Fig. 3.
Fig. 3. NFAT1 promotes IFN-γ production through a cell-intrinsic mechanism. / Both panels show that NFAT1−/− IL-4−/− T cells produce less IFN-γ, regardless of whether or not they are mixed with NFAT1+/+ IL-4−/− T cells. (A) Intracellular staining for IFN-γ–producing cells. CD4+ T cells from NFAT1+/+ IL-4−/− and NFAT1−/− IL-4−/− mice were left unlabeled or were labeled with the fluorescent tracker dye CFSE, then differentiated by culturing them separately or as a 1:1 mixture with anti-CD3 and IL-2 for 4 days. After restimulation for 4 hours with PMA and ionomycin, IFN-γ production was analyzed at a single-cell level by intracellular cytokine staining, without further separation of the mixed-cell populations. Numbers on the right refer to the percentage of cells in each quadrant, relative to the total number of NFAT1+/+ IL-4−/− (italics) or NFAT1−/− IL-4−/− (nonitalics) cells. Note that cell division during the differentiation period can be followed over 6 to 7 generations by the decrease of CFSE fluorescence intensity. Note also that CFSE labeling does not cause significant changes in the proportion of cells expressing IFN-γ. (B) Levels of secreted IFN-γ. Cells were treated as in panel A, except that mixed populations were separated by FACS sorting immediately before they were restimulated. Twenty-four hours after restimulation, IFN-γ secreted into the supernatants was analyzed by ELISA. (left) Cells were cultured separately. Again, note that CFSE labeling does not cause significant changes in levels of IFN-γ production. (right) Cells were cultured as mixed populations and separated by sorting; the labeled populations are indicated. Note that cells in the mixed cultures, which underwent the sorting procedure, showed a considerable decrease in their levels of IFN-γ production regardless of whether they expressed or lacked NFAT1 (compare the scale of the y-axis in the left and right panels). Nevertheless, the relative decrease in IFN-γ expression by NFAT1−/− IL-4−/− versus NFAT1+/+ IL-4−/− T cells was maintained even after cell sorting (right panel).

NFAT1 promotes IFN-γ production through a cell-intrinsic mechanism.

Both panels show that NFAT1−/− IL-4−/− T cells produce less IFN-γ, regardless of whether or not they are mixed with NFAT1+/+ IL-4−/− T cells. (A) Intracellular staining for IFN-γ–producing cells. CD4+ T cells from NFAT1+/+ IL-4−/− and NFAT1−/− IL-4−/− mice were left unlabeled or were labeled with the fluorescent tracker dye CFSE, then differentiated by culturing them separately or as a 1:1 mixture with anti-CD3 and IL-2 for 4 days. After restimulation for 4 hours with PMA and ionomycin, IFN-γ production was analyzed at a single-cell level by intracellular cytokine staining, without further separation of the mixed-cell populations. Numbers on the right refer to the percentage of cells in each quadrant, relative to the total number of NFAT1+/+ IL-4−/− (italics) or NFAT1−/− IL-4−/− (nonitalics) cells. Note that cell division during the differentiation period can be followed over 6 to 7 generations by the decrease of CFSE fluorescence intensity. Note also that CFSE labeling does not cause significant changes in the proportion of cells expressing IFN-γ. (B) Levels of secreted IFN-γ. Cells were treated as in panel A, except that mixed populations were separated by FACS sorting immediately before they were restimulated. Twenty-four hours after restimulation, IFN-γ secreted into the supernatants was analyzed by ELISA. (left) Cells were cultured separately. Again, note that CFSE labeling does not cause significant changes in levels of IFN-γ production. (right) Cells were cultured as mixed populations and separated by sorting; the labeled populations are indicated. Note that cells in the mixed cultures, which underwent the sorting procedure, showed a considerable decrease in their levels of IFN-γ production regardless of whether they expressed or lacked NFAT1 (compare the scale of the y-axis in the left and right panels). Nevertheless, the relative decrease in IFN-γ expression by NFAT1−/− IL-4−/− versus NFAT1+/+ IL-4−/− T cells was maintained even after cell sorting (right panel).

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