Figure 6.

In vitro addition of iron to splenocytes decreases the number of proliferating CD8⁺ T cells (CFSE low) (A), negatively affects perforin degranulation in CD8⁺ cytotoxic T cells (B) and significantly reduces the CD8⁺ T cell dependent lysis of target cells (C). (A, B) Splenocytes isolated from tumor-naive C57Bl/6N mice were stimulated with plate-bound anti-CD3 antibodies and iron in form of iron citrate (FeC₆H₅O₇; non-transferrin bound iron, NTBI) was added. Proliferation of CD8⁺ T cells was measured by flow cytometry depending on CFSE 72h after culture start. Data are presented as Pie Plots (mean ± SEM) n = 4. Perforin was stained intracellularly as described in Materials and Methods n=5. Statistical significance was determined by a two-tailed T-test and corrected for multiple comparisons with the Benjamini–Hochberg method. (C) The capability of iron treated and non-iron treated CD8⁺ T cells to lyse target cells was measured with a chromium release assay as described in Material and Methods. Representative flow cytometry results and summary plots are shown (mean ± SEM). Statistical significance was determined by 2-way ANOVA. The results of Tuckey post-hoc-test are presented in the plots: ns: not significant, *p < 0.05, **: p < 0.01. control, Fe (ratio 15:1) n = 5, control, Fe (ratio 30:1) n = 4. The results of ANOVA are presented in Materials and Methods/Specific statistical data analyzed in main figures.