Interferon regulatory factor 8 integrates T-cell receptor and cytokine-signaling pathways and drives effector differentiation of CD8 T cells Fumi Miyagawaa, Hong Zhanga, Atshushi Terunumaa, Keiko Ozatob, Yutaka Tagayac,1,2,3, and Stephen I. Katza,1,3 aDermatology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; bLaboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; and cMetabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Edited* by Thomas A. Waldmann, National Cancer Institute, National Institutes of Health, Bethesda, MD, and approved June 19, 2012 (received for review January 26, 2012) We recently demonstrated that differentiation of cytotoxic T cells differentiation (27). Nevertheless, the role of IRF8 in CD8 T requires cooperation between T-cell receptor (TCR)/costimulation cells remains elusive; IRF8KO mice manifest impaired CD8 and γc-cytokines. Here we demonstrate that the transcription fac- T-cell responses against certain viruses (22). Thus, we embarked tor IFN regulatory factor 8 (IRF8) is expressed in CD8 T cells by the on a detailed assessment of the role of IRF8 in CD8 T cells. Here, we demonstrate the critical need for the convergence of combination of these two signals. More importantly, depletion of γ IRF8 in these cells abrogated the differentiation of naive CD8 T cells c-Jak3 and TCR/costimulation-signaling pathways in the tran- scription of IRF8 and demonstrate that removal of IRF8 cripples into effector cells in an experimental graft-vs.-host disease mouse fi − model. We also show that IRF8 seems to not operate upstream of CD8 effector T cells. Associated with these ndings, IRF8 OT-I cells failed to induce GvHD in our model (28). We also show other critical factors such as T-bet and eomesodermin, which have that either IRF8 action is independent of that of T-bet and been implicated in effector maturation. Collectively, our work Eomes (9–11) or IRF8 may operate downstream of these factors, shows that IRF8 integrates the TCR/costimulation and γc-cyto- – but not by way of them, and therefore propose that IRF8 be kine signaling pathways and mediates the transition of naive added to the roster of critical regulators for the differentiation of CD8 T cells to effector cells, thus identifying IRF8 as one of the naive CD8 T cells into effector cells. IMMUNOLOGY molecular regulators of CD8 T-cell differentiation. Results γ common c-cytokine | T-cell receptor signaling | CD8 effector Suboptimal Antigen Stimulation and a γc-Cytokine Cooperatively differentiation | transcription factors | Jak3 Generate Functional CD8 T Cells. We previously developed an ex- perimental model of GvHD (28) and reported that cooperation D8 T cells are essential for the adaptive immune response of γc-cytokine(s) and Ag/TCR-signaling events is indispensable Cagainst various intracellular pathogens and tumors. A typical for disease development. Briefly, a membrane-bound form of CD8 T-cell response consists of three phases: clonal expansion of chicken OVA was expressed in mice under the K14 promoter. antigen (Ag)-specific cells and acquisition of effector functions, Adoptive transfer of syngeneic OT-I cells (CD8+Vα2+Vβ5+) contraction of the effector cells through apoptosis, and genera- that recognize OVA in these mice causes GvHD-like pathogenic tion of long-lived memory cells (1–4). The acquisition of effector changes (28). In a strain with lower OVA copy number (K14- functions is critical for the control of intracellular pathogens and mOVAlow mice), GvHD did not occur spontaneously after tumors and is accompanied by the production of cytotoxic mol- adoptive transfer of OT-I cells but occurred when cells were ecules, perforin and granzyme, as well as two main cytokines, injected with IL-15 (and other selective γc-cytokines) (13) (Fig. IFN-γ and tumor necrosis factor (TNF) (5–7). S1A). Furthermore, deletion of IL-15 by crossing K14-mOVAhigh The transition from naive to effector CD8 T cells requires mice (mice with high OVA copy number) with IL-15KO mice marked changes in gene expression (8) mediated by transcription abrogated disease (13). These observations led us to postulate factors (5). The T-box transcription factors, T-bet (9, 10) and that TCR and γc-cytokine signals cooperate physiologically to eomesodermin (Eomes) (11), are the best-described regulators enable full maturation of effector T cells. To test this hypothesis, of this process in CD8 T cells (11, 12). serially diluted OVA–peptide was cultured with OT-I cells in the We previously reported that transgenic mice expressing oval- high presence or absence of IL-15. Amounts greater than 10 pg/mL of bumin (OVA) protein by the keratin 14 promoter (K14-mOVA the peptide induced a maximal proliferative response by OT-I mice) develop graft-vs.-host disease (GvHD) after the transfer of cells, and 0.3 pg/mL was a suboptimal dose of peptide that in- OT-I CD8 cells, whereas similar transgenic mice expressing much A B low duced OT-I proliferation (Fig. 1 and Fig. S1 ). However, at lower OVA copy numbers (K14-mOVA mice) did not (13, 14). doses between 0.03 and 3 pg/mL of peptide, the cellular pro- We also demonstrated that the onset of GvHD in this model γ liferative response was greatly enhanced by the presence of IL- is dependent only on CD8 T cells. The injection of c-cytokines 15, and combinatorial treatment of 0.3 pg/mL of peptide and (15), especially IL-15, could create GvHD in K14-mOVAlow mice upon OT-I cell transfer, whereas K14-mOVAhigh mice on an IL-15KO background failed to develop GvHD upon OT-I cell transfer (13). These data suggested a critical requirement Author contributions: F.M., K.O., Y.T., and S.I.K. designed research; F.M., H.Z., and A.T. γ performed research; F.M., H.Z., and A.T. analyzed data; and F.M., K.O., Y.T., and S.I.K. for c-cytokines in the effector differentiation of CD8 T cells wrote the paper. in vivo. We then hypothesized that concurrent signaling events fl consisting of the γc-pathway and the T-cell receptor (TCR)/ The authors declare no con ict of interest. costimulation pathway are critical for the transition of naive *This Direct Submission article had a prearranged editor. CD8 T cells into effector cells. 1Y.T. and S.I.K. contributed equally to this work. In this study, we sought a molecular integrator of these two 2Present address: Division of Basic Science and Vaccine Research, Institutes of Human pathways in the effector differentiation of CD8 T cells and Virology, University of Maryland School of Medicine, Baltimore, MD 21201. identified IFN regulatory factor 8 (IRF8). IRF8 belongs to the 3To whom correspondence may be addressed. E-mail: [email protected] or yta- IFN regulatory factor family (16–18) and critically controls the [email protected]. lineage commitment between the myeloid and B cells (19–26). In This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. addition, IRF8 controls a silencing program for Th17 cell 1073/pnas.1201453109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1201453109 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Fig. 1. Increased expression of IRF8 correlates with full effector function of OT-I cells and disease activity in K14-mOVA mice. (A) Synergistic effects of IL-15 and Ag peptide on the growth response of OT-I cells. Purified OT-I cells were cultured at 1.25 × 105 cells/mL (4-mL culture) in the presence of the same number of irradiated BMDC (peptide-pulsed or nonpulsed). Cells were counted on day 5 and divided by 5 × 105 to convert to a fold increase. Data are pooled from three independent experiments (error bars, SEM). (B) Synergistic effects of peptide and IL-15 in IFN-γ. Stimulation: peptide, 0.3 pg/mL of SIINFEKL; IL-15, 15 nM; peptide + IL-15, 0.3 pg/mL of SIINFEKL + 15 nM of IL-15. OT-I cells (2.5 × 104) were cultured with peptide-pulsed or nonpulsed BMDCs (2.5 × 104)inthe presence or absence of IL-15. Three days later, production of IFN-γ in the supernatants was determined by an ELISA (error bars, SEM). *P < 0.05 between groups. Data are representative of two independent experiments with duplicates in each experiment. (C) Venn diagram depicting the overlap and distinction of gene expression between OT-I cells stimulated with three different treatments (peptide, IL-15, and peptide + IL-15 as in B) for 6 or 12 h (adjusted P < 0.05; fold change > 1.5). Gene expression in each of the stimulated OT-I cells was measured by DNA microarray analysis to a single reference (untreated naive OT-I cells). (D) Induction of IRF8mRNA requires cooperation of cytokine and Ag-peptide stimulation: peptide, 0.3 pg/mL of SIINFEKL; IL-15, 15 nM; peptide + IL-15, 0.3 pg/mL of SIINFEKL + 15 nM of IL-15. OT-I cells were cultured as for A but harvested for RNA extraction 6 or 12 h later. Data represent the microarray assessment of the IRF8mRNA expression. An asterisk indicates adjusted P < 0.05 vs. untreated OT-I cells. 15 nM of IL-15 induced maximum proliferation (Fig. 1A and Fig. number of genes induced by the peptide alone dropped to 311 S1B). These combination treatments also induced IFN-γ pro- genes (Fig. 1C), suggesting the rather transient nature of Ag/ duction (Fig. 1B), indicating that OT-I cells gained effector costimulatory signaling under these conditions.