Figure 5.
Figure 5. Antigen-specific synapse formation induces STAT4 signaling and IFN-γ neosynthesis in T cells. (A) DCs were pretreated with TLR agonist, loaded with 10 nM of OVA peptide, and mixed with OT-I cells. After 30 min of incubation, cells were lysed and analyzed by Western blotting using an antibody against pSTAT4. Control lanes (1–4) are T cells alone (1) or incubated with soluble IL-12 (2) and DCs alone not stimulated (3) or stimulated (4) with TLR agonist. Lanes 5–8 are DCs coincubated with T cells in different conditions as indicated in the table. (B) Detection of pSTAT4 by intracellular FACS analysis. DCs were either left untreated or stimulated with TLR agonist (TLR), loaded (pep) or not with OVA peptide, and mixed with OT-I for 30 min. Cells were fixed and labeled intracellularly with anti-pSTAT4 Alexa Fluor 488 antibody. The MFI was determined by gating on isolated T cells (gate on T cells) or on DC–T cell doublets (gate on DC–T cell) as described in experimental procedure. Data represent the mean values ± SEM (subtracted for the isotype control values) obtained in three independent experiments. (C) Control DCs or DCs treated with colchicine were used in the assay described in B. Data show the MFI ± SEM of pSTAT4 signal determined on T cells engaged in doublets in four independent experiments (P < 0.01; Student’s t test). (D) DC–T cell antigen-specific interactions trigger IFN-γ neosynthesis in T cells. DCs activated by TLR agonist and loaded with peptide were allowed to interact with OT-I for 2 h, fixed, and labeled. Confocal images show polarized IL-12 (red) and DC-MTOC (blue) facing IFN-γ staining around the T cell MTOC (green). (E) Quantification of IFN-γ neosynthesis in T cells engaged in antigen-specific synapses was measured by intracellular FACS analysis under different conditions (as specified). Values plotted indicate the percentage of IFN-γ+/CD8+ T cells gated in the region of DC–T cell doublets (isolated T cells show no IFN-γ signal), subtracted for the isotype control (one of three experiments with identical results is presented). (F) Early expression of IFN-γ in T cells correlates to DC-MTOC polarization at the IS. Cells were labeled as in (D) and quantified. Bars show the distribution of IFN-γ–positive T cells in synapse with DCs that present (polarized) or not (not polarized) the MTOC facing the T cell membrane. The data comes from quantification of >100 cells in three independent experiments (**, P = 0.052).

Antigen-specific synapse formation induces STAT4 signaling and IFN-γ neosynthesis in T cells. (A) DCs were pretreated with TLR agonist, loaded with 10 nM of OVA peptide, and mixed with OT-I cells. After 30 min of incubation, cells were lysed and analyzed by Western blotting using an antibody against pSTAT4. Control lanes (1–4) are T cells alone (1) or incubated with soluble IL-12 (2) and DCs alone not stimulated (3) or stimulated (4) with TLR agonist. Lanes 5–8 are DCs coincubated with T cells in different conditions as indicated in the table. (B) Detection of pSTAT4 by intracellular FACS analysis. DCs were either left untreated or stimulated with TLR agonist (TLR), loaded (pep) or not with OVA peptide, and mixed with OT-I for 30 min. Cells were fixed and labeled intracellularly with anti-pSTAT4 Alexa Fluor 488 antibody. The MFI was determined by gating on isolated T cells (gate on T cells) or on DC–T cell doublets (gate on DC–T cell) as described in experimental procedure. Data represent the mean values ± SEM (subtracted for the isotype control values) obtained in three independent experiments. (C) Control DCs or DCs treated with colchicine were used in the assay described in B. Data show the MFI ± SEM of pSTAT4 signal determined on T cells engaged in doublets in four independent experiments (P < 0.01; Student’s t test). (D) DC–T cell antigen-specific interactions trigger IFN-γ neosynthesis in T cells. DCs activated by TLR agonist and loaded with peptide were allowed to interact with OT-I for 2 h, fixed, and labeled. Confocal images show polarized IL-12 (red) and DC-MTOC (blue) facing IFN-γ staining around the T cell MTOC (green). (E) Quantification of IFN-γ neosynthesis in T cells engaged in antigen-specific synapses was measured by intracellular FACS analysis under different conditions (as specified). Values plotted indicate the percentage of IFN-γ+/CD8+ T cells gated in the region of DC–T cell doublets (isolated T cells show no IFN-γ signal), subtracted for the isotype control (one of three experiments with identical results is presented). (F) Early expression of IFN-γ in T cells correlates to DC-MTOC polarization at the IS. Cells were labeled as in (D) and quantified. Bars show the distribution of IFN-γ–positive T cells in synapse with DCs that present (polarized) or not (not polarized) the MTOC facing the T cell membrane. The data comes from quantification of >100 cells in three independent experiments (**, P = 0.052).

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