Figure 7
Figure 7. MKK6EE expression enhances human (hu)DC maturation and expands MELAN-A–specific CD8 T cells in vitro. (A). The structure of the lentivector constructs used for transduction of huDCs is shown on top. The ubiquitin promoter and IiOVA gene were replaced with the CMV promoter and GFP gene. Human DCs were transduced with dual lentivectors coexpressing MKK6EE with GFP and grown for 6 days at 37°C. Surface staining was performed for the indicated DC marker. Histogram plots gated on GFP-positive transduced DCs are shown. The number of events is represented as a function of log fluorescence intensity for the DC markers shown. I indicates the distribution from the isotype control. The horizontal line indicates the cells considered to be positive, which is set to exclude 95% of cells stained with the isotype control. Percentage of positive DCs and mean fluorescent intensities are indicated within each histogram. LPS indicates huDCs treated with LPS overnight previous to surface staining. GFP indicates huDCs transduced with lentivectors encoding only GFP. Levels of DC maturation markers were estimated from 3 independent experiments by surface staining and flow cytometry, as shown in the histograms above. Mean fluorescent intensities for each marker from GFP-expressing transduced huDCs were represented as percentages compared with levels after LPS treatment. Means are shown as column graphs with error bars (SD), for the indicated markers within the graph. Lentivectors encoding the indicated activators used for transductions are shown on the bottom of the graphs. Selected statistical comparisons between indicated samples and GFP-expressing huDCs are shown. *, significant differences (P < .05). (B) The structure of the lentivector constructs used for transduction of huDCs is shown on top. The ubiquitin promoter and IiOVA gene were replaced with the CMV promoter and MELAN-A gene. Expression of the indicated activators and MELAN-A was confirmed by immunoblot using FLAG-specific or HA-specific antibodies, as indicated on the left. kDa, kilodaltons. (C) Dot-plots from CD8 T cell cultures representing fluorescence intensity from APC-conjugated MELAN-A–specific MHC I pentamer as a function of CD8 fluorescence intensity in logarithmic scale. These T cell cultures were stimulated with immature huDCs loaded with MELAN-A peptide, cytokine-matured peptide-loaded huDCs and huDCs transduced with lentivectors coexpressing MKK6 K82A or MKK6EE with MELAN-A, as shown within each graph. The percentage of events within each quadrant and the absolute number of CD8 T cells are shown. (D) The graph on the left shows CD8 T cell numbers in cultures with the indicated huDC stimulator cells. The graph on the right shows MELAN-A–specific CD8 T-cell numbers in cultures with the indicated huDC stimulator cells. Each column represents the mean from 3 independent DC-lymphocyte cultures with error bars (SD). Relevant statistical comparisons are shown within each graph between the indicated samples and CD8 T-cell expansion by cytokine-matured peptide-loaded huDCs, and highly significant (***P < .001) differences are shown in the graphs. iDCp indicates immature huDCs loaded with class I MELAN-A peptide; mDCp indicates cytokine-matured huDCs loaded with class I MELAN-A peptide; K82A represents huDCs coexpressing MKK6 K82A with MELAN-A; MKK6 represents huDCs coexpressing MKK6 EE with MELAN-A.

MKK6EE expression enhances human (hu)DC maturation and expands MELAN-A–specific CD8 T cells in vitro. (A). The structure of the lentivector constructs used for transduction of huDCs is shown on top. The ubiquitin promoter and IiOVA gene were replaced with the CMV promoter and GFP gene. Human DCs were transduced with dual lentivectors coexpressing MKK6EE with GFP and grown for 6 days at 37°C. Surface staining was performed for the indicated DC marker. Histogram plots gated on GFP-positive transduced DCs are shown. The number of events is represented as a function of log fluorescence intensity for the DC markers shown. I indicates the distribution from the isotype control. The horizontal line indicates the cells considered to be positive, which is set to exclude 95% of cells stained with the isotype control. Percentage of positive DCs and mean fluorescent intensities are indicated within each histogram. LPS indicates huDCs treated with LPS overnight previous to surface staining. GFP indicates huDCs transduced with lentivectors encoding only GFP. Levels of DC maturation markers were estimated from 3 independent experiments by surface staining and flow cytometry, as shown in the histograms above. Mean fluorescent intensities for each marker from GFP-expressing transduced huDCs were represented as percentages compared with levels after LPS treatment. Means are shown as column graphs with error bars (SD), for the indicated markers within the graph. Lentivectors encoding the indicated activators used for transductions are shown on the bottom of the graphs. Selected statistical comparisons between indicated samples and GFP-expressing huDCs are shown. *, significant differences (P < .05). (B) The structure of the lentivector constructs used for transduction of huDCs is shown on top. The ubiquitin promoter and IiOVA gene were replaced with the CMV promoter and MELAN-A gene. Expression of the indicated activators and MELAN-A was confirmed by immunoblot using FLAG-specific or HA-specific antibodies, as indicated on the left. kDa, kilodaltons. (C) Dot-plots from CD8 T cell cultures representing fluorescence intensity from APC-conjugated MELAN-A–specific MHC I pentamer as a function of CD8 fluorescence intensity in logarithmic scale. These T cell cultures were stimulated with immature huDCs loaded with MELAN-A peptide, cytokine-matured peptide-loaded huDCs and huDCs transduced with lentivectors coexpressing MKK6 K82A or MKK6EE with MELAN-A, as shown within each graph. The percentage of events within each quadrant and the absolute number of CD8 T cells are shown. (D) The graph on the left shows CD8 T cell numbers in cultures with the indicated huDC stimulator cells. The graph on the right shows MELAN-A–specific CD8 T-cell numbers in cultures with the indicated huDC stimulator cells. Each column represents the mean from 3 independent DC-lymphocyte cultures with error bars (SD). Relevant statistical comparisons are shown within each graph between the indicated samples and CD8 T-cell expansion by cytokine-matured peptide-loaded huDCs, and highly significant (***P < .001) differences are shown in the graphs. iDCp indicates immature huDCs loaded with class I MELAN-A peptide; mDCp indicates cytokine-matured huDCs loaded with class I MELAN-A peptide; K82A represents huDCs coexpressing MKK6 K82A with MELAN-A; MKK6 represents huDCs coexpressing MKK6 EE with MELAN-A.

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