes with 100 μL stop solution (1M H₃PO₄) provided with the ELISA Kits. Plates were washed with PBS plus 0.05% Tween20 in-between incubations. Assay diluent provided by manufacturer or RPMI medium (Sigma) was used as negative controls and specific standard proteins were used as positive controls. Standard reconstitutions and curves were generated as per manufacturer’s instructions for each assay. Optical density values were obtained using a microplate reader set to 450 nm (Bio-Rad iMark Microplate reader). The derived TGFβ, granzyme B, TNFα, and IFNγ concentrations (ng/mL) were determined using specific standard curve-derived formulas. The derived TGFβ concentrations (ng/mL) were multiplied by 5 to correct for dilution.