Fig. 7

Blockade of iron-uptake via transferrin-TfR axis negatively affects T cell adhesion via integrins and consequently T cell: APC conjugate formation. A, B Immunoblot with a phosphorylation-specific antibody against PAK1/2 (T423 and T402, respectively). A Jurkat T cell activation was accomplished with soluble anti-CD3ε + anti-CD28 for the indicated times before cell lysis. Data from 3 independent experiments. Left: Band intensity quantification. Right: Relevant parts of one representative immunoblot. B Expanded primary T cells were treated with anti-TfR or left untreated. Activating antibodies (anti-CD3ε + anti-CD28) were added for the indicated times before lysis. PAK1/2 phosphorylation was detected in T cells of a fraction of donors. An exemplary immunoblot from “responder” T cells is shown. C Levels of surface LFA-1 measured using an antibody against the beta-subunit CD18 (ITGb2) on Jurkat T cells treated as indicated. D Maximum adhesive potential via LFA-1 upon 30-min treatment with 1 mM MnCl₂ forcing every LFA-1 surface molecule into high affinity conformation. E Adhesion of 30-min activated control and anti-TfR treated Jurkat T cells on ICAM-1 coating. F Adherent anti-TfR or untreated Jurkat T cells on ICAM-I coating upon 30-min activation with soluble anti-CD3ε and anti-CD28 relative to the maximum adhesion capacity induced by addition of 1 mM MnCl₂. G Quantification of specific activation-induced conjugate formation between anti-TfR treated or untreated Jurkat T cells and SEE-pulsed Raji B cells. Jurkat T and Raji B cells were stained with CTV and CFSE, respectively, with dye switching between experiments. Cells were mixed in a 1:1 ratio and conjugates formed for 15 min at 37 °C in a pellet. Data displayed as % of Jurkat T cells that formed conjugates with Raji B cells. Unspecific conjugates (Jurkat with un-pulsed Raji B cells) were subtracted for each corresponding sample. Error bars indicate mean ± SEM. Statistical significance determined with two-way ANOVA (A, C), or one-sample t-test (B) or with unpaired two-tailed Student’s t-test (D–G). *p < 0.05; ***p ≤ 0.001; n.s—not significant