Tpl2 is an IL-12–inducible gene in human and mouse cells. (A, left) Human PBMC were cultured for 3 d in RPMI containing 1 μg/ml PHA followed by an additional day of culture in 40 U/ml IL-2 to maximize expression of IL-12R (). The cells were then rested for 12 h in serum-free medium, followed by stimulation with medium alone (lane 1), IL-2 (lane 2), IL-4 (lane 3), IL-7 (lane 4), IL-12 (lane 5), or IL-15 (lane 6) at 10 ng/ml for 6 h, and Tpl2 mRNA expression was determined by Northern blot analysis using a Tpl2-specific probe. (A, right) A kinetic analysis of Tpl2 mRNA induction was performed by Northern blot analysis of 10 ng/ml IL-12–stimulated human PHA blasts. Data are representative of two independent experiments. White lines indicate that intervening lanes have been spliced out. (B) Human cord blood CD4⁺ T cells were cultured under Th1 or Th2 inducing conditions, as described in Materials and methods, and analyzed for Tpl2 message level by real-time PCR. Data are representative of two separate experiments. (C–E) For analysis of Tpl2 expression in mouse cells, CD4⁺ T cells were purified from mouse spleens. Cells were then grown for 3 d on plates coated with anti-CD3 and -CD28, rested for 5 h in cytokine-free medium, restimulated with IL-12 or IFN-γ (C), IL-12 (D), or IL-23 (E) for 4 h, and subsequently analyzed for Tpl2 expression by real-time PCR. Mouse cells analyzed were from WT (C and E; n = 3 replicate experiments) or IFN-γ–deficient mice (D; representative of two biological replicates from one experiment). (F) For transcriptional analysis of Tpl2 in response to TCR signals, 2 × 10⁶ CD4⁺ T cells were stimulated in 2 ml of media on a 12-well plate coated with anti-CD3 and -CD28 for the indicated times and analyzed for Tpl2 expression by real-time PCR. Data are representative of two independent experiments. Error bars represent SD from replicates within a representative experiment.

Tpl2 is a direct Stat4 target gene. (A) The Stat4 dependency of Tpl2 induction by IL-12 was examined by stimulating WT or Stat4-deficient mouse CD4⁺ T cell blasts with IL-12 for 4 h and analyzing Tpl2 mRNA induction. n = 3 replicate experiments. (B) ChIP using an anti-Stat4 antibody or nonimmune rabbit serum (NRS) was analyzed quantitatively by real-time PCR using primers designed around putative Stat sites within 1 kb upstream of the Tpl2 transcriptional start site. n = 4 independent experiments. (C) To further test the specificity of the binding, we compared the binding to the promoter region to that seen within an intronic region of the gene (intron 5). n = 1 experiment. Error bars represent SD from replicates within a representative experiment.

Tpl2 is required for optimal IL-12–induced IFN-γ production. (A and B) IL-12–induced IFN-γ production was analyzed in WT and Tpl2-deficient CD4⁺ T cells. CD4⁺ T cells isolated from WT or Tpl2-deficient mouse LN and spleens were activated and expanded on anti-CD3– and -CD28–coated flasks for 3 d. Cells were then rested for 5 h in cytokine-free medium before stimulation with 0–10 ng/ml IL-12 alone or in combination with 50 U/ml IL-2 or 10 ng/ml IL-18 for 24 h. Cytokine production was measured in cell culture supernatants using cytometric bead array (A) or intracellular cytokine staining (B). (C) For intracellular cytokine staining, rested cells were restimulated with IL-12 and IL-18 for 24 h with the addition of Golgi transport inhibitor for the last 5 h of culture. Whole splenocytes were stimulated with soluble anti-CD3 for 48 h, and the production of IFN-γ was measured by cytometric bead array. A, n = 3 experiments; B, n = 2 experiments; C, n = 3 experiments. Error bars represent SD from replicates within a representative experiment.

Tpl2 deficiency leads to impaired Th1 differentiation in vitro. Naive CD44^loCD62L^hi CD4⁺ T cells were purified by FACS sorting and cultured with immobilized anti-CD3 and -CD28 plus the appropriate cytokines and anti-cytokine antibodies for 7 d to generate Th0, Th1, Th2, or Th17 T helper lineages. Efficiency of polarization was determined based on IFN-γ, IL-4/IL-13, or IL-17 production, as measured by intracellular cytokine staining for IFN-γ and IL-13 or IL-4 (A), cytometric bead array for IFN-γ (B), real-time PCR for IFN-γ mRNA (C), and ELISA for IL-17 protein (D). Data shown for A–C are representative of at least three independent experiments; data in D are representative of two experiments. Error bars indicate SEM; *, P < 0.05.

Tpl2 regulates Stat4 and T-bet levels. (A) CD4⁺ T cell blasts were generated over 3 d of culture on immobilized anti-CD3 and -CD28. Expression of IL-12 receptor β1 and β2 were determined by real-time PCR or surface staining. n = 3 separate experiments each. (B) Cells were then stimulated with IL-12 for the indicated times. Whole cell lysates were analyzed for ERK (not depicted) or Stat4 activation by Western blotting. n = 3 independent experiments. (C) TCR-dependent regulation of Stat4 was determined by culturing 2 × 10⁶ cells in 2 ml of media on a 12-well plate coated with anti-CD3 and -CD28 for the indicated times. Stat4 expression was determined by real-time PCR analysis. Error bars represent SD from replicates within a representative experiment. n = 2 independent experiments. (D) ChIP was performed to determine whether Tpl2 deficiency affected IL-12–induced Stat4 binding to the Ifng promoter. Cells were activated and rested as in A, plated at 10⁶/ml in 10-cm petri dishes, and stimulated for 1 h with 10 ng/ml IL-12. Immunoprecipitation was performed using anti-Stat4 antibody or nonimmune rabbit serum, and binding to the Ifng promoter was analyzed by real-time PCR. Data represent means and SE of the mean of three independent experiments. (E, left) Purified CD4⁺ T cells were activated with anti-CD3 and -CD28 for 3 d and analyzed by intracellular staining for T-bet and Gata3 expression. n = 2 independent experiments with a total of three individual mice. (E, right) Additionally, purified CD4⁺ T cells were analyzed for Gata3 and T-bet mRNA expression by real-time PCR relative to an 18S control, and the ratio of Gata3/T-bet message is shown. The ratio of Gata3/T-bet was calculated by ΔCt method. Data represent means and SEM for a total of four mice per group in two separate experiments. *, P < 0.05. (F) Tpl2-dependent TCR signaling events were analyzed in WT and Tpl2-deficient mice. Five million cells/point were stimulated with biotinylated anti-CD3 on ice for 15 min before cross-linking with streptavidin at 37°C for the indicated times. Western blots were probed with antibodies recognizing phospho-ERK, total ERK, and actin. Data are representative of two independent experiments.

Tpl2 is required for normal host responses to T. gondii. WT or Tpl2-deficient mice were infected with 20 cysts of the avirulent T. gondii strain ME49 and analyzed for host responses during acute and chronic stages of infection. Both WT and Tpl2^−/− mice survived the infection for >8 wk. To explore their immune responses further, mice were killed on day 7 after infection and several parameters were analyzed, including systemic cytokine levels in serum (A), LN recall responses to STAg (B), and proportion of infected PECs (C and D). Arrowheads in D indicate intracellular T. gondii parasites. Host responses of chronically infected mice were assessed at week 5 by quantifying the numbers of encysted bradyzoites in the brain (E). Rag2-deficient mice were reconstituted with 10 million purified CD4⁺ T cells and infected with T. gondii >1 wk later. Survival of reconstituted infected mice was monitored (F). Serum levels of IFN-γ were also determined 7 d after infection (G). Data indicate means from at least seven mice per group from two separate infections for A, B, and G and are from four mice per group in C, E, and F, respectively. Error bars indicate standard errors of the mean.