Figure 3. Cytotoxicity is impaired in Stx11^-/- CTLs and NK cells, which display a phenotype comparable with that of WT counterparts.

(A) cDNA was prepared from ex vivo-sorted DX5⁺CD3⁻ or CD8⁺ T cells from WT or Stx11^-/- splenocytes and PCR was performed using gene-specific primers for Stx11 or Actin. Neg ctrl, negative control, omission of cDNA in PCR reaction; Pos ctrl, positive control, WT LPS stimulated BMM cDNA. Shown is one representative experiment out of two performed. (B) Western blot analysis of stx11 protein expression in naive NK and CD8⁺ T cells. Pos ctrl, membrane fractions of HEK cells transfected with a construct expressing murine STX11. Shown is one representative of three independent experiments. (C) NK cell specific lysis was assessed by release of cytoplasmic lactate dehydrogenase in vitro from YAC-1 cells co-incubated with IL-15 treated WT or Stx11^-/- splenocytes, at various effector/target ratios (E:T). Prior to the assay, an aliquot of effector cells was analyzed by flow cytometry to determine the relative percentages of NK cells, thus a splenocyte:target ratio of 50:1 corresponds to an NK cell:target ratio of 3.25:1. Results are expressed as mean + SD of three samples and are representative of three independent experiments. (D) CTL mediated cytotoxicity was assessed against P815 cells, in the presence of anti-CD3ε antibody, co-incubated with WT or Stx11^-/- CD8⁺ T cells stimulated for 2 rounds with anti-allo stimulation, at various effector/target ratios (E:T). Results are expressed as mean + SD of three samples and are representative of two independent experiments. (E, F) Flow cytometry analysis of WT or Stx11^-/- IL-15 treated NK cells and CTL stimulated for 2 rounds with anti-allo stimulation. Plots are gated on (E) SSC and NKp46⁺ or (F) SSC and CD8⁺ T cells. Values in plots indicate percent of positive cells. Shaded histograms show marker expression as indicated, bold lines show the corresponding isotype control. (E) Plots are representative of three independent experiments (n=8-9 mice in total). (F) Plots are representative of two independent experiments (n=5 mice in total). (G) Intracellular staining for Granzyme A (GrzA), -B (GrzB) or perforin of WT or Stx11^-/- NK cells treated with IL-15 (gated on NK1.1⁺CD3^- cells). Shaded histograms show marker expression as indicated (mean fluorescence intensity displayed), bold lines show the corresponding isotype control. Data shown are representative of three to four independent experiments. (H) Western blot for perforin and intracellular staining for GrzA and GrzB of WT or Stx11^-/- CD8⁺ T cells stimulated for 3 rounds of anti-allo stimulation (gated on CD8⁺ T cells). Shaded histograms show marker expression as indicated (mean fluorescence intensity displayed), bold lines show the corresponding isotype control. Data shown are representative of three to four independent experiments.