Annexin 5

Anonymised healthy donor blood samples were purchased from Cambridge Bioscience UK and mononuclear cells extracted. These were incubated with various concentrations of cladribine with or without 250 μM deoxycytidine to neutralise DCK activity [20 (link)]. Cells were stained with CD3-PE, CD19-PECy5 and CD27-BV205, Annexin V (Invitrogen, Loughborough, UK) and 4′,6-diamidino-2-phenylindole (DAPI) to detect live (Annexin V−, DAPI−), early (Annexin V+ , DAPI−) and late stage (Annexin V+, DAPI+) apoptosis. Cells were analysed using flow cytometry.

Cell toxicity was assessed for single drugs and the combination of different LRA families in previously isolated CD4⁺ T cells from three independent uninfected donors. CD4⁺ T cells were pre-incubated with the pan-caspase inhibitor Q-VD-OPh for 2h. Afterwards, CD4⁺ T cells (200,000 cells per well) were incubated for 22h with the compounds. Then, cells were stained with the apoptotic marker Annexin V (PE, Biolegend) and a viability dye (LIVE/DEAD fixable Violet Dead Cell Stain kit, Invitrogen) in order to identify the following stages of cell death: live cells (Annexin V^- Viability^-), early apoptotic cells (Annexin V⁺ Viability^-), late apoptotic+necrotic cells (Annexin V⁺ Viability⁺) and total cell death (Annexin V^- Viability⁺). In addition, different surface markers, including CD3 (Pe-Cy7, BD Biosciences), CD4 (AF700, BD Biosciences), CD45RO (BV605, Biolegend) and CD27 (FITC, Biolegend), were used to identify drug toxicity induced in the different CD4⁺ T cell subpopulations. The CD4⁺ T cell subsets were identified as follows: Naïve (T_NA) and Stem Cell Memory (T_SCM) (CD3⁺CD4⁺CD27⁺ CD45RO^-), Central (T_CM) and Transitional Memory (T_TM) (CD3⁺CD4⁺CD27⁺ CD45RO⁺), Effector Memory (T_EM) (CD3⁺CD4⁺CD27^- CD45RO⁺) and Terminally Differentiated cells (T_TD) (CD3⁺CD4⁺CD27^- CD45RO^-).

Cell apoptosis was analyzed by flow cytometry, as described before^{68 (link)}. Naïve T cells were activated under specified normoxic or hypoxic conditions. On day 2.5, cells were harvested, washed once with DPBS and again with 1× Annexin V binding buffer before staining with Annexin V (1:50, Thermo Fisher, #17–8007) and 7-AAD (1:200, Sigma, #129935) or propidium iodide (1:50, Thermo Fisher, #00–6990-42) in 1× Annexin V binding buffer for 30 min at room temperature. Cells were acquired by Attune NxT Flow Cytometer and data were analyzed by FlowJo.