Galectin-3 prevents TCR synapse formation in late-stage T cells.

<p>(A) CD7⁻ and CD7⁺ T cells were activated by incubation with anti-CD3 mAb plus anti-human CD28 mAb or as control by an isotype-matched IgG1 (medium). Cells were stained for TCR-alpha/beta (green), CD7 (blue), CD3 (red) and for galectin-3 (yellow). Immunofluorescence was visualized by a LSM. Alternatively cells were stained with mAbs specific to CD7 and subsequently recorded for gal-3 expression by staining with the anti-human gal-3mAb or as control by an isotype-matched IgG and analyzed by flow cytometry. A representative experiment out of five is shown. Galectin-3 specific signals were quantitatively recorded by a LSM as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#s4" target="_blank">Materials and Methods</a>. A minimum of 100 cells for each data point was recorded. (B) To monitor location of galectin-3 in lipid raft formation, isolated CD7⁻ and CD7⁺ subsets of CD8⁺ CD45RO⁺ T cells were incubated with or without swainsonine for 24 hrs and then activated for various time intervals (1 min until 20 min) on coverslips coated with the agonistic anti-CD3 mAb plus anti-CD28 mAb. T cell stimulation was stopped with paraformaldehyde and cells were stained with anti-TCR-alpha/betamAb (blue), anti-CD7 mAb (red) and anti-galectin-3 mAb (yellow) together with CtxB (green) for lipid rafts staining. Immunofluorescence was visualized by a LSM and quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#s4" target="_blank">Materials and Methods</a>. T cell staining after 5 min stimulation of one representative experiment out of five is exemplarily shown. (C) CD7⁻ and CD7⁺ subsets of CD8⁺ CD45RO⁺ T cells were incubated with or without swainsonine and activated as described in (B). Frequencies of cells producing IFN-gamma and showing degranulation indicated by CD107a was monitored by flow cytometry. Data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#pone-0030713-g003" target="_blank">Fig. 3</a> represent the mean ± SEM of five experiments and were compared using a paired t-test. * p<0.05. FU: fluorescence unit; MFI: mean fluorescence intensity.</p

TCR synapse formation is impaired in late-stage T cells.

<p>CD8⁺ CD45RO⁺ T lymphocytes were incubated on coverslips coated with the agonistic anti-CD3 mAb (UCHT-1) plus anti-CD28 mAb (CD28.2) or with the anti-CD2 mAbs (L303.1) and (L304.1) plus the anti-CD28 mAb (CD28.2). Cells were activated for various time intervals (1 min until 20 min), incubation was stopped by addition of paraformaldehyde, and cells were stained with TCR-alpha/betamAb (green) plus CD7 mAb (blue) or alternatively with CD2 mAb (blue) plus CD7 mAb (green) together with cholera toxin B (CtxB) (white) for lipid raft staining. Cells were analyzed on a LSM 510 with 630× microscope magnification. A minimum of 100 cells for each data point was recorded. Representative images after 0 min, 1 min, 5 min, 10 min and 20 min stimulation out of five independent experiments are shown. Synapse formation intensity (CTxB intensity) was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#s4" target="_blank">Materials and Methods</a>. A minimum of 100 cells of each cell population on each coverslip was recorded. Data represent mean scores from five experiments ± standard error of the mean and were compared using a paired t-test. * p<0.05. FU: fluorescence unit.</p