Stat4 is activated in response to IL-12. Most functions of IL-12, including the induction of IFN-γ, are compromised in the absence of Stat4. Since the precise role of Stat4 in IFN-γ induction has not been established, experiments were conducted to examine Stat4 activation of IFN-γ and other genes required for cytokine-induced expression of IFN-γ. We first examined IL-12 signaling components. Basal expression of IL-12Rβ1 and IL-12Rβ2 is decreased in Stat4-deficient cells compared with that in control cells. However, IL-12 was still capable of inducing equivalent phosphorylation of Jak2 and Tyk2 in wild-type and Stat4-deficient activated T cells. We have further determined that other cytokine signaling pathways that induce IFN-γ production are defective in the absence of Stat4. IL-18 induces minimal IFN-γ production from Stat4-deficient activated T cells compared with control cells. This is due to defective IL-18 signaling, which results from the lack of IL-12-induced, and Stat4-dependent, expression of the IL-18R. Following IL-12 pretreatment to induce IL-18R, wild-type, but not Stat4-deficient, activated T cells demonstrated IL-18-induced NF-κB DNA-binding activity. In addition, IL-12-pretreated Stat4-deficient activated T cells have minimal IFN-γ production followed by stimulation with IL-18 alone or in combination with IL-12 compared with control cells. Thus, Stat4 activation by IL-12 is required for the function of multiple cytokine pathways that result in induction of IFN-γ.

T lymphocyte growth is regulated by the cyclin-dependent kinase inhibitor p27Kip1. Mice deficient in p27Kip1 have increased proliferative responses to multiple cytokines, including IL-2, IL-4, and IL-12, but not to anti-CD3. In the absence of p27Kip1, T cells proliferate faster than control cells, as evidenced by increased [3H]thymidine uptake, increased cell growth and division, and an increased number of cells in S phase. Importantly, this regulation is specific for p27Kip1 in T cells, because hyperproliferation of T cells from mice deficient in p21Cip1/Waf1 was not observed. In vivo, there is an expansion of activated/memory CD4+ cells in p27Kip1-deficient mice before and after immunization. Furthermore, Ag-stimulated spleen cells from immunized p27Kip1-deficient mice demonstrated increased proliferative responses to IL-2 and increased secretion of IFN-γ. Although IL-4 stimulated proliferative responses are diminished in Stat6-deficient T cells, activated T cells from mice doubly deficient in both p27Kip1 and Stat6 recover normal proliferative responses to IL-4. Together, these data firmly support a role for p27Kip1 as a negative regulator of cytokine-stimulated T cell growth.

The in vivo function of Th cell subsets is largely dependent on the ability of differentiated CD4+ T cells to be recruited to specific sites and secrete restricted sets of cytokines. In this paper we demonstrate that Th1 and Th2 cells secrete discrete patterns of chemokines, small m.w. cytokines that function as chemoattractants in inflammatory reactions. Th2 cells secrete macrophage-derived chemokine and T cell activation gene 3, and acquisition of this pattern of expression is dependent on Stat6. In contrast, Th1 cells secrete lymphotactin and RANTES, though unlike IFN-γ, expression of these chemokines is independent of Stat4. We further show that supernatants from activated Th2 cells preferentially induce the chemotaxis of Th2 over Th1 cells, corresponding with Stat6-dependent expression of CCR4 and CCR8 in Th2 cells. These data provide the basis for restricted and direct T cell-mediated cellular recruitment to sites of inflammation.