Differential Strength of Mechanical Forces between APC:T-cell Conjugates.

<p>(<b>A</b>) Schematic illustration of AFM experiments. A T-cell-mounted AFM cantilever was placed above a DC or B-cell that was firmly attached to a glass cover slip. The T-cell was then brought into contact with the target cell. Interaction forces were measured by the deflection of the cantilever after a pre-defined contact time. (<b>B</b>) Corresponding force-distance curve of DC:T-cell (black) or B:T-cell (red) interactions. F represents maximal interaction force (double arrow). (<b>C</b>) Interaction forces of DC:T-cell (black) or B:T-cell conjugates (red) in the absence (-Ova) or presence of Ova peptide (+Ova) for contact time of ∼1–3 sec and 3 min. *p<0.01, unpaired <i>t</i>-test. Bars indicate mean ± s.e.m (n>10 pairs of cells). For each condition, OT-I T-cells were isolated from >3 independent experiments.</p

Cytokine secretion by DC:T-cell and B:T-cell co-cultures.

<p>(<b>A</b>) and (<b>B</b>): secretion of IL-2 (left) and IFN-γ (right) after 1 day co-culture of CD8+ OT-I T-cells with DCs or B-cells pre-pulsed or not with Ova peptide (10 pg–10 µg/ml). *p<0.01; **p<0.001, unpaired <i>t</i>-test. Bars indicate mean ± s.e.m. Data are representative of three independent experiments.</p

B-cells and DCs exhibit a similar phenotype.

<p>B-cells (Red) and DCs (Black) were stained with antibodies against H-2K^b (MHC class I), H-2K^b/Ova (pMHC), CD11c, CD19, CD54 (ICAM-1), CD80 (B7-1) and CD86 (B7-2). Filled histograms: isotype controls; Unfilled histograms: staining with antibody. Data are representative of three independent experiments.</p

Contribution of CD80 and CD86 to Interaction Forces and Cytokine Secretion.

<p>(<b>A</b>) Interaction forces; (<b>B</b>) IL-2 and (<b>C</b>) IFN-γ production of DC:T-cell or B:T-cell conjugates in the presence of blocking antibodies against CD80 and/or CD86. DCs or B-cells were pre-pulsed with Ova peptides (+Ova, 10 ng/ml) before antibody blocking. All force measurements were conducted with contact time of 3 min. *p<0.01; unpaired <i>t</i>-test. For each condition, OT-I T-cells were isolated from >3 independent experiments. αCD80: blocking antibody targeting CD80; αCD86: blocking antibody targeting CD86; αCD80/86: αCD80 and αCD86 simultaneously; IgG: isotype controls for both αCD80 and αCD86.</p