Figure 7.
Figure 7. Cdc42 depletion in DCs selectively impairs IL-12–dependent events in T cells. (A) Control and Cdc42-silenced cells activated by TLRs agonist and loaded (peptide) or not loaded (no peptide) with 10 nM of class I peptide were mixed with OT-I cells for 30 min and lysed to analyze the levels of activated STAT4 by W.B. Numbers indicate the intensity values (ratio pSTAT4 peptide/pSTAT4 no peptide normalized for actin levels) obtained from density scans. (B) Neosynthesis of IFN-γ in T cells engaged in synapse with control or Cdc42-silenced cells. DCs treated as in A were incubated for 2 h, fixed, and analyzed for intracellular IFN-γ in T cells engaged in synapse (gated on DC–T cell doublets) by FACS. Values represent average of three independent experiments (*, P = 0.0133). (C) Conjugate formation is not affected by Cdc4 silencing. 105 control or Cdc42-silenced DCs were loaded with graded doses of peptide as indicated, labeled with SNARF and mixed with 105 CFSE labeled OT-I for 20 min at 37°C. The number of DC–T cell doublets was measured by FACS gating on the double-positive events and normalized to the number of T cells. Values shown are mean ± SD of three experiments. (D) T cells proliferation and survival are affected by Cdc42 silencing in DCs. 104 control (control-siRNA) or Cdc42-depleted (Cdc42-siRNA) DCs were TLR stimulated, loaded with graded doses of peptide, and mixed with OVA-specific OT-I cells that had been prelabeled with CFSE. (left) Day 3 CFSE dilution profiles at the indicated doses of peptide. (right) The plot shows the total number of cells recovered at day 3. One of three independent experiments with similar results is shown. (E) Dot plots show the percentage of IFN-γ–producing cells at day 3 after priming, when T cells were primed using control or Cdc42-depleted DCs. Data show one representative of two experiments. Cell culture supernatants were harvested at day 3, and the levels of IFN-γ were measured by ELISA (values are mean ± SD of three independent experiments (**, P = 0.001). IFN-γ levels secreted by T cells are inhibited by DCs expressing dominant-negative Cdc42. Cdc42WT or the dominant-negative Cdc42N17 were stimulated as in D and mixed with OT-I. Bars show the levels of IFN-γ in day 3 cell culture supernatants. Data are the means ± SD of three independent experiments (**, P = 0.0017). (G) Expression of a rescue construct partially restores IFN-γ levels. DCs were cotransfected with control or Cdc42-specific siRNA, plus a plasmid encoding a Cdc42 resistant to silencing (rescue). Cells were treated as in F. Bars shows values of IFN-γ in day 3 cell culture supernatants from 2 independent experiments (means ± SD; *, P = 0.0385, **, P = 0.0068).

Cdc42 depletion in DCs selectively impairs IL-12–dependent events in T cells. (A) Control and Cdc42-silenced cells activated by TLRs agonist and loaded (peptide) or not loaded (no peptide) with 10 nM of class I peptide were mixed with OT-I cells for 30 min and lysed to analyze the levels of activated STAT4 by W.B. Numbers indicate the intensity values (ratio pSTAT4 peptide/pSTAT4 no peptide normalized for actin levels) obtained from density scans. (B) Neosynthesis of IFN-γ in T cells engaged in synapse with control or Cdc42-silenced cells. DCs treated as in A were incubated for 2 h, fixed, and analyzed for intracellular IFN-γ in T cells engaged in synapse (gated on DC–T cell doublets) by FACS. Values represent average of three independent experiments (*, P = 0.0133). (C) Conjugate formation is not affected by Cdc4 silencing. 105 control or Cdc42-silenced DCs were loaded with graded doses of peptide as indicated, labeled with SNARF and mixed with 105 CFSE labeled OT-I for 20 min at 37°C. The number of DC–T cell doublets was measured by FACS gating on the double-positive events and normalized to the number of T cells. Values shown are mean ± SD of three experiments. (D) T cells proliferation and survival are affected by Cdc42 silencing in DCs. 104 control (control-siRNA) or Cdc42-depleted (Cdc42-siRNA) DCs were TLR stimulated, loaded with graded doses of peptide, and mixed with OVA-specific OT-I cells that had been prelabeled with CFSE. (left) Day 3 CFSE dilution profiles at the indicated doses of peptide. (right) The plot shows the total number of cells recovered at day 3. One of three independent experiments with similar results is shown. (E) Dot plots show the percentage of IFN-γ–producing cells at day 3 after priming, when T cells were primed using control or Cdc42-depleted DCs. Data show one representative of two experiments. Cell culture supernatants were harvested at day 3, and the levels of IFN-γ were measured by ELISA (values are mean ± SD of three independent experiments (**, P = 0.001). IFN-γ levels secreted by T cells are inhibited by DCs expressing dominant-negative Cdc42. Cdc42WT or the dominant-negative Cdc42N17 were stimulated as in D and mixed with OT-I. Bars show the levels of IFN-γ in day 3 cell culture supernatants. Data are the means ± SD of three independent experiments (**, P = 0.0017). (G) Expression of a rescue construct partially restores IFN-γ levels. DCs were cotransfected with control or Cdc42-specific siRNA, plus a plasmid encoding a Cdc42 resistant to silencing (rescue). Cells were treated as in F. Bars shows values of IFN-γ in day 3 cell culture supernatants from 2 independent experiments (means ± SD; *, P = 0.0385, **, P = 0.0068).

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