Fig. 5

T cell activation requires iron supply via transferrin-TfR system. A Effectivity of transferrin uptake blockade with anti-TfR (10 μg/ml) in activated Jurkat T cells. Cells were pre-treated with anti-TfR or not, subsequently incubated at 37 °C for 90 min with 20 μg/ml transferrin-Alexa647 and analysed using flow cytometry. B Levels of intracellular labile iron pool (LIP) in anti-TfR-treated versus untreated, activated Jurkat T cells as determined by staining with the iron sensor dye Calcein-AM. Lower fluorescence intensity corresponds to higher LIP levels. C Levels of intracellular reactive oxygen species (ROS) in anti-TfR-treated versus untreated, 5-min activated Jurkat T cells as determined by staining with the ROS sensor dye CellROX green. D Upregulation of CD69 upon activation on plate-bound anti-CD3ε + anti-CD28. Jurkat T cells were pre-treated or not with anti-TfR and subsequently incubated for 20 h in coated wells for activation. Anti-TfR treatment was maintained throughout the whole experiment where applicable. Surface CD69 expression was determined by staining with anti-CD69-PE and subsequent flow cytometry. E Effectivity of transferrin uptake blockade with anti-TfR (10 μg/ml) in 20-h activated primary T cells (activated on plate bound anti-CD3ε + anti-CD28). Cells were pre-treated with anti-TfR or not, subsequently incubated at 37 °C for 90 min with 30 μg/ml transferrin-Alexa647 and analysed using flow cytometry. F Upregulation of CD69 upon activation on plate-bound anti-CD3ε + anti-CD28. Primary T cells were pre-treated or not with anti-TfR and subsequently incubated for 20 h in coated wells for activation. Anti-TfR treatment was maintained throughout the whole experiment where applicable. Surface CD69 expression was determined by staining with anti-CD69-PE and subsequent flow cytometry. G Upregulation of CD25 upon activation on plate-bound anti-CD3ε + anti-CD28. Primary T cells were pre-treated or not with anti-TfR and subsequently incubated for 96 h in coated wells for activation. Anti-TfR treatment was maintained throughout the whole experiment where applicable. Surface CD25 expression was determined by staining with anti-CD25-APC and subsequent flow cytometry. Data points represent individual experiments. Statistical significance determined with unpaired two-tailed Student’s t-test (A, D–G) or one-sample t-test (B, C). *p < 0.05; **p ≤ 0.01; ***p ≤ 0.001; no indication—not significant