Figure 4. Stathmin−/− T cells have impaired MTOC polarization and are defective in cytolysis.

(A) Stathmin knockout or littermate control T cells were purified, rested for 30 minutes and plated on anti-CD3 coated glass coverslips for the indicated time points. Cells were fixed, permeabilized and stained with anti-αβ-tubulin and imaged using confocal microscopy. Data are represented as mean + SEM and more than 50 cells per condition were scored in 3 experiments. All KO values are significantly different from WT (P<0.001, Unpaired t test). (B) Rested T cells from stathmin knockout OT-1 or littermate OT-1 controls were conjugated with peptide-pulsed CFSE-labeled RMA-S cells onto glass coverslips for 5 minutes at room temperature. The cells were then fixed, permeabilized, stained with Hoechst and anti-αβ-tubulin, and analyzed using confocal microscopy. Data are represented as mean + SEM and more than 50 cells per condition were scored in 3 experiments. P value < 0.001 (Unpaired t test). Images represent cells scored as polarized or not polarized. Scale bars =10μm. (C) Cytolysis by stathmin KO or littermate control CTL was assayed using standard ⁵¹Cr assay. Cells were plated at indicated E:T ratios and incubated for 4–6 hours. Supernatants were counted on a MicroBeta counter and specific lysis was calculated. Differences in specific lysis between stathmin wild-type and knockout CTL are significant (p<0.001, Unpaired t-test) at all E:T ratios. Graph is representative of 2 experiments. (D, E) Degranulation was assayed by LAMP-1 staining during 45 minutes of OT-1+ stathmin knockout or control T cell incubation with peptide-loaded RMA-S cells. Graphs show mean + SEM and are representative of 4 experiments.