Adoptive transfer of CD8+ T cells cultured with Cl-IB-MECA mediates enhanced tumor suppression in melanoma-bearing mice.

<p><b>A</b>) C57Bl6j mice were s.c. inoculated with 2.5×10⁵cells B16-F10 cells/mouse. 10 days after B16 injection, mice received a single p.t. injection of Cl-IB-MECA (20 ng/mouse) or 1×10⁶ CD8+ T cells/mouse treated with Cl-IB-MECA (0.1 µg/ml) or 1×10⁶ untreated CD8+ T cells/mouse or PBS (Ctr). <b>B</b>) Tumor volume (mm³) measured in control mice (Ctr, n = 11) and mice receiving a single dose of Cl-IB-MECA (n = 13) or adoptively transferred with Cl-IB-MECA-treated CD8+ T cells (n = 14) or with untreated CD8+ T cell (n = 10). <b>C</b>) Increased survival of melanoma-bearing mice receiving Cl-IB-MECA (p<0.05) or CD8+ T cell transfer cultured with Cl-IB-MECA (p<0.001) compared with control groups (n = 5/group). Data are from three independent experiments and represent mean ± SEM. Statistical differences were determined by one way ANOVA and Student's t test, as appropriate. ***p<0.001. Comparison of survival between groups was performed using long-rank test.</p

<i>In vivo</i> effectiveness of CD8+ T cell transfer after Cl-IB-MECA treatment is associated with increased apoptosis rate, granzyme B and TNF-α release into melanoma lesions.

<p><b>A</b>) and <b>B</b>) TNF-α and granzyme B levels, respectively, detected into tissue homogenates from mice adoptively transferred with Cl-IB-MECA-stimulated CD8+ T cells or Cl-IB-MECA-treated mice. <b>C</b>) Representative pictures of melanoma cryosections Tunel stained (FITC) and stained with PI (red). Positive and negative controls are also provided. <b>D</b>) Quantitative analysis of Tunel+ cells detected in melanoma sections. Results are expressed as mean ± SEM (n = 5/group). Data are from two independent experiments and represent mean ± SEM, n = 6 in each experiment. Statistical difference was determined by one way ANOVA. *p<0.05, **p<0.01, ***p<0.001.</p

Neutralization of TNF-α abrogated the anti-tumor effect of Cl-IB-MECA-treated CD8+ T cells.

<p><b>A</b>) Tumor volume (mm3) in mice receiving anti-TNF-α mAb (dashed lines) or isotype IgG control (continuous lines) and injected with Cl-IB-MECA or PBS or CD8+ T cells treated or not with Cl-IB-MECA. <b>B</b>) Percentage of CD11c+CD207 high cells in the tissue of mice described above. <b>C</b>) Representative dot plot is shown. <b>D</b>) and <b>E</b>) Expression of CD80 and MHC I, respectively, on CD11c+CD207 high cells in the tissue of mice described above. Data are from two independent experiments and represent mean ± SEM, n = 9 for each experiment. Statistical difference was determined by one way ANOVA. *p<0.05, **p<0.01, ***p<0.001.</p