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ARTICLE |
CORRESPONDENCE William E. Paul: wpaul{at}niaid.nih.gov
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Naive CD4+ T cells can differentiate into at least two major distinct phenotypes, Th1 and Th2 cells, which are characterized by polarized patterns of cytokine production. Many factors have been reported to play a role in differentiation to the Th1 and Th2 phenotypes. Among these, the set of cytokines present during the priming period is particularly important. The presence of IL-4 is critical for in vitro Th2 differentiation (1, 2), whereas in vitro Th1 differentiation depends heavily on IFN-
Naive CD4+ T cells are capable of producing IL-4 when they are stimulated with peptide/MHC class II complex on APCs; this endogenously produced IL-4 was shown to be sufficient for Th2 differentiation. When no polarizing cytokines are added to a culture, strength of signal through TCR may control Th cell differentiation (13). Thus, priming for IL-4producing cells often is observed when a low concentration of antigen-derived peptide is used, whereas high concentrations of peptide were reported to induce IFN-producing cells (14). Similarly, priming with ligands that interact with TCR less energetically than a WT peptide, so-called "altered peptide ligands," often favors Th2 differentiation (15).
The basis of the "signal strength" effect on biasing differentiation to Th1 or Th2 subset has not been clarified, although evidence for the involvement of NFAT (16), extracellular signal-regulated kinase (ERK), and activated protein-1 (AP-1) (17) has been presented. We have reexamined the "dose effect" on priming for Th cell differentiation using rigorously purified naive CD4+ T cells from TCR transgenic mice. We show that low concentrations of peptide induce IL-4independent induction of IL-4 mRNA beginning at 1416 h after stimulation. This early IL-4 mRNA expression is associated with IL-4independent early GATA-3 expression and is dependent on IL-2. However, IL-2 is not essential for early GATA-3 expression. Moreover, naive CD4+ T cells from GATA-3 conditional KO mice fail to express IL-4 mRNA in response to TCR stimulation indicating that early IL-4 transcription is GATA-3 dependent. High concentrations of peptide inhibit early IL-4 mRNA expression by abrogating early GATA-3 induction and by blocking IL-2R-mediated signaling, as shown by the failure of STAT5 phosphorylation, although IL-2 is produced abundantly. This high concentrationmediated inhibition is reversed by the blockade of ERK pathway by an inhibitor of mitogen-activated protein kinase kinase (MEK) allowing the cells to express GATA-3 and to respond to IL-2. This results in the restoration of early IL-4 mRNA expression and subsequent development into high-rate IL-4producing cells. We conclude that TCR-mediated control of Th2 differentiation is dependent upon early IL-4 transcription determined by the independent actions of GATA-3 and IL-2, and that the dose effect reflects dose-dependent control of early transcription of GATA-3 and high dose desensitization of the IL-2R mediated by TCR-induced ERK activation.
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RESULTS |
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IFN- was produced by T cells cultured with splenic DCs at low and high concentrations of peptide; antiIL-12 did not block the development of IFN-
producing cells, but antiIFN-
did (Fig. 1 B). When P13.9 cells were used, few, if any, IFN-
producing cells appeared, even without neutralization of IFN-
or IL-12 (Fig. 1 C). Thus, splenic DCs and P13.9 cells differ in their capacity to induce naive CD4+ T cells to develop into IFN-
producers, even in the absence of IL-12. Because splenic DCs were shown recently not to produce IFN-
(18), this suggests that the APCs differ in their intrinsic capacity to stimulate naive CD4+ T cells to produce "early" IFN-
. P13.9 cells did stimulate priming for IFN-
production if small numbers of CD44high (memory) line 94 CD4+ T cells were present (Fig. 1 C). The latter cells presumably served as a source of "early" IFN-
upon stimulation (unpublished data).
High peptide concentration-induced activation is inhibitory for TCR-induced early IL-4 mRNA expression
In subsequent experiments, P13.9 cells were used as APCs, because the antigen concentration effects held when splenic DCs or P13.9 cells were used to present pPCC. We examined the early expression of IL-4 mRNA in naive line 94 CD4+ T cells primed with various concentrations of pPCC. IL-4 mRNA was first detectable at 14 h in cells that were stimulated with 0.01 µM pPCC, and increased in level of expression by 2024 h (Fig. 2 A). Increasing the concentration of pPCC to 0.1 µM diminished the amount of IL-4 mRNA by >10-fold, and IL-4 mRNA was barely detectable when 10 µM pPCC was used for priming. This early IL-4 mRNA expression was independent of IL-4 as shown by the failure of antiIL-4 to diminish the level of IL-4 mRNA in cells that were stimulated for 24 h with 0.01 µM pPCC (Fig. 2 C).
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IFN- mRNA levels were extremely low throughout the first 24 h of culture; there was no significant difference in IFN-
mRNA expression among cell populations that were stimulated with different concentrations of pPCC (Fig. 2 A). IL-2 mRNA was induced by 6 h of culture and showed an entirely different concentration dependence from that of IL-4 mRNA (Fig. 2 A). IL-2 mRNA was maximal at 1 and 10 µM and >10-fold lower at 0.01 µM pPCC.
Expression of IL-4 and GATA-3 after 24 h is IL-4 dependent
Culturing naive line 94 CD4+ T cells with 0.01 µM peptide in the presence of antiIL-4 did not affect the amount of IL-4 or GATA-3 mRNA at 24 h. However, the presence of antiIL-4 prevented the increase in IL-4 and GATA-3 mRNA levels that were observed at 48 and 72 h when "early" IL-4 was not neutralized (Fig. 2 C). Furthermore, if IL-4 was added from the outset of the culture, the amount of IL-4 and GATA-3 mRNA that was expressed by the cells at all time points throughout 72 h was increased further (unpublished data). These results indicate that, in cells cultured under neutral conditions, the early "IL-4independent" IL-4 that is produced is essential for the continued production of IL-4 and the continued expression of GATA-3 by these cells. AntiIL-4 reduced the frequency of cells producing IL-4 in response to challenge with pPCC (0.01 µM) at 72 h from 35.6 to 4.8% (Fig. 2 D). This implied that the further induction of IL-4 by endogenous "early" IL-4 is essential for the differentiation of naive CD4+ T cells into cells that are capable of secreting large amounts of IL-4.
IL-2 is essential for early IL-4, but not GATA-3, induction
Adding antiIL-2 and anti-CD25 to cells that were cultured under neutral conditions completely inhibited the early (24 h) appearance of IL-4 mRNA but had no inhibitory effect on early GATA-3 mRNA (Fig. 2 C) or protein expression (not depicted). At 48 h, IL-4 mRNA still was not observed and GATA-3 mRNA levels failed to increase (Fig. 2 C), presumably because of the lack of IL-4, which is essential for the further increase of GATA-3 mRNA (19). The effect of neutralizing IL-2 on early IL-4 mRNA expression could not be accounted for by its effects on cell growth, because there was no cell division during the first 24 h of cultureas measured by CFSE dilutionand cell yield at 24 h was not affected by the addition of antiIL-2 and anti-CD25 (unpublished data). Together, these results imply that early IL-4 production depends on the induction of GATA-3 and on an IL-2mediated event.
GATA-3deficient naive CD4+ T cells fail to produce IL-4 in response to in vitro challenge
Although GATA-3 was demonstrated to play a critical role in Th2 differentiation (1922), it has remained unclear whether GATA-3 is involved in IL-4independent early IL-4 production. To address this question, we crossed mice that were homozygous for a "floxed" Gata3 gene (22) onto CD4-Cre transgenic mice (Gata3f/fCD4-Cre mice) (23), so that the Gata3 gene could be deleted in peripheral T cells. These mice possess few CD4+ T cells (not depicted), and most of those that are present exhibit a memory or effector/memory phenotype (Fig. 3 A). We purified CD44lowCD62LhighCD4+ T cells from these mice and from mice that were homozygous for the floxed gene without CD4-Cre (Gata3f/f) (Fig. 3 A). Such "naive" cells derived from Gata3f/fCD4-Cre mice failed to express IL-4 mRNA 24 h after stimulation, whereas those derived from Gata3f/f mice expressed considerable amounts of IL-4 mRNA in response to immobilized anti-CD3 and anti-CD28; this IL-4 mRNA expression was not inhibited by antiIL-4 (Fig. 3 B). Thus, deletion of GATA-3 prevented early expression of IL-4 mRNA in response to TCR-mediated stimulation. Cells from Gata3f/fCD4-Cre mice were equivalent to those from Gata3f/f mice in their capacity to induce IL-2 mRNA in response to anti-CD3 and anti-CD28 (Fig. 3 B), which indicated that loss of GATA-3 does not influence the degree of T cell activation.
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Moreover, blockade of the ERK pathway allowed cells that were stimulated with high concentrations of peptide to respond to IL-2. Thus, naive line 94 CD4+ T cells without U 0126 pretreatment failed to show IL-2driven STAT5 phosphorylation at 24 h, although they did express phospho-STAT5 at 48 h (Fig. 5 D). By contrast, in cells that were pretreated with U 0126, a small, but significant, degree of IL-2driven STAT5 phosphorylation was observed at 24 h; phosphorylation was enhanced further at 48 h (Fig. 5 D).
These results indicate that ERK activation, as a result of stimulation with high concentrations of peptide, suppresses early GATA-3 mRNA induction and IL-2Rmediated signaling, which jointly result in the failure of "high-dose" cells to produce early IL-4. Stimulation of line 94 CD4+ T cells with 10 µM pPCC induced striking phosphorylation of ERK, as shown by flow cytometric analysis with an antiphospho-ERK antibody (Fig. 6; open line graphs). By contrast, little or no ERK phosphorylation was detected in cells that were stimulated with 0.01 µM pPCC at any time from 2 min to 2 h after stimulation (Fig. 6; open line graphs). Pretreatment of line 94 CD4+ T cells with U 0126 blocked TCR-induced ERK phosphorylation in response to low and high concentrations of pPCC (Fig. 6; shaded line graphs).
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c-Maf was shown to play an important role in IL-4 production; its expression is induced by TCR stimulation (24, 25). Therefore, it was possible that blocking the ERK pathway might down-regulate TCR-induced c-Maf expression at low peptide concentration, causing the failure to sustain IL-4 mRNA expression in the late phase of activation. However, we did not observe any decrease in c-Maf mRNA expression in cells that were pretreated with U 0126 and stimulated with 0.01 µM pPCC (Fig. 7 B). Moreover, the level of c-Maf mRNA expression in cells that were stimulated with 10 µM pPCC was higher than that in cells with 0.01 µM pPCC (Fig. 7 B). Thus, it is unlikely that c-Maf is involved in TCR-induced early IL-4 production or that inhibition of c-Maf accounts for U 0126-mediated diminution in late IL-4 production at low peptide concentration.
Exogenous IL-2 rescues U 0126-mediated suppression of Th2 priming at low peptide concentration
The diminished priming for high-rate IL-4producing cells at low peptide concentration seemed to be accounted for by the diminution in IL-2 production by cells that were treated with the MEK inhibitor. This occurred at high and low peptide concentrations; however, the "inhibited" amounts of IL-2 mRNA at high peptide concentration still exceeded the "uninhibited" amounts of IL-2 mRNA in cells that were stimulated with 0.01 µM pPCC (Fig. 7 A). To determine whether U 0126-mediated suppression of IL-4 production in "low-dose" stimulated cells was due to diminution in IL-2 production, we supplemented these cultures with different concentrations of IL-2 at 24 h of stimulation. Addition of 100 U/ml of IL-2 at 24 h restored IL-4 and GATA mRNAs at 48 and 72 h. This restoration was IL-4 dependent, which implied that IL-2 addition allowed endogenous IL-4 production, which in turn, was essential to up-regulate IL-4 and GATA-3 mRNA (Fig. 8 A). Addition of IL-2 at 24 h also restored priming for high-rate IL-4producing cells that were pretreated with U 0126 and stimulated with 0.01 µM peptide. 30 units of IL-2 caused partial restoration, whereas 100 units fully restored priming for IL-4producing capacity. IL-2mediated restoration of priming for high-rate IL-4producing cells was blocked completely by the inclusion of antiIL-4 (Fig. 8 B).
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MEK inhibition does not restore IL-4 production simply by lowering global T cell activation
Lck is activated in response to the ligation of TCR with antigen peptideMHC class II complex, and plays an important role in the signal transduction through TCR. It was shown that agonistic peptide-induced activated ERK phosphorylates Ser59 of Lck and prevents it from inactivation by the SHP-1 phosphatase; this "protected" Lck is detectable as a 59-kD protein on SDS-PAGE (26). It is possible that blockade of the ERK pathway by a MEK inhibitor simply decreases the level of T cell activation by diminishing Lck activity, and allows naive CD4+ T cells that are stimulated with high concentrations of peptide to express early IL-4 mRNA. To determine whether the MEK inhibitor exerted its effect by simply diminishing the degree of T cell activation in response to 10 µM pPCC, we compared the relative amount of p59lck in cells that were stimulated with various concentrations of pPCC with that in cells that were pretreated with U 0126 and stimulated with 10 µM pPCC (Fig. 9 A). The relative amount of p59lck increased in a peptide concentration-dependent manner, and reached a plateau at 1 µM pPCC. U 0126 diminished the amount of p59lck in cells that were stimulated with 10 µM pPCC to an amount that was comparable to that induced by 0.1 µM pPCC. However, there were substantially greater amounts of p59lck in U 0126-pretreated cells that were stimulated with 10 µM pPCC compared with cells that were stimulated with 0.01 µM pPCC in the absence of inhibitor. Because the levels of IL-4 and GATA-3 mRNA in U 0126-pretreated cells that were stimulated with 10 µM pPCC were significantly higher than those in DMSO-pretreated cells that were stimulated with 0.1 µM pPCC and were comparable to those in cells that were stimulated with 0.01 µM pPCC (Fig. 9 B), it is unlikely that the effects of the MEK inhibitor can be ascribed to a global inhibition of T cell activation.
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DISCUSSION |
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The requirement of GATA-3 for early IL-4 transcription is based on the evidence that naive (CD44lowCD62Lhigh) CD4+ T cells purified from spleen and lymph nodes of Gata3f/fCD4-Cre mice failed to induce early IL-4 mRNA when stimulated with immobilized anti-CD3 and anti-CD28, whereas such T cells from Gata3f/f mice expressed considerable amounts of early "IL-4independent" IL-4 mRNA. Moreover, there is much indirect evidence that early GATA-3 expression is required for low concentrationmediated induction of IL-4. Thus, the dose response curve of early GATA-3 and early IL-4 mRNA expression were the same, and GATA-3 mRNA appeared slightly earlier than did IL-4 mRNA (unpublished data). Although GATA-3 has been reported to play an important role in Th2 differentiation (1922), our current work is the first to demonstrate clearly that GATA-3 is responsible for TCR-induced, IL-4independent early IL-4 production by naive CD4+ T cells.
We have not clarified the mechanism by which TCR-mediated signaling induces early GATA-3 expression. Das et al. (29) reported that CD4+ T cells from NF-B p50-deficient mice failed to express GATA-3 in the nucleus 96 h after stimulation with anti-CD3 and anti-CD28, and that these cells are impaired markedly in Th2 polarization. They also showed that CD4+ T cells that were pretreated with an inhibitor of NF-
B p50 did not express GATA-3 in the nucleus at 96 h. However, it remains unclear whether early GATA-3 induction is dependent on NF-
B p50. Previous reports have demonstrated that TCR-induced NF-
B activation is down-stream of protein kinase C
activation (30), and that the degree of protein kinase C
activation is directly dependent on the strength of TCR signaling (31). However, given the fact that early GATA-3 expression was suppressed in a peptide concentrationdependent manner, the degree of NF-
B activation may not account for TCR-induced early GATA-3 expression.
We showed in the present report that blockade of the ERK pathway allowed naive CD4+ T cells that were stimulated with high concentrations of peptide to induce GATA-3 mRNA and to express its protein in the nucleus. Komine et al. (32) reported that retroviral infection with Runx1 cDNA dramatically diminished the frequency of IL-4producing cells by inhibiting GATA-3 mRNA expression under neutral and Th2-skewing conditions. This raised the possibility that the peptide concentration effect might be mediated by preferential induction of Runx1 at high peptide concentration. However, our real-time PCR analysis showed no significant difference in Runx1 mRNA levels between cells that were stimulated with low and high concentrations of peptide at 1224 h of stimulation (unpublished data). Moreover, U 0126 pretreatment did not alter the levels of Runx1 mRNA expression at high peptide concentration. Thus, it is unlikely that Runx1 is responsible for high peptide concentrationmediated suppression of early GATA-3 expression.
Jorritsma et al. (17) showed that treatment of naive AND TCR transgenic CD4+ T cells with a MEK inhibitor (PD98059) during priming with relatively high concentrations (5 µg/ml) of moth cytochrome c peptide resulted in an increased ratio of nuclear JunB homodimers to JunB/c-Fos heterodimers that was capable of binding to the IL-4 promoter upon recall challenge with moth cytochrome c peptide. Therefore, they concluded that robust ERK activation inhibits IL-4 transcription by altering the pattern of distinct AP-1 complexes. However, we observed that 10 µM pPCC induced significantly higher nuclear JunB expression than did 0.01 µM pPCC at 12 to 24 h of priming, and that pretreatment with U 0126 did not alter the pattern of JunB expression (unpublished data). Moreover, 0.01 and 10 µM pPCC induced comparable levels of nuclear c-Fos expression, and pretreatment with U 0126 did not alter the pattern of c-Fos expression at low and high pPCC concentrations (unpublished data). Therefore, although it is possible that cells that have completed their differentiation to Th1 or Th2 phenotype have different AP-1 complexes that are capable of binding to the IL-4-promoter, it is unlikely that the formation of such different complexes is responsible for the peptide concentrationdependent early IL-4 production.
LeGros et al. (1) originally demonstrated that IL-4 and IL-2 are required for priming of CD4+ T cells to develop into IL-4producing cells; however, the precise role of IL-2 in Th2 priming was not clarified. Recently, Cote-Sierra et al. (5) showed that IL-2 mediates its effect by stabilizing the accessibility of the Il4 gene after 48 h of priming. In the present report, we found that when naive line 94 CD4+ T cells were primed with 0.01 µM pPCC, induction of early IL-4 mRNA expression required IL-2, whereas early GATA-3 mRNA expression was independent of IL-2. We also observed that naive CD4+ T cells from IL-2/ 5C.C7 TCR transgenic RAG2/ mice failed to express "24-h" IL-4 mRNA in response to 0.01 µM pPCC, and that addition of exogenous IL-2 restored IL-4 mRNA expression. By contrast, IL-2/ cells expressed levels of GATA-3 mRNA that were comparable to WT cells; addition of exogenous IL-2 did not enhance its expression (Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20051304/DC1). Although the importance of STAT5, especially STAT5a, in Th2 differentiation has been reported (33, 34), it remains to be elucidated whether IL-2driven STAT5 activation is involved in early IL-4 production.
In conclusion, peptide concentrationmediated strength of TCR signaling regulates early IL-4 production by naive CD4+ T cells by controlling the levels of ERK activation which, in turn, influence early GATA-3 expression and responsiveness to IL-2. Weak and transient ERK activation that is induced by low concentrations of peptide allows naive CD4+ T cells to express early GATA-3 and to respond to endogenous IL-2, both of which are required for early IL-4 production. This endogenously produced early IL-4 is required for priming of CD4+ T cells to develop into high-rate IL-4producing (Th2) cells. By contrast, intense and sustained ERK activation that is induced by high concentrations of peptide inhibits early GATA-3 expression and transiently desensitizes the IL-2R; this results in the failure of naive CD4+ T cells to produce early IL-4 and to undergo subsequent Th2 differentiation.
Biologically, these results imply that low concentration challenge of individuals without overt activation of Toll-like receptors would lead to preferential priming of naive CD4+ T cells to develop into a Th2 phenotype, and thus, predispose to allergic inflammatory response. Bottomly and colleagues argued that low concentrations of LPS may be required to allow mice to develop an antigen-stimulated airway hypersensitivity response in vivo (35, 36). Addition of LPS (10 ng/ml) did not alter the overall antigen concentration effects described here (unpublished data). This suggests that peptide concentration/strength of TCR signaling may be a central element in determining in vivo priming for Th2 responses, and thus, bias toward allergic inflammation.
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MATERIALS AND METHODS |
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Culture media.
RPMI 1640 was purchased from Biosource and supplemented with 10% FCS, 2-ME, glutamine, penicillin, streptomycin, and sodium pyruvate (referred to as cRPMI).
Reagents.
FITC-anti-CD3, -CD8, -CD11b, -CD16/32, -CD24, -CD25, -B220, -NK1.1, -DX5, -Ly-6C/6G, -I-Ak, -I-Ab; AlexaFluor 488anti-CD4; PEanti-CD62L, IL-4, goat-antimouse Ig, -streptavidin; CyChrome-anti-CD44, -CD4; APCanti-CD4, IFN-; biotinylated anti-CD11c; purified antiphospho-STAT5 (clone 47), -ZAP70 (clone 29); neutralizing antiIL-2; blocking anti-CD25; rat IgG1; and rat IgG2a for isotype controls were purchased from BD Biosciences. AntiIL-4, antiIL-12, antiIFN-
, anti-CD3, and anti-CD28 were purified from ascites by Harlen. Antiphospho-ERK mAb (E10) was purchased from Cell Signaling Technology. Mitomycin C, SB 203580, and SP 600125 were purchased from Calbiochem. AntiGATA-3 mAb (HG3-31) was purchased from Santa Cruz Biotechnology, Inc. CFSE and AlexaFluor 647 donkey-antimouse IgG were purchased from Invitrogen. U 0126 and DAPI were purchased from Promega and Sigma-Aldrich, respectively. pPCC (residues 88-104; KAERADLIAYLKQATAK) was synthesized by American Peptide Company.
Isolation of naive CD4+ T cells and splenic DCs.
CD4+ T cells were isolated from lymph nodes of line 94 mice or line 110 mice by negative selection using FITC-conjugated mAbs to CD8, B220, I-Ak, CD16/32, NK1.1, and CD24, followed by incubating with anti-FITC Microbeads (Miltenyi Biotec) and passing through a MACS LS column (Miltenyi Biotec). Isolated cells were stained with PEanti-CD62L and CyChrome anti-CD44, and then CD44lowCD62Lhigh CD4+ T cells were sorted by FACS Vantage III SE (Becton Dickinson) and used as naive CD4+ T cells. In some experiments, naive CD4+ T cells were labeled with 1.25 µM CFSE before use.
When we used Gata3f/fCD4-Cre mice and Gata3f/f mice, CD4+ T cells were isolated from spleen and lymph nodes by passing through a Mouse T Cell Enrichment Column (R&D Systems), followed by negative selection as described above, except FITC-anti-I-Ab was used instead of FITC-anti-I-Ak, and FITCanti-DX5, -CD11b and -Ly-6C/6G were added. Isolated cells were stained with PEanti-CD62L, Cy-Chromeanti-CD44, and APCanti-CD4. CD4+CD44lowCD62Lhigh cells were sorted by FACS Vantage III SE (Becton Dickinson) and used as naive CD4+ T cells.
DCs were isolated as follows. Spleens of B10.A mice were treated with Liberase Brendzyme 2 and DNase I (Roche Applied Science), and CD11c+ cells were isolated by positive selection using anti-CD11c (N418) Microbeads (Miltenyi Biotec) and a MACS LS column. Isolated cells were stained with biotinylated anti-CD11c (HL3) followed by PE-streptavidin and FITCanti-DX5, anti-NK1.1, and anti-CD3. CD11c+, DX5, NK1.1, and CD3 cells were sorted by FACS Vantage III SE (Becton Dickinson), and used as splenic DCs.
Priming of naive CD4+ T cells.
Naive CD4+ T cells (5 x 105) from line 94 mice or line 110 mice were cultured in 1 ml of cRPMI in a 48-well plate with P13.9 fibroblast cells (1.25 x 105) stably expressing I-Ek, CD80, and CD54 (37) that had been treated with 25 µg/ml mitomycin C and loaded with 0.00110 µM pPCC, or with splenic DCs (5 x 104) in the presence of 0.00110 µM pPCC. In some experiments, naive CD4+ T cells were pretreated with U 0126 (MEK inhibitor), SB 203580 (p38 MAPK inhibitor), SP 600125 (JNK inhibitor), or DMSO (Sigma-Aldrich) at 37°C for 1 h. The final concentration of DMSO was 0.03%; this amount of DMSO did not affect T cell growth or viability (unpublished data). When indicated in figure legends, the following were added to the priming culture: neutralizing mAbs to IL-2, IL-4, IL-12, or IFN-; blocking mAb to CD25 (10 µg/ml for all mAbs); or isotype-matched control mAbs.
Naive CD4+ T cells (1 x 105) from Gata3f/fCD4-Cre mice or Gata3f/f mice were stimulated with immobilized anti-CD3 (1 µg/ml) and anti-CD28 (3 µg/ml) in the presence of recombinant human IL-2 (100 U/ml) with or without antiIL-4 (10 µg/ml).
Quantitative RT-PCR.
Stimulated cells were lysed in TRIzol (Invitrogen) and total RNA was isolated using RNeasy Midi Kit (QIAGEN) following the manufacturer's instructions. One microgram of total RNA was reverse-transcribed to cDNA using SuperScript II First-Strand Synthesis System for RT-PCR (Invitrogen). Quantitative PCR was performed on a 7900HT sequence detection system (Applied Biosystems). The primer and probe sets for detecting murine IL-2, IL-4, and IFN- (FAM-MGB probe), and TaqMan Ribosomal RNA Control Reagents for detecting the 18S ribosomal RNA (VIC-MGB probe) were purchased from Applied Biosystems. The sequences of primer and MGB probe for GATA-3 (34) and c-Maf (38) are described elsewhere. The levels of mRNA for cytokines and transcription factors were normalized to that of 18S ribosomal RNA.
Intracellular staining for IL-4/IFN-, phospho-STAT5, and phospho-ERK.
Primed T cells were harvested 72 h after stimulation, washed extensively with HBSS, and cultured overnight in 2 ml of cRPMI containing 100 U/ml rhIL-2 in a 24-well plate. Cells were harvested, washed with HBSS, and restimulated with immobilized anti-CD3 and anti-CD28 (3 µg/ml of each mAb) for 6 h. For the last 2 h, 2 µM monensin (Calbiochem) was added. Cells were harvested; fixed with 4% paraformaldehyde (PFA); permeabilized with 0.5% Triton X-100 and 0.1% BSA in PBS; and stained with PEanti-IL-4, CyChromeanti-CD4, and APCanti-IFN-.
For phospho-STAT5 staining, 1 x 106 line 94 CD4+ T cells that were pretreated with U 0126 (3 µM) or DMSO were stimulated with 2.5 x 105 P13.9 cells that were preloaded with 0.01 or 10 µM pPCC in the presence or absence of antiIL-2 plus anti-CD25. Cells were harvested 24 and 48 h after stimulation; fixed and permeabilized as described above; and then stained with antiphospho-STAT5 mAb followed by FITC-anti-CD25 (7D4), PE-antimouse Ig, and CyChromeanti-CD4. CD25 expression was detected on cell surface, not in cytoplasm, by a confocal microscope (unpublished data).
For phospho-ERK staining, 1 x 106 line 94 CD4+ T cells that were pretreated with U 0126 (3 µM) or DMSO were added to 2.5 x 105 P13.9 cells that were preloaded with 0.01 or 10 µM pPCC in a 1.5-ml microcentrifuge tube, spun at 12,000 rpm for 10 s, and incubated at 37°C. Cells were fixed with 4% PFA at various time points, permeabilized as described above, and stained with antiphospho-ERK mAb followed by PE-antimouse Ig and CyChromeanti-CD4.
Stained cells were acquired by a FACSCalibur (Becton Dickinson); the data were analyzed using a CELLQuest software (Becton Dickinson) by gating on CD4+ T cells.
Confocal microscopic analysis for GATA-3 protein expression.
U 0126 (3 µM) or DMSO-pretreated line 94 CD4+ T cells (1 x 106) were stimulated with P13.9 cells that were preloaded with 0.01 or 10 µM pPCC. Cells were harvested 20 h after stimulation, mounted on a coverslip, and fixed with 4% PFA. Cells were permeabilized as described above, and stained with antiGATA-3 mAb followed by AlexaFluor 488anti-CD4, AlexaFluor 647 donkeyantimouse IgG, and DAPI. Stained cells were visualized with a Leica TCS-NT/SP confocal microscope with a 63x oil immersion objective. 20 to 30 z sections separated by 0.2 µm were acquired. Reconstitutions of images were performed by using an Imaris software system (BitPlane) and Photoshop software (Adobe Systems).
Western blot analysis for p56/p59lck.
Line 94 CD4+ T cells were stimulated as described in "phospho-ERK staining." After 20 and 60 min of incubation, culture media was removed, and cells were treated with 40 µl of lysis buffer (1% NP-40, 10 mM Tris-HCl, 2 mM EDTA, 140 mM sodium chloride, 1 mM sodium orthovanadate) freshly supplemented with protease inhibitors (Complete Mini; Roche Applied Science) for 30 min on ice. After removal of debris by centrifugation at 12,000 rpm for 15 min, 20 µl of 3 x SDS sample buffer (Cell Signaling Technology) was added to whole cell lysates, and 20 µl of aliquots were used for electrophoresis on an 8% polyacrylamide gel (Invitrogen). Fractionated proteins were transferred onto a nitrocellulose membrane (Bio-Rad Laboratories). Lck was detected with rabbit anti-Lck polyclonal serum (BD Biosciences) followed by horseradish peroxidaselabeled goat antirabbit IgG (Bio-Rad), and visualized with SuperSignal West Dura Extended Duration Substrate (Pierce Chemical Co.). To confirm an equal loading, the probed membrane was stripped with Restore Western Blot Stripping Buffer (Pierce Chemical Co.) and reprobed with anti-ZAP70 mAb (BD Biosciences) followed by horseradish peroxidaselabeled goat antimouse IgG (Bio-Rad), and visualized with SuperSignal West Pico Extended Duration Substrate (Pierce Chemical Co.).
Online supplemental material.
Figs. S1 and S2 show the dot plot analysis for CFSE versus IL-4 and the histograms of CFSE dilution corresponding to Fig. 1, B and C, respectively. Fig. S3 shows the failure of IL-2/ naive line 94 CD4+ T cells to produce early IL-4 in response to low concentrations of pPCC. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20051304/DC1.
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Acknowledgments |
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H. Yamane was a recipient of the Post-Doctoral Fellowship from Uehara Memorial Foundation.
The authors have no conflicting financial interests.
Submitted: 28 June 2005
Accepted: 10 August 2005
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References |
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