Stimulation of CD4+ T cells and NK cells

CD4⁺T cells were isolated from frozen PBMCs. All CD4⁺T cells were positively selected with a CD4⁺T cell isolation kit (Miltenyi Biotec, Germany), yielding CD4⁺T cell populations at a purity of 96–99%. Purified CD4⁺T cells were stimulated with 4 µg/mL plate-bound anti-human CD3 (OKT3) monoclonal antibody (mAb; eBioscience, San Diego, CA, USA) and 4 µg/mL soluble anti-human CD28 (CD28.2) mAb (Becton Dickinson, Le Pont De Claix France) in the presence of recombinant human IL-2 (Proleukine, Chiron, Amsterdam, 100 U/mL) and recombinant human interferon alpha-2a (Roferon-A) and IFNλ2 (Biotechne, UK) at the indicated dose^{19 (link)}. After 5 days of culture, CD38 and CD25 expression and the frequency of 7-AAD⁺ cells were measured by flow cytometry on stimulated CD4⁺T cells. Natural killer (NK) cells were isolated from PBMCs. NK cells were negatively selected with the NK cell isolation kit (Miltenyi Biotec, Germany), yielding NK cell populations at a purity of 96–99%. NK cells were stimulated with IL-15 (Miltenyi Biotec, 10 ng/mL), IL-2 (Proleukine, Chiron, Amsterdam 100 U/mL), and recombinant human interferon alpha-2a (Roferon-A) at the indicated dose. After 3 days of culture, expression of CD56, CD95, and NKG2D was measured by flow cytometry (Supplementary Table 2a).