By
From the * Department of Pathology, Washington University School of Medicine, St. Louis, Missouri
63110; and the Department of Inflammation/Autoimmune Diseases Hoffmann La-Roche Inc.,
Nutley, New Jersey 07110
The developmental commitment to a T helper 1 (Th1)- or Th2-type response can significantly
influence host immunity to pathogens. Extinction of the IL-12 signaling pathway during early
Th2 development provides a mechanism that allows stable phenotype commitment. In this report we demonstrate that extinction of IL-12 signaling in early Th2 cells results from a selective
loss of IL-12 receptor (IL-12R) 2 subunit expression. To determine the basis for this selective
loss, we examined IL-12R
2 subunit expression during Th cell development in response to
T cell treatment with different cytokines. IL-12R
2 is not expressed by naive resting CD4+
T cells, but is induced upon antigen activation through the T cell receptor. Importantly, IL-4
and IFN-
were found to significantly modify IL-12 receptor
2 expression after T cell activation. IL-4 inhibited IL-12R
2 expression leading to the loss of IL-12 signaling, providing an
important point of regulation to promote commitment to the Th2 pathway. IFN-
treatment
of early developing Th2 cells maintained IL-12R
2 expression and restored the ability of these cells to functionally respond to IL-12, but did not directly inhibit IL-4 or induce IFN-
production. Thus, IFN-
may prevent early Th cells from premature commitment to the Th2 pathway. Controlling the expression of the IL-12R
2 subunit could be an important therapeutic
target for the redirection of ongoing Th cell responses.
In chronic immune responses, cytokine production by
CD4+ T cells may polarize toward either a Th1 or Th2
response (1). Th1 cytokines, such as IFN- Recently, reversibility of Th2 responses has been examined both in vivo and in vitro (14). Th2 responses developing during infection by Leishmania major of susceptible
BALB/c mice were found to revert to healing Th1-type responses upon treatment with IL-12 and the antibiotic compound Pentostam (14) or under certain conditions in models employing T cell transfers into scid mice (15). These
studies suggest that emerging Th2 responses may be reversible. In vitro analysis of TCR-transgenic naive T cells showed that emergent Th2 responses become unresponsive
to IL-12 and effectively resist reversal to Th1 phenotype
(16, 17). We partially characterized the mechanism of in
vitro resistance of Th2 cells to IL-12 as a defect in proximal
IL-12 signaling (17). T cells activated in vitro for 3 d under
strongly polarizing conditions (IL-4 and anti-IL-12) were
unable to phosphorylate Jak2, Stat1, Stat3, and Stat4 in response to IL-12 (17). Expression of the IL-12R Recently, a second component of the IL-12 receptor
(IL-12R)1 was identified and cloned. This component, the
IL-12 receptor Cytokines and Antibodies.
Recombinant human IL-2 was provided by Takeda (Osaka, Japan), recombinant murine IL-4 by
Genzyme (Cambridge, MA), recombinant murine IL-12 by
Hoffmann-La Roche (Nutley, NJ), and recombinant murine
IFN- Medium and Peptides.
T cells were maintained in IMDM
(Washington University Tissue Culture Support Center, St. Louis,
MO) supplemented as described (17). OVA peptide from chicken
ovalbumin (residues 323-339) was synthesized on an Applied
Biosystems model 430 peptide synthesizer (Foster City, CA).
Animals.
Mice transgenic for the DO11.10 T Cell Purification and T Cell Cultures.
Mel-14hi/CD4+ T cells
were isolated from spleens of 4-6 wk old unimmunized DO11.10
TCR-transgenic mice on a FACS® Vantage cell sorter as described
(28) yielding purities of >98%. 2.5 × 105 FACS®-sorted Mel14hi/CD4+ DO11.10 T cells were stimulated in 2 ml cultures
with 0.3 µM OVA peptide presented by irradiated BALB/c splenocytes (2,000 rads, 6 × 106/well) in the presence of 10 U/ml
IL-12 and 10 µg/ml anti-IL-4 (11B11) to promote Th1 phenotype development, 200 U/ml IL-4 and 3 µg/ml anti-IL-12
(TOSH) to promote Th2 phenotype development, or the combination of 10 U/ml IL-12 and 200 U/ml IL-4. At 72 h the cells
were expanded threefold in fresh medium. These differentiated Th cells were harvested on day 7, washed, and counted. 1.25 × 105 T cells were restimulated with OVA peptide and BALB/c
splenocytes with or without addition of cytokines or anti-cytokine
antibodies as indicated in the figure legends. Supernatants were
collected after 48 h and analyzed by capture ELISA for IFN- and lymphotoxin, promote phagocytic and inflammatory responses,
whereas Th2 cytokines like IL-4, IL-5, and IL-6 promote
allergic and eosinophilic responses and provide specific B cell
help associated with IgE isotype switching (1). Cytokines
present in the early stages of antigen driven CD4+ T cell
activation help to determine the specific pattern of Th phenotype that develops (4). Th1 development is enhanced
when naive T cells are activated in the presence of IL-12
(5, 6). IFN-
assists Th1 development initially through a
mechanism consistent with promoting the ability of naive
T cells to respond to IL-12 (7, 8). Conversely, Th2 cells
develop when IL-4 but not IL-12 is present during activation of naive T cells (5, 9). In addition, cross regulation between these subsets takes place, so that development of one
subset is inhibited by cytokines produced by the other (2, 4).
This mechanism provides one way for responses to become
self-reinforcing and helps to stabilize the emergence of polarized phenotypes. For example, the Th2 cytokines interleukin 4 (IL-4) and IL-10 suppress Th1 development by
inhibiting production of IFN-
and IL-12 (10), whereas
IFN-
has been thought to selectively limit the outgrowth
of Th2 cells (11).
1 subunit
was similar in Th1 and Th2 cells, suggesting that this receptor subunit was not responsible for lack of IL-12 signaling
in Th2 cells. Also, both Th1 and Th2 cells expressed similar levels of the relevant kinases, Jak2 and Tyk2 and the STAT proteins Stat1, Stat3, and Stat4 (17). Thus, the precise molecular basis for the signaling defect remained unclear.
We also compared loss of IL-12 responsiveness in T cells from
Balb/c (L. major susceptible) and B10.D2 (L. major resistant) strains and found a correlation between resistance and
the maintenance of T cell IL-12 responsiveness. Thus, when
stimulated without addition of cytokines or anti-cytokine
antibodies, B10.D2 T cells maintained IL-12 responsiveness whereas Balb/c T cells lost IL-12 responsiveness (19).
We suggested that prolonging the period of IL-12 responsiveness in an emerging T cell response may allow resistant
strains to reverse the early Th2-type response toward a protective Th1 response due to the action of IL-12 generated during the later stages of infection (19, 20). However, the precise molecular basis for the defect in IL-12 signaling still remained unclear (19).
2 subunit, is necessary for IL-12 signaling
through the Jak/STAT pathway. In the present report, we
now show that the basis for the IL-12 signaling defect in
Th2 cells is the specific down regulation of this newly
identified IL-12 receptor component, the IL-12R
2 subunit. To determine the basis for this selective loss, we examined IL-12R
2 subunit expression in response to T cell
treatment with cytokines. IFN-
treatment of Th cells developing in Th2-inducing conditions induced the expression of IL-12R
2 mRNA and restored the ability of these
T cells to functionally respond to IL-12. IFN-
may thus
prevent early Th cells from premature commitment to the
Th2 pathway. These results help to resolve some of the discrepancies reported regarding the reversibility of ongoing murine Th2 responses in vivo and in vitro. They may also
help to explain some of the differences observed between
murine and human Th2 cells.
by Genentech (South San Francisco, CA). Anti-IL-12 mAb (TOSH) was supplied by Drs. C.S. Tripp and E.R. Unanue
(Washington University School of Medicine, St. Louis, MO) (22),
anti-IFN-
mAb (H22) by Dr. R.D. Schreiber (23), and polyclonal rabbit antiserum specific for Stat4 was provided by Dr.
James Darnell (Rockefeller University, New York, NY) (24, 25).
The anti-phosphotyrosine reagent RC20 was purchased from
Transduction Laboratories (Lexington, KY). Anti-IL-4 mAb 11B11
has been described (26).
-TCR (27)
were maintained on the BALB/c background. Female BALB/c mice
were purchased from Harlan Sprague Dawley (Indianapolis, IN).
and
IL-4 (29).
Immunoprecipitation and Western Blot Analysis. Analysis of Stat4 tyrosine phosphorylation was performed as described (30). In brief, total cellular lysates of 1.5-2.5 × 107 T cells were immunoprecipitated with anti-Stat4 antisera and resolved by SDS-PAGE. After transfer to nitrocellulose, blots were probed with RC20 (1:2,500). Blots were stripped and reprobed with anti-Stat4 antisera (1:3,000).
Northern Blot Analysis.
Total T cell RNA was isolated from
Th1 and Th2 cells using RNAzol RNA isolation solvent (TelTest, Friendswood, TX). Northern blot analysis was performed
with 15 µg of total RNA per lane, and membranes were sequentially
probed with the full-length murine IL-12 receptor 1 subunit
cDNA (31), the full-length murine IL-12 receptor
2 subunit
cDNA and the cDNAs for GAPDH (32) or pHE7 (33).
Mel-14hi CD4+ T cells from DO11.10 TCRtransgenic mice were purified and activated with antigen in
the presence of IL-12 and antibodies to IL-4, or IL-4 and
antibodies to IL-12, for 7 d to generate polarized Th1 or
Th2 populations (17). Subsequently, IL-12R expression was
analyzed by Northern blotting on day 7 and 9 after secondary activation (Fig. 1 A). Th1 cells expressed the IL-12R 2 mRNA at high levels on these days. In contrast, IL-12R
2 mRNA was completely absent in Th2 cells harvested at
these same time points. As we previously observed (17),
IL-12R
1 mRNA was present in both Th1 and Th2 cells
(Fig. 1 A, top).
To determine how rapidly expression of IL-12R 2
mRNA subsided during Th2 development, we induced Th1
and Th2 differentiation from naive CD4+ T cells and analyzed IL-12R
2 expression on days 3, 5, and 7 after primary activation. To test expression in naive T cells, we obtained naive CD4+ T cells by cell sorting and prepared
mRNA for Northern analysis (Fig. 1 B). Neither IL-12R
1 nor IL-12R
2 mRNA was detectable in naive T cells
(Fig. 1 B). On days 3, 5, and 7 after primary activation, developing Th1 cells expressed high levels of both the IL-12R
1 and IL-12R
2 mRNA (Fig. 1, B and C). On day 3, Th2 cells expressed IL-12R
1 mRNA at comparable or
slightly higher levels than Th1 cells, but expressed only very
low levels of IL-12R
2 mRNA (Fig. 1 C). By days 5 and 7 after primary activation IL-12R
2 mRNA was undetectable in Th2 cells (Fig, 1 C).
We next asked what conditions controlled the maintenance
of IL-12 signaling in developing Th cells. IL-4 was thought
to dominate IL-12 for effects on T helper phenotype development (5, 34), since the addition of IL-4 and IL-12 together led to Th2 development both in TCR-transgenic
and anti-CD3 driven systems (5, 34). We first wished to
determine if it was the lack of IL-4 in primary cultures that
allowed the maintenance of IL-12 responsiveness in developing Th1 cells. Thus, we activated naive T cells in the presence of IL-4 and IL-12 together in the primary culture and assessed IL-12 responsiveness on day 5 after primary stimulation (Fig. 2). Surprisingly, these T cells were able to phosphorylate Stat4 upon IL-12 treatment (Fig. 2, middle), similar to Th1 cells (Fig. 2, top), but unlike IL-12 unresponsive
Th2 cells (Fig. 2, bottom). This maintenance of Stat4 phosphorylation correlated with functional IL-12 responses in
these T cells as measured by IL-12-induced IFN- production (Fig. 3). T cells arising from stimulation in IL-4
and IL-12 together showed significant IL-12 induced IFN-
production during restimulation (Fig. 3, top). Restimulation of these cells without exogenously added IL-12 led to
production of 180 U/ml IFN-
, while addition of IL-12
increased IFN-
production to 400 U/ml. Because these T
cells arose in IL-4, they acquired the Th2-type property of
producing IL-4 and IL-10, two cytokines which inhibit
IFN-
production (35, 36). In this system, IL-10 could also
be produced by other cells used as APCs. Neutralization of
IL-4 and IL-10 revealed a significantly increased IL-12-
mediated induction of IFN-
, from less than 50 U/ml of
IFN-
produced upon restimulation in the absence of IL-12
to 1,300 U/ml of IFN-
produced in the presence of IL-12
(Fig. 3, top). In contrast, the control Th2 cells produced
very low amounts of IFN-
(20 U/ml) upon restimulation in the presence of IL-12, a level which was not increased
by neutralization of IL-4 and IL-10 (Fig. 3, bottom). Taken
together, these results therefore suggest that the presence of
IL-12 during primary T cell activation, not the absence of
IL-4, was responsible for maintenance of functional responsiveness to IL-12.
We suspected that IL-12 was not the primary stimulus
for induction of the IL-12R 2 subunit, since a cytokine
cannot signal unless its receptor is already expressed at some
level on the cell surface. Previously, IFN-
has been shown
to be required for IL-12-induced Th1 development from
naive BALB/c T cells (7, 34). IL-12 is known to induce
IFN-
production from CD4+ and CD8+ T cells, as well
as NK cells. Since these cells are present in the irradiated
splenocytes used as APCs, we suspected that IFN-
could
be inducing the expression of the IL-12R
2 subunit. To test this possibility, combinations of IL-12, IFN-
and IL-4, or the corresponding neutralizing antibodies were added
during primary T cell activation and the IL-12 responsiveness of these developing Th cells was analyzed by measuring IL-12-induced Stat4-phosphorylation on day 5 after
activation (Fig. 4). Th cells activated in the presence of
both IL-12 and IL-4 maintained IL-12-induced Stat4 phosphorylation (Fig. 4, lane 4). The maintenance of IL-12 responsiveness on these cells was completely dependent on the
production of endogenous IFN-
during primary activation,
since neutralization of IFN-
abolished IL-12-induced Stat4
phosphorylation (Fig. 4, lane 5). Under Th2 inducing conditions (IL-4 and anti-IL-12), Th2 cells lost the ability to
phosphorylate Stat4 in response to IL-12 (Fig. 4, lane 7).
However, addition of IFN-
to this culture restored IL-12induced Stat4 phosphorylation (Fig. 4, lane 9). Finally, under Th1 inducing conditions (IL-12 and anti-IL-4), T cells
retained the ability to phosphorylate Stat4 in response to
IL-12 in a manner that was independent of IFN-
(Fig. 4,
lanes 1-3). In summary, (a) the presence of IL-4 during
primary activation inhibits IL-12 signaling in developing
Th cells, and (b) IFN-
is able to override this inhibition
and restore IL-12 signaling in early Th cells.
IL-4 and IFN-
To test whether IFN--induced restoration of IL-12
signaling involved induction of the IL-12R
2 subunit, we
performed Northern blot analysis of Th cells derived under
the various conditions described in Fig. 4. T cells activated
in the presence of both IL-4 and IL-12 expressed the IL-12R
2 mRNA (Fig. 5, lane 4). This expression was dependent
on endogenous IFN-
production, since neutralization of
IFN-
in the primary culture led to the disappearance of
IL-12R
2 mRNA (Fig. 5, lane 3). T cells arising from
stimulation in the presence of IL-4, anti-IL-12 mAb and
exogenous IFN-
added during primary activation expressed
high levels of the IL-12R
2 mRNA (Fig. 5, lane 6). Addition of anti-IFN-
mAb blocked expression of IL-12R
2 mRNA (Fig. 5, lane 5). Th1 cells, arising from stimulation in the presence of IL-12 plus anti-IL-4 mAb, expressed
IL-12R
2 mRNA independently of IFN-
(Fig. 5, lanes 1 and 2). Thus, in each case, IL-12-inducible Stat4 phosphorylation (Fig. 4) correlated with expression of the IL-12R
2 mRNA (Fig. 5).
To examine whether IFN- treatment of developing Th
cells during primary culture altered functional responses to
IL-12, we measured the IL-12-dependent IFN-
production in developing Th cells derived under the same conditions used in Fig. 5. Additionally, since production of endogenous IL-4 and IL-10 production by these cells directly
inhibits IFN-
production, we neutralized these cytokines
to more clearly assess the true potential of these cells for
IL-12 induced IFN-
production. T cells arising from
stimulation in IL-4 plus anti-IL-12 plus IFN-
responded
to IL-12 by producing 550 U/ml of IFN-
in the secondary stimulation (Fig. 6, top), whereas addition of anti-IFN-
mAb led to cells producing less than 50 U/ml of IFN-
upon restimulation (Fig. 6, bottom). For T cells treated with
IL-4 and IL-12 during primary activation, IFN-
in the
primary culture was required for IL-12 induced IFN-
production upon secondary stimulation (Fig. 6, compare column 3 in top and bottom). For Th1 cells, arising from
stimulation in the presence of IL-12 and anti-IL-4 mAbs,
IFN-
was not required for subsequent development of IL-12
responsiveness (Fig. 6, compare column 1 in top and bottom). Thus, IFN-
regulates IL-12 responsiveness with the
same pattern as it regulates IL-12R
2 expression in developing Th cells (Fig. 5).
The data presented in this report provide a molecular basis for the previously described IL-12 unresponsiveness of
Th2 cells and help explain several discrepancies that become apparent when comparing murine and human Th2
cells. Expression of the IL-12R 2 subunit is required for
recruitment and activation of the STAT proteins involved
in IL-12 signaling (37). When IL-4 is neutralized by antibodies, TCR-activation alone is sufficient for inducing IL-12R
2 expression. However, when even low levels of
IL-4 are present, expression of the IL-12R
2 is inhibited.
Thus, during in vitro Th2 development, IL-12R
2 expression is strongly inhibited by the IL-4 that is added to
induce Th2 development. This inhibition leads to loss of
IL-12 signaling capacity and helps to stabilize the emergent
Th2 response against reversal of phenotype upon subsequent IL-12 exposure. Loss of IL-12 responsiveness may
thus be an early step in the commitment of T cells to the
Th2 pathway.
Regarding the role of IFN- in Th development, we
find that IFN-
overrides the IL-4-induced inhibition of
IL-12R
2 expression. Thus, in T cells arising from stimulation with IL-4 and anti-IL-12 mAbs (i.e., fully Th2 inducing conditions), IFN-
treatment during primary activation restored IL-12R
2 expression and functional IL-12
responsiveness. These T cells could produce IL-4 and IL-10 but retained the capacity for IL-12-induced IFN-
production. The ability of Th2 cells to respond to IL-12 has
previously been described for human (38, 39), but not murine Th2 cells (16, 17). This discrepancy can now be explained as follows. Murine Th2 cells lose expression of the
IL-12R
2 due to the absence of IFN-
in Th2 cultures.
Human Th2 cells, on the other hand, express low but
functional levels of the IL-12R
2 subunit, and human
IL-12 R
2 expression is induced by IFN-
rather than
IFN-
(Francesco Sinigaglia, personal communication).
Elucidation of the molecular basis for this distinct form of
regulation between these two species will require the direct
examination of the promoter/enhancer regions of the respective IL-12R
2 gene.
Our data also further clarify the role of IFN- in Th1
development. Results from several previous reports (7, 40)
indicated that IFN-
, although by itself not sufficient, was
clearly required for IL-12-induced Th1 development from
naive T cells. Other studies however did not identify such a
requirement, even though similar TCR-transgenic experimental systems were used (41). This apparent discrepancy
can now be explained by the established difference in IL-4
production that occurs in the two experimental mouse strains used. Studies that did not identify this requirement used
TCR-transgenic mice on the C57/BL6 background in which
very little IL-4 is produced (19, 28). The substantial amount
of IL-4 produced by the BALB/c strain inhibits expression
of IL-12R
2 and imposes the observed requirement for
IFN-
that allows IL-12R
2 expression (Guler, M., N. Jacobson, U. Gubler, K. Murphy, manuscript in preparation). In the C57 or B10 backgrounds, the absence of IL-4 allows
IL-12R
2 expression to occur independently of IFN-
.
In this report IFN- was not required for the expression
of the IL-12 receptor
2 subunit on T cells differentiated to
the Th1 phenotype with IL-12 and anti-IL-4. This result
may be due to the complete absence of IL-4 in these primary cultures, thus artificially creating C57/BL6 or B10-like
conditions. Moreover, when Th1 differentiation was induced using either IL-12 alone or IL-12 plus a low level of
IL-4 (2-5 U/ml) in the primary stimulation, a requirement
for IFN-
during the primary activation for Th1 development was observed (data not shown). Thus a requirement for IFN-
during Th1 development may only be evident
in situations where low levels of IL-4 are present. In contrast, when IL-4 is absent during primary stimulation IFN-
is not required for maintenance of the IL-12R
2 subunit
on developing Th cells.
Finally, our results may be relevant for understanding responses to certain intracellular pathogens. For example, resistance to L. major in various strains of mice is complex and
probably controlled by several genetic loci. The present study
implies that cytokines could act to promote either susceptibility or resistance to pathogens by their distinct actions on
IL-12R 2 expression. IL-4 inhibits expression of the
IL-12R
2 subunit and imposes a requirement for IFN-
to maintain IL-12 responsiveness. Thus, when IFN-
production is limited, IL-4 production in early responses may
critically inhibit IL-12R
2 expression, with significant impact on development of protective responses. In strains
that produce IL-4 early, IFN-
production may be critical
for inducing IL-12R
2 expression on T helper cells to allow for IL-12-induced Th1 differentiation. NK cells may
be an important in vivo source of such early IFN-
production, since IFN-
can be induced by the action of IL-12
and TNF on NK cells (42). As suggested by studies of the
development of resistance to L. major (43, 44), NK cells
may thus play an important role in Th1 development.
Address correspondence to Kenneth M. Murphy, Department of Pathology, Washington University School of Medicine, Campus Box 8118, 660 S. Euclid Ave., St. Louis, MO 63110.
Received for publication 25 November 1996 and in revised form 8 January 1997.
1 Abbreviation used in this paper: IL-2R, IL-12 receptor.We thank Drs. E. Unanue and R. Schreiber for helpful discussions and reagents. We thank Dr. J. Darnell for his gift of anti-Stat antiserum.
This work was supported by National Institutes of Health grants AI34580, AI31238, and AI39676 and a grant from the American Cancer Society. S.J. Szabo was supported by training grant CA09547.
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