Isolation and Expansion of CD8+ Central Memory T Cells

CD8⁺CD45RO⁺CD62L⁺ central memory T cells (T_CM) were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors (Puget Sound Blood Center, Seattle, WA) by negative selection using CD8 isolation kits and CD45RA microbeads and positive selection with CD62L microbeads (Miltenyi Biotec). Following isolation, CD8⁺ T_CM were stimulated with anti CD3/CD28 Dynabeads (Life Technologies) and transduced at a multiplicity of infection (MOI) of 3 on the third day of culture. EGFRt⁺ T_CM subsets were enriched by immunomagnetic selection using biotinylated Erbitux and anti-biotin microbeads (Miltenyi Biotec), then expanded as previously described [44 (link)]. CD8⁺ T_CM were maintained in RPMI medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/mL recombinant human interleukin 2 (IL-2) and 1 ng/mL recombinant human interleukin 15 (IL-15).