stim ulus NS IFN-a NS IFN-a PBL-T cells Kit225 cells

B

STAT4 p2

STAT4 p i

ST A T l-a

STATl-p

IFN-a

STAT3-a

STAT3-P

Figure 5.1) IFN -a activates STA Tl, STAT3 and STAT4 >vith different kinetics in

h u m a n T l y m p h o c y t e s . - A ) IFN-a-induced STAT4 DNA-binding in PBL-T and Kit225 cells. B)

Kinetics o f IFN-a-induced DNA-binding o f STA Tl, STATS and STAT4. 20 x 10^ PBL-T cells (A, B) or Kit225 cells (A) were left unstimulated (NS) or stimulated with 10^ U / ml o f IF N -a for 10 minutes or indicated times. Proteins were affinity precipitated using the GRR (A) or the GAS-STAT4 (B) oligonucleotide, resolved on SDS-PAGE and immunoblotted with anti-STAT4 C- (A) or anti-STAT4 C- fbllowed by sequential reprobing with anti-STATl pan and anti-STAT3 pan.

and Figure 5.1 B upper panel). We have shown that STAT4 translocates to the nucleus in response to IL-12 in human T lymphocytes (Chapter 3, Figure 3.8 A). To examine if IFN-a regulates the cellular localisation of STAT4, cytoplasmic and nuclear cell lysates were prepared from quiescent and IFN -a stimulated Kit225 cells (Figure 5.2 A) and PBL-T cells (data not shown). Western blot analysis shows that the cellular localisation of STAT4 is regulated very similarly by IFN -a and IL-12 (Chapter 3, Figure 3.8 A and Figure 5.2 A). A low amount of STAT4 is present in the nucleus of unstimulated cells. Upon 18 minutes of IFN -a stimulation, the basal nuclear levels of STAT4 are clearly increased. The presence o f both STAT4pl and STAT4p2 can be noted. The amount of nuclear STAT4 is further increased with longer (60 minutes) IFN-a stimulation. STAT4p2 is detected predominantly in the nucleus o f IFN -a stimulated T cells.

Studies o f the post-translational modification o f STAT4 in response to IL-12 indicated that STAT4 serine phosphorylation is not required for nuclear translocation (Chapter 3, Figure 3.9 C). To test if this is a general characteristic of STAT4 activation, we studied the effect of the H7 serine/ threonine kinase inhibitor on IFN-a-regulated STAT4 nuclear translocation. Pre-treatment of T cells with H7 blocks IFN-a-induced generation of STAT4p2 (Figure 5.2 B). The effect of H7 is more pronounced at shorter time points, in a similar way as for IL-12-induced STAT4p2. A kinetics assay reveals that H7 blocks approximately 90% of IFN-a- induced STAT4p2 at 15 minutes, while only around 60% inhibition is seen at 60 minutes stimulation time (Figure 5.2 B). However, the cellular fractionation experiment shows that H7 prevents accumulation o f STAT4p2 but STAT4pl molecules can still translocate into the nucleus o f IFN-a-stimulated PBL-T cells (Figure 5.2 C). These data confirm that, independent of stimulus, STAT4 serine phosphorylation is not required for STAT4 nuclear translocation.

97 Kd—

IFN-a (time) ^

lysate

cytoplasmic

nuclear

STAT4 p2

STAT4 +

STAT4 p i

B

_{STAT4 p2}

STAT4 p i

H7

H-7

stimulus

lysate

cytoplasmic

nuclear

STAT4 p2

STAT4 +

STAT4 p i

Figure 5.2) IFN -a regulates the cellular localisation of STAT4.- iFN -a-induced nuclear translocation o f STAT4 (A). Effect o f the H7 serine/ threonine kinase inhibitor on IFN -a- induced STAT4 DNA binding (B) and nuclear translocation (C). Kit225 cells B) and PBL-T cells (C) were left unstimulated (NS) or stimulated with 10^ U/ ml IF N -a for 18 minutes or the indicated times. Proteins were GRR-affinity precipitated from 20 x 10^ cells (B) or acetone precipitated from cytoplasmic (5 x 10^ cells) or nuclear (10 x 10^ cells) lysates (A, C), resolved on SDS-PAGE and immunoblotted with anti-STAT4 C-. In B and C cells were incubated with 200 pM H-7 for 30 minutes prior to stimulation. Results are representative o f at least two independent experiments.

5.2.4 The IFN-a/ STAT4 response is more transient than the IL-12/ STAT4

response

IL-12 and IFN-a have been shown to drive Thl cell differentiation in human in vitro systems [77, 89]. However, patients with impaired IL-12 responses and consequent Thl cell differentiation deficiencies suffer firom recurrent Mycoplasma sp. and Salmonella sp. infections [67-69, 72]. Thus, IF N -a is not able to fully compensate for the lack of IL-12 responses in human in vivo systems. STAT4 activation is essential for Thl cell differentiation [7, 8]. Differences in the regulation of STAT4 by IL-12 and IFN -a could explain the inability of IF N -a to induce full Thl cell responses in vivo.

Using oligonucleotide affinity precipitation, we directly compared the early kinetics of activation of STAT4 by IL-12 and IFN -a (Figure 5.3 A). In agreement with the data shown in Chapter 3 and Figure 5.1 B, the initial kinetics of STAT4 activation as well as the generation of STAT4pl and STAT4p2 are very similar in response to IL-12 and IF N -a (Figure 5.3 A). Both responses are rapid. DNA-bound STAT4 molecules appear at 2-5 minutes and remain present for at least 60 minutes upon stimulation. Equivalent amounts of STAT4pl and STAT4p2 can be seen at 10-20 minutes and STAT4p2 is the predominant form at longer stimulation times in response to either cytokine. The IFN -a/ STAT4 response is slightly stronger as compared to the IL-12/ STAT4 response, but no major differences in the kinetics of STAT4 activation in response to IL-12 and IFN -a were seen.

Th cell differentiation is a long-term process. The constant presence o f Thl or Th2 driving cytokines and derived signals seems to be required to achieve full commitment to a specific Th phenotype. Therefore, the long-term kinetics of the IL-12/ STAT4 and IFN-a/ STAT4 responses where compared in PBL-T cells. An ohgonucleotide affinity precipitation assay shows that IL-12 activation of STAT4

is more sustained than the IFN -a /STAT4 response (Figure 5.3 B). High levels of activated STAT4 are detected for at least nine hours after IL-12 stimulation. In contrast active STAT4 molecules are seen in cells that have been stimulated with IFN -a for three hours but not at longer time points (Figure 5.3 B). This transience of the IFN -a/ STAT4 response was consistent and was seen to last for only three to four hours (Figure 5.3 B and data not shown). These data show that the IFN-a/ STAT4 response is more transient than the IL-12/ STAT4 response in human PBL- T cells. The possible mechanisms involved in this differential regulation of STAT4 are the focus of the following sections.

5.2.5 IFN-a does not have a negative effect on PBL-T cell survival

The transient IFN -a/ STAT4 response in PBL-T cells could reflect a negative effect of IFN -a on T cell survival. To test this possibility we compared the viability of PBL-T cells incubated for six hours in the absence or presence o f IFN -a, IL-2 and IL-12 (Figure 5.4). Propidium iodide staining and flow cytometry analysis were used to determine cell death. The percentage of dead cells is comparable (around 12 %) in IFN-a, IL-2 and IL-12 stimulated cells and is lower than in cells that were left unstimulated (-20 %) (Figure 5.4). Therefore, IFN -a does not have a negative effect on PBL-T cell survival, at least within the time in which the IFN -a/ STAT4 response decays. In fact, the reverse tendency was seen, T cells incubated with IFN -a had an enhanced survival when compared to unstimulated cells (Figure 5.4 and data not shown). These data indicate that the transient IFN -a/ STAT4 response is not caused by IFN -a induced cell death.