1. Regulation of interferon-γ gene expression by nuclear factor of activated T cells
by Alexander Kiani, Francisco J. Garcı́a-Cózar, Ivonne Habermann, Stefanie Laforsch, Toni Aebischer, Gerhard Ehninger, and Anjana Rao
Blood
Volume 98(5):
September 1, 2001
©2001 by American Society of Hematology

2. Reduced IFN-γ production by NFAT1−/−IL-4−/− T-helper cells
Reduced IFN-γ production by NFAT1−/−IL-4−/− T-helper cells.(A) NFAT1−/− IL-4−/− T cells show diminished levels of IFN-γ mRNA.
Reduced IFN-γ production by NFAT1−/−IL-4−/− T-helper cells.(A) NFAT1−/− IL-4−/− T cells show diminished levels of IFN-γ mRNA. CD4+ T cells from NFAT1+/+ IL-4−/− and NFAT1−/−IL-4−/−mice were cultured for 4 days with anti-CD3 and IL-2, rested for 48 hours, and restimulated with anti-CD3. Differentiation and restimulation were performed under neutral conditions, without the addition of antibodies and cytokines other than IL-2. Six hours after restimulation, total cellular RNA was isolated and analyzed by RNase protection assay for transcript levels of the indicated cytokines. (B) NFAT1−/− IL-4−/− T cells show diminished production of IFN-γ protein. NFAT1+/+ IL-4−/− and NFAT1−/−IL-4−/− CD4+ T cells were cultured as in panel A. Supernatants were collected 24 hours after restimulation, and levels of IFN-γ and IL-2 were determined by ELISA. Values are expressed as the means ± SEM of 9 (IFN-γ) or 3 (IL-2) independent experiments.
Alexander Kiani et al. Blood 2001;98:
©2001 by American Society of Hematology

3. IL-4 promotes Th2 differentiation of NFAT1−/− IL-4−/− T cells
IL-4 promotes Th2 differentiation of NFAT1−/− IL-4−/− T cells.(A) IL-4 treatment up-regulates mRNAs encoding Th2 cytokines and down-regulates IFN-γ mRNA in both NFAT1−/−IL-4−/− (▨) and NFAT1+/+IL-4−/− (▪) cells.
IL-4 promotes Th2 differentiation of NFAT1−/− IL-4−/− T cells.(A) IL-4 treatment up-regulates mRNAs encoding Th2 cytokines and down-regulates IFN-γ mRNA in both NFAT1−/−IL-4−/− (▨) and NFAT1+/+IL-4−/− (▪) cells. CD4+ T cells from NFAT1+/+ IL-4−/− and NFAT1−/−IL-4−/− mice were cultured for 4 days with anti-CD3 and IL-2 in the presence of IL-4 ( U/mL), rested for 48 hours, and restimulated with anti-CD3. Six hours after restimulation, total cellular RNA was isolated and analyzed by RNase protection assay for transcript levels of the indicated cytokines. (B) IL-4 treatment up-regulates IL-5 production and down-regulates IFN-γ production in both NFAT1−/− IL-4−/− and NFAT1+/+ IL-4−/− cells. NFAT1+/+IL-4−/− and NFAT1−/− IL-4−/−CD4+ T cells were cultured as in panel A. Supernatants were collected 24 hours after restimulation, and levels of IFN-γ and IL-5 were determined by ELISA. Values are expressed as the means ± SEM of 3 independent experiments.
Alexander Kiani et al. Blood 2001;98:
©2001 by American Society of Hematology

4. NFAT1 promotes IFN-γ production through a cell-intrinsic mechanism
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.
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).
Alexander Kiani et al. Blood 2001;98:
©2001 by American Society of Hematology

5. Decreased production of IFN-γ by NFAT1−/− IL-4−/− T cells cannot be explained by overexpression of Maf or GATA-3.CD4+ T cells from NFAT1+/+IL-4−/− and NFAT1−/− IL-4−/−mice were cultured for 4 days with anti-CD3 and IL-2, either under neutral conditions (lan...
Decreased production of IFN-γ by NFAT1−/− IL-4−/− T cells cannot be explained by overexpression of Maf or GATA-3.CD4+ T cells from NFAT1+/+IL-4−/− and NFAT1−/− IL-4−/−mice were cultured for 4 days with anti-CD3 and IL-2, either under neutral conditions (lanes 1 and 2) or in the presence of 1000 U/mL IL-4 to induce Th2 differentiation (lanes 3 and 4). After harvesting the cells, total RNA was extracted, and Northern blot analysis was performed as described in “Materials and methods.” Ethidium bromide staining of the 28S bands confirmed equal loading of RNAs isolated from NFAT1−/− IL-4−/− and NFAT1+/+IL-4−/− cells (data not shown). Results of 1 of 2 identical experiments are shown.
Alexander Kiani et al. Blood 2001;98:
©2001 by American Society of Hematology

6. NFAT1 is required for optimal IFN-γ production by T cells at all stages of T-cell differentiation.NFAT1 is required for optimal IFN-γ production by naive and differentiated T-helper cells.
NFAT1 is required for optimal IFN-γ production by T cells at all stages of T-cell differentiation.NFAT1 is required for optimal IFN-γ production by naive and differentiated T-helper cells. NFAT1+/+IL-4−/− and NFAT1−/− IL-4−/−CD4+ T cells were sorted by FACS for high expression of Mel-14 and were cultured with plate-bound anti-CD3 and IL-2 for 2 consecutive rounds of differentiation, in the absence (top panels) or presence (bottom panels) of IL-12. Before differentiation (stimulation 1, left panel) and after the first (stimulation 2, middle panels) and second (stimulation 3, right panels) rounds of differentiation, cells were stimulated for 48 hours (stimulation 1) or 24 hours (stimulations 2 and 3) with plate-bound anti-CD3, after which IFN-γ was analyzed in the supernatants of the cells. Note that y-axis scales representing IFN-γ production are different in all panels.
Alexander Kiani et al. Blood 2001;98:
©2001 by American Society of Hematology

7. Decreased resistance of NFAT1−/−IL-4−/− mice to infection with L major.
Decreased resistance of NFAT1−/−IL-4−/− mice to infection with L major. (A) NFAT1−/−IL-4−/− and NFAT1+/+ IL-4−/−female mice (10 mice per group) were infected with promastigotes ofL major in the left hind footpad. Footpad thickness was recorded at the indicated times using a metric caliper, and the lesion size was calculated as the difference between the injected and the uninjected footpads. Values are shown as means ± SD. (B) NFAT1−/− IL-4−/− and NFAT1+/+IL-4−/− mice (3 mice per group) were infected with promastigotes of L major as in panel A. Eleven weeks after infection, the number of parasites in the draining popliteal lymph nodes was determined as described in “Material and methods.” Values are shown as means ± SD. KO, knockout mice; DKO, double-knockout mice.
Alexander Kiani et al. Blood 2001;98:
©2001 by American Society of Hematology

8. NFAT1 regulates the acute phase of IFN-γ production in T-helper cells
NFAT1 regulates the acute phase of IFN-γ production in T-helper cells.(A) Reduced IFN-γ production by freshly isolated NFAT1−/− IL-4−/− T-helper cells in response to stimulation with anti-CD3 but not IL-12/IL-18.
NFAT1 regulates the acute phase of IFN-γ production in T-helper cells.(A) Reduced IFN-γ production by freshly isolated NFAT1−/− IL-4−/− T-helper cells in response to stimulation with anti-CD3 but not IL-12/IL-18. NFAT1+/+IL-4−/− (▪) and NFAT1−/−IL-4−/− (▨) CD4+ T cells were stimulated either with plate-bound anti-CD3 (left panel) or a combination of IL-12 and IL-18 (right panel) for 48 hours, after which IFN-γ was analyzed in the supernatants of the cells. Shown is a representative of 3 (left panel) or 5 (right panel) independent experiments. (B) The selective NFAT inhibitor GFP-VIVIT suppresses IFN-γ production by a murine T-cell clone. Cl.7W2 cells were transfected with plasmids encoding either GFP or GFP-VIVIT. The next day the cells were either left unstimulated or were stimulated for 5 hours with PMA and ionomycin. IFN-γ production was analyzed at a single-cell level by intracellular cytokine staining. Dot blots from 1 of 3 similar experiments are shown. Thresholds for IFN-γ production were set using the unstimulated control. Note that each panel shows transfected (GFP-positive) and nontransfected (GFP-negative) cells of the same sample.
Alexander Kiani et al. Blood 2001;98:
©2001 by American Society of Hematology