MouseCD4+ andCD8+ T cells were obtained from the spleens and lymph nodes of C57Bl/6 mice. To obtain single-cellsuspensions of T cells, organs were first homogenized through a cell strainer in ACK lysis buffer and passed through a magnetic column labeled with negative selection microbeads (Miltenyi) following manufacturers’ instructions. CD8a+ and CD4 + T cell isolation kits (Miltenyi, Refs130–104-075 and130–104-454) were used to isolate mouse T cells. The same procedure was performed to purify OT-1CD8+ T cells from CD45.1 OT-1 mice. Purified lymphocytes were cultured in complete RPMI medium containing 10% FBS.
Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor’s buffy coats by a centrifugation gradient in Ficoll-paque(GH Healthcare) and then,CD4+ andCD8+ T cells were purified using negative selection microbeads through a magnetic column. CD8a+ and CD4 + T cell isolation kits (Miltenyi130–096-495, and130–096-533) were used. Human T cells were cultured in Ex Vivo serum free medium (Gibco) containing 10% human serum AB (Sigma). The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of the Universidad de Navarra (protocol R-131.16 (Ref 2019.162)).
Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor’s buffy coats by a centrifugation gradient in Ficoll-paque(GH Healthcare) and then,CD4+ andCD8+ T cells were purified using negative selection microbeads through a magnetic column. CD8a+ and CD4 + T cell isolation kits (Miltenyi130–096-495, and130–096-533) were used. Human T cells were cultured in Ex Vivo serum free medium (Gibco) containing 10% human serum AB (Sigma). The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of the Universidad de Navarra (protocol R-131.16 (Ref 2019.162)).