Fig 2: Fast transferrin-TfR recycling axis is demarked by phosphatidylserine. A Representative confocal images at 3 different z-levels at (0 µm) and above (0.44 µm and 0.88 µm) the immunological synapse of activated Jurkat T cells expressing Flot1/2-GFP and the PS-sensor LactC2-mCherry. B Nearest neighbour analysis between green and red channel identifying % of vesicles positive for both colours (= 320 nm apart; % Flot1 vesicles also containing Flot2, % Flot1/2 vesicles positive for LactC2 (PS) and random points to each other) in GFP and mCherry as described in [15]. C Representative confocal images of fixed, 20-min activated WT (upper) or FlotKO (lower) Jurkat T cells expressing PS sensor LactC2-GFP, PI(3)P-sensor 2 × FYVE-GFP or PI(4)P-sensor OSBP-PH-GFP. Scale bar = 5 µm. D Mean fluorescence dispersion of GFP channel in WT and FlotKO Jurkat T cells. E Representative time series of confocal imaging of an activated Jurkat T cell expressing the PS-sensor Lact-C2-mCherry during addition of transferrin-Alexa488. Error bars indicate mean ± SEM. Statistical significance determined with one-way ANOVA (B) or unpaired two-tailed Student’s t-test (D). Data points represent single live-imaged cells (B) or means of independent experiments (D). **p < 0.01; ****p = 0.0001, n.s.—not significant

Fig 3: T cell activation stimulates uptake and recycling of transferrin and TfR. A Transferrin uptake stimulation upon T cell activation. Jurkat T cells were equilibrated at 37 °C in the presence of transferrin-Alexa647 and subsequently left untouched (resting), or 30 min treated with non-stimulating solution (0 min activated) or 30 min stimulated with activating antibodies anti-CD3e + anti-CD28. Uptake was stopped on ice and cells were analysed for transferrin-Alexa647 uptake. Displayed is the fold-change of Alexa647 signal relative to untouched (resting) controls. B Transiently boosted TfR recycling upon T cell activation. Jurkat T cells were incubated with biotinylated anti-TfR for 90 min to label all TfR pools with antibodies. Then, surface-exposed anti-TfR-biotin was blocked with unlabelled streptavidin. To assess TfR recycling upon activation, cells were activated with soluble activating antibodies (anti-CD3e + anti-CD28) or left untreated for the indicated times. Antibody-labelled TfR delivered to the surface was detected with Pacific Blue-labelled streptavidin. Fold change of bound streptavidin-Pacific Blue in activated T cells is depicted relative to the corresponding resting cells. C Increased surface TfR levels upon T cell activation correlates with boosted TfR recycling. Jurkat T cells were activated with soluble activating antibodies (anti-CD3e + anti-CD28) or left untreated for the indicated times and immediately fixed with 3.7% paraformaldehyde (PFA). Surface TfR was then stained with biotinylated anti-TfR, which was detected with streptavidin-Pacific Blue. Data points and error bars indicate mean of n = 4 independent experiments and standard error of the mean (SEM). D Increased surface TfR levels upon activation of expanded primary human T cells. Expanded primary T cells were activated with soluble activating antibodies (anti-CD3e + anti-CD28) or left untreated for the indicated times and immediately fixed with 3.7% PFA. Surface TfR was then stained with biotinylated anti-TfR, which was detected with streptavidin-Pacific Blue. Data points indicate mean of n = 3 independent experiments ± SEM. E Increased TfR recycling upon T cell activation. Expanded primary human T cells were treated as described in B. Results were analysed and depicted as in B. Statistical significance determined with unpaired two-tailed Student’s t-test (A), one-way ANOVA (B, E) or two-way ANOVA (C, D). **p < 0.01; ****p = 0.0001, no indication—not significant

Fig 4: 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-CD3e + 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-CD3e + 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-CD3e + 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-CD3e + 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

Fig 5: CD47, CD71, and EpCAM Are Expressed on Ovarian Cancer-Derived EVs(A) Flow cytometry of the beads covered with IgG and antibodies against CD71, CD47, PALP, or MUC18 incubated with the medium conditioned by Kuramochi cells (palmtdTomato positive) (charts representative of three experiments).(B) Fraction of palmtdTomato-positive beads depending on CD47 and CD71 expression on EVs from the panel of five cell lines (n = 3).(C) mbGluc activity from beads with EVs captured by antibodies against MUC18, CD47, and CD71 from conditioned medium of Kuramochi cells (following removal of cells; n = 3).(D) Fraction of palmtdTomato-positive beads depending on antigen expression on EVs from DF30 and OV90 cell lines (n = 3).(E) Diagram of in vivo experiment to measure capture of EVs from serum of OVCAR5 tumor-bearing animal (left) and mbGluc activity from beads with EVs depending on the antigen expression (right) (n = 3 animals, two serum samples were processed from each animal, bioluminescence read in three to five technical replicates).(F) EVs were isolated by differential centrifugation and filtration steps from ascites from patients with ovarian cancer (n = 3) and with benign conditions such as cirrhosis or hepatitis (n = 3). Proteins were resolved by SDS-PAGE and stained with antibodies to EpCAM, CD63, and B(beta)-actin and detected with chemiluminescence.See also Figure S3.