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Background: Inhibition of G-protein βγ (Gβγ) signaling was found previously to enhance T cell receptor (TCR)-stimulated increases in interleukin 2 (IL-2) mRNA in CD4 + T helper cells, suggesting that Gβγ might be a useful drug target for treating autoimmune diseases, as low dose IL-2 therapy can suppress autoimmune responses. Because IL-2 may counteract autoimmunity in part by shifting CD4 + T helper cells away from the Type 1 T helper cell (TH1) and TH17 subtypes towards the TH2 subtype, the purpose of this study was to determine if blocking Gβγ signaling affected the balance of TH1, TH17, and TH2 cytokine mRNAs produced by CD4 + T helper cells. Methods: Gallein, a small molecule inhibitor of Gβγ, and siRNA-mediated silencing of the G-protein β 1 subunit (Gβ 1 ) were used to test the effect of blocking Gβγ on mRNA levels of cytokines in primary human TCR-stimulated CD4 + T helper cells. Results: Gallein and Gβ 1 siRNA decreased interferon-γ (IFN-γ) and IL-17A mRNA levels in TCR-stimulated CD4 + T cells grown under TH1-promoting conditions. Inhibiting Gβγ also decreased mRNA levels of STAT4, which plays a positive role in TH1 differentiation and IL-17A production. Moreover, mRNA levels of the STAT4-regulated TH1-associated proteins, IL-18 receptor β chain (IL-18Rβ), mitogen-activated protein kinase kinase kinase 8 (MAP3K8), lymphocyte activation gene 3 (LAG-3), natural killer cell group 7 sequence (NKG7), and oncostatin M (OSM) were also decreased upon Gβγ inhibition. Gallein also increased IL-4, IL-5, IL-9, and IL-13 mRNA levels in TCR-stimulated memory CD4 + T cells grown in TH2-promoting conditions. Conclusions: Inhibiting Gβγ to produce these shifts in cytokine mRNA production might be beneficial for patients with autoimmune diseases such as rheumatoid arthritis (RA), Crohn’s disease (CD), psoriasis, multiple sclerosis (MS), and Hashimoto’s thyroiditis (HT), in which both IFN-γ and IL-17A are elevated.

Background: The role of cAMP in regulating T cell activation and function has been controversial. cAMP is generally known as an immunosuppressant, but it is also required for generating optimal immune responses. As the effect of cAMP is likely to depend on its cellular context, the current study investigated whether the mechanism of activation of Gα s and adenylyl cyclase influences their effect on T cell receptor (TCR)-stimulated interleukin-2 (IL-2) mRNA levels. Methods: The effect of blocking G s -coupled receptor (G s PCR)-mediated G s activation on TCR-stimulated IL-2 mRNA levels in CD4 + T cells was compared with that of knocking down Gα s expression or inhibiting adenylyl cyclase activity. The effect of knocking down Gα s expression on TCR-stimulated cAMP accumulation was compared with that of blocking G s PCR signaling. Results: ZM-241385, an antagonist to the G s -coupled A 2A adenosine receptor (A 2A R), enhanced TCR-stimulated IL-2 mRNA levels in primary human CD4 + T helper cells and in Jurkat T cells. A dominant negative Gα s construct, Gα s DN3, also enhanced TCR-stimulated IL-2 mRNA levels. Similar to G s PCR antagonists, Gα s DN3 blocked G s PCR-dependent activation of both Gα s and Gβγ. In contrast, Gα s siRNA and 2’,5’-dideoxyadenosine (ddA), an adenylyl cyclase inhibitor, decreased TCR-stimulated IL-2 mRNA levels. Gα s siRNA, but not Gα s DN3, decreased TCR-stimulated cAMP synthesis. Potentiation of IL-2 mRNA levels by ZM-241385 required at least two days of TCR stimulation, and addition of ddA after three days of TCR stimulation enhanced IL-2 mRNA levels. Conclusions: G s PCRs play an inhibitory role in the regulation of TCR-stimulated IL-2 mRNA levels whereas Gα s and cAMP can play a stimulatory one. Additionally, TCR-dependent activation of Gα s does not appear to involve G s PCRs. These results suggest that the context of Gα s /cAMP activation and the stage of T cell activation and differentiation determine the effect on TCR-stimulated IL-2 mRNA levels.