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Department of Inflammation/Autoimmune Diseases, Hoffmann-La Roche Inc., Nutley, NJ 07110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production. Therefore,
IL-12, in binding to the high affinity IL-12R, interacts with
IL-12Rß1 primarily via regions on the IL-12 p40 subunit and with
IL-12Rß2 via 20C2-reactive, heterodimer-specific regions of IL-12 to
which the p35 and p40 subunits both contribute. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by both T and NK cells
(7, 8, 9, 12). IL-12 mediates its biologic activities through binding to
specific cell surface receptors. This IL-12R was initially
characterized on PHA-activated lymphoblasts and IL-2-activated NK cells
(13, 14). These cells display at least two classes of sites that bind
125I-labeled human IL-12
(125I-huIL-12)2
with Kd values of 5 to 20 pM and 2 to 6 nM (15, 16). The IL-12R has recently been shown to be composed of at
least two protein subunits, IL-12Rß1 and IL-12Rß2 (15, 17, 18).
Each of the identified IL-12R subunits is a member of the cytokine
receptor superfamily, with the most pronounced homologies to gp130 and
two other ß-type cytokine receptors (19), the receptors for
lymphocyte inhibitory factor and granulocyte CSF. Individually,
huIL-12Rß1 and huIL-12Rß2 expressed in COS-7 cells bind
125I-huIL-12 with a Kd of about 5
nM, corresponding to the low affinity binding seen in PHA-activated
lymphoblasts. In addition to this low affinity binding site, cells
expressing both huIL-12R subunits exhibit high affinity
125I-huIL-12 binding (Kd =
50 pM) and IL-12 responsiveness (17, 18).
Bioactive IL-12 is a heterodimer composed of a p40 and a p35
protein subunit. Homodimeric mouse IL-12 p40 subunit
(mo(p40)2) is a potent mouse IL-12 antagonist that has been
shown to block 125I-huIL-12 binding to huIL-12Rß1, but
not to high affinity IL-12R, on PHA-activated human lymphoblasts (20).
Anti-huIL-12Rß1 mAbs have been shown to specifically inhibit
huIL-12-induced proliferation of PHA-activated lymphoblasts,
development of LAK activity, and production of IFN- from resting
PBMC (16), demonstrating that huIL-12Rß1 is necessary for huIL-12
signaling. In addition, the huIL-12 heterodimer-specific mAb 20C2 has
been shown to inhibit huIL-12-induced proliferation of PHA-activated
lymphoblasts, but does not inhibit low affinity
125I-huIL-12 binding to huIL-12Rß1-transfected COS-7
cells (21). The present study demonstrates that IL-12 inhibitors can be
grouped into two classes that differ in their ability to block huIL-12
interaction with either huIL-12Rß1 or huIL-12Rß2, and that these
two classes of IL-12 inhibitors can synergistically inhibit the
multiple interactions between IL-12 and its functional receptor
complex.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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COS-7 cells were obtained from the American Type Culture Collection (Rockville, MD) and were transfected with pEF-BOS expression constructs (22) encoding human IL-12Rß1 (15) and/or IL-12Rß2 (18). In the experiments shown, COS-7 cells (40 x 106) were transfected with 25 µg of IL-12Rß2-containing and/or 2.5 µg of IL-12Rß1-containing pEF-BOS DNA by electroporation using a Bio-Rad Electroporator (Bio-Rad Laboratories, Richmond, CA) at 250 µF and 350 V and a 0.4-cm cuvette as previously described (23). The 10:1 ratio of huIL-12Rß2:huIL-12Rß1 expression construct DNA was used to help compensate for the greater expression efficiency of huIL-12Rß1 compared with that of huIL-12Rß2 (18). Purified recombinant huIL-12 and mo(p40)2 were provided by F. Podlaski and A. Stern (Department of Inflammation/Autoimmune Diseases, Hoffmann-La Roche, Nutley, NJ). Radioiodination of huIL-12 was performed using IodoGen (Pierce, Rockford, IL) as previously described (24) and yielded 125I-huIL-12 with a sp. act. of about 4,000 cpm/fmol. The anti-huIL-12Rß1 mAbs 2B10 and 2-4E6 have been described previously (16). The heterodimer-specific anti-huIL-12 mAb 20C2 has also been previously described (21, 25).
125I-huIL-12 binding assays
Binding assays were conducted using COS-7 cells harvested 48 to 72 h after transfection as described previously (15). Briefly, 1 x 105 COS-7 cells were incubated with various concentrations of 125I-huIL-12 in the absence (total binding) and the presence (nonspecific binding) of 10 µg/ml huIL-12 for 90 min at 22°C. Cell-associated radioactivity was determined by centrifuging the cells through oil and measuring the radioactivity present in the cell pellet. Analysis of the binding data by the method of Scatchard (26) was performed using the nonlinear regression LIGAND equilibrium binding data analysis program (27). The number of receptor sites per cell was calculated assuming that all transfected COS-7 cells were expressing equal numbers of IL-12Rs. Therefore, we are actually reporting an average number of receptor sites per cell. For huIL-12Rß1, about 90 to 100% of the transfected COS-7 cells appear to express huIL12Rß1 by flow cytometric analysis with anti-huIL-12Rß1 mAbs. Anti-huIL-12Rß2 mAbs are not currently available, and therefore the transfection efficiency for huIL-12Rß2 in COS-7 cells is not known.
IL-12-induced proliferation and IFN- production assays
IL-12-stimulated proliferation was determined using
PHA-activated lymphoblasts as described previously (28). All
proliferation assays were conducted in triplicate. IFN- production
by PBMC was measured in the presence of 20 U/ml rIL-2 and 200 U/ml
huIL-12 as described previously (29). At these concentrations, neither
IL-2 nor IL-12 alone elicited IFN- secretion. IFN- cultures were
conducted in quadruplicate, and IFN- was measured using a specific
ELISA as previously described (29).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The functional, high affinity IL-12R is composed of at least two
protein subunits, huIL-12Rß1 and huIL-12Rß2. As a first step in the
investigation of the interaction of IL-12 with its receptor complex,
the abilities of various inhibitors of IL-12 to interfere with IL-12
binding to transiently transfected COS-7 cells expressing either
huIL-12Rß1 or huIL-12Rß2 were studied. Both the ß1 and ß2
receptor subunits, when expressed in COS-7 cells, bound
125I-huIL-12 (Fig. 1
)
with low affinity (Kd = 5 nM), in
agreement with previously reported results (15, 18). As expected, mAb
2B10, which recognizes huIL-12Rß1 (16), blocked
125I-huIL-12 binding to COS-7 cells expressing
huIL-12Rß1. Treatment with 2B10 inhibited 125I-huIL-12
binding by 78 ± 5% at all concentrations of
125I-huIL-12 examined. Increasing the concentration of 2B10
did not result in further inhibition of 125I-huIL-12
binding; however, treatment with a combination of two
anti-huIL-12Rß1 mAb, 2B10 and 2-4E6, did result in complete
inhibition of 125I-huIL-12 binding to
huIL-12Rß1-transfected COS-7 cells (data not shown). In contrast,
2B10 had no effect on 125I-huIL-12 binding to COS-7 cells
expressing huIL-12Rß2 (Fig. 1). Isotype control Abs had no
significant effect on 125I-huIL-12 binding to COS-7 cells
expressing huIL-12Rß1 or huIL-12Rß2 (data not shown). Similarly,
mouse p40 homodimer (mo(p40)2), a known IL-12 antagonist
(20, 30, 31), was able to completely inhibit 125I-huIL-12
binding to huIL-12Rß1-transfected COS-7 cells (Fig. 2) as previously reported (20).
Interestingly, mo(p40)2 had no effect on
125I-huIL-12 binding to huIL-12Rß2-transfected COS-7
cells (Fig. 2). This demonstrates that mo(p40)2 interacts
primarily with the ß1 chain of the IL-12R complex.
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).
In contrast to 2B10 and mo(p40)2, 20C2 increased binding of
125I-huIL-12 to huIL-12Rß1-transfected COS-7 cells. A
similar increase in 125I-huIL-12 binding to
huIL-12Rß1-transfected COS-7 cells was observed when the
nonneutralizing mAb 4D6, directed against the huIL-12 p40 subunit (32),
was used (data not shown), suggesting that the observed increase in
125I-huIL-12 binding was due to cross-linking of
125I-huIL-12 molecules, which then bound to the cell
surface. In contrast, 20C2 clearly inhibited the binding of
125I-huIL-12 to huIL-12Rß2-transfected COS-7 cells,
demonstrating that the binding of 20C2 to huIL-12 prevents the
interaction of huIL-12 with the ß2 receptor subunit.
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In agreement with previously reported results (18), coexpression
of huIL-12Rß1 and huIL-12Rß2 in COS-7 cells creates both high
(Kd = 50 pM; 3,000 sites/cell) and low
(Kd = 7 nM; 115,000 sites/cell) affinity
125I-huIL-12 binding sites (Fig. 4). Scatchard analysis and nonlinear
regression curve fitting of 125I-huIL-12 binding in the
presence of mAb 20C2 demonstrated that 20C2 treatment blocked high
affinity 125I-huIL-12 binding to the cotransfected cells,
leaving the low affinity binding sites
(Kd = 8 nM) largely unaffected (Fig. 4).
This is in agreement with the previously reported ability of 20C2 to
block high affinity, but not low affinity, binding of
125I-huIL-12 to PHA-activated human lymphoblasts
(21).3 Similarly, in three
independent experiments, mo(p40)2 treatment blocked high
affinity 125I-huIL-12 binding, whereas low affinity
125I-huIL-12 binding (Kd = 1
nM) was blocked 82 ± 8% by mo(p40)2 treatment (Fig. 4). Inhibition of low affinity 125I-huIL-12 binding by
mo(p40)2 is presumably due to inhibition of
125I-huIL-12 binding to the large number of huIL-12Rß1
homodimers present on the cotransfected COS-7 cells (as shown in Fig. 2
, upper panel), with the residual
125I-huIL-12 binding measured in the presence of
mo(p40)2 presumably due to low affinity binding to the
relatively few huIL-12Rß2 homodimers present on the cotransfected
COS-7 cell surface. The ability of mo(p40)2 to block high
affinity binding to rIL-12R on cotransfected COS-7 cells contrasts with
its previously reported (20) inability to inhibit binding of
125I-huIL-12 to naturally occurring, high affinity IL-12R
on PHA-activated human lymphoblasts.
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). In a series of three independent experiments, 2B10 treatment (25
µg/ml) blocked 78 ± 6% of low affinity
125I-huIL-12 binding, but had little, if any, effect on
high affinity binding to the cotransfected COS-7 cells (24 ± 21%
inhibition). Increasing the concentration of 2B10 did not increase its
effect on high or low affinity binding (data not shown). Isotype
control Abs had no effect on the binding of 125I-huIL-12 to
the cotransfected COS-7 cells (data not shown). Similar experiments
conducted with PHA-activated human lymphoblasts demonstrated that 2B10
partially inhibited 125I-huIL-12 binding. However, the low
levels of binding that remained upon 2B10 treatment made it difficult
to evaluate the Kd of the remaining binding
sites (data not shown). In contrast, combinations of 20C2 and
mo(p40)2 or 20C2 and 2B10 completely blocked binding of
125I-huIL-12 to PHA-activated human lymphoblasts.
Inhibition of IL-12-induced proliferation and IFN-
production
The abilities of the various IL-12 inhibitors, alone and in
combination, to block IL-12 bioactivity were examined next.
IL-12-induced proliferation of PHA-activated lymphoblasts is a commonly
used measure of IL-12 bioactivity (28). Consistent with its failure to
inhibit high affinity IL-12R binding, 2B10 alone was a poor inhibitor
of IL-12-induced proliferation (Fig. 5A), in agreement with
previous results (16). Although 20C2 alone was able to inhibit
IL-12-induced proliferation, the combination of 20C2 with 2B10 acted
synergistically to potently inhibit IL-12 bioactivity (Fig. 5A). Similarly, the combination of
mo(p40)2 and 20C2 synergistically inhibited IL-12-induced
lymphoblast proliferation (Fig. 5B). Control mouse
and rat Abs showed no significant effect on IL-12-induced lymphoblast
proliferation (16).
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(6). Therefore, we investigated the effects of the various classes of
inhibitors on IL-12-induced IFN- production from human PBMC. In the
absence of IL-12 stimulation, IFN- was not detected. Whereas 500
ng/ml mo(p40)2 and 20 ng/ml 20C2 alone each had no effect
on IL-12-stimulated IFN- production, the combination of these two
inhibitors synergistically decreased IFN- production by 80% (Fig. 6A). Similar
synergistic inhibitory properties of 2B10 and 20C2 were observed (Fig. 6B), demonstrating that the two classes of IL-12
antagonists can synergistically inhibit both IL-12-induced
proliferation and IFN- production. Control mouse and rat Abs showed
no significant effect on IL-12-induced IFN- production
(16).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mAb 20C2 has been shown to be largely specific for the human IL-12 p75 heterodimer (25, 32), but has also been reported to react weakly with the IL-12 p40 subunit (25). We have found that the 20C2 mAb can interact weakly with the human p40 homodimer, but did not observe binding to purified human p40 monomer. Moreover, the p35 subunit also appears to contribute to the epitope recognized by 20C2 (M. Gately and P. Ling, unpublished observations). Previous studies have shown that 20C2 inhibits IL-12-induced proliferation of PHA-activated human lymphoblasts without inhibiting low affinity binding of 125I-huIL-12 to huIL-12Rß1-transfected COS-7 cells (21).
In addition to 20C2, we used mAb 2B10 to examine IL-12/IL-12R interactions. We have previously reported that 2B10 recognizes huIL-12Rß1, and although by itself it is not inhibitory, the combination of anti-huIL-12Rß1 mAbs 2-4E6 and 2B10 inhibits IL-12-induced proliferation of PHA-activated lymphoblasts (16). Extension of previous studies of mouse p40 homodimer, which demonstrated potent inhibition of the biologic activities of mouse IL-12-induced, but not huIL-12-induced, proliferation of PHA-activated human lymphoblasts (20), was also conducted.
Using transfected COS-7 cells expressing either huIL-12Rß1 or huIL-12Rß2, two classes of IL-12 inhibitors were identified based on their ability to interfere with the binding of 125I-huIL-12 to these receptor subunits. mAb 2B10 and mo(p40)2 blocked binding to huIL-12Rß1, but not to huIL-12Rß2. This extends the previous observation that mo(p40)2 interacts primarily with the IL-12Rß1 subunit of either the human (20) or mouse (33) IL-12R by demonstrating that mo(p40)2 does not interact strongly with the huIL-12Rß2 subunit. Whereas both mo(p40)2 and 2B10 interfere with the interactions between huIL-12 and huIL-12Rß1, mo(p40)2 appears to be a more effective inhibitor, since 2B10 can only inhibit about 80% of the low affinity binding of 125I-huIL-12 to huIL-12Rß1-transfected COS-7 cells, whereas mo(p40)2 completely blocks low affinity binding to huIL-12Rß1.
In contrast to mo(p40)2 and 2B10, anti-huIL-12 heterodimer-specific mAb 20C2 recognizes an epitope on huIL-12 that interacts with huIL-12Rß2 but not huIL-12Rß1, as demonstrated by the selective inhibition of 125I-huIL-12 binding to huIL-12Rß2-transfected COS-7 cells. At lower huIL-12 concentrations (<2 nM), 20C2 completely blocks this low affinity binding.
Studies using huIL-12Rß1/ß2-cotransfected COS-7 cells support the
hypothesis that direct interaction of IL-12 with both receptor subunits
is required for high affinity IL-12 binding. Inhibition of
125I-huIL-12 interaction with either the ß1 or the ß2
subunit by mo(p40)2 or 20C2, respectively, eliminated the
observed high affinity 125I-huIL-12 binding. Low affinity
binding, however, presumably representing interaction of
125I-huIL-12 with the uninhibited receptor subunit,
remained. Simultaneous treatment with both types of inhibitors, e.g.,
20C2 and mo(p40)2, completely inhibited
125I-huIL-12 binding (data not shown). In agreement with
the lower efficacy of 2B10 to inhibit the huIL-12/huIL-12Rß1
interactions discussed above, treatment with 2B10 was unable to
interfere with high affinity binding to the cotransfected cells,
although low affinity binding was decreased by 2B10 treatment. The
lower efficacy of 2B10 to block huIL-12/huIL-12Rß1 interactions can
also explain why a combination of anti-huIL-12Rß1 mAb 2B10 and
2-4E6 is required to inhibit huIL-12 bioactivity (16). Consistent with
the important roles of IL-12 binding to both the ß1 and ß2 receptor
subunits, the two classes of IL-12 inhibitors can act synergistically
to inhibit IL-12 bioactivity. Combinations of the huIL-12Rß1
inhibitor mo(p40)2 or 2B10 and the huIL-12Rß2 inhibitor
20C2 synergistically inhibit IL-12-stimulated PHA-activated human
lymphoblast proliferation and IL-12-induced IFN- production from
human PBMC.
It is interesting to note that whereas mo(p40)2 can inhibit high affinity binding of 125I-huIL-12 to huIL-12Rß1/ß2-cotransfected COS-7 cells, mo(p40)2 is not a potent inhibitor of high affinity binding of 125I-huIL-12 to PHA-activated human lymphoblasts or of PHA-activated human lymphoblast proliferation (20). In addition, the high affinity binding of 125I-huIL-12 to cotransfected COS-7 cells has a Kd of about 50 pM (18), whereas PHA-activated human lymphoblasts and the human Kit225/K6 cell line exhibit high affinity 125I-huIL-12 binding with a Kd of about 5 to 20 pM (15) (R. Chizzonite, unpublished observations). This suggests that cotransfected COS-7 cells do not exactly mimic the native high affinity IL-12R found on activated T or NK cells. In agreement with the idea that differences exist between cells expressing the native IL-12R and cells expressing cotransfected ß1 and ß2 receptor subunits, cotransfected Ba/F3 cells expressing huIL-12Rß1 and huIL-12Rß2 are IL-12 responsive (17, 18), and mo(p40)2 is a potent inhibitor of huIL-12-induced proliferation in these cotransfected Ba/F3 cells (D. H. Presky and M. K. Gately, unpublished observations). It is still unknown whether these differences are due to an additional, as yet unidentified, component of the high affinity IL-12R complex on PHA-activated lymphoblasts or to differences in protein processing in the various cell types.
In conclusion, we have identified two classes of IL-12 antagonists that
differentially inhibit the interaction of IL-12 with the IL-12Rß1 and
IL-12Rß2 subunits of the high affinity IL-12R complex. These two
classes of IL-12 inhibitors can function to synergistically block
IL-12-stimulated proliferation and IFN- production. Overall, the
results suggest that binding of IL-12 to the high affinity IL-12R
complex involves multiple interaction sites, including direct
interactions with both the ß1 and ß2 receptor subunits. IL-12
appears to interact with huIL-12Rß1 primarily via domains on the
IL-12 p40 subunit and with huIL-12Rß2 via a heterodimer-specific
region of IL-12 to which the IL-12 p40 and p35 subunits may both
contribute. We are currently exploring whether simultaneous inhibition
of the multiple sites of interaction between IL-12 and its receptor
complex can be exploited to design potent IL-12 antagonists.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: huIL-12, human interleukin-12; mo(p40)2, mouse interleukin-12 p40 subunit homodimer.
3 R. Chizzonite, T. Truitt, P. Nunes, B. Desai, A. Chua, A. Stern, M. Gately, and U. Gubler. Low and high affinity receptors for IL-12 on human T cells: Characterization by IL-12 and anti-receptor antibody binding. Submitted for publication.
Received for publication February 13, 1997. Accepted for publication November 1, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by natural killer cell stimulatory factor: characterization of the responder cells and synergy with other inducers. J. Exp. Med. 173:869.
s, ßs, and kinases of cytokine receptor complexes. Cell 74:587.[Medline]
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