Fig. 3

In vivo apoptosis of tumor-infiltrating CD8⁺ T cells 4 days after transfer. a Mice bearing induced (n = 14) or transplanted (n = 13) tumors (500 mm³) received adoptive transfer of TCRP1A CD8⁺ T cells as in Fig. 2. Four days later, tumors were analyzed by FACS ex vivo for CD8⁺ T cells among living cells. b–d Draining lymph nodes (LN), spleens and tumors from tumor-bearing mice were analyzed 4 days after adoptive transfer of TCRP1A CD8⁺ T cells by ex vivo FACS staining for CD8 and H-2L^d-P1A tetramers b, CD69 c, and with Annexin V d (for b–d: n = 75 mice for induced tumors, n = 75 mice for T429.11, n = 32 mice for T429.6, n = 12 for tumor-free mice). e H-2L^d-P1A tetramer-negative CD8⁺ T cells infiltrating induced tumors or spleens from tumor-free mice were stained for Annexin V. Mice were identical to b–d (induced tumors: n = 75, spleens from tumor-free mice: n = 12). f Mice bearing induced (n = 4) or transplanted (n = 3) tumors (500 mm³) were transferred with activated TCRP1A CD8⁺ T cells. Four days after transfer, they received an i.v. injection of FLIVO (inhibitor-based pan-caspase probe) 4 h before killing. Apoptosis of TCRP1A CD8⁺ T cells was evaluated ex vivo by FACS staining for FLIVO. Mice receiving a non-targeting FLIVO control dye showed no staining of TCRP1A CD8⁺ T cells. g Slices (300 µm) of fresh tumor tissues were incubated with CMAC-stained TCRP1A CD8⁺ T cells for 24 h. Cryosections (7 μm) were stained for apoptosis using inhibitor-based active pan-caspase marker FLICA, and scanned with a MIRAX digital microscope. Data were quantified using Biopix software (n = 5 mice/group; three sections analyzed per mouse). h TiRP mice bearing either pigmented (Mela) or unpigmented (Amela) induced tumors were treated and analyzed as in b–d (n = 20 mice/group). Results are expressed as mean + s.e.m. Unpaired t-test, two-tailed a–h, *P < 0.05, **P < 0.01, ***P < 0.001. ****P < 0.0001