IL-27 Suppresses CD28-Medicated IL-2 Production through Suppressor of Cytokine Signaling 3

Mice

BALB/c mice were purchased from Japan SLC. Mice transgenic (Tg) for αβ TCR recognizing OVA_323–339 (DO11.10; BALB/c background) (13) were provided by Dr. T. Yoshimoto (Hyogo College of Medicine, Hyogo, Japan). STAT1^+/− and STAT1^−/− mice (14) of a mixed background of 129/Sv and C57BL/6 were provided by Dr. R. D. Schreiber (Washington University, St. Louis, MO). STAT1-deficient mice (14) of 129/Sv background and wild-type 129/Sv mice were purchased from Taconic Farms. Mice lacking STAT3 specifically in T cells (Lck-Cre/STAT3^flox/flox) were generated by mating STAT3^flox/flox mice (15), in which the STAT3 gene is flanked by two loxP sites, and Lck-Cre Tg mice (16) (purchased from Center for Animal Resources and Development), in which the Cre recombinase transgene is regulated by T cell-specific Lck promoter. STAT3^flox/flox, Lck-Cre/STAT3^flox/+, or Lck-Cre/STAT3^+/+ mice were used as control mice. STAT4-deficient mice (17) and T-bet-deficient mice (18) of BALB/c background were purchased from The Jackson Laboratory. All animal experiments were performed in accordance with our institutional guidelines.

Cells

Naive CD4⁺ T cells and a mouse T cell hybridoma cell line 2B4, provided by Dr. T. Saito (RIKEN Research Center for Allergy and Immunology, Kanagawa, Japan), were cultured in RPMI 1640 medium supplemented with 10% FBS and 50 μM 2-ME. PLAT-E, a packaging cell line provided by Dr. T. Kitamura (University of Tokyo, Tokyo, Japan) (19), was maintained in DMEM supplemented with 10% FBS.

Reagents

Anti-CD3 (145-2C11), anti-IL-2 (S4B6), anti-IL-4 (11B11), anti-IFN-γ (XMG1.2), and anti-Thy1.2 (30-H12) were purchased from American Type Culture Collection. Anti-CD28 (37.51) and mouse rIL-2 were obtained from BD Biosciences. Anti-CD25 (PC61.5) and FITC-anti-rat IgG were obtained from eBioscience. Anti-STAT1, anti-STAT3, anti-STAT5, and T-bet were purchased from Santa Cruz Biotechnology. Anti-phosphotyrosine (anti-pY)-STAT1, anti-pY-STAT3, and anti-pY-STAT5 were obtained from Cell Signaling Technology. Anti-SOCS3 and anti-actin were purchased from Medical Biological Laboratories and Sigma-Aldrich, respectively. Mouse rIL-12 was obtained from R&D Systems. Human rIL-2 and mouse rIFN-γ were provided by Shionogi.

Preparation of purified rIL-27 protein

Recombinant IL-27 was prepared as a soluble tagged fusion protein by flexibly linking EBV-induced gene 3 to p28 as described previously (20).

Preparation of naive CD4⁺ T cells

Primary T cells were purified by passing spleen cells depleted of erythrocytes through nylon wool. The flow-through fraction was incubated with biotin-conjugated anti-CD8α, anti-B220, anti-Mac-1, anti-Ter-119, and anti-DX5, followed by incubation with anti-biotin magnetic beads (Miltenyi Biotec) and passed through a magnetic cell sorting column (Miltenyi Biotec); the negative fraction was collected (CD4⁺ T cells, >95%). These purified T cells were then incubated with anti-CD62L magnetic beads (Miltenyi Biotec), and the positive fraction was collected as purified naive CD4⁺ T cells (CD62L⁺ cells, >99%).

IL-2 and IFN-γ production assays

Naive CD4⁺ T cells (1 × 10⁵ cells/ml) from DO11.10 Tg mice were stimulated with 1 μM OVA_323–339 peptide and irradiated T/NK cell-depleted BALB/c spleen cells (1 × 10⁶ cells/ml) in the presence or the absence of IL-27 (10 ng/ml) for various times. T/NK cell-depleted spleen cells were prepared as follows. Spleen cells depleted of erythrocytes were incubated with anti-Thy1.2, followed by incubation with anti-rat IgG magnetic beads (Miltenyi Biotec) together with anti-DX5 magnetic beads (Miltenyi Biotec) and passed through a magnetic cell-sorting column. The negative fraction was used as T/NK cell-depleted spleen cells. Naive CD4⁺ T cells (5 × 10⁵ cells/ml) were stimulated with plate-coated anti-CD3 (2 μg/ml) and anti-CD28 (0.5 μg/ml) in the presence or the absence of IL-27 and/or IL-12 (10 ng/ml) for various times. 2B4 cells (2 × 10⁵ cells/ml) were stimulated with plate-coated anti-CD3 (0.03 μg/ml) and anti-CD28 (0.5 μg/ml) in the presence or the absence of IL-27 for 16 h. Culture supernatant was collected and analyzed for IL-2 and/or IFN-γ production by ELISA (21).

RT-PCR analysis

Total RNA was extracted by using a guanidine thiocyanate procedure, cDNA was prepared using oligo(dT) primer and SuperScript reverse transcriptase (Invitrogen Life Technologies), and RT-PCR was performed using Taq DNA polymerase as described previously (22). Cycle conditions were 94°C for 40 s, 60°C for 20 s, and 72°C for 40 s. Primers used for hypoxanthine phosphoribosyltransferase (HPRT) were described previously (23). The following primers were also used; SOCS3 sense primer, 5′-TTGTCGGAAGACTGTCAACG-3′; SOCS3 antisense primer, 5′-GAGAGTCCGCTTGTCAAAGG-3′; WSX-1 sense primer, 5′-ACCCAAATGAAGCCAGACAC-3′; WSX-1 antisense primer, 5′-CACACAAGGTCTTGGGTCCT-3′; gp130 sense primer, 5′-AGTCTGGGTGGAAGCAGAGA-3′; and gp130 antisense primer, 5′-CTTGGTGGTCTGGATGGTCT-3′.

Quantitative RT-PCR analysis

cDNA synthesis was performed as described above. Real-time PCR was performed on an ABI 7500 (Applied Biosystems). PCR primers and probes for mouse SOCS3 and HPRT in the TaqMan Rodent Control Reagents and TaqMan Gene Expression Assays (Applied Biosystems), respectively, were used according to the manufacturer’s instructions. PCR parameters are as recommended for the TaqMan Universal PCR Master Mix kit (Applied Biosystems). Triplicate samples of 2-fold serial dilutions of cDNA were assayed and used to construct the standard curves.

Preparation of 2B4 transfectants

SOCS3 cDNA was isolated by RT-PCR using total RNA prepared from Con A-activated spleen cells and was confirmed by sequencing. Antisense SOCS3 cDNA (24) was generated using standard PCR methods and subcloned into p3xFLAG-CMV-10 vector (Sigma-Aldrich). 2B4 cells were then transfected with the antisense SOCS3 expression vector or the empty vector as a control by electroporation and selected with geneticin (G418).

Retroviral infection

Wild-type SOCS3, dominant-negative SOCS3(F25A) (25, 26), and antisense SOCS3 cDNAs were generated using standard PCR methods and subcloned into a bicistronic retroviral vector pMX-IRES/EGFP (27), provided by Dr. T. Kitamura. The PLAT-E cell line was transfected with the resultant vectors or the empty vector as control by using FuGene 6 (Roche) and cultured to generate the retroviral supernatant. 2B4 cells were then infected with the supernatant as described previously (28) and purified by sorting using a FACSVantage (BD Biosciences).

Proliferation assay

Naive CD4⁺ T cells (1 × 10⁶ cells/ml) were stimulated with plate-coated anti-CD3 (2 μg/ml) and anti-CD28 (0.5 μg/ml) in the presence or the absence of IL-27. After 3 days, stimulated cells were recovered and washed. Resultant cells (1 × 10⁵ cells/ml) were cultured in the presence of human IL-2 for 48 h and pulsed with [³H]thymidine for the last 24 h.

Western blotting

Cells were lysed in a lysis buffer containing protease inhibitors, and resultant cell lysates were separated by SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride membrane (Millipore) as described previously (28). The membrane was blocked, probed with primary Ab and then with the appropriate secondary Ab conjugated to HRP, and visualized with the ECL detection system (Amersham Biosciences) according to the manufacturer’s instructions.

IL-27 inhibits CD28-mediated IL-2 production in naive CD4⁺ T cells stimulated with Ag plus APC and also with plate-coated anti-CD3 plus anti-CD28

We and other groups previously reported that IL-27 induces T-bet and subsequent IL-12β2 expression in naive CD4⁺ T cells and synergizes with IL-12 in IFN-γ production (1, 3, 4, 6). Although T-bet transcriptionally up-regulates IFN-γ production, it was originally demonstrated to down-regulate IL-2 production as well (7). In addition, WSX-1-deficient CD4⁺ T cells were reported to overproduce IL-2 (5). Therefore, we first examined the effect of IL-27 on primary IL-2 production. Naive CD4⁺ T cells from DO11.10 Tg mice were stimulated with OVA_323–339 peptide and irradiated T/NK cell-depleted BALB/c spleen cells in the presence or the absence of IL-27 (10 ng/ml) for various times, and culture supernatant was collected and analyzed for IL-2 production by ELISA. The Ag-specific IL-2 production in naive CD4⁺ T cells gradually increased with time, and IL-27 greatly inhibited IL-2 production (Fig. 1⇓A). Furthermore, naive CD4⁺ T cells from wild-type BALB/c mice were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence of IL-27 and/or IL-12 for various times and analyzed for IL-2 and IFN-γ production (Fig. 1⇓, B and C). As reported previously (1, 3, 4, 6), IL-27 induced T-bet expression (data not shown) and synergistic IFN-γ production with IL-12 (Fig. 1⇓C). In marked contrast, IL-27 clearly inhibited IL-2 production in a dose-dependent manner, whereas IL-12 failed to affect IL-2 production, but appeared to enhance the inhibitory effect of IL-27 (Fig. 1⇓, B and D). Without costimulation by anti-CD28, IL-2 production was not detected under the experimental conditions (Fig. 1⇓E). In the presence of anti-CD28, greater production of IL-2 was observed with the higher dose of anti-CD3 used for plate coating. IL-27 inhibited IL-2 production independently of the dose of anti-CD3. These results suggest that IL-27 inhibits CD28-mediated IL-2 production in naive CD4⁺ T cells stimulated with Ag plus APC and also with plate-coated anti-CD3 plus anti-CD28.

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FIGURE 1.

IL-27 inhibits CD28-mediated IL-2 production in naive CD4⁺ T cells stimulated with Ag plus APC and also with plate-coated anti-CD3 plus anti-CD28. A, Inhibition of Ag-specific IL-2 production by IL-27. Naive CD4⁺ T cells from DO11.10 Tg mice were stimulated with OVA_323–339 peptide and irradiated T/NK cell-depleted BALB/c spleen cells in the presence or the absence of IL-27 (10 ng/ml) for various times. B and C, Inhibition of IL-2 production, but augmentation of IFN-γ production in the presence of IL-12 by IL-27. Naive CD4⁺ T cells were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence or the absence of IL-27 (10 ng/ml) and/or IL-12 (10 ng/ml) for various times. D, Dose-dependent inhibition of IL-2 production by IL-27. Naive CD4⁺ T cells were stimulated with plate-coated anti-CD3, anti-CD28, and various concentrations of IL-27 for 48 h. E, Inhibition of IL-2 production by IL-27 independently of the dose of anti-CD3 used for plate coating. Naive CD4⁺ T cells were stimulated with various doses of plate-coated anti-CD3 and IL-27 (10 ng/ml) in the presence or the absence of anti-CD28 for 48 h. Culture supernatant was collected and assayed for IL-2 and/or IFN-γ production in triplicate by ELISA. Data are shown as the mean ± SD. Similar results were obtained in three to five independent experiments.

IL-27 induces SOCS3 expression in naive CD4⁺ T cells stimulated with plate-coated anti-CD3 and anti-CD28

Recently, it was reported that the expression of SOCS3 in early T cell activation influences the ability of IL-2 production mediated by CD28 costimulation (25). In addition, IFN-γ and IL-6 activate STAT1 and STAT3, respectively, both of which lead to SOCS3 induction. Therefore, we next examined the effect of IL-27 on SOCS3 expression. Naive CD4⁺ T cells were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence or the absence of IL-27 for various times, total RNA was prepared and analyzed for SOCS3 and HPRT mRNA expression by RT-PCR and real-time PCR (Fig. 2⇓). As reported previously (25), primary unstimulated CD4⁺ T cells expressed a significant level of SOCS3 mRNA, and the expression rapidly decreased after stimulation. However, in the presence of IL-27, SOCS3 expression was quickly recovered and increased; this pattern appeared to correlate with the inhibition of IL-2 production (Fig. 1⇑B). These results suggest that IL-27 induces SOCS3 expression in naive CD4⁺ T cells stimulated with plate-coated anti-CD3 and anti-CD28, implying that SOCS3 may play a role in the exertion of inhibitory effects by IL-27.

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FIGURE 2.

IL-27 induces SOCS3 expression in naive CD4⁺ T cells stimulated with plate-coated anti-CD3 and anti-CD28. Naive CD4⁺ T cells were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence or the absence of IL-27 (10 ng/ml) for various times. Total RNA was prepared and analyzed for mRNA expression of SOCS3 and HPRT as a control by RT-PCR and real-time PCR. Similar results were obtained in six independent experiments.

STAT1, but not STAT3, STAT4, and T-bet, is required for the inhibition of CD28-mediated IL-2 production by IL-27, which is highly correlated with the induction of SOCS3 expression

To further explore the correlation between inhibition of IL-2 production and induction of SOCS3 expression by IL-27 and also which IL-27 downstream signaling molecule is required for the inhibition of CD28-mediated IL-2 production, we next used various mice lacking each of these signaling molecules, T-bet, STAT1, STAT3, and STAT4. Naive CD4⁺ T cells were prepared from these knockout mice and respective control mice and stimulated with plate-coated anti-CD3 and anti-CD28 in the presence or the absence of IL-27. After 48 h, culture supernatant was collected and analyzed for IL-2 production by ELISA. Total RNA was also prepared and analyzed for SOCS3 and HPRT mRNA expression by RT-PCR. Inhibition of IL-2 production by IL-27 was still observed in STAT3-, STAT4-, and T-bet-deficient naive CD4⁺ T (Fig. 3⇓, B–D). However, in STAT1^−/− naive CD4⁺ T cells, IL-2 production was hardly inhibited by IL-27 compared with that in STAT1^+/− naive CD4⁺ T cells (Fig. 3⇓A). Consistent with these results, the induction of SCOS3 mRNA expression was still observed in STAT3-, STAT4-, and T-bet-deficient naive CD4⁺ T cells, but not in STAT1-deficient naive CD4⁺ T cells (Fig. 3⇓). These results suggest that the inhibition of IL-2 production is highly correlated with the induction of SOCS3 expression, and that STAT1 is required for inhibition of CD28-mediated IL-2 production and induction of SOCS3 expression by IL-27, although STAT3, STAT4, and T-bet are not essential to them.

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FIGURE 3.

STAT1, but not STAT3, STAT4, or T-bet, is required for the inhibition of CD28-mediated IL-2 production by IL-27, which is highly correlated with the augmentation of SOCS3 expression. Naive CD4⁺ T cells lacking each of the IL-27 downstream signaling molecules, STAT1 (A), STAT3 (B), STAT4 (C), and T-bet (D), and their control cells were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence or the absence of IL-27 (10 ng/ml) for 48 h. Culture supernatant was collected and assayed for IL-2 production in triplicate by ELISA. Data are shown as the mean ± SD. Total RNA was also prepared and analyzed for mRNA expression of SOCS3 and HPRT as a control by RT-PCR. Similar results were obtained in at least three independent experiments.

Similar inhibition of IL-2 production and induction of SOCS3 expression by IL-27 are observed in a T cell hybridoma cell line 2B4 as well as in primary naive CD4⁺ T cells

To clarify a role for SOCS3 in the inhibition of CD28-mediated IL-2 production by IL-27, we next used a CD4⁺ T cell hybridoma cell line, 2B4, instead of primary naive CD4⁺ T cells. We first confirmed that 2B4 cells express both IL-27R subunits, gp130 and WSX-1, which were determined by RT-PCR (data not shown), and that 2B4 cells are responsive to IL-27, resulting in activation of STAT1 and STAT3, which was detected by Western blotting using anti-pY-STAT1 and anti-pY-STAT3 (data not shown). Then, 2B4 cells were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence of IL-27. Culture supernatant was collected after 16 h and analyzed for IL-2 production by ELISA (Fig. 4⇓A). Although stimulation with plate-coated anti-CD3 alone induced the production of significant amounts of IL-2, the addition of anti-CD28 further enhanced IL-2 production. Consistent with the results obtained using primary naive CD4⁺ T cells (Fig. 1⇑D), IL-27 efficiently inhibited the CD28-mediated IL-2 production to the level obtained after stimulation with plate-coated anti-CD3 alone in a dose-dependent manner. Moreover, total RNA was prepared after the 3-h stimulation and analyzed for mRNA expression of SOCS3 by RT-PCR and real-time PCR (Fig. 4⇓B). Correlated with the inhibition of IL-2 production, SOCS3 mRNA expression was induced in the presence of IL-27. Thus, the inhibition of CD28-mediated IL-2 production and the induction of SOCS3 expression by IL-27 were observed not only in primary naive CD4⁺ T cells, but also in a T cell hybridoma cell line, 2B4.

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FIGURE 4.

Similar inhibition of IL-2 production and augmentation of SOCS3 expression by IL-27 are observed in a T cell hybridoma cell line 2B4 as well as in primary naive CD4⁺ T cells. A, 2B4 cells were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence or the absence of various concentrations of IL-27 for 16 h. Culture supernatant was then collected and assayed for IL-2 production in triplicate by ELISA. Data are shown as the mean ± SD. B, Total RNA was also prepared after the stimulation for 3 h in the presence or the absence of IL-27 (10 ng/ml) and was analyzed for mRNA expression of SOCS3 and HPRT as a control by RT-PCR and real-time PCR. Similar results were obtained in at least three independent experiments.

Forced expression of antisense SOCS3 or dominant negative SOCS3(F25A) in a T cell line blocks the inhibition of CD28-mediated IL-2 production by IL-27

Because a T cell line is more suitable for gene transduction, we next generated 2B4 cells devoid of functional SOCS3 expression by transducing antisense SOCS3 or dominant negative SOCS3(F25A). We first prepared 2B4 cells expressing antisense SOCS3 and its empty vector as a control by transfecting and selection with geneticin (G418). Resultant stable transfectants (two clones each) were then stimulated with plate-coated anti-CD3 and anti-CD28 in the presence or the absence of IL-27. After stimulation for 3 h, total RNA was prepared and analyzed for mRNA expression of SOCS3 by RT-PCR. Augmentation of SOCS3 mRNA expression by IL-27 was barely observed in 2B4 transfectants expressing antisense SOCS3, although augmentation was clearly observed in 2B4 transfectants expressing control vector (Fig. 5⇓A). Correlated with the inability to augment SOCS3 mRNA expression, inhibition of IL-2 production by IL-27 was not detected in 2B4 transfectants expressing antisense SOCS3, although the inhibition was clearly detected in 2B4 transfectants expressing control vector (Fig. 5⇓A).

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FIGURE 5.

Forced expression of antisense SOCS3 or dominant negative SOCS3(F25A) in a T cell line blocks the inhibition of CD28-mediated IL-2 production by Il-27. 2B4 stable transfectants (two clones each) expressing antisense SOCS3 or control vector (A), and 2B4 cells, expressing dominant negative SOCS3(F25A), antisense SOCS3, wild-type SOCS3, or control vector (B), which were prepared by retrovirus-mediated gene transduction, were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence or the absence of IL-27 (10 ng/ml) for 16 h. Culture supernatant was then collected and assayed for IL-2 production in triplicate by ELISA. Data are shown as the mean ± SD. Total RNA was also prepared after the stimulation for 3 h and was analyzed for mRNA expression of SOCS3 and HPRT as a control by RT-PCR. Expression of SOCS3 at the protein level in these unstimulated cells was analyzed by Western blotting with anti-SOCS3 and anti-actin (C). Similar results were obtained in at least three independent experiments.

We also prepared 2B4 cells expressing dominant negative SOCS3(F25A), which contains a point mutation in the kinase inhibitory region of SOCS3 (25, 26), antisense SOCS3, and control vector by retrovirus-mediated gene transduction, followed by purification with sorting, and analyzed the ability of IL-27 to inhibit CD28-mediated IL-2 production in these 2B4 cells as described above. The expression of dominant negative SOCS3(F25A) and wild-type SOCS3 was confirmed by Western blotting using anti-SOCS3, although endogenous expression of SOCS3 was hardly detected (Fig. 5⇑C). Augmentation of SOCS3 mRNA by IL-27 was observed in 2B4 cells expressing control vector and dominant negative SOCS3(F25A), although almost no augmentation of SOCS3 mRNA expression was observed in 2B4 cells expressing antisense SOCS3 (Fig. 5⇑B). Constitutive expression of SOCS3 mRNA was observed in 2B4 cells expressing wild-type SOCS3. In 2B4 cells expressing dominant negative SOCS3(F25A) and antisense SOCS3, but not in those expressing control vector, CD28-mediated IL-2 production was hardly inhibited by IL-27. In contrast, in 2B4 cells expressing wild-type SOCS3, augmentation of IL-2 production by anti-CD28 itself was not observed regardless of the presence or the absence of IL-27.

Taken together, these results suggest that IL-27 inhibits CD28-mediated IL-2 production, and that SOCS3, whose expression is induced by IL-27, plays a critical role in the inhibitory effect.

IL-27 inhibits IL-2-mediated cell proliferation and STAT5 activation without affecting CD25 expression

When naive CD4⁺ T cells were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence or the absence of IL-27 for 3 days and then expanded in medium containing IL-2 for 3 more days, we initially noticed that recovery of the cell number in the presence of IL-27 appears to be less than that in the absence of IL-27 (data not shown). This implies that IL-27 may affect IL-2-mediated cell proliferation in addition to IL-2 production. Therefore, we finally investigated the effect of pretreatment with IL-27 on IL-2 responses. Naive CD4⁺ T cells were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence or the absence of IL-27 for 3 days, and then these cells were washed and analyzed for the responsiveness to IL-2 by determining IL-2-mediated cell proliferation and tyrosine phosphorylation of STAT5, which is a critical signaling molecule activated by IL-2 (Fig. 6⇓, A and B, respectively). Naive CD4⁺ T cells stimulated in the absence of IL-27 efficiently proliferated in response to increasing amounts of IL-2. In contrast, pretreatment with IL-27 reduced IL-2-mediated cell proliferation dose-dependently. The pretreatment also inhibited IL-2-induced STAT5 phosphorylation in STAT1^+/+ naive CD4⁺ T cells, but not in STAT1^−/− naive CD4⁺ T cells. This is consistent with the finding that STAT1 is required for induction of SOCS3 expression by IL-27 (Fig. 3⇑). We then examined the effect of IL-27 on the induction of CD25 (IL-2Rα) expression by FACS analysis. Stimulation with plate-coated anti-CD3 and anti-CD28 greatly enhanced CD25 expression on naive CD4⁺ T cells, whereas comparable induction of CD25 expression was observed in the presence and the absence of IL-27 (Fig. 6⇓C). These results suggest that IL-27 inhibits IL-2-mediated cell proliferation and STAT5 activation without affecting CD25 expression as well as IL-2 production.

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FIGURE 6.

IL-27 inhibits IL-2-mediated cell proliferation and STAT5 activation without affecting CD25 expression. A, Inhibition of IL-2-mediated proliferation by pretreatment with IL-27. Naive CD4⁺ T cells were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence of various concentrations of IL-27 for 3 days, then these cells were washed and analyzed for the responsiveness to IL-2 by measuring IL-2-mediated proliferation. Cells were restimulated with various concentrations of human IL-2 for 48 h and were pulsed with [³H]thymidine for the last 24 h. B, Inhibition of IL-2-induced STAT5 activation by pretreatment with IL-27. STAT1^+/+ and STAT1^−/− (129/Sv background) naive CD4⁺ T cells were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence of IL-27 (10 ng/ml) for 3 days, then these cells were washed, rested overnight, and restimulated with mouse IL-2 (10 ng/ml) for 20 min. STAT5 activation was analyzed by Western blotting with anti-pY-STAT5 and anti-total STAT5. C, No effect of IL-27 on up-regulation of CD25 expression. Naive CD4⁺ T cells were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence or the absence of IL-27 (10 ng/ml) for various times and analyzed for cell surface expression of CD25 by FACS using anti-CD25 (solid line) and control rat IgG (plain line with shading). Similar results were obtained in at least two independent experiments.