Cytofix

For assessment of the phosphorylation of MLKL and VZV antigen expression, uninfected and VZV-infected HT-29s were seeded onto coverslips and allowed to adhere overnight. Cells were then treated with T+S+V, or DMSO as a control, for 7–8 h. Cells were then washed in PBS and fixed with cytofix (BD). Cells were permeabilised with ice cold methanol for 10 mins, then washed in Tris buffered saline (TBS). Blocking was performed using 20% normal donkey serum (NDS, Sigma-Aldrich) then primary antibodies (diluted in 10% NDS) incubated overnight at 4 ^oC.
For detection of VZV antigens alone cells were fixed with cytofix (BD biosciences), washed in PBS and permeabilised with 0.1% Triton X-100 for 10 mins (Sigma-Aldrich). Blocking was performed using 20% normal donkey serum (NDS, Sigma-Aldrich) then primary anti-VZV antibodies (diluted in 10% NDS) incubated for 1 hr at room temperature. In both cases bound primary antibodies were detected using species-specific Alexa Fluor 488 or 594 conjugated secondary antibodies. Cells were then mounted in Prolong Gold antifade containing 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (Life Technologies). Imaging was performed using a Zeiss Axioplan 2 upright microscope with an Axiocam camera (Carl Zeiss). Counting for pMLKL was performed by randomly imaging 10–20 non-overlapping regions of each slide and manually counting cells.

T cell apoptosis was investigated by stimulating 5x10⁵ engineered T cells 1:1 with antigen-loaded microbeads or MM.1S tumor cells for 24, 48, or 72 hrs in the presence of DMSO or LCL161 at indicated concentrations. Afterwards, cells were immediately stained with live/dead fixable viability dye at room temperature for 20 minutes, fixed with BD Cytofix, and stained for phosphatidylserine and CD3 surface expression on ice for 60 min. Cells were analyzed by flow cytometry and populations were defined as live (PS^- live/dead^-), early apoptotic (PS⁺ live/dead^-), and late apoptotic (PS⁺ live/dead⁺), refer to Supplemental Figure 13 for gating strategy.
To investigate AICD in TAC T cells, cells were stimulated as above with antigen-loaded microbeads for 48 hrs in the presence of DMSO or LCL161. Cells were stained with live/dead fixable viability dye at RT, fixed with BD Cytofix, stained for phosphatidylserine on ice for 60 min, permeabilized with BD Phosflow perm/wash I as per manufacturers guidelines, then stained intracellularly for active caspase 3. Percent apoptotic in live cells was defined as live/dead^- PS⁺ active-caspse3⁺, refer to Figure 6B for gating strategy.