Figures & data
Figure 1. Ag-specific effector CD8+ T cell IFN-γ production is temperature sensitive. Effector Pmel-1 CD8+ T cells were generated by pulsing Pmel-1 splenocytes with 0.1 µg/mL gp10025-33 peptide (at 37°C) and 30 IU of rIL-2 for 5 days. (A) Effector Pmel-1 CD8+ T cells were incubated at 33°, 37°, and 39.5°C for 6 h and re-stimulated with C57BL/6 splenocytes pulsed with 0.1 µg/mL gp10025-33 peptide. Supernatants were removed after 18 h of co-incubation at 37°C and analysed for IFN-γ by ELISA. (B) Effector Pmel-1 CD8+ T cells were incubated at 33°, 37°, or 39.5°C for 2 to 6 h and activated with pulsed C57BL/6 splenocytes. Supernatants were analysed for IFN-γ by ELISA 18 h after pulsing. (C) Effector Pmel-1 CD8+ T cells were incubated at 33°, 37°, or 39.5°C for 6 h and stimulated with varying concentrations of gp10025-33 peptide pulsed C57BL/6 splenocytes. Results are reported as the mean ± SD. These results are representative of two independent experiments (p < 0.05, #vs 37°C and *vs 33°C).
Figure 2. Mild hyperthermia enhances Ag-specific CD8+ cytotoxicity. Effector Pmel-1 CD8+ T cells were incubated at 33°, 37°, or 39.5°C for 6 h and then co-incubated with Cr51 labelled EL-4 (gp100 negative) or B16.F10 (gp100 positive) target cells for 4 h at 37°C. Supernatant was collected and percentage lysis was determined by chromium release by lysed target cells. Results are reported as the mean ± SD. These results are representative of two independent experiments.
Figure 3. Mild hyperthermia does not affect new protein synthesis but induces GM1 clustering in the plasma membrane. (A) Effector Pmel-1 CD8+ T cells were incubated at 33°, 37°, or 39.5°C for 6 h in the presence of 10 µm cycloheximide then stimulated with gp10025–33 peptide pulsed C57BL/6 splenocytes for 18 h. Supernatants were analysed for IFN-γ production by ELISA. (B) Effector cells were incubated at 33°, 37°, 39.5°C for 6 h and adhered onto alcian blue coated coverslips, fixed with paraformaldehyde. Cells were stained with FITC-CTxB, visualised for clustering by fluorescent microscopy, and (C) quantified. Results are reported as the mean ± SD. These results are representative of two independent experiments (p < 0.05, #vs 37°C and *vs 33°C).
Figure 4. Mild hyperthermia does not affect effector CD8+ T cell activation when TCR signalling is bypassed. Effector Pmel-1 CD8+ T cells were incubated at 33°, 37°, 39.5°C for 6 h and activated with 0.5 µg/mL of ionomyocin and varying concentrations of PMA for 18 h at 37°C. Supernatants were collected and analysed for IFN-γ by ELISA. Results are reported as the mean ± SD. These results are representative of two independent experiments.
Figure 5. Effector CD8+ T cell Ag-specific signalling is enhanced by mild hyperthermia. Cells were incubated at 33°, 37°, or 39.5°C for 6 h and stimulated with 0.1 µg/mL gp10025–33 peptide pulsed C57BL/6 splenocytes at 37°C. Stimulation was varied between 0–30 min. (A–D) Expression of phosphorylated LAT and PKCθ were determined by western blot. (A, C) Western blots. (B, D) Densitometry was performed using total β-actin levels as background controls. These results are representative of two independent experiments.
Figure 6. Effector CD8+ T cell IFN-γ transcription is regulated by temperature. Effector CD8+ T cells were incubated at 33°, 37°, 39.5°C for 6 h and stimulated with 0.1 µg/mL gp10025–33 peptide pulsed C57BL/6 splenocytes for 0–2 h at 37°C. RNA was isolated and cDNA was synthesised. mRNA levels were assessed by real-time PCR. All message levels are relative to GAPDH controls and experimental gene expression is relative to cells activated with null peptide. Results are reported as the mean ± SD. These results are representative of two independent experiments (p < 0.05, #vs 37°C and *vs 33°C).
