Fig. 4.

LPL Nb-expressing T cells are unable to form a normal IS. a, b Jurkat T cells transfected with EGFP, LPL Nb5-EGFP or LPL Nb9-EGFP were incubated for 40 min with SEE-pulsed (+SEE) or unstimulated (−SEE) superantigen-presenting cells (Raji cells) and stained for actin (phalloidin-alexa 594, red) (a) or L-plastin (Alexa-594, red) (b). Transfected Jurkat cells are depicted in green (EGFP) and the nuclei were stained with DAPI. The pictures were acquired with an epifluorescence microscope and are representative for three independent experiments. Bar 10 μm. c PBMCs expressing GSN Nb11 or LPL Nb5 were primed with SEE and conjugated with SEE-pulsed Raji cells as described and stained for L-plastin (alexa-594, red) or actin (phalloidin-alexa 594, red). In the merged pictures, PBMCs expressing the EGFP-tagged nanobodies are shown in green and the Raji’s are shown in magenta (far red). The pictures were acquired with a laser scanning confocal microscope and are representative for three independent experiments. Bar 10 μm. d Confocal images of conjugated Jurkat cells expressing LPL Nbs or EGFP. Left panel Raji cells are shown in magenta (far red) and transfected Jurkat cells in green (EGFP). Right panel Z-dimensional acquisition for actin (alexa-594, red) and EGFP (green) staining, followed by projection on the Z-axis. Bar 5 μm. e Representation of the relative actin intensity profile as a function of the length (μm) measured on the Z-dimensional acquisition (as described in d, right panel) for EGFP (solid line) and LPL Nb5 (dashed line). f Bar graph of the IS length (measured with the method described in b) obtained for different conjugated cells (n = 20) in each condition from three independent experiments. g Representation of the cell–cell contact area. The area was calculated from the two different values (L and Z); L being the value obtained in b, and Z being the actin length measurement obtained from the z-dimensional acquisitions (described in “Materials and Methods”). h Representation of the IS area of conjugated cells (n = 20) for EGFP, GSN Nb11, LPL Nb5 or LPL Nb9 transfected cells from three independent experiments. Error bars mean ± SEM. Unpaired t tests were performed to observe statistical differences in the area of the IS between cells expressing EGFP and cells expressing a nanobody (*p < 0.05; **p < 0.01; ***p < 0.001)