Immunogenicity of CML LSCs in vitro. (A) Expression of MHC class I, MHC class II, CD80, CD86, PD-L1, and PD-L2 on H8 CML LSCs. 1 representative plot of 3–10 is shown. (B and C) ³H-thymidine assays. (B) 3 × 10³ FACS-purified, irradiated (10 Gy) LSC stimulators were co-incubated with 1.5 × 10⁴ MACS-purified CD8⁺ T cell responders from BL/6 mice infected with LCMV 6 wk earlier. One representative experiment out of two is shown. (C) 3 × 10³ FACS-purified, irradiated (10 Gy) LSC stimulators were co-incubated with 1.5 × 10⁴ MACS-purified CD8⁺ T cell responders from naive p14 TCR transgenic animals. One representative experiment out of five is shown. (D–E) In vitro killing assay. (D) 10⁴ H8 LSCs FACS-purified from H8 CML mice at day 18 after transplantation were co-incubated with 2 × 10⁴ naive BL/6 splenocytes (ctrl) or p14 effector splenocytes (p14) overnight, followed by transfer into methylcellulose. Colonies were enumerated 7 d later. One representative experiment out of two is shown. (E) 10⁴ FACS-purified H8 or BL/6 LSCs from H8 or BL/6 CML mice (20 d after CML induction) were co-incubated with 0, 10³, 10⁴, 10⁵, 10⁶, or 10⁷ MACS-purified naive CD8⁺ T cells (ctrl) or p14 effector CD8⁺ T cells (p14) overnight, followed by transfer into methylcellulose in duplicates. Colonies were enumerated 7 d later. Percent killing was calculated in relation to culture condition without CD8⁺ T cells (0). Data are displayed as mean ± SEM. Statistics: Student’s t test (B–D), two-way ANOVA (E). *, P < 0.05; **, P < 0.005; ***, P < 0.0001. Isotype controls, blue lines; stainings, red lines. E:T, effector/target ratio; LSCs, leukemia stem cells; Resp., responders; Stim., stimulators.