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The Journal of Immunology, 2006, 177: 5195-5203.
Copyright © 2006 by The American Association of Immunologists, Inc.

Distinct Characteristics of Murine STAT4 Activation in Response to IL-12 and IFN-{alpha}1

Lisa S. Berenson*, Maya Gavrieli*, J. David Farrar2,*, Theresa L. Murphy* and Kenneth M. Murphy3,*,{dagger}

* Department of Pathology and Center for Immunology, and {dagger} Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The role of type I IFN in Th1 development, STAT4 activation, and IFN-{gamma} production in murine T cells has remained unresolved despite extensive examination. Initial studies indicated that IFN-{alpha} induced Th1 development and IFN-{gamma} production in human, but not murine, T cells, suggesting species-specific differences in signaling. Later studies suggested that IFN-{alpha} also induced Th1 development in mice, similar to IL-12. More recent studies have questioned whether IFN-{alpha} actually induces Th1 development even in the human system. In the present study, we compared the capacity of IL-12 and IFN-{alpha} to induce Th1 differentiation, STAT4 phosphorylation, and IFN-{gamma} production in murine T cells. First, we show that IFN-{alpha}, in contrast to IL-12, cannot induce Th1 development. However, in differentiated Th1 cells, IFN-{alpha} can induce transient, but not sustained, STAT4 phosphorylation and, in synergy with IL-18, can induce transient, but not sustained, IFN-{gamma} production in Th1 cells, in contrast to the sustained actions of IL-12. Furthermore, loss of STAT1 increases IFN-{alpha}-induced STAT4 phosphorylation, but does not generate levels of STAT4 activation or IFN-{gamma} production achieved by IL-12 or convert transient STAT4 activation into a sustained response. Our findings agree with recent observations in human T cells that IFN-{alpha}-induced STAT4 activation is transient and unable to induce Th1 development, and indicate that IFN-{alpha} may act similarly in human and murine T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The differentiation of naive CD4+ T cells toward the Th1 phenotype involves several cytokines and transcription factors (1, 2, 3, 4). One of these cytokines, IL-12, promotes Th1 development through activation of the transcription factor STAT4 (5), which is required for optimal Th1 responses in vivo (6, 7). Other cytokines, including members of the IFN family, have been examined for their ability to induce Th1 differentiation in human and murine T cells (8, 9, 10, 11, 12). For some time, the actions of type I IFN, such as IFN-{alpha}, were thought to differ between the human and murine system. While IFN-{alpha} was claimed to drive Th1 development in human T cells (8, 9, 10), type I IFN did not drive Th1 development in mouse T cells (11, 12, 13). Type I IFN were thought to induce STAT4 phosphorylation in human T cells, but not murine Th1 cells (14, 15). However, it was recently reported that IFN-{alpha} can activate STAT4 in murine T cells responding to lymphocytic choriomeningitis virus (LCMV)4 infection (16) or already differentiated Th1 cells (13), although these studies did not demonstrate the ability of IFN-{alpha} to induce Th1 differentiation. A potential species-specific STAT4 activation by IFN-{alpha} was proposed to involve a mutation in the murine STAT2 gene that would diminish STAT4 recruitment (15, 17). However, STAT2–/– leukocytes show levels of IFN-{alpha}-induced STAT4 phosphorylation comparable to wild-type (WT) cells (18).

We recently re-evaluated the actions of IFN-{alpha} in murine T cells. We observed that IFN-{alpha} did not induce Th1 differentiation in a murine Ag-specific system despite detectable, although low, levels of STAT4 activation (13). Also, a recent study of IL-12 and IFN-{alpha} signaling in human T cells reported that IL-12 induced much stronger Th1 development than IFN-{alpha}, and that IFN-{alpha}-induced STAT4 phosphorylation was transient compared with IL-12 (19). Transient STAT4 activation in response to IFN-{alpha} stimulation was also reported in anti-CD3-stimulated human T cells (20) and in human NK cells (21).

In this report, we compared IL-12 and IFN-{alpha} for their ability to activate STAT4 and induce Th1 development and IFN-{gamma} production in murine CD4+ T cells. First, we confirm that IFN-{alpha} does not induce Th1 development in naive CD4+ T cells. Furthermore, IFN-{alpha} activation of STAT4 and production of IFN-{gamma} is transient in already differentiated Th1 cells. The transient activation of STAT4 by IFN-{alpha} is not due to cytokine consumption or loss of the IFN-{alpha} receptor 1 (IFNAR1) and does not interfere with IL-12-induced STAT4 activation. Finally, while STAT1–/– Th1 cells treated with IFN-{alpha} showed increased STAT4 phosphorylation and IFN-{gamma} production compared with WT cells, the loss of STAT1 did not allow for sustained STAT4 activation to be induced by IFN-{alpha}, consistent with its inability to induce Th1 development in STAT1–/– T cells (13). These findings suggest that the actions of type I IFN are similar between human and mouse T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

BALB/c, DO11.10 transgenic (22), DO11.10 STAT1–/– (23), DO11.10 STAT4–/– (23), and DO11.10 mouse/human (m/h) STAT2 knock-in (KI) mice (24) were as described previously. All animal studies were approved by the Washington University Animal Studies Committee.

Abs and reagents

Purified anti-CD3{epsilon}, PE-conjugated anti-mouse IFN-{gamma}, allophycocyanin-conjugated anti-mouse IFN-{gamma}, PE-Cy5-conjugated anti-mouse CD4, and allophycocyanin-conjugated anti-mouse CD4 were purchased from BD Biosciences. IL-12 (Genetics Institute) was used at 10 U/ml and IL-18 (Molecular Biological Laboratories) at 50 ng/ml. IFN-{alpha} (murine rIFN-{alpha}A; PBL Biomedical Laboratories) was used at 1000 U/ml or as indicated.

Cell purifications and activation

DO11.10 CD4+ T cells were purified using CD4+ MACS beads (Miltenyi Biotec) and cultured with irradiated BALB/c splenocytes, 0.3 µM OVA, IFN-{gamma} (200 U/ml, a gift from R. D. Schreiber, Washington University, St. Louis, MO), anti-IL-4 (11B11 hybridoma supernatant) (25), IL-12, and IL-2 (40 U/ml; Takeda). All cells were restimulated on day 7 with irradiated BALB/c splenocytes, 0.3 µM OVA, IL-2 (40 U/ml), and Th1-inducing cytokines (IL-12, IFN-{gamma}, and anti-IL-4). After 2 wk of Th1 differentiation, cells were assayed for IFN-{gamma} production and passed weekly with irradiated BALB/c splenocytes, 0.3 µM OVA, IL-2, and anti-IL-4. Before use in experiments, resting T cells were purified by Histopaque (Sigma-Aldrich) gradient.

For experiments using naive cells, CD4+ T cells were isolated as described above from DO11.10 splenocytes and stimulated with anti-CD3 (10 µg/ml), IFN-{gamma}, and anti-IL-12 (TOSH hybridoma supernatant) (26) for various times as indicated.

For experiments using m/h STAT2 KI cells (24), DO11.10 or DO11.10 m/h STAT2 KI splenocytes were stimulated for 4 h at 37°C with IFN-{alpha} or anti-IFN-{alpha} Ab (1 µg/ml; PBL Biomedical Laboratories). RNA was prepared and Affymetrix microarrays performed as described previously (27).

Western blotting

Immunoprecipitation and Western blotting were performed on lysates from 15 x 106 T cells as described previously (13).

Intracellular STAT4 protein staining

Intracellular STAT4 protein staining was adapted from previous reports (28, 29, 30, 31). Briefly, resting T cells were stimulated with the indicated cytokines at 107 cells/ml in 48-well plates at 37°C; stimulation was stopped with ice-cold PBS. Cells were fixed with 2% paraformaldehyde, permeabilized with ice-cold 100% methanol, and stained with 0.5 µg of primary Ab (rabbit anti-STAT4 or anti-phosphoSTAT4, Zymed; or anti-STAT1, Santa Cruz Biotechnology) or isotype control (rabbit IgG; Caltag Laboratories) in staining buffer (0.5% BSA in PBS with 0.5% saponin). Secondary Ab (FITC-conjugated goat anti-rabbit; Caltag Laboratories) was added at a final concentration of 0.2 µg/ml in staining buffer. Anti-CD4 Abs were added. Cells were washed and immediately analyzed on a FACSCalibur (BD Biosciences) and data analysis used FlowJo (Tree Star).

For microscopy, a Zeiss LSM510 confocal system was used. Cells were stained with TO-PRO-3 (Molecular Probes) during secondary staining.

Cytokine quantification

Resting Th1 cells were stimulated at 2 x 106 cells/ml in 48-well plates. For intracellular cytokine staining (ICS), brefeldin A (1 µg/ml; Epicentre Technology) was added for the final 4 h. If ELISA were performed, supernatants were removed before addition of brefeldin A. ICS and ELISA were performed as described previously (13).

IFNAR1 Staining

Resting Th1 cells were stimulated with the indicated cytokines for 4 h and stained with biotinylated anti-IFNAR1 (MAR1-5A3 (32), a gift from R. D. Schreiber and K. C. Sheehan, Washington University, St. Louis, MO) or control isotype (GIR-208 (32), a gift from R.D. Schreiber and K.C. Sheehan) followed by streptavidin-PE (Caltag Laboratories).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Transient STAT4 phosphorylation in Th1 cells in response to IFN-{alpha}

Previous results suggested that IFN-{alpha} can induce STAT4 tyrosine phosphorylation in Th1 cells (16), but is quantitatively less potent than IL-12 (13). In agreement, we find that IFN-{alpha} induces STAT4 tyrosine phosphorylation in DO11.10 Th1 cells, but at levels that appear much less than the amount of STAT4 phosphorylation induced by IL-12 (Fig. 1A). To better quantify this difference, we established an assay to measure STAT4 tyrosine phosphorylation using intracellular phosphoprotein staining (pICS) (Fig. 1B). This method allows for more precise measurement of STAT4 tyrosine phosphorylation than allowed by Western blot analysis. We examined the levels of STAT4 tyrosine phosphorylation (pSTAT4) after 30 min of stimulation with IL-12 or IFN-{alpha} (Fig. 1B). We found that while both IL-12 and IFN-{alpha} induced STAT4 phosphorylation, IL-12 clearly induced much higher levels than were induced by IFN-{alpha}, in agreement with our IP/Western blot analysis (Fig. 1A). Specifically, the background pSTAT4 staining has a median fluorescence intensity (MFI) of 4, which is induced by IL-12 to 25, but is induced by IFN-{alpha} to only an MFI of 11.


Figure 1
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  		FIGURE 1. Transient STAT4 activation in response to IFN-{alpha} stimulation in Th1 cells. A, Resting DO11.10 Th1 cells were stimulated for 30 min with IL-12 or IFN-{alpha} or left unstimulated. Cell lysates were immunoprecipitated with anti-STAT4 and blotted with anti-phosphotyrosine, stripped, and reprobed with anti-STAT4; this experiment was performed five times. B, Resting DO11.10 Th1 cells were stimulated for 30 min with IL-12 or IFN-{alpha} or left unstimulated. The shaded region represents unstimulated cells, dashed line the IFN-{alpha}-treated cells, and solid line the IL-12-treated cells; this experiment was performed seven times. C, Resting DO11.10 Th1 cells were stimulated with the indicated cytokine for 30 min or 6 h or left unstimulated before pICS for pSTAT4. The shaded region represents unstimulated cells, and the solid line represents cells after cytokine stimulation; this experiment was performed three times. D and E, Resting DO11.10 Th1 cells were stimulated with either IL-12 (triangles) or IFN-{alpha} (squares) for up to 1 h (D) or up to 7 h (E). Data are pICS for STAT4 and pSTAT4 and are represented as the ratio of the MFI of the pSTAT4 to STAT4 signal; these experiments were each performed three times.

 
A recent report demonstrated that in human T cells, IFN-{alpha} induced only transient STAT4 activation, in contrast to sustained activation induced by IL-12 (19). To test if a similar phenomenon occurs in murine T cells, we compared STAT4 phosphorylation induced by IL-12 and IFN-{alpha} at 30 min and 6 h (Fig. 1C). The level of pSTAT4 induced by IL-12 remained the same at both times, whereas pSTAT4 was induced by IFN-{alpha} at 30 min, but returned to background levels at 6 h (Fig. 1C, right panels).

Our previous study had examined STAT4 activation by IFN-{alpha} only after 30 min of stimulation (13). Thus, we wanted to identify the time point at which maximal levels of pSTAT4 are induced by IFN-{alpha} and IL-12 signaling (Fig. 1, D and E). For this, we determined the levels of intracellular STAT4 protein and pSTAT4 at various times and calculated the ratio of pSTAT4 to STAT4 to correct for any changes in total levels of STAT4 (Fig. 1, D and E). We found maximal pSTAT4 occurs by 30 min after IL-12 and IFN-{alpha} stimulation, which then remained stable for 1 h. Following this, pSTAT4 induced by IFN-{alpha} gradually returned to background by 4 h after stimulation, whereas the pSTAT4 induced by IL-12 was still substantially above background even at 7 h (Fig. 1E). Thus, pSTAT4 levels induced by IL-12 diminished slightly over time (Fig. 1E), while pSTAT4 levels induced by IFN-{alpha} returned to background levels by 4 h after stimulation (Fig. 1E). A similar course of STAT4 activation by IFN-{alpha} was reported for human T cells (19).

To test whether the phosphorylation of STAT4 induced by IFN-{alpha} resulted in the functional nuclear translocation of STAT4, we conducted confocal microscopy (Fig. 2). In unstimulated T cells, STAT4 staining was distributed throughout the cell. STAT4 was localized to the nucleus after 30 min and after 6 h of stimulation with IL-12. In contrast, in response to IFN-{alpha} treatment, STAT4 was localized to the nucleus after 30 min, but returned to a nonnuclear distribution at 6 h (Fig. 2). In summary, IFN-{alpha} induces transient nuclear translocation of STAT4 compared with sustained nuclear translocation induced by IL-12.


Figure 2
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  		FIGURE 2. IFN-{alpha} induces transient nuclear translocation of STAT4. Resting Th1 cells were stimulated for 30 min or 6 h with IL-12 or IFN-{alpha} or left unstimulated. Intracellular staining for STAT4 was conducted for analysis for confocal microscopy. Representative images from three independent experiments are shown.

 
IFN-{alpha} induces transient IFN-{gamma} production in differentiated Th1 cells

We had previously reported that even though IFN-{alpha} appeared to activate STAT4 by Western analysis, we observed no induction of IFN-{gamma} production after 21 h of stimulation with IFN-{alpha} and IL-18 (13). Since we now find that IFN-{alpha}-induced STAT4 activation is transient (Fig. 1E), we re-examined the time course of IFN-{gamma} production by ICS in Th1 cells treated with IL-12 and IL-18, or IFN-{alpha} and IL-18 (Fig. 3A). Treatment with IL-12 and IL-18 rapidly induced high levels of intracellular IFN-{gamma} production that were sustained as long as at 21 h after stimulation. In contrast, treatment with IFN-{alpha} and IL-18 induced IFN-{gamma} production that peaked at 6 h, and gradually declined thereafter (Fig. 3A). IL-12 and IL-18 induced IFN-{gamma} production in 94% of CD4+ cells after only 4 h, and in 99% of cells at all later time points examined (Fig. 3A). In contrast, IFN-{alpha} induced IFN-{gamma} production in 52% of cells at 4 h of stimulation (Fig. 3A) peaking at 86% 6 h after stimulation and declining thereafter. We also examined the accumulation of IFN-{gamma} secreted in response to IL-12 and IL-18 or IFN-{alpha} and IL-18 (Fig. 3B). IFN-{gamma} induced by IL-12 and IL-18 treatment gradually accumulated to ~3300 ng/ml after 17 h of culture (Fig. 3B, {blacksquare}). By contrast, very little IFN-{gamma} accumulated in response to IFN-{alpha} and IL-18 treatment, and was never higher than 175 ng/ml (Fig. 3B, {square}). In summary, IL-12 and IL-18 synergize to induce sustained IFN-{gamma} production, whereas IFN-{alpha} and IL-18 induce transient IFN-{gamma} production, which accumulated to significantly lower levels.


Figure 3
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  		FIGURE 3. IFN-{alpha} induces transient, not sustained, IFN-{gamma} production in differentiated Th1 cells. A, Resting DO11.10 Th1 cells were stimulated with the indicated cytokines for the indicated times. Brefeldin A was added for the last 4 h of stimulation before ICS. Numbers indicate percentage of CD4+ cells expressing intracellular IFN-{gamma} staining above background; this experiment was performed five times. B, Resting DO11.10 Th1 cells were stimulated with IL-12 and IL-18 ({blacksquare}), IFN-{alpha} and IL-18 ({square}), or IL-12, IFN-{alpha}, and IL-18 (Figure 3) for the indicated times, and ELISA analysis was performed. IFN-{gamma} production observed at 2 h was below 25 ng/ml for the IL-12 and IL-18 and IL-12, IFN-{alpha}, and IL-18 conditions. This experiment was repeated three times.

 
The data in Fig. 3, A and B, illustrate an important point regarding use and comparison of ICS and ELISA data. While the MFI of the IFN-{gamma} signal induced by IL-12 and IL-18 reaches levels in the third and fourth decades of the logarithmic histogram, the MFI signal induced by IFN-{alpha} and IL-18 resides within the second decade. Even though there is still a sizable percentage of cells that show induction of IFN-{gamma} by IFN-{alpha}, as indicated in the quadrant values in Fig. 3A, there is a much less indication of IFN-{gamma} production when the linear values of IFN-{gamma} production are evaluated by ELISA. Thus, particularly when only a single time point is analyzed, it may be important to evaluate cytokine production by ELISA as well as ICS, and to also consider the MFI of the ICS signal in addition to the gated percentages of cells.

We considered whether the transient IFN-{alpha}-induced STAT4 phosphorylation might result from induction of a suppressor, such as suppressor of cytokine signaling (SOCS) proteins which could inhibit the tyrosine kinase activity directed at STAT4 (33). To test for such a mechanism, we treated Th1 cells with IL-12, IL-18, and IFN-{alpha} simultaneously to test whether IFN-{alpha} might inhibit IFN-{gamma} induced by IL-12 and IL-18 (Fig. 3, A and B,

Formula

). No evidence of inhibition of IFN-{gamma} production was seen with the additional IFN-{alpha} treatment. This result does not exclude the potential for involvement of inhibitory proteins in termination of IFN-{alpha} signaling, but does show that any such mechanism does not act in a dominant manner to terminate other cytokine signaling pathways.

IFN-{alpha}-induced IFN-{gamma} production is STAT4-dependent

While IFN-{gamma} production induced by IL-12 and IL-18 is STAT4 dependent (34), it is conceivable that IFN-{alpha} and IL-18 might induce IFN-{gamma} production through a STAT4-independent signaling pathway. As a control, we showed that STAT4–/– CD4+ cells differentiated under Th1 conditions cannot produce IFN-{gamma} in response to IL-12 and IL-18, but can produce IFN-{gamma} in response to PMA and ionomycin (Fig. 4), which bypasses the signaling requirement for STAT4 as previously reported (35). We therefore compared IFN-{gamma} production by WT and STAT4–/– T cells in response to IL-12 and IL-18, or to IFN-{alpha} and IL-18 treatment at 4 and 21 h (Fig. 4). IL-12 and IL-18 treatment induced IFN-{gamma} in 52% of WT T cells at 4 h, which remained constant at 51% at 21 h. In contrast, STAT4–/– T cells show no IFN-{gamma} induction, as expected. IFN-{alpha} and IL-18 induced IFN-{gamma} in 30% of WT T cells after 4 h of stimulation. However, <1% of the STAT4–/– T cells produced IFN-{gamma} after 4 or 21 h of IFN-{alpha} stimulation (Fig. 4, bottom panels). In summary, IFN-{alpha}-induced IFN-{gamma} production is transient and STAT4-dependent, and so is consistent with the short-lived STAT4 activation induced by IFN-{alpha}.


Figure 4
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  		FIGURE 4. IFN-{alpha}-induced IFN-{gamma} production is STAT4-dependent. Resting DO11.10 Th1 or STAT4–/– DO11.10 Th1 cells were stimulated for 4 or 21 h with IL-12 and IL-18 or IFN-{alpha} and IL-18. PMA and ionomycin treatment was for 4 h. Brefeldin A was added for the last 4 h of stimulation. Numbers indicate percentage of CD4+ cells, which have IFN-{gamma} staining levels above isotype control staining; this experiment was performed twice.

 
Transient STAT4 activation by IFN-{alpha} is not caused by cytokine consumption or receptor down-regulation

We previously demonstrated that amounts of IFN-{alpha} as high as 50,000 U/ml were not capable of inducing Th1 differentiation in vitro (13). However, we wished to confirm that IFN-{alpha} was not simply being consumed during the experiment, causing only the appearance of inactivity. Purified DO11.10 CD4+ T cells were stimulated with OVA/APCs, IL-2, and IL-12 or IFN-{alpha}. Additionally, aliquots of fresh IFN-{alpha} were added at the indicated times. T cells treated with IL-12 at the initial stimulation were 40% positive for IFN-{gamma} production upon restimulation at day 7 (Fig. 5A). In contrast, even repeated additions of IFN-{alpha} did not induce IFN-{gamma} to >13% of cells (Fig. 5A), only slightly above background. To directly test for inactivation of IFN-{alpha}, we incubated Th1 cells with IL-12 or IFN-{alpha} at 37°C. These culture supernatants were harvested at 6 h when IFN-{alpha}-induced STAT4 phosphorylation has abated, placed on resting Th1 cells for 30 min, and STAT4 phosphorylation in these Th1 cells measured. The pSTAT4 level induced by IFN-{alpha} was identical regardless of the conditions in which the IFN-{alpha} had been previously incubated (Fig. 5B, right panels). As a control, similar findings were observed with IL-12 (Fig. 5B, left panels). Thus, consumption or inactivation of IFN-{alpha} is unlikely to explain the lack of Th1 differentiation by IFN-{alpha}.


Figure 5
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  		FIGURE 5. Transient responses to IFN-{alpha} are not caused by cytokine consumption or receptor down-regulation. A, Naive DO11.10 CD4 cells were stimulated with OVA, APC, IL-2 and the indicated cytokine. IL-12 was added only on day 0, while IFN-{alpha} was added on the indicated days. Cells were harvested on day 7 and stimulated for 4 h with PMA and ionomycin. Data are the percentage of CD4+ T cells producing IFN-{gamma} above the level observed in unstimulated cells; this experiment was performed twice. B, IL-12 or IFN-{alpha} was added at 37°C to resting DO11.10 Th1 cells (10 x 106/ml) or to media alone for 6 h (primary cytokine incubation conditions). Supernatants were harvested and used to stimulate resting DO11.10 Th1 cells (secondary stimulation). Cytokines that had not been preincubated (none) were used as a control. Secondary stimulation was for 30 min and pICS was performed for pSTAT4. Gray peaks represent pSTAT4 levels of unstimulated cells, and solid lines represent pSTAT4 in cytokine treated cells; this experiment was performed twice. C, Resting DO11.10 Th1 cells were stimulated for 4 h with the indicated cytokine, and staining for IFNAR1 was conducted. Gray peaks are isotype staining of CD4+ cell controls. Dashed lines are levels of IFNAR1 after 4 h of staining, and solid lines are unstimulated cells; this experiment was performed twice.

 
Previous studies in the human system have suggested that down-regulation of IFNAR1 might cause transient IFN-{alpha} signaling (19, 36), but IFNAR1 is not well-characterized in the murine system. We examined surface IFNAR1 expression in murine Th1 cells following treatment with IFN-{alpha} or IL-12 for 4 h at 37°C (Fig. 5C). Treatment of cells for 4 h of stimulation with IL-12 or IFN-{alpha} caused little change in IFNAR1 expression (Fig. 5C, compare dashed and solid lines).

Loss of STAT1 augments but does not sustain transient IFN-{alpha} induction of IFN-{gamma}

STAT1 may regulate the ability of IFN-{alpha} to activate STAT4 (37, 38). We have previously demonstrated that IFN-{alpha} induces greater STAT4 phosphorylation in STAT1–/– Th1 cells compared with WT Th1 cells, but is still unable to induce Th1 differentiation in STAT1–/– T cells (13). To assess whether STAT1 expression controls transient STAT4 activation, we used pICS to examine pSTAT4 induced by IFN-{alpha} in STAT1–/– T cells at various times after stimulation. IFN-{alpha} and IL-12 induced similar levels of pSTAT4 at early times in STAT1–/– T cells (Fig. 6A). However, over a long time course, STAT4 phosphorylation still diminished more rapidly in response to IFN-{alpha} treatment compared with sustained IL-12 stimulation in STAT1–/– T cells (Fig. 6B), even though this was increased compared with the response induced by IFN-{alpha} in WT cells (compare with Fig. 1E). Therefore, although IFN-{alpha} is capable of inducing STAT4 phosphorylation more robustly in a STAT1–/– Th1 cell than in a WT cell, it is by no means equivalent to the outcome of IL-12 treatment in either STAT1–/– or WT T cells.


Figure 6
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  		FIGURE 6. Loss of STAT1 in T cells augments, but does not sustain, IFN-{alpha}-induced pSTAT4 and IFN-{gamma}. A and B, Resting STAT1–/– DO11.10 Th1 cells were stimulated with IL-12 (triangles) or IFN-{alpha} (squares) for the indicated times (A and B), and pICS for STAT4 and pSTAT4 was performed. Data are represented as the ratio of the MFI of the pSTAT4 to STAT4; this experiment was performed twice. C, Resting STAT1–/– DO11.10 Th1 cells were stimulated with IL-12 and IL-18 ({blacksquare}) or IFN-{alpha} and IL-18 ({square}) for the indicated times, and ELISA for IFN-{gamma} was done; this experiment was performed twice. D, Resting STAT1–/– or WT DO11.10 Th1 cells were stimulated with the indicated cytokines for the indicated times. Numbers indicate percentage of CD4+ cells staining for intracellular IFN-{gamma} above levels of unstimulated cells; this experiment was performed three times.

 
We next determined if IFN-{gamma} production induced by IFN-{alpha} and IL-18 in STAT1–/– Th1 cells was also transient, as observed in WT Th1 cells. In STAT1–/– T cells, IFN-{alpha} synergized with IL-18 to induce amounts of IFN-{gamma} that were slightly less than induced by IL-12 and IL-18 (Fig. 6C); IL-18 alone was incapable of inducing IFN-{gamma} production (data not shown). However, by 17 h of cytokine stimulation, IL-12 and IL-18 induced more than two-fold higher IFN-{gamma} production than IFN-{alpha} and IL-18 treatment, consistent with greater STAT4 phosphorylation induced by IL-12 compared with IFN-{alpha} at later time points (Fig. 6B). Over a longer time course of cytokine stimulation, we observed sustained IFN-{gamma} production induced by IL-12 and IL-18 in both WT and STAT1–/– T cells, in contrast to a gradual loss of IFN-{gamma} induced by IFN-{alpha} and IL-18 in both WT and STAT1–/– T cells (Fig. 6D). Intracellular IFN-{gamma} induced by IFN-{alpha} and IL-18 was 95% after 6 h of stimulation but decreased to 36% after 54 h, in contrast to 99% at this time in response to IL-12 and IL-18. Thus, loss of STAT1 augments, but does not sustain, STAT4 phosphorylation and IFN-{gamma} production in response to IFN-{alpha}.

Naive cells respond to IL-12 more rapidly than to IFN-{alpha}

So far, we have examined IFN-{alpha}-induced STAT4 activation only in already differentiated Th1 cells. We next characterized STAT4 activation by IFN-{alpha} in naive CD4+ T cells. It is known that naive CD4 T cells cannot immediately induce STAT4 phosphorylation in response to IL-12 due to the absence of the IL-12Rbeta2 (39). However, it is not known if naive T cells, which can rapidly respond to IFN-{alpha} for activation of STAT1/2 (40), can also induce STAT4 phosphorylation immediately. First, we asked if and when IFN-{alpha} can activate STAT4 during primary stimulation of naive T cells. Purified CD4+ T cells were stimulated with anti-CD3 for various lengths of time and STAT4 phosphorylation measured in response restimulation with IL-12 or IFN-{alpha}; neither cytokine had been present during the initial activation. During the initial stimulation, IL-12 was neutralized to eliminate any bias toward Th1 differentiation, but IFN-{gamma} was added to allow normal induction of IL-12Rbeta2 (39). Neither IL-12 nor IFN-{alpha} could induce STAT4 phosphorylation in naive T cells after 24 h of activation (Fig. 7A). However, IL-12 was able to activate pSTAT4 after 48 h of initial activation (Fig. 7A), consistent with our previous study (39). IFN-{alpha} was first able to activate low levels of STAT4 phosphorylation only after 72 h in culture. The level of pSTAT4 induced by IFN-{alpha} was less than that induced by IL-12 after 72 h. Maximum STAT4 activation by IFN-{alpha} was seen after 96 h of primary T cell activation. Furthermore, the STAT4 phosphorylation induced by IFN-{alpha} was transient, since it had returned to background by 6 h of IFN-{alpha} treatment (data not shown). In summary, primary T cells are unable to induce STAT4 phosphorylation in response to IFN-{alpha} until after 3 days of stimulation.


Figure 7
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  		FIGURE 7. IFN-{alpha} does not activate STAT4 in naive CD4+ T cells until 3 days following primary stimulation. A, Purified CD4+ T cells were stimulated with anti-CD3 (10 µg/ml), anti-IL-12, and IFN-{gamma}, with cells removed from anti-CD3 stimulation after 48 h. Cells were restimulated after the indicated length of time after initial activation with IL-12 or IFN-{alpha} for 30 min, and pICS for pSTAT4 was performed. This experiment was performed twice. B, Purified CD4+ T cells were stimulated with anti-CD3 (10 µg/ml) for 0, 24, or 48 h before harvest and staining for IFNAR1; resting Th1 cells were also used. IFNAR1 staining is solid lines; dashed lines are staining with isotype control. MFI are of IFNAR1 staining; this experiment was performed twice. C, Freshly purified CD4+ T cells or resting Th1 cells were stimulated with IFN-{alpha} for 30 min or left unstimulated before staining for STAT1. This experiment was performed twice.

 
We wished to determine whether the inability of naive cells to respond to IFN-{alpha} was due to insufficient levels of IFNAR1. We activated freshly purified CD4+ T cells for 0, 24, or 48 h with anti-CD3 and then stained the cells with an anti-IFNAR1 Ab (Fig. 7B). We find that the level of IFNAR1 is relatively low in resting naive CD4+ T cells, and increases with activation, and that the level of IFNAR1 expression after 48 h of activation is approximately equivalent to that observed on a resting Th1 cell line, as indicated by the MFI measurements (Fig. 7B). We next determined if naive cells were capable of responding to IFN-{alpha} stimulation. Naive CD4+ T cells or resting Th1 cells were stimulated for 30 min with IFN-{alpha} or left unstimulated before pICS for STAT1. Cells were examined to determine whether STAT1 was present in the cell cytoplasm or had undergone nuclear translocation into the cell nucleus (Fig. 7C). We found that naive CD4+ T cells did not respond to the IFN-{alpha} stimulation, as indicated by the lack of STAT1 nuclear translocation (Fig. 7C, top row), in contrast to the clear STAT1 nuclear translocation that was observed in resting Th1 cells in response to IFN-{alpha} (Fig. 7C, bottom row). Conceivably, the low levels of IFNAR1 expression by naive CD4+ T cells may explain the lack of response to IFN-{alpha} before activation, but additional work will be required to determine whether this is the only basis for this result.

Similar genes induced by IFN-{alpha} in WT and m/h STAT2 KI cells

Previous results suggested that a minisatellite in murine STAT2 might prevent IFN-{alpha} activation of STAT4 in murine T cells (17). We have generated mice with a chimeric STAT2 containing the c-terminal portion of human STAT2 (m/h STAT2 KI mice) that removes the region encoded by the minisatellite (24). T cells from these mice were still unable to differentiate into Th1 cells in response to IFN-{alpha} treatment (24), similar to WT murine T cells (13). Since our data demonstrates that IFN-{alpha} induces transient STAT4 activation, we wished to examine splenocytes from these mice to determine how the global gene expression differed after IFN-{alpha} treatment, compared with WT splenocytes. We found surprisingly few differences between the genes that were induced by IFN-{alpha} after 4 h in DO11.10 m/h STAT2 KI cells when compared with WT DO11.10 cells (Table I, Fig. 8). Many genes known to be induced by IFN-{alpha}, such as 2'-5' oligoadenylate synthetase 2 (41), show very similar levels of inducibility in the m/h STAT2 KI cells (Table I). We also compared the level of induction of ~90 IFN-{alpha}-inducible genes between WT and m/h STAT2 KI cells. This was done by plotting the fold increase in gene expression induced by IFN-{alpha} treatment in WT vs the increase occurring in m/h STAT2 KI cells (Fig. 8). We found that these genes as a group were induced similarly in both populations, as evident by regression line that showed a slope of 1.05. In conclusion, the minisatellite insertion in STAT2 does not appear to play a significant role in altering the pattern of gene induction by IFN-{alpha}.


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  		Table I. Similar fold induction of IFN-{alpha}-inducible gene expression in WT and m/h STAT2 KI micea

 

Figure 8
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  		FIGURE 8. IFN-{alpha} induces similar genes in WT and m/h STAT2 KI cells. Splenocytes from unimmunized DO11.10 or DO11.10 m/h STAT2 KI mice were stimulated for 4 h with IFN-{alpha} or anti-IFN-{alpha}. RNA was prepared, and microarray analysis was performed. Shown are the fold expression levels for all genes that were induced by IFN-{alpha} in WT cells >4-fold over untreated cells. Fold induction in WT cells is shown on the horizontal axis, and fold induction in m/h STAT2 KI cells on the vertical axis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study extends a study in human T cells that demonstrated that IFN-{alpha} does not drive Th1 development because it cannot sustain STAT4 phosphorylation (19). Earlier studies had suggested that both IL-12 and type I IFN induced Th1 development in human T cells (8, 9, 10), but that only IL-12 drove Th1 differentiation in murine T cells (11, 12). Later, this supposed difference between human and mouse was suggested to derive possibly from the inability of murine STAT2 to recruit STAT4 to the type I receptor due to an insertional mutation in the mouse STAT2 gene (15, 17). However, STAT2 knockout mice were recently reported to maintain STAT4 phosphorylation in response to IFN-{alpha} in lymphocytes (18). Our recent study demonstrated that this ability of IFN-{alpha} to phosphorylate STAT4 was not sufficient for inducing Th1 differentiation in murine T cells (13), in agreement with a report in the human system that IFN-{alpha} cannot drive Th1 development due to the transient nature of STAT4 phosphorylation (19).

To further examine this issue, we have systematically evaluated the quantitative and kinetic parameters of IFN-{alpha}-induced STAT4 activation in murine T cells. We find that IFN-{alpha} induces only transient STAT4 phosphorylation, does not drive Th1 differentiation even with repeated stimulation by IFN-{alpha}, and can only sustain transient IFN-{gamma} production in synergy with IL-18. Additionally, we find no contribution of the minisatellite in murine STAT2 in causing any global differences in IFN-{alpha}-induced gene expression.

This study has confirmed that type I IFN do not drive Th1 development in murine T cells, consistent with previous work in the murine system (11, 13) and in agreement with more recent findings in the human system (19). Importantly, earlier conclusions regarding the ability of IFN-{alpha} to induce Th1 differentiation in human T cells (8, 42) were not based on comparisons with IL-12 or conducted under conditions that ensured an absence of IL-12 activity. This study is the first to demonstrate in murine T cells that IFN-{alpha} induces transient STAT4 phosphorylation and transient IFN-{gamma} production, similar to the recent findings in human T cells (19). These results suggest that human and murine CD4+ T cell responses to type I IFN are similar regarding activation of STAT4.

Previous work suggested that the reported differences in IFN-{alpha} responsiveness between humans and mice might be due to the presence of a minisatellite in the carboxy terminus (15, 17). The carboxy terminus of STAT2 was thought to recruit STAT4 to IFNAR in murine cells for phosphorylation by receptor-associated kinases (43, 44). To determine whether this mutation alters the global pattern of IFN-{alpha}-induced genes between humans and mice, we have compared splenocytes from WT mice and m/h STAT2 KI mice in which this minisatellite has been deleted (24). We found a similar pattern of global gene expression induced by IFN-{alpha} in both types of cells. While the correction of this mutation restored STAT4 activation in response to IFN-{alpha} in fibroblasts (17), it did not alter IFN-{alpha}-induced gene expression in lymphocytes and so it is unlikely to be a cause for species-specific differences in STAT4 activation in T cells. Indeed, differential responsiveness to type I IFN between fibroblasts and macrophages has been reported in STAT2–/– mice (45), suggesting that the cell type can influence IFN-{alpha} signaling. In addition, there is evidence for a STAT2-independent pathway for IFN-{alpha}-induced STAT4 phosphorylation in murine T cells (18).

Our observation of transient STAT4 phosphorylation induced by IFN-{alpha} explains the limited amount and duration of IFN-{gamma} induced by IFN-{alpha} in Th1 cells. While IL-12 induces sustained STAT4 phosphorylation and causes production of IFN-{gamma} in synergy with IL-18, IFN-{alpha} induces transient STAT4 phosphorylation and causes much less accumulation by comparison. The cause of the transient nature of IFN-{alpha}-induced STAT4 activation is still unclear. We find no evidence for IFNAR1 down-regulation with continued IFN-{alpha} stimulation, and find no evidence for consumption of IFN-{alpha} during the assays. Whatever the mechanism, it appears to be a process that is intrinsic to the IFNAR pathway, since simultaneous treatment of IFN-{alpha} along with IL-12 and IL-18 treatment showed no inhibition of IFN-{gamma} production compared with IL-12 and IL-18 alone.

We have previously reported that IFN-{alpha} cannot induce Th1 differentiation in either WT or STAT1–/– CD4+ T cells (13). However, the quantity and duration of STAT4 phosphorylation induced by IFN-{alpha} is increased in STAT1–/– T cells compared with WT T cells, consistent with previous reports (37). Even in STAT1–/– T cells, however, IL-12 still induces greater STAT4 phosphorylation and IFN-{gamma} production than induced by IFN-{alpha}. Importantly, STAT4 phosphorylation in response to IFN-{alpha} treatment remains transient in STAT1–/– Th1 cells, suggesting that interaction with STAT1 is not the causes of transient IFN-{alpha} signaling. However, increased STAT4 phosphorylation in STAT1–/– T cells may indicate competition between STAT1 and STAT4 at the IFNAR.

While these data can be interpreted to reflect quantitative and qualitative differences in STAT4 activation by IL-12 and IFN-{alpha}, it is also possible that IL-12 might activate other signaling pathways that either contribute to STAT4 signaling or to induction of STAT4 targets that are not activated by IFN-{alpha}. For example, if IL-12 activated an alternative pathway that modified STAT4 in a manner other than tyrosine phosphorylation, then such a modification could influence STAT4 activity or stability in a manner promoting Th1 development that is not exhibited by IFN-{alpha} signaling. A potential candidate, for instance, would be differential activation of MAPK pathways that either modify or cooperate with STAT4 activity, but further work is needed to identify such pathways.

Finally, we examined naive T cells for STAT4 phosphorylation induced by IFN-{alpha}. We find that IFN-{alpha} is unable to induce STAT4 phosphorylation until 3 days after primary activation. In contrast, IL-12 becomes able to induce STAT4 phosphorylation by day 1 to day 2 after primary activation. This result might suggest that IFN-{alpha} cannot activate STAT4 early enough or for long enough in a primary response to efficiently drive Th1 development. IFN-{alpha} is induced acutely in response to viral infection and exerts important actions on cells in the first day of infection (46). Our results suggest that little STAT4 activation would occur in T cells in the first 48 h of infection in response to the IFN-{alpha}, which could result from low IFNAR1 expressed by naive CD4+ T cells. Further study will be needed to determine the mechanism underlying the differences in STAT4 activation by IL-12 and IFN-{alpha}.


   Acknowledgments
 
We thank M. Himmelmann for secretarial support, V. Grigura for technical assistance, C. Lindsley for advice on experimental design, and R. Akilesh for confocal microscopy suggestions and assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by the Howard Hughes Medical Institute (to K.M.M.) and grants from the National Institutes of Health (P01 AI31238 and P50 HL54619; to K.M.M.). Back

2 Current address: Center for Immunology and Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390. Back

3 Address correspondence and reprint requests to Dr. Kenneth M. Murphy, Department of Pathology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: murphy{at}pathbox.wustl.edu Back

4 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; ICS, intracellular cytokine staining; IFNAR, IFN-{alpha} receptor; KI, knock-in; m/h, mouse/human; MFI, median fluorescence intensity; pICS, intracellular phosphoprotein staining; pSTAT4, phosphorylated STAT4; SOCS, suppressors of cytokine signaling; WT, wild type. Back

Received for publication March 27, 2006. Accepted for publication July 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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