IL-27 induces Stat1, -3, -4, and -5 phosphorylation. CD4⁺ cells purified from TCCR^+/+ or TCCR^-/- splenocytes were treated with the indicated cytokines for 15 min in serum-free medium. Cell lysates were immunoprecipitated (IP) with Stat1 or Stat3 antibodies. Immunoprecipitates or cell lysates (for Stat4, -5, and -6 detection) were submitted to SDS/PAGE, followed by Western blotting (WB) with antibodies specific for the indicated phosphorylated Stat proteins. To control for equal protein loading, blots were stripped and reprobed with antibodies specific for the indicated total Stat proteins. Horizontal lines indicate the position of a 98-kDa marker.

Stat1 is not required for the IL-27-induced proliferation of CD4⁺ cells. CD4⁺ cells purified from Stat1^+/+ (□) or Stat1^-/- (▪) splenocytes were activated in triplicate wells with plate-bound anti-CD3ε and anti-CD28 antibodies in complete medium supplemented with anti-mIL-2 antibodies and increasing concentrations of mIL-27. [³H]Thymidine (1 μCi per well; 1 Ci = 37 GBq) was added during the last 24 h of this 3-day assay. Results are shown as the fold increase in [³H]thymidine incorporation in the presence of IL-27 compared with its absence (16,478 and 22,857 cpm in Stat1^+/+ and Stat1^-/- cells, respectively, in the absence of IL-27).

IL-27 does not induce significantly the production of IFNγ by differentiated CD4⁺ T cells. CD4⁺ T cells purified from TCCR^+/+ or TCCR^-/- splenocytes were activated as in Fig. 2 in the indicated conditions in the absence (white bars) or presence (gray bars) of IL-27. Three days after activation, cells were expanded in medium containing mIL-2 only. Cells were collected on day 6 or 7 and washed, and 10⁶ cells were restimulated with plate-bound anti-CD3ε antibodies. Cytokines secreted in the supernatant 24 h after restimulation were measured in duplicate by ELISA. (A) IFNγ production. (B) IL-4 production. Results shown are representative of three independent experiments.

IL-27-mediated induction of T-bet does not require Stat1, but IL-27-mediated suppression of GATA-3 is strictly dependent on Stat1. CD4⁺ T cells purified from Stat1^+/+ or Stat1^-/- splenocytes were activated as in Fig. 2 in the absence (white bars) or presence (gray bars) of IL-27. Before activation or 48 h later, cells were collected for total RNA preparation. Levels of rpl19, T-bet, IL-12R β2, and GATA-3 mRNAs were determined as indicated in Fig. 2. Results shown are arbitrary units normalized for rpl19 expression and are representative of two independent experiments. Fold inductions (for T-bet expression and IL-12R β2) or suppressions (for GATA-3 expression) between relevant samples are indicated. Error bars represent 1 SD. P values between data points that are discussed in the text as significantly different are <0.05. (A) T-bet mRNA levels. (B) IL-12R β2 mRNA levels. (C) GATA-3 mRNA levels.

IL-27 induces T-bet and IL-12R β2 and suppresses GATA-3 in developing CD4⁺ T cells. (A–C) CD4⁺ T cells purified from TCCR^+/+ or TCCR^-/- splenocytes were activated with plate-bound anti-CD3ε and anti-CD28 antibodies in the absence (white bars) or presence (gray bars) of IL-27 in T_H1 conditions (IL-12 plus anti-IL-4 plus IL-2), T_H2 conditions (IL-4 plus anti-IL-12 plus anti-IFNγ plus IL-2), or neutral (N) conditions (anti-IL-12 plus anti-IFNγ plus anti-IL-4 plus IL-2). Before activation or 48 h later, cells were collected for total RNA preparation. Levels of rpl19 (a ribosomal housekeeping gene), T-bet, IL-12R β2, and GATA-3 mRNAs were determined in duplicate by real-time RT-PCR and compared with levels measured in serial dilutions of a reference RNA. Results shown are arbitrary units normalized for rpl19 expression and are representative of three independent experiments. Error bars represent 1 SD. P values between data points that are discussed in the text as significantly different are <0.02. (A) T-bet mRNA levels. (B) IL-12R β2 mRNA levels. (C) GATA-3 mRNA levels. Fold suppressions between relevant samples are indicated. (D) CD4⁺ T cells purified from TCCR^+/+ or TCCR^-/- splenocytes were activated as in A–C. Forty-eight hours after activation, cells activated in the absence (filled gray histograms) or presence (open histograms) of mIL-27 were stained with anti-mCD4 and anti-mIL-12R β2 antibodies. Histograms are gated on live CD4⁺ cells.

Effects of IL-27 on primary NK cells. (A) IL-27 does not induce the proliferation of primary NK cells. A total of 2 × 10⁶ peripheral blood mononuclear cells from a healthy human donor were seeded in complete medium in the absence or presence of increasing concentrations of hIL-15 (□) or hIL-27 (•). Percentages of NK cells (CD56⁺/CD3^- cells) were determined by FACS analysis before the initiation of the cultures and 7 days later, when cells were collected and counted. Results are indicated as the fold increase in total NK cell numbers in the presence of cytokines over the fold increase in the absence of cytokine (background: 0.54-fold in the absence of cytokine). (B) IL-27, like IL-12 and IL-15, induces T-bet expression in primary NK cells. DX5⁺ NK cells purified from TCCR^+/+ (white bars) or TCCR^-/- (gray bars) splenocytes were cultured in vitro for 44 h in the absence or presence of the indicated cytokines. Cells were collected for total RNA preparation, and rpl19 and T-bet mRNA levels were measured by real-time RT-PCR as indicated in Fig. 2. nd, not detectable. (C) IL-27, unlike IL-12 and IL-15, does not induce IFNγ production by primary NK cells. Supernatants from cultures of DX5⁺ TCCR^+/+ NK cells described in B were collected after 44 h, and IFNγ production was measured by ELISA. nd, not detectable. (D) IL-27, unlike IL-12 and IL-15, does not increase the cytotoxic activity of primary NK cells. DX5⁺ NK cells purified from TCCR^+/+ splenocytes were cultured in vitro for 18 h in the absence (⋄) or presence of mIL-27 (•), hIL-15 (□), or mIL-12 (▵). Cells were collected and incubated for 4 h with ⁵¹Cr-labeled NK-sensitive YAC-1 target cells at the indicated NK-to-target ratios. Specific lysis of YAC-1 cells was determined by measuring ⁵¹Cr released in the supernatants.