Interferon regulatory factor 8 integrates T-cell receptor and cytokine-signaling pathways and drives effector differentiation of CD8 T cells

Abstract

We recently demonstrated that differentiation of cytotoxic T cells requires cooperation between T-cell receptor (TCR)/costimulation and γc-cytokines. Here we demonstrate that the transcription factor IFN regulatory factor 8 (IRF8) is expressed in CD8 T cells by the combination of these two signals. More importantly, depletion of IRF8 in these cells abrogated the differentiation of naive CD8 T cells into effector cells in an experimental graft-vs.-host disease mouse model. We also show that IRF8 seems to not operate upstream of other critical factors such as T-bet and eomesodermin, which have been implicated in effector maturation. Collectively, our work shows that IRF8 integrates the TCR/costimulation and γc-cytokine–signaling pathways and mediates the transition of naive CD8 T cells to effector cells, thus identifying IRF8 as one of the molecular regulators of CD8 T-cell differentiation.

CD8 T cells are essential for the adaptive immune response against various intracellular pathogens and tumors. A typical CD8 T-cell response consists of three phases: clonal expansion of antigen (Ag)-specific cells and acquisition of effector functions, contraction of the effector cells through apoptosis, and generation of long-lived memory cells (1–4). The acquisition of effector functions is critical for the control of intracellular pathogens and tumors and is accompanied by the production of cytotoxic molecules, perforin and granzyme, as well as two main cytokines, IFN-γ and tumor necrosis factor (TNF) (5–7).

The transition from naive to effector CD8 T cells requires marked changes in gene expression (8) mediated by transcription factors (5). The T-box transcription factors, T-bet (9, 10) and eomesodermin (Eomes) (11), are the best-described regulators of this process in CD8 T cells (11, 12).

We previously reported that transgenic mice expressing ovalbumin (OVA) protein by the keratin 14 promoter (K14-mOVA^high mice) develop graft-vs.-host disease (GvHD) after the transfer of OT-I CD8 cells, whereas similar transgenic mice expressing much lower OVA copy numbers (K14-mOVA^low mice) did not (13, 14). We also demonstrated that the onset of GvHD in this model is dependent only on CD8 T cells. The injection of γc-cytokines (15), especially IL-15, could create GvHD in K14-mOVA^low mice upon OT-I cell transfer, whereas K14-mOVA^high mice on an IL-15KO background failed to develop GvHD upon OT-I cell transfer (13). These data suggested a critical requirement for γc-cytokines in the effector differentiation of CD8 T cells in vivo. We then hypothesized that concurrent signaling events consisting of the γc-pathway and the T-cell receptor (TCR)/costimulation pathway are critical for the transition of naive CD8 T cells into effector cells.

In this study, we sought a molecular integrator of these two pathways in the effector differentiation of CD8 T cells and identified IFN regulatory factor 8 (IRF8). IRF8 belongs to the IFN regulatory factor family (16–18) and critically controls the lineage commitment between the myeloid and B cells (19–26). In addition, IRF8 controls a silencing program for Th17 cell differentiation (27). Nevertheless, the role of IRF8 in CD8 T cells remains elusive; IRF8KO mice manifest impaired CD8 T-cell responses against certain viruses (22). Thus, we embarked on a detailed assessment of the role of IRF8 in CD8 T cells.

Here, we demonstrate the critical need for the convergence of γc-Jak3 and TCR/costimulation-signaling pathways in the transcription of IRF8 and demonstrate that removal of IRF8 cripples CD8 effector T cells. Associated with these findings, IRF8⁻OT-I cells failed to induce GvHD in our model (28). We also show that either IRF8 action is independent of that of T-bet and Eomes (9–11) or IRF8 may operate downstream of these factors, but not by way of them, and therefore propose that IRF8 be added to the roster of critical regulators for the differentiation of naive CD8 T cells into effector cells.

Results

Suboptimal Antigen Stimulation and a γc-Cytokine Cooperatively Generate Functional CD8 T Cells.

We previously developed an experimental model of GvHD (28) and reported that cooperation of γc-cytokine(s) and Ag/TCR-signaling events is indispensable for disease development. Briefly, a membrane-bound form of chicken OVA was expressed in mice under the K14 promoter. Adoptive transfer of syngeneic OT-I cells (CD8⁺Vα2⁺Vβ5⁺) that recognize OVA in these mice causes GvHD-like pathogenic changes (28). In a strain with lower OVA copy number (K14-mOVA^low mice), GvHD did not occur spontaneously after adoptive transfer of OT-I cells but occurred when cells were injected with IL-15 (and other selective γc-cytokines) (13) (Fig. S1A). Furthermore, deletion of IL-15 by crossing K14-mOVA^high mice (mice with high OVA copy number) with IL-15KO mice abrogated disease (13). These observations led us to postulate that TCR and γc-cytokine signals cooperate physiologically to enable full maturation of effector T cells. To test this hypothesis, serially diluted OVA–peptide was cultured with OT-I cells in the presence or absence of IL-15. Amounts greater than 10 pg/mL of the peptide induced a maximal proliferative response by OT-I cells, and 0.3 pg/mL was a suboptimal dose of peptide that induced OT-I proliferation (Fig. 1A and Fig. S1B). However, at doses between 0.03 and 3 pg/mL of peptide, the cellular proliferative response was greatly enhanced by the presence of IL-15, and combinatorial treatment of 0.3 pg/mL of peptide and 15 nM of IL-15 induced maximum proliferation (Fig. 1A and Fig. S1B). These combination treatments also induced IFN-γ production (Fig. 1B), indicating that OT-I cells gained effector function. These results suggest that partial-to-low T-cell responses by suboptimal challenges of Ag can develop into a full response by the concomitant presence of certain γc-cytokines, an observation that mirrors our previous in vivo results (13). Thus, we conclude that γc-signaling, but not that of a particular γc-cytokine, is required to affect these changes. We also observed that similar cooperation between a γc-cytokine and the TCR/costimulation pathways operates in polyclonal CD8 T cells.

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) in the 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.

Transcription Factor IRF8 Potentially Integrates γc-Cytokine and TCR/Costimulation Signals.

To determine the intracellular events by which γc-cytokine signaling cooperates with TCR/costimulation, we conducted microarray analyses using cDNAs prepared from OT-I cells stimulated by (1) Ag alone (peptide 0.3 pg/mL) (2), cytokine alone (IL-15 15nM), or (3) a combination of both (Fig. 1A, arrows). Costimulation by CD28 was always present because we used bone marrow dendritic cells (BMDC) as antigen presenting cells (APC). This dose of peptide, although suboptimal as assessed by the proliferative response of OT-I cells (Fig. 1A), caused the induction of 2,825 transcripts at 6 h of stimulation (Fig. 1C), and IL-15 stimulation induced 2,889 transcripts. When these two signals were combined, induction of 2,088 genes was observed, but the combination led to the induction of only 137 distinct transcripts. The picture dramatically changed at 12 h; the number of genes induced by the peptide alone dropped to 311 genes (Fig. 1C), suggesting the rather transient nature of Ag/costimulatory signaling under these conditions. Cytokine signaling seemed more persistent (1,064 genes remain induced), but the combination of two pathways induced 921 distinct genes (Fig. 1C), making the majority of the genes in this pool unique only to the combinatorial signal. Among the 921 genes, 144 genes were persistently detected in the combination group at both 6- and 12-h time points, whereas 748 genes were newly induced at 12 h. Because neither cytokine alone nor peptide alone led to the robust proliferation of, and production of IFN-γ by, OT-I cells (Fig. 1 A and B), we reasoned that a critical transcription factor(s) that causes the differentiation of effector CD8 T cells via de novo protein synthesis may be found in this gene pool of 144 genes (Table S1). The results indicate the initiation of a robust transcription network in CD8 T cells upon activation. As a first step to delineate such a network, we identified transcription factors in the pool of induced genes (Table S1). We thus identified IRF8 and c-jun and chose IRF8 for subsequent study (Fig. 1D and Table S1).

Other key transcription factors known to control CD8 T-cell effector functions, such as T-bet and Eomes, were strongly and persistently induced by IL-15 alone, suggesting that IRF8 and T-bet/Eomes may belong to different categories (Fig. S1C). We do not know, however, if a strong Ag stimulation alone would induce these two factors (Discussion). The microarray results were validated by real-time PCR (Fig. S1D, Table S2) and Western blot analysis (Fig. S1E).

To determine whether our observation could be extrapolated to polyclonal CD8 T cells, we treated CD8 T cells with suboptimal doses of αCD3mAb and IL-15, and they expressed higher levels of IRF8mRNA than either treatment alone (Fig. S1F). OT-I cells stimulated with suboptimal doses of αCD3mAb+IL-15 (Fig. S1G) responded similarly in experiments depicted in Fig. S1 D and F. Thus, we hypothesized that the induction of IRF8 could be linked to the acquisition of effector functions by CD8 T cells.

Reasonable Correlation of IRF8 Expression with Disease Activity.

We next asked whether IRF8 expression correlates with the activation status of OT-I cells and the clinical course of GvHD in K14-mOVA mice. To this end, 2 d after OT-I cells were injected into congenic (CD45.2) K14-mOVA^low, K14-mOVA^high, and IL-15 treated K14-mOVA^low mice, we sorted the transferred OT-I cells by CD45.1 expression (Fig. S1A). Cells from K14-mOVA^high mice expressed the highest levels of IRF8 followed by those sorted from K14-mOVA^low mice treated with IL-15 and those sorted from K14-mOVA^low mice (Fig. S1H). Isolation of injected OT-I cells at earlier time points yielded too few cells for assessment although they may express higher IRF8 expression than the 48hr-OT-I cells. Nonetheless, this result suggests that the up-regulation of IRF8 correlates with effector function and disease activity in these K14-mOVA mice.

IRF8 Is an Activation Molecule in CD8 T Cells.

IRF8 is the only IRF family member whose expression is confined to immune cells. However, its expression in CD8 T cells has not been extensively studied. Our results (Fig. S2 A and B) demonstrate the activation-associated expression of IRF8 in OT-I cells. Similar results were obtained with polyclonal CD8 T cells (Fig. S2C), confirming that IRF8 is expressed in CD8 T cells upon activation.

Effector Maturation of CD8 T Cells Is Dependent on the Integration of Jak-STAT and TCR/Costimulation-Signaling Pathways via IRF8.

Our studies suggested that concurrent signaling through γc-cytokine and TCR/costimulation pathways may be critical in the differentiation of functional effector CD8 T cells via IRF8. This finding prompted us to determine whether blocking either of these pathways individually would abrogate these outcomes. To test this hypothesis, we first used a Jak3 inhibitor, CP-690,550 (hereafter referred to as CP) (29), to block endogenous Jak3-STAT signaling upon T-cell activation. Cells stimulated by high doses of Ag without exogenous cytokine with various concentrations of CP were tested for IRF8mRNA expression. CP blocked the induction of IRF8mRNA in these cells (Fig. 2A). Moreover, this blockade was seen with polyclonal CD8 T cells (Fig. S2D). Notably, CP did not inhibit the proliferation of OT-I cells (Fig. 2B) nor did it inhibit the induction of the expression of CD44 (Fig. 2C and Fig. S2E), excluding nonspecific toxic effects of this inhibitor. Interestingly, the induction of CD25 and CD69 was inhibited by CP (Fig. 2 D and E and Fig. S2E) in agreement with the previous suggestion that Jak3-STAT5 regulates the expression of these genes (30, 31). Moreover, the CP treatment reduced the amount of IFN-γ production by OT-I cells (Fig. 2F). These results suggest that the γc-signaling is intrinsic to the immunologic activation of CD8 T cells for the full generation of functional effector cells and that IRF8 could be critical in the integration of the TCR/costimulation and Jak-STAT–signaling pathways. Likewise, we then used a Zap70 inhibitor, piceatannol. Piceatannol inhibited IRF8 expression (Fig. 3A) and proliferation (Fig. 3B) and IFN-γ production (Fig. 3C) in OT-I cells in a dose-dependent manner in agreement with our proposed model for the requirements for IRF8 (Fig. S3).

Fig. 2.

Specific inhibition of γc-signaling compromises the induction of IRF8. (A) IRF8mRNA levels in OT-I cells cultured in the presence of CP-690,550. Two million cells/2 mL were stimulated with plate-bound αCD3mAb (10 μg/mL) and αCD28mAb (5 μg/mL) in the presence of various concentrations of CP or DMSO (vehicle) for 6 h. The IRF8mRNA level was determined by real-time PCR. Fold changes in reference to naive cells are shown. Data are pooled from two independent experiments with triplicates in each experiment (error bars, SEM). (B) Proliferation of OT-I cells was not affected by CP. CFSE-labeled OT-I cells were cultured as above with or without CD3/CD28 stimulation for 2 d. Cells were gated on CD8Vα2 cells. Data are representative of two independent experiments. (C–E) Expressions of activation markers on cultured OT-I cells in the presence of CP. OT-I cells were cultured as in A for 2 d and stained for Vα2, Vβ5, CD44, CD25, and CD69. Cells were gated by Vα2 and Vβ5. Data shown are representative of three independent experiments. The mean fluorescence intensity (MFI) of Fig. S2E was determined by flow cytometry. (F) IFN-γ production from OT-1 cells in the presence of CP. OT-I cells (1 × 10⁵) were cultured in 96-well plate as in A, and production of IFN-γ was determined by ELISA at the 48-h time point. Data are representative of two independent experiments with duplicates in each experiment (error bars, SEM).

Fig. 3.

Inhibition of the TCR signaling abrogates the induction of IRF8. (A) IRF8mRNA levels in OT-I cells cultured in the presence of piceatannol. OT-I cells were cultured as in Fig. 2A but with piceatannol or DMSO for 6 h. Data are pooled from two independent experiments with triplicates in each experiment (error bars, SEM). (B) Dose-dependent inhibition of proliferation of OT-I cells by piceatannol. CFSE-labeled OT-I cells were cultured as in Fig. 2B but with piceatannol. Data are representative of two independent experiments. (C) IFN-γ production from OT-1 cells is impaired in the presence of piceatannol. OT-I cells were cultured as in Fig. 2F but with piceatannol or DMSO. Data are representative of two independent experiments with duplicates in each experiment (error bars, SEM).

Deletion of IRF8 from OT-I Cells Attenuates GvHD Responses in Vivo.

To determine if IRF8 is directly involved in the effector function of OT-I cells, we generated OT-I mice on an IRF8KO background. Significant numbers of Vα2⁺Vβ5⁺OT-I cells were present in all lymphoid organs of OT-I/IRF8KO mice, indicating that IRF8 is dispensable for the development of CD8 T cells (Fig. S4). Like the IRF8KO mice (22), OT-I/IRF8KO mice also had lymphadenopathy and splenomegaly and manifested increased percentages of Gr-1⁺CD11b⁺ granulocytes (Fig. S4). We assessed the acquisition of effector functions of IRF8⁻OT-I cells in vivo using our K14-mOVA^high mice (13, 28). Effector maturation of OT-I cells was examined by the production of IFN-γ and by the manifestation of cytotoxicity against skin. Disease activity correlates well with the activation status of injected OT-I cells (13). IRF8⁻OT-I cells failed to cause GvHD in K14-mOVA^high mice as judged by the lack of apparent weight loss and pathological skin lesions (Fig. 4 A and B). Histology of the ears showed that IRF8⁻OT-I cells caused no pathological changes (Fig. 4C). These data strongly indicate that IRF8 critically controls processes that lead to effector differentiation of OT-I cells in vivo.

Fig. 4.

IRF8⁻OT-I cells induce less severe GvHD than do OT-I cells after adoptive transfer into K14-mOVA^high mice. (A) Kinetic change of body weight of OT-I–injected K14-mOVA^high mice. One million naive OT-I or IRF8⁻OT-I cells were injected into K14-mOVA^high mice. The mice were weighed daily, and skin lesions were monitored for 14 consecutive days. Pooled results from two (of six) independent experiments are shown (n = 9 each; error bars, SEM). **P < 0.001 and *P < 0.05. (B) Development of skin lesions in K14-mOVA^high mice. Photos were taken on day 14 after injection of cells. (C) Histology of the ears of K14-mOVA^high mice on day 14 after injection of cells. Magnification: 20×.

Relationship Between IRF8 and T-bet/Eomes.

T-bet and Eomes have been shown to be critical in the functional activation and effector differentiation of CD8 T cells (9–11). Because the role of IRF8 defined in this study somewhat resembles those of T-bet/Eomes, we asked whether or not IRF8 operates dependently on these T-box factors. As described earlier, microarray analysis suggested that the transcriptional requirement for the T-box factors (γc-signal alone) is different from that for IRF8 (both γc-cytokine and TCR/costimulation). Real-time PCR experiments revealed that the induction of T-bet (Fig. S5 A and B, Table S2) and Eomes (Fig. S5 A–D, Table S2) did not differ much in the presence or absence of IRF8, suggesting that either IRF8 operates completely independently of these T-box factors or at least IRF8 is not situated upstream of T-box factors. Furthermore, the Jak3 inhibitor, but not the Zap 70 inhibitor, blocked T-bet in a dose-dependent manner (Fig. S5 C and D).

IRF8⁻OT-I Cells Have Full Proliferative Capability but Produce Less IFN-γ.

Thus far our studies have defined a unique role of IRF8 in the in vivo activation of naive CD8 T cells. We next determined which compartment of T-cell activation might be controlled by IRF8 in vivo. First, we injected OT-I and IRF8⁻OT-I cells into K14mOVA^high mice and monitored their proliferation. Carboxyfluorescein diacetate succinimidyl ester (CFSE) experiments showed that OT-I cells proliferated similarly in the presence or absence of IRF8 in response to OVA (Fig. S6A), suggesting that IRF8 does not directly control the clonal expansion of the cells. Consistently, IRF8KO mice do not manifest any deficit in their T-cell compartments. These pieces of data strongly suggest that γc-cytokine and TCR/costimulation pathways are integral components enabling the functional maturation of CD8 T cells and that IRF8 functions as the molecular bridge integrating these two distinct pathways.

The total number of OT-I and IRF8⁻OT-I cells in secondary lymphoid organs did not differ between K14-mOVA^high mice injected with OT-I or with IRF8⁻OT-I cells (Fig. S6B). However, when assessing activation markers, injected OT-I cells exhibited an activated phenotype (CD25^highCD44^highCD62L^low) whereas in IRF8⁻OT-I cells there was less up-regulation of CD25 and greater expression of CD62L (Fig. S6C), suggesting that the lack of IRF8 selectively compromised the activation state of OT-I cells. Thus, IRF8 seems to be involved in the phenotypic maturation of CD8 T cells, but is dissociated from the clonal expansion. This finding is reminiscent of the effect of the Jak3 inhibitor CP that only impaired activation marker expression of OT-I cells without affecting their proliferative response to the Ag (Fig. 2 B–E). Next, we determined the relevance of IRF8 in the effector function of CD8 T cells. IFN-γ serum levels and intracellular staining were significantly lower in the K14-mOVA^high injected with IRF8⁻OT-I cells (Fig. 5A and Fig. S7A). In contrast, TNF-α, another effector cytokine of CD8 T cells, was produced equally in both OT-I and IRF8⁻OT-I cells (Fig. S7B), suggesting that the transcriptional regulation of IFN-γ and TNF-α is distinct. These results strongly suggest that IRF8 is critically and selectively involved in the acquisition of effector functions by CD8 T cells.

Fig. 5.

IRF8 mediates IFN-γ induction and killing activity in vivo. (A) Serum IFN-γ concentrations on day 5 after adoptive transfer. One million OT-I or IRF8⁻OT-I cells were transferred into WT or K14-mOVA^high mice. Five days later, the serum IFN-γ level was measured by an ELISA (n = 5/group; error bars, SEM). Data were pooled from two independent experiments. (B) In vivo killing activity of OT-I cells or IRF8⁻OT-I cells (5 d after they were adoptively transferred into K14-mOVA^high mice) was assessed by flow cytometry. CFSE intensity of cells in the mice was determined 6 h after injection of a 1:1 mixture of SIINFEKL-pulsed splenocytes labeled with a high concentration of CFSE and unpulsed splenocytes labeled with a low concentration of CFSE. Numbers above the bracketed lines indicate the percentage of CFSE^high cells. Data are representative of two independent experiments.

IRF8 Controls Cytotoxicity.

We next determined whether or not IRF8 deletion impairs the Ag-dependent cytotoxicity of OT-I cells. To address this question, we used an in vivo cytotoxic T lymphocyte (CTL) assay. IRF8⁻OT-I cells in K14-mOVA^high mice showed modest, but significantly lower, cytotoxicity than OT-I cells under the same conditions (Fig. 5B). The reduction in IFN-γ production and of the killing ability seen at day 5 is likely to be relevant to the lack of development of GvHD in K14-mOVA^high mice because their kinetics paralleled that of the clinical symptoms of K14-mOVA^high mice injected with OT-I cells (Fig. 4A). It should be noted that Perforin and Granzyme mRNAs were also detected in the CD8 effector T cells, and like IRF8, both are inhibited by the Jak3 and Zap 70 inhibitors (Fig. S5 C and D, Table S2). Collectively, these results suggest that IRF8 is one of the critical factors in the development of effector CD8 T cells from naive precursors.

Discussion

Here we demonstrate (i) that γc-signaling concurrent or cooperating with TCR/costimulation is indispensable for the differentiation of effector CD8 T cells and (ii) that these two signaling cascades converge on IRF8, making this protein one of the critical signal integrator molecules in this process. Many studies have shown that TCR stimulation, per se, is insufficient for successful activation/maturation of T cells. Costimulatory molecules including CD28, CD40L, and OX40 have been integrated into the equation to account for missing signals (32). However, our study suggests that γc-cytokine signaling constitutes another critical element (i.e., the γc-Jak axis) in addition to the combination of TCR and costimulatory signals. Due to the dramatic depletion of major lymphoid cells in γc^−/− and Jak3^−/− mice (33–35), it has been difficult to precisely define the contribution of γc-cytokine signaling upon Ag-induced T-cell activation in the periphery.

Our in vitro experiments showed that the γc-cytokine effect becomes visible only at suboptimal doses of the Ag. However, the microarray analysis showed that even this nonproliferative TCR stimulation (peptide/MHC I and CD28) induced nearly 3,000 genes in agreement with previous observations that the outcome of T-cell activation (i.e., proliferation, cytokine production, cytokinesis, cytotoxicity, etc.) is hierarchically ordered depending on the intensity of the TCR signaling (36, 37). Moreover, the CP experiment implied the inherent need for γc-cytokine stimulation even at the point of T-cell activation in response to high doses of Ag. However, the combination of γc-cytokine and low-dose pMHC/TCR seems to qualitatively change the nature of the T-cell activation, as opposed to quantitatively amplifying the signaling, because the kinetics and repertoire of the gene activation dramatically changed (more lingering effect and newer genes differentially expressed) when γc-cytokine and pMHC/TCR stimulation were combined.

In vivo, T-cell activation occurs in a special milieu that is full of environmental factors including select γc-cytokines (i.e., IL-7 and -15). It is very likely that some T cells encounter relatively low levels of Ag and would still differentiate into functional effector cells with the γc-cytokines provided by the microenvironment. It is possible that CD8 T cells stimulated by high doses of Ag and those by concurrent cytokine/low-dose Ag may differentiate into different types of effector/memory T cells.

Which γc-cytokine may be involved in this function? Some γc-cytokines such as IL-2 or IL-4 are produced as a consequence of, and only after, T-cell activation (15). However, two γc-cytokines, IL-7 and IL-15, are produced by nonlymphoid cells (15, 38). Furthermore, we and others have demonstrated that IL-15 can be surface-born via IL-15Rα by dendritic cells (DCs) activated through TLR (the IL-15 transpresentation paradigm) (39, 40). Knowing that TLR signaling could facilitate the capacity of Ag to present to T cells by the same DCs, it is plausible that DCs can concurrently present a γc-cytokine and Ag to neighboring CD8 T cells. Our in vivo and in vitro data clearly support this proposal.

Here we have demonstrated that IRF8 is an important integrator of TCR/costimulation and γc-signaling. Moreover, IRF8 seems to contribute to the phenotypic maturation and functionality of CD8 effector T cells because IRF8⁻OT-I cells demonstrate a less diverse array of surface Ags upon activation and failed to induce GvHD when transferred into K14-mOVA^high mice, although IRF8⁻OT-I cells maintained a similar capacity of clonal expansion as OT-I cells.

Naturally, differentiation of naive CD8 T cells into effector and/or memory cells is transcriptionally regulated. Recent studies implicated the involvement of two related factors belonging to the T-box transcription factor family, T-bet and Eomes (5, 7), in the differentiation of CD8 T cells. In addition, several other factors have also been identified in this process, including Stat4, Stat5, Notch2, Blimp-1, and Bcl6 (7). Furthermore, Runx3 and Notch1 control Eomes expression (7). Thus, the formation of a network of transcription factors appears to support the differentiation of effector/memory T cells. Our studies suggest that IRF8 is an important and nonredundant component of this network. We also determined whether IRF8 controls CD8 T-cell differentiation via these transcription factors. Real-time PCR analyses demonstrated that the expression of the transcripts encoding T-bet, Eomes, and Runx3 was not affected by the deletion of IRF8 from OT-I cells, suggesting that IRF8 does not induce these factors. Further analysis will determine if T-bet and Eomes control IRF8 expression.

The concurrent response of γc-cytokine and TCR/costimulation to low doses of Ag may present another intriguing scenario for in vivo CD8 T-cell differentiation. Activation of CD8 T cells in autoimmunity and chronic viral infection may be dependent on a lesser amount of circulating Ag than that in acute immunologic activation. Such chronic disease conditions may also likely cause continuous production of γc-cytokines, in particular those of IL-7 and -15, by environmental cells. Assuming that the IRF8-mediated pathway would represent activation of CD8 T cells associated with a low dose of Ag in combination with select γc-cytokines, IRF8 may be the key player during chronic activation of CD8 T cells whereas T-box factors could primarily control acute CD8 T-cell activation. As a corollary, depletion of IRF8 should have a more profound effect in blocking pathological activation of CD8 T cells in autoimmunity and chronic viral infection and may present an opportunity for unique clinical intervention for these diseases.

Materials and Methods

Mice.

K14-mOVA mice (13, 28) and IRF8KO mice (22) have been described previously. OT-I/IRF8KO mice were generated by backcrossing OT-I mice with IRF8KO mice. These mice were housed in a clean conventional facility and bred and used in accordance with protocols approved by the Animal Care and Use Committee of the National Cancer Institute (41).

Microarray Gene Profiling Analysis.

OT-I cells were treated with OVA–peptide alone, IL-15 alone, or OVA–peptide + IL-15 for 6 or 12 h, and total RNA was extracted. Treated OT-I cells were then compared with naive OT-I cells that were cultured for 6 or 12 h without stimuli using Mouse Genome 430 2.0 array (Affymetrix). The details are described in SI Materials and Methods.

The protocols for the purification of cells and adoptive transfer, cell culture and cell growth assessment, ELISA, real-time PCR, antibodies, flow cytometry, CFSE labeling, intracellular cytokine staining, in vivo CTL assay, and histological analysis are described in detail in SI Materials and Methods.

Statistical Analysis.

Data were compared using a Student’s t test. Values of P < 0.05 were considered a significant difference.