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The Journal of Immunology, 2000, 164: 2861-2865.
Copyright © 2000 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Ectopic Expression of the IL-12 Receptor-ß2 in Developing and Committed Th2 Cells Does Not Affect the Production of IL-4 or Induce the Production of IFN-{gamma}1

Victoria L. Heath2,*, Louise Showe{dagger}, Chad Crain*, Franck J. Barrat*, Giorgio Trinchieri{ddagger} and Anne O’Garra*

* Department of Immunobiology, DNAX Research Institute, Palo Alto, CA 94304; {dagger} Wistar Institute, Philadelphia, PA 19104; and {ddagger} Schering-Plough, Dardilly, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The IL-12 receptor-ß2 (IL-12Rß2) chain is expressed on Th1 cells and lost upon differentiation to the Th2 phenotype. This has been suggested as the basis for commitment of Th1 cells, because early differentiated Th2 cells do not reverse their phenotype and do not produce IFN-{gamma} on restimulation in the presence of IL-12. In this study, we ectopically expressed the IL-12 receptor-ß2 (IL-12Rß2) bicistronically with enhanced green fluorescent protein by retroviral infection in developing and committed Th2 cells. Restimulation of Th2 cells expressing this ectopic IL-12Rß2 in the presence of IL-12 led to levels of IL-4 production similar to those in control Th2 cells. The expression of IL-12Rß2 in Th2 cells did not lead to significant levels of IFN-{gamma} production, although IL-12-mediated STAT signaling and proliferation were restored. Thus, although the IL-12Rß2 and IL-12-dependent STAT4 activation are required for Th1 responses, activation of this pathway is not sufficient to restore a Th1 phenotype in developing or committed Th2 cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Upon activation CD4+ T cells produce IL-2 and can then differentiate into discrete subsets of T cells, named Th1 and Th2 cells, producing distinct patterns of cytokines such as IFN-{gamma} and TNF-ß or IL-4, IL-5, and IL-13, respectively (1, 2, 3, 4, 5). Th1 and Th2 cells play an important role in the eradication of different pathogens but have also been implicated in various immune pathologies (2, 4, 6). IL-12 is a dominant factor for induction of Th1 development (7, 8, 9, 10). On binding to its functional high affinity receptor (11), consisting of the IL-12 receptor-ß1 (IL-12Rß1)3 and IL-12 receptor-ß2 (IL-12Rß2), IL-12 activates STAT1, STAT3, and STAT4 in Th1 cells, leading to the production of IFN-{gamma} (12, 13, 14, 15, 16, 17). The requirement for IL-12 signaling for Th1 responses in vivo has been confirmed in mice with targeted deletion of the IL-12 p40 (18), IL-12Rß1 (19), and STAT4 (20, 21) genes. IL-12-induced STAT3 and STAT4 activation was found not to occur in Th2 cells (13, 14, 22); subsequently, they were shown to lack expression of the IL-12Rß2 chain (14, 16). It was thus proposed that extinction of the IL-12 signaling during early Th2 development provides a mechanism that allows stable phenotype commitment (14, 16).

To address the role of the IL-12Rß2 in Th phenotype commitment, we ectopically expressed the IL-12Rß2 in developing and committed Th2 cells restoring IL-12-mediated STAT activation. IL-12Rß2-expressing Th2 cells cultured even in the presence of IL-12 produced levels of IL-4 comparable to those of the control Th2 cells and did not produce significant levels of IFN-{gamma}. This suggests that the absence of IL-12-dependent STAT4 signaling is not required for Th2 commitment and does not explain the inability of Th2 cells to make IFN-{gamma}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Mice and T cell clones

Naive CD4+ T cells from DO11.10 {alpha}ß-TCR-transgenic mice (23) were stimulated weekly with OVA323–339 and APC under Th1 or Th2 conditions as previously described (24). Clones used were the HDK1, a keyhole limpet hemocyanin-specific Th1 clone (25), and D10.G4.1, a conalbumin-specific Th2 clone (26).

Cytokines, Abs and Ag

Recombinant cytokines were used as follows: mouse IL-4 and mouse IL-2 (DNAX, Palo Alto, CA), mouse IL-12 (PharMingen, San Diego, CA), and mouse IL-18 (PeproTech, Rocky Hill, NJ). Monoclonal anti-cytokine mAbs and mAbs used for T cell preparation were as described (24). Finally, antisera used for immunoassays were anti-phosphotyrosine (clone 4G10) and anti-STAT4 (Upstate Biotechnology, Lake Placid, NY).

Cell culture and retroviral transduction

The protocol for retroviral infection has been described in detail (24). Retrovirally infected T cells were sorted on the basis of enhanced green fluorescent protein (EGFP) (24) and analyzed directly or polarized for a further week. T cells (5 x 104 in 200 µl) were then stimulated with or without cross-linked anti-CD3 (10 µg/ml) and soluble anti-CD28 (1 µg/ml) in medium alone, IL-12 (5 ng/ml), IL-18 (10 ng/ml), or a combination of both. [3H]Thymidine (1 µCi/well) was added for the last 4 h of a 24-h incubation for measurement of DNA synthesis. Supernatants were harvested at 48 h and tested for IL-4 and IFN-{gamma} by immunoassay (27).

Cloning and plasmid construction

The IL-12Rß2 (11) was cloned from a mouse spleen cDNA library and inserted into the pMX-IRES-EGFP vector, which has been previously described (24), using EcoRI and NotI restriction sites.

Biochemical analyses

T cells (5 x 106–107) were rested in RPMI containing 2% serum for 5 h, cells were then activated for 15 min with IL-12 at 50 ng/ml, and cytoplasmic and nuclear extracts were prepared as described (28). Immunoprecipitation of STAT4 from cytoplasmic extracts and immunoblots were performed as described (29). EMSAs were performed as described using a 32P-labeled m67 SIE probe (30).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Ectopic expression of IL-12Rß2 in developing and committed Th2 cells does not result in IFN-{gamma} production or down-regulation of IL-4

To determine its effects on developing Th2 cells, we introduced the mouse IL-12Rß2 gene using retroviral infection as previously described (24). The retroviral IL-12Rß2 was bicistronically linked to EGFP (RV-IL-12Rß2-EGFP), allowing for the detection and sorting of IL-12Rß2-expressing cells (Fig. 1GoA). RV-IL-12Rß2-EGFP, or an RV-EGFP control encoding EGFP alone, were transduced into {alpha}ß-TCR-transgenic CD4+ DO.11.10 T cells on days 1 and 2 after antigenic stimulation and cultured under Th2 conditions (Fig. 1GoB). Approximately 30–50% of cells were positive for EGFP expression after 7 days (Fig. 1GoC). EGFP-positive cells were purified by flow cytometry to >98% and restimulated directly for analysis of cytokine production in supernatants, or cultured for another week under Th2 conditions (Fig. 1GoB). Control Th1 cells (developed by stimulation as above, but under Th1 conditions) restimulated with anti-CD3 plus anti-CD28 alone produced high levels of IFN-{gamma}, and these levels were enhanced as expected by addition of IL-12, IL-18, or both IL-12 and IL-18 (Fig. 2GoA) (30). In contrast, Th2 cells ectopically expressing IL-12Rß2 produced little to no detectable IFN-{gamma} on restimulation with anti-CD3 and anti-CD28 (Fig. 2GoA) or with APC plus OVA323–339 (data not shown). On addition of IL-12, minute quantities of IFN-{gamma} were produced (<0.5 ng/ml) which were not seen in the Th2 or EGFP Th2 controls; however, this amount was insignificant when compared with the level of IFN-{gamma} production by Th1 cells. Addition of IL-18, either alone or in combination with IL-12, upon restimulation with anti-CD3 and anti-CD28, did not augment IFN-{gamma} production in IL-12Rß2-EGFP Th2 cells, suggesting either that the IL-18R is not expressed, or that the cells are refractory to IL-18 signaling (30). Essentially identical results were obtained when RV-IL-12Rß2-EGFP-infected Th2 cells were examined 2 weeks after polarization (data not shown), or with an RV-IL-12Rß2-EGFP-infected Th2 clone, D10, which produced little to no IFN-{gamma} on restimulation in the presence of IL-12 or IL-12 and IL-18 (Fig. 2GoB). Furthermore, the ectopic expression of the IL-12Rß2 in developing or committed Th2 cells had little to no effect on the levels of IL-4 produced on restimulation as compared with the Th2 controls. An enhanced level of IL-4 was occasionally observed, in the presence of IL-12 in both developing and committed Th2 cells infected with the RV-IL-12Rß2-EGFP, above that of the RV-EGFP-infected control Th2 cells. This may be attributable to an increased proliferation of IL-12Rß2-EGFP-Th2 cells in response to IL-12 (Fig. 3GoC). This demonstrated that ectopic expression of the IL-12Rß2 chain of the IL-12 receptor was not sufficient to maintain or restore the ability of Th2 cells to produce IFN-{gamma}, even when restimulated in the presence of IL-12.



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  		FIGURE 1. Experimental design. A, Map of the retroviral vector pMXI-EGFP and pMXI IL-12Rß2-EGFP; B, experimental outline showing the timing of retroviral infection and analysis of 1- and 2-wk polarized IL-12Rß2-expressing Th2 cells or Th2 clones; C, flow cytometry plots to show EGFP positivity of unsorted and sorted RV-EGFP-Th2- and RV-IL-12Rß2-EGFP-Th2-infected cells. LTR, long terminal repeat; IRES, internal ribosomal entry site.

 


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  		FIGURE 2. Developing and committed IL-12Rß2-expressing Th2 cells are not induced to produce IFN-{gamma} on restimulation with IL-12, and levels of IL-4 production are unchanged. A, Naive CD4+ DO11.10 were stimulated with splenic APC, OVA323–339 peptide under Th2 conditions and retrovirally infected with RV-EGFP or RV-IL-12Rß2 EGFP on days 1 and 2. At day 7, EGFP cells were sorted and restimulated with anti-CD3 and anti-CD28 with no cytokine addition, IL-12, IL-18, or IL-12 and IL-18 as described in Materials and Methods. Supernatants were harvested at 48 h, and levels of IL-4 and IFN-{gamma} were determined by ELISA. B, D10 cells were transduced with either RV-EGFP or RV-IL-12Rß2, on days 1 and 2 after restimulation. EGFP+ cells were sorted and used in a bioassay as described in A. The HDK Th1 cell clone was included for a comparison.

 


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  		FIGURE 3. Expression of IL-12Rß2 reconstitutes IL-12 signaling in Th2 cells. A, 1-wk polarized Th1 and Th2 cells and retrovirally infected IL-12Rß2-EGFP-Th2 or EGFP-Th2 cells were sorted (purity, >98% EGFP) and used immediately for a signaling experiment. Of these cells, 107 were rested for 5 h in medium containing low serum and then divided into two; these were either left untreated or activated with IL-12 for 15 min. Nuclear extracts were prepared, protein content was measured, and 0.5 µg was used in an EMSA assay with a 32P m67 SIE probe. B, Parental D10 cells, IL-12Rß2-EGFP-D10, EGFP-D10 cells, and HDK1 cells were rested for 5 h in medium containing low serum and then activated with IL-12. STAT4 was immunoprecipitated (ip STAT4) from cytoplasmic extracts and immunoblotted an anti-phosphotyrosine (APT)-specific mAb; blots were stripped and reprobed with anti-STAT4 to show protein loading. C IL-12Rß2-EGFP-D10 or EGFP-D10 cells (104/200 µl) were incubated with medium, IL-12, IL-18, or IL-12 and IL-18 for 24 h. [3H]Thymidine (1 µCi/well) was added for the last 4 h before harvesting and measuring DNA synthesis.

 
Previous studies have indicated that IL-4 down-regulates, whereas IL-12, IFN-{gamma} (31), and/or IFN-{alpha} selectively up-regulate the expression of the IL-12Rß2 (16, 32). Furthermore, Szabo et al. (14) showed that culture of Th2 cells with IFN-{gamma} could maintain their expression of the IL-12Rß2 and their functional responsiveness to IL-12. These Th2 cells (cultured in IFN-{gamma}) could activate STAT4 in response to IL-12 and produce IFN-{gamma} on restimulation (14). Taken together, these studies and our present findings suggest that IFN-{gamma} may be achieving more than just the restoration of IL-12-induced signaling. Indeed, IFN-{gamma} does activate other pathways which are required for Th1 development, such as IRF-1 (33, 34) and possibly other Th1 commitment factors. Th cells derived in IL-4 plus IL-12 also maintained IL-12Rß2 expression and produced IFN-{gamma} upon restimulation in the presence of IL-12, and this was augmented by anti-IL-4 and anti-IL-10 mAbs (14). It is not clear, however, whether IL-4 and IL-10 down-regulate APC-derived factors and/or receptors or transcription factors on Th cells required for IFN-{gamma} production.

Ectopic expression of the IL-12Rß2 restores IL-12-induced STAT signaling in Th2 cells

It was important to determine that ectopic expression of the IL-12Rß2 chain in Th2 cells led to expression of a functional high affinity IL-12 receptor that was able to activate STAT molecules. This was tested in 1-wk polarized Th1, Th2, EGFP-Th2, and IL-12Rß2-EGFP-Th2 cells by subsequent activation with IL-12 and preparing nuclear extracts that were used in an EMSA assay with an M67-SIE probe. STAT binding complexes are clearly seen only in Th1 cells and the IL-12Rß2-EGFP-Th2 cells on activation with IL-12, and not in the Th2 or the EGFP-Th2 control cells (Fig. 3GoA). The multiple bands represent STAT1, -3, and -4 complexes (Fig. 3GoA), as described (13). Furthermore, we show that STAT4 is phosphorylated only in IL-12Rß2-EGFP-D10 and Th1 cells, and not in parental D10 and EGFP-D10 controls treated with IL-12 (Fig. 3GoB). IL-12 also induced proliferation in the IL-12Rß2-EGFP-D10 cells (Fig. 3GoC), providing further evidence that the expression of the IL-12Rß2-EGFP alone restores IL-12-induced signaling (11). Thus, we show that restoration of IL-12 signaling is sufficient for the induction of IL-12-induced proliferation in Th2 clones but not to reconstitute significant levels of IFN-{gamma} production in Th2 cells. Another example of the uncoupling of STAT4 signaling and IFN-{gamma} production is the ability of IFN-{alpha} to activate STAT4 but not IFN-{gamma} production by human T cells (16, 17).

Th2 cells expressing the IL-12Rß2 ectopically cannot be induced to switch to a Th1 phenotype

Although the ectopic expression of the IL-12Rß2 in Th2 cells did not result in significant IL-12-induced IFN-{gamma} production on restimulation, it seemed possible that the expression of this receptor led to a more plastic Th population, which could be induced to produce IFN-{gamma} on stimulation for an extended period in the presence of IL-12. Polarized IL-12Rß2-EGFP-Th2 cells were restimulated in the presence of IL-12 and anti-IL-4 mAb for a further 7 days; however, they still did not produce significant levels of IFN-{gamma} on restimulation, similar to the EGFP controls (Fig. 4Go). These cells showed little to no change in the levels of IL-4 production (data not shown). The inability of the IL-12-induced STAT signaling pathway to restore the production of the Th1-specific cytokine IFN-{gamma} in developing Th2 cells appears to be in contrast to the action of the reciprocal Th2-specific STAT6 signaling pathway. Ectopic expression and activation of STAT6 signaling in developing and 1-wk polarized (although not committed) Th1 cells resulted in the induction of the Th2-specific cytokines, IL-4, IL-5, and IL-13, and down-regulation of IFN-{gamma} (35). This asymmetry between Th1 vs Th2 regulation may result from a greater need to regulate the Th1 pathway, which can lead to significant immunopathology. Our findings that reconstitution of IL-12 signaling alone does not restore the induction of IFN-{gamma} production by Th2 cells may explain data from Ouyang et al. (36) that a STAT4-inducible, Th1-specific transcription factor, ERM, was unable to induce the production of IFN-{gamma} in Th cells developed under Th2 or even neutral conditions. Thus, although IL-12-induced STAT4 activation is clearly required for a robust Th1 response (18, 19, 20, 21), it appears not to be sufficient to induce IFN-{gamma} in developing Th2 cells.



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  		FIGURE 4. IL-12Rß2-expressing Th2 cells cannot be switched to a Th1 phenotype. A, Schematic of the experimental design. B, Developing Th2 cells were retrovirally infected on days 1 and 2, and EGFP+ cells were sorted on day 7. These cells were then driven for an additional week in either Th1 or Th2 conditions and restimulated with anti-CD3 and anti-CD28 to reveal their phenotype. Supernatants were harvested at 48 h, and levels of IFN-{gamma} were determined by ELISA.

 
It has been proposed that the extinction of IL-12 signaling via down-regulation of the IL-12Rß2 is key in preventing IL-12-induced IFN-{gamma} production by Th2 cells (13, 14, 16). Our data suggest that this is not the only checkpoint in the regulation of commitment to a Th2 phenotype and that restoration of the IL-12-dependent STAT4 signaling pathway neither diminishes the production of IL-4 nor results in significant IFN-{gamma} production by developing or committed Th2 cells.


   Acknowledgments
 
We thank Dr. Nicholas Cacalano for technical advice and Drs. Naoko Arai, Hirokazu Kurata, Margaret Steel, Robert Coffman, James Johnston, and Michael Tomlinson for advice and critical review of the manuscript.


   Footnotes
 
1 DNAX Research Institute is supported by the Schering-Plough Research Institute. Back

2 Address correspondence and reprint requests to Dr. Victoria Heath, DNAX Research Institute, 901 California Avenue, Palo Alto, CA 94304-1104. E-mail address: Back

3 Abbreviations used in this paper: IL-12Rß1, IL-12 receptor-ß1; IL-12Rß2, IL-12 receptor-ß2; EGFP, enhanced green fluorescent protein. Back

Received for publication November 30, 1999. Accepted for publication January 21, 2000.


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 Introduction
 Materials and Methods
 Results and Discussion
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J. D. Ahlers, I. M. Belyakov, S. Matsui, and J. A. Berzofsky
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R. Nishikomori, S. Gurunathan, K. Nishikomori, and W. Strober
BALB/c Mice Bearing a Transgenic IL-12 Receptor {{beta}}2 Gene Exhibit a Nonhealing Phenotype to Leishmania major Infection Despite Intact IL-12 Signaling
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M.-N. Avice, M. Rubio, M. Sergerie, G. Delespesse, and M. Sarfati
CD47 Ligation Selectively Inhibits the Development of Human Naive T Cells into Th1 Effectors
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T Cells from Human Allergen-Induced Late Asthmatic Responses Express IL-12 Receptor {beta}2 Subunit mRNA and Respond to IL-12 In Vitro
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D. E. Jones, L. U. Buxbaum, and P. Scott
IL-4-Independent Inhibition of IL-12 Responsiveness During Leishmania amazonensis Infection
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