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IMMUNOBIOLOGY
From the Center for Hematologic Oncology, Department of
Adult Oncology, Dana-Farber Cancer Institute, Department of Medicine,
Brigham and Women's Hospital, Harvard Medical School, Boston, MA.
Interleukin-12 (IL-12) plays a critical role in modulating the
function of T lymphocytes and natural killer cells. IL-12 has potent
antitumor effects in animal models, mediated primarily by its ability
to enhance cytolytic activity and secretion of interferon- Interleukin-12 (IL-12) is a heterodimeric cytokine
produced primarily by antigen-presenting cells.1,2 IL-12
has a variety of immunoregulatory effects on both T cells and natural
killer (NK) cells, including the induction of interferon- Previous studies have demonstrated that a variety of other cytokines
modulate the response of cells to IL-12. These effects occur through
modulation of different components of the IL-12 signaling pathway. For
example, the expression of IL-12R The ability of IL-12 to enhance the cytotoxicity of NK cells and
cytotoxic T lymphocytes (CTLs) probably plays a role in the therapeutic
potential of IL-12 in patients with cancer. Preclinical studies in
animal models demonstrated that administration of IL-12 resulted in
regression of established tumors and activation of antitumor
immunity.15-18 It was also demonstrated that treatment with a single dose of IL-12 augmented the cytolytic activity and proliferation of lymphocytes in cancer patients.19
However, significant antitumor effects were not observed in patients
treated with IL-12.20,21 The production of IFN- Cytokines, antibodies, and reagents
Enrichment and culture of human T cells
Immunofluorescence analysis of IL-12R expression CD3-activated T cells were cultured with or without indicated cytokines for 3 days. Cells were labeled with IgG1 isotype control, anti-IL-12R 1 antibody (12R44) or anti-IL-12R2 antibody
(5A7). Cells were then labeled with secondary antibody PE-conjugated goat antimouse IgG1. The expression of IL-12R1 and 2
was analyzed on a Coulter EPICS XL flow cytometer.
Western blotting and Northern blot analysis of STAT4 messenger RNA Whole-cell extracts were prepared and Western blot was performed as described previously.23 Total RNA was prepared from activated T cells that were cultured with or without cytokines for 24 hours. We used 20 µg total RNA in Northern blot analysis. The probe used for Northern blotting was generated by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA from activated T cells and 2 STAT4-specific primers designed according to the STAT4 complementary DNA (cDNA) sequence in GenBank (Accession L78440). Reverse transcription and PCR were performed as described previously.24Immunoprecipitation of STAT4 protein Whole-cell extracts from 20 × 106 untreated or treated cells were prepared, and the STAT4 protein was immunoprecipitated with 10 µg STAT4 antibody as described previously.23Nuclear extracts and electrophoretic mobility shift assay Activated T cells were cultured with or without cytokines in anti-CD3-coated flasks for 24 hours. Cells were then treated with IL-12 for 20 minutes. Nuclear extracts were prepared and the electrophoretic mobility shift assay (EMSA) was performed as previously described.23 The STAT-binding oligonucleotides used, 5'-GAGCCTGATTTCCCCGAAATGATGAGC-3' and its complement, are derived from the IFN- -responsive factor 1 gene promoter.25
IFN- in all supernatants was
determined by IFN- enzyme-linked immunosorbent assay (ELISA)
(Endogen, Boston, MA).
Proliferation assay CD3-activated T cells were cultured with or without cytokines in anti-CD3-coated flasks for 2 days. Cells were then washed and plated at 30 000 cells per well in triplicate into 96-well U-bottom plates and cultured with or without IL-12. After 24 hours, cells were pulsed with 1 µCi per well of [3H]-thymidine (TdR) for 16 hours and harvested (EG&G Wallac, Terku, Finland). The incorporation of [3H]TdR was measured with a liquid scintillation counter (1205 Betaplate, Pharmacia LKB).
IL-12 up-regulates the expression of IL-12R 1 and 2 subunits, we investigated the effect of IL-12 on
the expression of 1 and 2 on activated T cells. CD3-activated T
cells were incubated with or without cytokines for 3 days, and the
expression of IL-12 receptor subunits was examined by flow cytometry.
As shown in Figure 1, the expression of
IL-12R2 was upregulated by IL-2 or IL-12 and by IL-2 plus IL-12.
IFN-, which was used as a control cytokine, did not affect the
expression of IL-12R2. In contrast, the expression of IL-12R1 was
significantly decreased in T cells treated with IL-12 or with IL-2 plus
IL-12 (Figure 1). The up-regulation of IL-12R2 by IL-2 and IL-12 and the down-regulation of IL-12R1 by IL-12 in activated T cells are
similar to what we observed previously in NK cells.11
These results indicate that in both cell populations the expression of
IL-12R1 and IL-12R2 is regulated through different mechanisms. However, IL-12-treated T cells still express significant levels of
IL-12R1, and the lower levels of IL-12R1 expression induced by
IL-12 may not have any functional consequence in these cells.
IL-12 down-regulates the expression of STAT4 in activated T cells STAT4 plays a major role in IL-12 signaling in both T cells and NK cells. Our previous studies demonstrated that IL-12 did not promote the expression of STAT4 in NK cells. Instead, IL-12 inhibits IL-2-induced STAT4 expression in this cell population.11 We therefore examined whether IL-12 activation affected the expression of various STAT proteins, especially STAT4, in activated T cells. Activated T cells were cultured with or without IL-2, IL-12, IL-2 plus IL-12, or IFN- for 4 days. The expression of STAT1, STAT4, and STAT5 at the
end of each day was examined by Western blot. Both STAT1 and STAT5
expression remained at the same level in T cells cultured with and
without cytokines (Figure 2, middle, lower panels, and data not shown). The appearance of a shift of the
STAT5 band is due to the phosphorylation of STAT5 induced by IL-2
(Figure 2, lower panel, lanes 2, 4, 7, and 9). Treatment with IL-12
also induced a shifted STAT4 band, which consists of tyrosine-phosphorylated STAT4 (Figure 2, upper panel, lanes 3, 4, 8, and 9).11,23 However, in contrast to STAT1 and STAT5, the
level of total STAT4 was significantly decreased in IL-12 or IL-2 plus
IL-12-treated T cells within 24 hours, and low levels of STAT4 protein
persisted for the 4 days we examined this phenomenon (Figure 2, upper
panel, lanes 3, 4, 8, and 9, and data not shown). IFN- did not
influence the expression of any of the STAT proteins examined.
Similarly to what we previously observed in NK cells, the expression of
STAT4 in these activated T cells was enhanced by IL-2 (Figure 2, top
panel).11 The effect of IL-12 on the expression of the 2 kinases involved in IL-12 signaling, Jak2 and Tyk2, was also examined,
and neither kinase was decreased at the protein level after IL-12
treatment (data not shown). This result indicated that among the
kinases and STATs that are involved in IL-12 signaling, the expression
of STAT4 protein was specifically down-regulated by IL-12. Because
STAT4 is the major STAT involved in IL-12 signaling, the
down-regulation of STAT4 expression by IL-12 may have functional
consequences on the subsequent T-cell responsiveness to IL-12.
Functional impact of IL-12-induced STAT4 degradation STAT4 plays a critical role in IL-12 signaling. To investigate the impact of IL-12-induced STAT4 down-regulation on T-cell functional responses to IL-12, we examined STAT4 DNA-binding activity in response to IL-12. Activated T cells were cultured with or without additional cytokines for 24 hours and then treated with IL-12. As shown in Figure 4, IL-12 induced significant STAT4 DNA-binding activity in T cells cultured with medium alone, IL-2, or IFN-, as indicated by the
formation of STAT4 and DNA complex (complex S). The addition of STAT4
antibody resulted in a new shifted complex, SS, formed by STAT4, DNA,
and anti-STAT4 antibody (Figure 3, lanes 6, 7, and 10). This confirmed that complex S represented the STAT4-DNA complex. However, no visible level of complex S was induced by IL-12 in T cells cultured with IL-12 or IL-2 plus IL-12 (Figure 3,
lanes 3 and 4). These results indicate that T cells treated with IL-12
have much less STAT4 DNA-binding activity in response to further IL-12
stimulation. We further examined other IL-12-induced functions in
activated T cells, including IFN- production and proliferation. In
response to IL-12 restimulation, T cells that were previously cultured
with IL-12, either alone or in combination with IL-2, displayed
significantly reduced IFN- production compared with T cells cultured
without cytokine or with IFN-. Similarly to what we observed in NK
cells, T cells treated with IL-2 showed enhanced IFN- production in
response to IL-12 (Figure
4A).11 Moreover, T cells
that were pretreated with IL-12, either alone or in combination with
IL-2, also showed significantly reduced proliferative response to IL-12
compared with T cells cultured in other conditions (Figure 4B). These
data indicate that treatment of activated T cells with IL-12 results in
significantly reduced functional responses to further IL-12
stimulation.
The effect of IL-12 treatment on the expression of STAT4 messenger RNA Having shown that IL-12 significantly reduces the expression of STAT4 protein in activated T cells, we undertook further experiments to examine the mechanism whereby STAT4 expression was reduced. First, we examined the expression of STAT4 messenger RNA (mRNA) in activated T cells by Northern blot. Although STAT4 protein is markedly reduced in T cells treated with IL-12 or IL-2 plus IL-12 (Figure 5, bottom panel), there was no significant reduction in the level of STAT4 mRNA in these cells compared with untreated T cells or T cells treated with IL-2 or IFN-
(Figure 5, top panel). These results suggest that IL-12 does not affect
the expression of STAT4 at the level of gene transcription.
Inhibition of proteasome-pathway degradation blocks IL-12-induced down-regulation of STAT4 protein The proteasome pathway has been shown to be involved in the turnover of tyrosine phospho-STATs activated in several signaling pathways.26-28 To examine whether the proteasome pathway is also involved in the down-regulation of STAT4, we examined the effect of a specific inhibitor of this pathway, MG-132, on the down-regulation of STAT4 in IL-12-treated T cells. As noted previously, STAT4 protein was decreased in IL-12-treated T cells compared with untreated or IL-2-treated T cells (Figure 6A, top panel). Pretreatment with DMSO had no effect on the decrease of STAT4 induced by IL-12. Preincubation with MG-132 did not alter the expression of STAT4 in control T cells or IL-2-treated T cells. However, MG-132 inhibited the reduction of STAT4 protein induced by IL-12 (Figure 6A, top panel). More importantly, there appeared to be an accumulation of the slowly migrating phosphorylated STAT4 band in these T cells. The more rapidly migrating band, which represents unphosphorylated STAT4, was not affected in comparison with IL-12-treated T cells without the inhibitor MG-132 (Figure 6A, top panel, lanes 4 to 6). To confirm that the STAT4 protein that was degraded through the proteasome pathway was indeed the tyrosine-phosphorylated STAT4, we immunoprecipitated STAT4 protein from untreated cells or IL-12-treated cells in the absence or presence of DMSO or MG-132 (Figure 6B). Similarly to what we observed in Figure 6A, treatment with IL-12 induced a reduction of STAT4 protein in the absence or presence of DMSO. The presence of MG-132 in IL-12-treated cells led to an accumulation of the slowly migrating STAT4 band (Figure 6B, top panel). Reprobing with phospho-tyrosine-specific antibody revealed that this slowly migrating STAT4 band represents the tyrosine-phosphorylated STAT4 protein (Figure 6B, bottom panel). These results suggest that the proteasome pathway is involved in the IL-12-induced STAT4 down-regulation. More importantly, the degradation appears to involve primarily the phosphorylated form of STAT4. Although previous studies have demonstrated that IL-12 also activates STAT1,23 MG-132 does not lead to any increase in phospho-STAT1 or phospho-STAT1 in IL-12-treated cells. MG-132 also did not result in accumulation of phospho-STAT1 induced by IL-2
(Figure 6A, bottom panel). These observations suggest that degradation
through the proteasome pathway specifically targets phospho-STAT4. The
delayed activation of this degradation process suggests that
phosphorylation of STAT4 alone may not be sufficient for the
degradation of STAT4. Other IL-12-induced factors may also be involved
in this process.
Down-regulation of STAT4 through the proteasome pathway is specific for IL-12 signaling IFN- is also known to induce phosphorylation of
STAT4.29 To investigate whether the down-regulation of
STAT4 is specific for IL-12, we examined the effect of MG-132 on
IFN--induced activation of STAT4. As shown in Figure
7, activated T cells were stimulated for
6 hours with IFN-, and results were compared with similar treatment
with IL-12. In contrast to IL-12-treated T cells, there was no visible
phospho-STAT4 band in the IFN--treated T cells (Figure 7, lanes 7 and 8), and there is a low level of phospho-STAT4 accumulation after
MG-132 treatment (Figure 7, lane 9). The unphosphorylated STAT4
continues to represent the predominant STAT4 band in cells treated with
MG-132 (Figure 7, lane 9). To understand the mechanism that leads to
the different effects of MG-132 on phospho-STAT4 induced by these 2 cytokines, we compared the kinetics of STAT4 activation. As shown in
Figure 8, IL-12-treated T cells
exhibited persistent activation of STAT4 as indicated by the appearance of a shifted STAT4 band throughout the time we tested. This was associated with a marked decrease of total STAT4 protein by 24 hours
(Figure 8, top panel). However, IFN- induced only a transient phosphorylation of STAT4, as indicated by the disappearance of the
shifted STAT4 band by 6 hours (Figure 8, panel 3). In contrast to
IL-12-treated T cells, the level of STAT4 protein in IFN--treated T cells was maintained at a similar level throughout the time we
observed. These observations suggest that although both IL-12 and
IFN- induce activation of STAT4, the degradation of phospho-STAT4 was specific for the IL-12 signaling pathway. This may be attributed to
the persistent activation of STAT4 induced by IL-12 rather than the
transient activation of STAT4 induced by IFN-.
In previous studies, it has been demonstrated that expression of
the IL-12 receptor can be modulated by other cytokines, such as IL-2
and IL-4, and that this provides an important mechanism for the
regulation of IL-12 responsiveness. IL-12 also modulates further
responsiveness to IL-12, but the mechanism whereby IL-12 autoregulation
occurs has not been defined. In the present study, we examined various
components of the IL-12 signaling pathway in activated human T cells
and confirmed that once these cells have been activated by IL-12, they
become resistant to further IL-12 stimulation. This specific
down-regulation of IL-12 signaling appears to be mediated primarily
through reduced levels of STAT4 protein, a critical component of the
IL-12 signaling pathway. Decreased levels of STAT4 following IL-12
activation do not appear to result from decreased STAT4 gene
transcription but from increased degradation of STAT4 protein. Initial
experiments suggest that this increased degradation specifically
targets the tyrosine-phosphorylated STAT4 protein that results from
IL-12-induced activation. Other STAT proteins and STAT4 activation
following IFN- Cytokine signaling through the Jak-STAT pathways involves the
phosphorylation of STAT proteins by Jak kinases, the translocation of
phospho-STATs to the nucleus, the dephosphorylation of STATs by
tyrosine phosphatase in the nucleus, and the return of the dephosphorylated STAT protein to the cytoplasm.27 Several
mechanisms have previously been shown to regulate this signaling
pathway. One mechanism is inhibition of STAT phosphorylation by members of the suppressor of cytokine signaling (SOCS) family. SOCS proteins are induced following cytokine stimulation, bind to Jak kinases, and
inhibit activated Jak kinases from further phosphorylation of STAT
proteins.30,31 This mechanism has been shown to
down-regulate IFN- Several studies have demonstrated that the proteasome pathway can be
involved in the turnover of phosphorylated STAT proteins. In previous
studies, addition of the proteasome-specific inhibitor MG-132 was shown
to prolong the tyrosine phosphorylation of STAT1, STAT4, STAT5, and
STAT6 induced by IFN- The depletion of cellular STAT4 in activated T cells markedly affects
the function of these cells and specifically reduces their ability to
secrete IFN- Although IL-12 has been shown to activate STAT1 in addition to STAT4,
our results demonstrated that neither STAT1 nor phospho-STAT1 was
depleted after IL-12 activation. This finding suggests that the
degradation process activated by IL-12 is specific for STAT4 and does
not affect other STAT proteins activated in the same pathway. In
addition, STAT4 was not depleted after IFN- The negative regulation of IL-12 signaling through down-regulation of
STAT4 expression may contribute to the ineffectiveness of IL-12 in
clinical trials in patients with cancer. In these studies, single doses
of IL-12 augment the cytolytic activity, secretion of INF-
Submitted November 1, 2000; accepted February 8, 2001.
Supported by National Institutes of Health grant CA41619. E.Z. is supported by the Lawrence and Susan Marx Immunotherapy Fellowship.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jerome Ritz, Dana-Farber Cancer Institute, 44 Binney St, Boston, MA 02115; e-mail: jerome_ritz{at}dfci.harvard.edu.
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© 2001 by The American Society of Hematology.
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Abstract
Introduction
