Here, we identify AP4 as the transcription factor that is induced by c-Myc and sustains activation of antigen-specific CD8+ T cells. virus or intracellular bacterium, antigen (Ag)-specific CD8+ T cells are primed by signals through the T cell receptor (TCR), co-stimulatory molecules and cytokine receptors and undergo rapid expansion, effector differentiation, and memory cell formation1, 2. In the priming phase, activated CD8+ T cells grow in size by Wortmannin increasing global gene transcription and protein translation and utilize aerobic glycolysis pathways to produce the energy and materials for biosynthesis prior to cell cycle entry3, 4, 5, 6, 7. Previous studies established that the transcription factor (TF) c-Myc is essential for the initiation of the global cellular activation processes in activated lymphocytes as well as cancer and embryonic stem cells8, 9, 10, 11. c-Myc is induced by signals through the TCR and IL-2 receptor (IL-2R)12 and is essential for the metabolic reprograming and cell growth of T cells13. During acute infection, CD8+ T cell expansion persists even after levels of Ag and inflammation wane4. While this persistent proliferation may be driven by residual Ag on Ag-presenting cells, other evidence suggests that optimally primed CD8+ T cells continue proliferation after Ag and cytokines decrease to sub-optimal concentrations14, 15, 16, 17. Expression of c-Myc is rapidly induced in activated T cells9, 13. Its expression, however, does not persist throughout the duration of T cell expansion9, 18. These findings suggest that other TFs maintain c-Myc-initiated cellular activation Rabbit Polyclonal to ZNF134 to maximize clonal expansion and effector differentiation of T cells during acute responses to pathogen infection. We hypothesized that TCR and cytokine receptor signals during the early stage of pathogen infection induce TFs, which program CD8+ T cells for a durable response. Among the cytokines established as important for CD8+ T cell responses, IL-2 sustains clonal expansion and possibly and gene repression24, 25, 26. Although mRNA expression was reduced modestly upon withdrawal of IL-2, AP4 protein expression was substantially diminished (Fig. 1b,c), suggesting that sustained AP4 expression requires IL-2R signals at both transcriptional and post-transcriptional levels. AP4 expression from retrovirus (RV) also required IL-2R stimulation (Fig. 1d), suggesting its expression regulated predominantly at the post-transcriptional level. The half-life of AP4 protein was two to three hours under permissive (+IL-2) or non-permissive (IL-2 neutralization) conditions (Fig. 1e), with its degradation Wortmannin mediated by the ubiquitin-proteasome pathway (Fig. 1f). AP4 expression was sustained by TCR stimuli or other gamma chain cytokines (IL-7 and IL-15) but not by IL-12 or type I interferons (Fig. 1g). These results suggest that a common pathway converging from TCR and IL-2R chain signaling sustains AP4 protein expression. Consistently, MEK and p38 MAPK inhibitors U0126 and SB203580, respectively, attenuated the accumulation of AP4 protein in the presence of TCR or IL-2R stimulation (Fig. 1h). To validate the roles of TCR and IL-2R in maintaining AP4 expression we examined AP4 levels in Ag-specific CD8+ T cells during acute infection with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV-Arm) (Fig. 1i). AP4 protein was expressed highly in Ag-specific CD8+ T cells on days 4 and 5 after infection. The level declined on days 6 and 7 as T cell expansion markedly slowed. In activated CD8+ T cells on day 4.5 after LCMV-Arm infection, AP4 protein was detected specifically in CD25Hi cells, but not in CD25Lo cells, despite similar mRNA levels in both subpopulations (Fig. 1j). Furthermore, Ag-specific CD8+ T cells lacking expressed reduced amounts of AP4 protein Wortmannin four days after LCMV-Arm infection (Fig. 1k). We conclude that AP4 is regulated post-transcriptionally in CD8+ T cells via signaling through TCR and IL-2R and =3). (b) qRT-PCR analysis showing expression levels of indicated genes in CD8+ T cells treated as in (a). Error bars, s.d. (= 2). (c) Immunoblot showing AP4 expression in CD8+ T cells treated as in (a). Phosphorylated STAT5 and -tubulin serve as controls. (= 4). (d) Immunoblot showing AP4 expression in IL-2-treated or IL-2-deprived = 2). (e) Immunoblot analysis of AP4 in IL-2-treated or IL-2-deprived CD8+ T cells in the presence of cycloheximide (CHX, 10 M) for indicated time. (= 2). (f) Immunoblot analysis of AP4 in IL-2-deprived CD8+ T cells in the presence of MG-132 (10 M) for indicated time. (= 2). (g) Immunoblot analysis of AP4 in CD8+ T cells stimulated with different concentrations of.