Signals from OX40 regulate nuclear factor of activated T cells c1 and T cell helper 2 lineage commitment

Abstract

T cell helper type 2 (Th2) differentiation is driven by a source of IL-4 receptor (IL-4R) that mobilizes IL-4R signaling pathways and the transcription factor GATA-3. Naïve CD4 cells can secrete IL-4 independently of IL-4R signals, but how this secretion is regulated is not understood. Here we demonstrate that costimulation through the tumor necrosis factor receptor family molecule OX40, in synergy with CD28, is essential for high levels of nuclear factor of activated T cells c1 to accumulate in the nucleus of a recently activated naïve T cell. This action is not dependent on either IL-4R or IL-2R signals and results in OX40 controlling initial naïve T cell IL-4 transcription. OX40 signals subsequently enhance nuclear GATA-3 accumulation through an IL-4R-dependent action, leading to Th2 differentiation. These data show that, in the absence of an exogenous source of IL-4, OX40 provides a critical synergistic and temporal signal with other noncytokine receptors to modulate nuclear factor of activated T cells c1 and to promote optimal Th2 generation.

Fig. 1.

OX40–OX40L interactions are required for Th2 differentiation. Naïve CD4 T cells from wild-type (filled circles) or OX40-deficient (open circles) OT-II mice were cultured with APCs and indicated doses of OVA-323–339. (A) IL-4 and IFN-γ recall responses measured at day 7 after Ag/APC restimulation of equal numbers of effector T cells for 24 h. (B) OX40 and OX40L expression on wild-type CD4 and CD11b⁺CD11c⁺ APCs after stimulation with 0.1 μM Ag for 36 h. The blank histograms indicate isotype control. The shaded histograms indicate positive staining. (C) Effect of OX40L blocking on recall cytokine responses of wild-type T cells cultured with 0.1 μM Ag. αOX40L, anti-OX40L Ab. All data are representative of at least three individual experiments.

Fig. 2.

Defective Th2 differentiation in vivo in OX40-deficient CD4 T cells. Naïve wild-type or OX40^−/− OT-II CD4 cells were transferred into Thy1.1 B6.PL hosts. Mice were immunized with OVA/alum, and draining lymph node cells were taken at 4 days. (A) The number of OT-II T cells measured by staining for Thy1.2. Data are the mean ± SE of three mice. (B and C) IL-4 and IFN-γ mRNA levels in purified Thy1.2⁺ cells measured by quantitative RT-PCR, normalized to hypoxanthine phosphoribosyltransferase (HPRT). Data are relative values with SD of triplicate PCR wells. Similar results were obtained at day 2.

Fig. 3.

IL-4 is a critical factor for OX40-driven Th2 differentiation. Naïve CD4 T cells from wild-type (filled symbols) or OX40-deficient (open symbols) OT-II mice were cultured with APCs and 0.1 μM OVA-323–339 as in Fig. 1.(A) Primary IL-2 production from naïve T cells stimulated for 1–5 days. (B and C) Recall IL-4 production at 7 days in the presence of IL-2 (10 ng/ml), IL-4 (10 ng/ml), anti-IL-4 or anti-IFN-γ (10 μg/ml), added at days 0 and 3. (D) Primary IL-4 and IFN-γ mRNA induction, in the presence or absence of anti-IL-4, 36 h after naïve T cell activation. mRNA levels in purified CD4 cells by RT-PCR, normalized to HPRT, are shown. Data are relative values with SD of triplicate PCR wells. Results are representative of at least two experiments.

Fig. 4.

PI3K, CN, and NFAT control OX40-mediated IL-4 and Th2 differentiation. Naïve CD4 T cells from wild-type OT-II mice were stimulated with 0.1 μM Ag/APCs as in Fig. 1. After 12 h of activation, LY (PI3K inhibitor), cyclosporin A (CsA) (CN inhibitor) (A and B), VIVIT peptide (NFAT inhibitor), VEET control peptide (C and D), or DMSO were added. T cells were harvested at 36 h to measure IL-4 mRNA (A and C) and at day 7 to measure IL-4 recall responses after Ag restimulation as in Fig. 1 (B and D).

Fig. 5.

OX40 directs IL-4-independent nuclear accumulation of NFATc1. Naïve CD4 T cells from wild-type and OX40^−/− OT-II mice were stimulated with 0.1 μM Ag/APCs in the presence or absence of anti-IL-4, as in Fig. 1. (A and B) Levels of NFATc1, NFATc2, and GATA-3 were examined in purified CD4 cells at 36 h by blotting cytoplasmic and nuclear extracts. Lamin B, control nucleus-specific protein, is shown. (C–F) Naïve wild-type CD4 cells were stimulated with anti-CD3/CD28 and IL-2 for 24 h. (C) OX40 expression at 24 h. (D) Nuclear NFATc1 at 24 h. Anti-IL-2R-α and anti-β blocking mAbs were added from the beginning of culture. (E and F) Nuclear NFATc1 at 28 h. At 24 h, CD4 cells were recultured without stimulation or with anti-OX40 (100 μg/ml) for 4 h in the presence of blocking Abs, CsA, or LY. Data are representative of at least two independent experiments.

Fig. 6.

OX40 triggering up-regulates IL-4 and nuclear NFATc1 in effector Th2 cells. CD4 T cells were cultured in Th2 skewing conditions. At day 6, live effector cells were restimulated with beads coated with anti-CD3 and anti-OX40. (A) OX40 expression at day 6. (B and E) IL-4 protein and (D and G) IL-4 mRNA, 4 h after stimulation. (C and F) Nuclear NFATc1 levels at 4 h. Th2 cells were preincubated for 2 h with cycloheximide (D), LY (E and F), CsA, VIVIT peptide, or VEET peptide (G), then restimulated with Ab-coated beads for 4 h.