Figure 5. CD40-CD40L is required for ACT effectiveness.

(A) CFSE-labeled, CD8⁺ T cells specific for either OVA or gp100 antigens were incubated with EG7 tumor slices from either WT or different KO mice as indicated. NO was detected by confocal microscopy of slices loaded with DAR-4M AM. NO-release levels were measured as mean of fluorescence and expressed as fold induction over control (without CD8). Mean ± s.e.m.; n=12 slices pooled from 3 independent experiments, ***p ≤0.001, **p ≤0.01, by using One Way ANOVA.

(B) CFSE-labeled, polyclonal CD8⁺ T cells specific for OVA were derived from immunized WT or E8I^cre x Cd40lg^flox/flox mice and were incubated with EG7 tumor slices from either WT or CD40L KO mice, as indicated. NO was detected as in A. Error bars, mean ± s.e.m.; n=12 slices pooled from 3 independent experiments, ***p ≤ 0.001, **p ≤ 0.01, by using One Way ANOVA and the Holm–Sidak method of correction for all pairwise multiple comparison.

(C) Survival percentages of WT, CD40 KO, and CD40L KO EG7 tumor-bearing mice untreated or treated with ACT (n=10). *p ≤ 0.05 *** p ≤ 0.001, logrank test.

(D) EG7 tumor-bearing RAG-deficient mice were reconstituted with CD8⁺ T lymphocytes isolated from spleens and lymph nodes of WT and CD40L KO, either EG7 tumor-bearing or tumor-free, mice. After 2 days, ACT with OVA-specific CD8⁺CD45.1⁺ T lymphocytes was performed. Tumor area at days 9, and 13 following ACT are reported. Horizontal lines represent means of n=7. **p ≤ 0.01, unpaired Student t-test.

See also figure S5.