Fig. 1.

Hyperthermia-treated apoptotic tumor cells are immunogenic. a EG7 cells were stained with an anti-OVA (dark line) or isotype control antibody (dotted line) and assessed by flow cytometry. b Heat (45 °C and 65 °C)-treated and control EG7 cells were stained with FITC-labeled anti-Annexin V antibody and PI and analyzed by flow cytometry. The gating for necrotic cells (45 °C and 65 °C) expressing Annexin V and PI was based upon the assessment of the live cells (37 °C). c Heat-treated EG7 cells were stained with an anti-HSP70 antibody (color lines) or isotype control antibody (dotted line) and analyzed by flow cytometry. Mean fluorescence intensity (MFI) numbers are indicated. d Heat-treated EG7 cells were stained with PI and an anti-HSP70 Ab and analyzed by flow cytometry. The gating for necrotic cells (45 °C and 65 °C) expressing both intracellular PI and intracellular HSP70 was based upon the assessment of the live cells (37 °C). The value in the right upper quadrant represents the percentage of apoptotic cells expressing HSP70. e Representative fluorescence images of heat-treated EG7 cells that were stained with PI and an anti-HSP70 Ab are shown. f Heat-treated EG7 tumor cells (1 × 10⁶ cells/mouse) were i.v. injected into mice (6 per group). At 7 days after the cell injection, blood samples from immunized mice were stained with OVA-specific PE-Tetramer and a FITC-labeled anti-CD8 antibody and analyzed by flow cytometry. The gating for OVA-specific CTLs stained with both FITC-labeled anti-CD8 antibody and PE-tetramer in mice immunized with heat (45 °C and 65 °C)-treated EG7 tumor cells was based upon the assessment of CTLs in the control PBS-treated mice. A total of 20,000 CD8⁺ T cells were counted. The value in each panel represents the percentage of OVA-specific CD8⁺ T cells among the total CD8⁺ T-cell population. *P < 0.05 versus the cohort of CD8⁺ T cells activated by 45 °C-treated EG7 (Student’s t test). One representative experiment out of two total experiments is shown