Apurinic/Apyrimidinic Endonuclease 1/Redox Factor-1 (Ape1/Ref-1) Modulates Antigen Presenting Cell-mediated T Helper Cell Type 1 Responses*

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ape1/Ref-1) is a multifunctional protein possessing DNA repair, redox control, and transcriptional regulatory activities. Although Ape1/Ref-1 plays multiple roles in the immune system, its functions in helper T (Th) cell activation and differentiation are largely unknown. In this study, the function of Ape1/Ref-1 in Th cell activation was analyzed using an Ape1/Ref-1 redox-specific inhibitor, E3330. When splenocytes from OT-II mice, which are ovalbumin (OVA)-specific T-cell receptor transgenic mice, were activated with OVA in the presence of E3330, the induction of IFN-γ-producing OT-II T cells was significantly increased. In contrast, E3330 did not enhance IFN-γ production from plate-bound anti-CD3 antibody-stimulated CD4+ T cells in the absence of antigen presenting cells (APCs). Furthermore, E3330-pretreated and OVA-pulsed APCs also enhanced the IFN-γ production from OT-II T cells. These results suggested that E3330 enhances Th1 responses by modifying APC function. E3330 did not alter the surface expression of MHC-II or the co-stimulatory molecules CD80 and CD86 on APCs. On the other hand, E3330 up-regulated the IL-12 p35 and p40 gene expression, and IL-12 surface retention, but decreased the IL-12 secretion from Toll-like receptor (TLR) ligand-stimulated APCs. These results were confirmed with Ape1/Ref-1 knockdown experiments. Taken together, our findings indicated that the suppression of Ape1/Ref-1 redox function leads to an increased cell surface retention of IL-12 and enhances Th1 responses. This is the first study to demonstrate that Ape1/Ref-1 modulates the IL-12 production and secretion from APCs and controls Th1 immune responses.

a redox control domain and a DNA repair domain (1)(2)(3). Ape1/Ref-1 functions in DNA base excision repair by virtue of its apurinic/apyrimidinic endonuclease activity, and in DNA proofreading by its exonuclease activity, and also modulates the DNA binding activity of several transcription factors, including nuclear factor-B, early growth response protein-1, p53, activator protein (AP)-1, cyclic response element-binding protein, and hypoxia-induced factor-1␣ (4,5), via its redox activity.
Ape1/Ref-1 plays several roles in the immune system. The redox activity of Ape1/Ref-1 is involved in CD40-mediated B cell activation through Pax5 transcription factor activation (6), whereas its AP endonuclease activity is essential for immunoglobulin class-switch recombination (7). Moreover, Ape1/ Ref-1 modulates TLR2-mediated inflammatory responses in primary keratinocytes by activating nuclear factor-B and hypoxia-induced factor-1␣, resulting in the expression of inflammatory cytokines and chemokines (8). Our group also reported that Ape1/Ref-1 is essential for IL-21-induced ERK1/2 signaling in a mouse pro-B cell line (9). Despite the accumulating evidence that Ape1/Ref-1 plays various roles in the immune system, its function in T cell immune responses has not been thoroughly investigated.

Inhibition of the Redox Function of Ape1/Ref-1 with E3330 Enhances the Induction of IFN-␥-Producing OT-II T Cells Upon
OVA Stimulation-To determine the role of the redox function of Ape1/Ref-1 in naïve helper T cell activation and differentiation, splenocytes from OT-II mice were stimulated with OVA and/or Pam3 CSK4 (Pam3) for 3 days in the presence or absence of E3330, and then analyzed by intracellular IFN-␥ staining and flow cytometry. Notably, E3330 significantly increased the induction of IFN-␥-producing OT-II T cells upon OVA, or OVA and Pam3 stimulation (Fig. 1, A and B). Secreted IFN-␥ in the culture medium, determined by cytometric bead array (CBA) analysis, was also up-regulated in E3330-treated splenocytes stimulated with OVA or OVA/Pam3 (data not shown). These results suggested that the redox function of Ape1/Ref-1 suppresses Th1 immune responses.
E3330 Has No Effect on the Anti-CD3/Anti-CD28 Antibody-mediated Induction of IFN-␥-producing CD4 ϩ T Cells in the Absence of APCs-The above findings prompted us to investigate whether E3330 acts directly on T cells. To address this question, we used anti-CD3 antibodies to stimulate CD4 ϩ T cells in the absence of APCs. Purified CD4 ϩ T cells were activated with plate-bound anti-CD3 antibodies and soluble anti-CD28 antibodies, and then cultured in the presence or absence of E3330. E3330 had no signifi-cant effect on the induction of IFN-␥-producing T cells in the absence of APCs (Fig. 2, A and B), suggesting that E3330 may affect APCs, but not T cells, during Th1 cell differentiation.

E3330 Enhances the Induction of OT-II IFN-␥-producing T Cells by Altering the Function of Antigen Presenting Cells-To
test the hypothesis that E3330 modulates OT-II T cell differentiation through effects on APCs, splenic DCs or BMDCs were pretreated with E3330 and pulsed with OVA or OVA-(323-339) peptide, respectively. The E3330 was then washed away, and the pretreated DCs were co-cultured with the OT-II T cells. The E3330-pretreated OVA-or OVA peptide-pulsed DCs induced more IFN-␥-producing CD4 ϩ OT-II T cells than did the control OVA or OVA peptide-pulsed DCs (Fig. 3, A and B). These results further suggested that E3330 enhances Th1 responses by altering APC function.
E3330 Has No Effect on the Expression of Co-stimulatory Molecules but Enhances IL-12 Expression on the Cell Surface of BMDCs-Next, we explored the mechanism by which E3330 modulated the APC function. In addition to the MHC-II-pre-  sented antigenic peptide, the B7 co-stimulatory molecules CD80 and CD86 expressed on APCs provide indispensable activation signals to naïve CD4 ϩ T cells (13). The treatment of BMDCs with E3330 alone had no significant effect on the cell surface expression of these molecules (Fig. 4). Although the TLR ligand LPS or Pam3 clearly up-regulated the expression of CD80 and CD86, and up-regulated MHC-II to a lesser extent, the addition of E3330 had no further effect on the expression of these molecules (Fig. 4).
Next, we examined the effect of E3330 on the TLR ligandstimulated expression of cytokines in BMDCs. Although treating BMDCs with LPS or Pam3 increased the secretion of IL-6, IL-12, and TNF-␣, the addition of E3330 did not further enhance the secretion of these cytokines. On the contrary, E3330 had a mild, but statistically significant inhibitory effect on the LPS-induced IL-12 level (Fig. 5). Because IL-12 plays a key role in Th1 differentiation, we further investigated the effect of E3330 on IL-12 expression. Notably, we found that E3330 significantly enhanced the Pam3-induced mRNA expression of Il12a and Il12b, which, respectively, encode the p35 and p40 subunits of IL-12 ( Fig. 6). Taken together, these results suggested that E3330 enhances the IL-12 mRNA expression, but suppresses the IL-12 protein secretion. IL-12p70 is reported to be localized to the cell surface membrane before its secretion (14,15). We therefore examined the cell surface IL-12 by flow cytometry analysis. As expected, E3330 significantly up-regulated the surface expression of IL-12 on BMDCs (Fig. 7,  A and B).
A genetically engineered membrane-bound form of IL-12 is reported to be active (16,17). To confirm that the membranebound IL-12 retains its cytokine function, we stimulated splenic CD4 ϩ T cells with various concentrations of plate-bound IL-12 and plate-bound anti-CD3 and anti-CD28 antibodies. The solid-phase IL-12 promoted the differentiation of CD4 ϩ T cells to IFN-␥-producing Th1 cells in a dose-dependent manner (Fig.  8). These results strongly suggested that instead of secreted, soluble IL-12, the IL-12 expressed on the APC surface enhanced Th1 differentiation.
E3330 Enhances the Pam3-induced Activity of p38 MAPK, an Upstream Regulator of IL-12 Gene Expression-To gain insight into the intracellular signaling mechanisms that mediate the E3330-enhanced activation of IL-12 genes in BMDCs, we focused on the mitogen-activated protein kinase (MAPK) signaling pathway, one of the most conserved signaling pathways in mammalian cells. MAPKs consist of three major subgroups: the p38 MAPKs, extracellular-regulated kinases (ERKs), and c-Jun N-terminal kinases (JNKs). The MAPKs play various roles in regulating Th1/Th2 balance (18), and p38 MAPK is specifically involved in the regulation of IL-12 gene expression (19). Therefore, we investigated the effect of E3330 on p38 MAPK phosphorylation in BMDCs, and found that E3330-pretreated BMDCs exhibited enhanced p38 MAPK phosphorylation after Pam3 stimulation (Fig. 9A). This finding indicated that the redox function of Ape1/Ref-1 might inhibit the TLR ligand-induced p38 MAPK activation and IL-12 gene expression in APCs.

Discussion
Here we demonstrated that Ape1/Ref-1 modulated the helper T cell activation and differentiation through the modifi-   NOVEMBER 4, 2016 • VOLUME 291 • NUMBER 45

JOURNAL OF BIOLOGICAL CHEMISTRY 23675
Because Ape1/Ref-1 is a multifunctional protein, it may regulate TLR signaling through molecules other than the p38 MAPK. For example, Ape1/Ref-1 is known to be required for AP-1 activity. Interestingly, TLR signaling-induced c-Fos expression and AP-1 activity appear to negatively regulate both IL-12 gene activations (20). Therefore, we analyzed the TLR ligand stimulation-induced activity of ERK1/2, which are upstream signaling molecules of c-Fos and AP-1. However, E3330 had no effect on this ERK1/2 activity (data not shown), suggesting that the inhibition of Ape1/Ref-1 by E3330 mainly affects the p38 MAPK signaling in the BMDCs.
IL-12 has both secreted and membrane-bound forms (14,15). Given the divergent effects of E3330 on cell surface retention and secretion of IL-12 from BMDCs, we hypothesized that the inactivation of the redox activity of Ape1/Ref-1 may inhibit shedding of IL-12 from the cell surface. IL-6 receptor, which shares sequence homology with IL-12p40 (21), is shed from the cell surface by a disintegrin and metalloprotease 17 (ADAM17), a member of the ADAM metalloprotease family that was originally identified as TACE (TNF-␣ converting enzyme), or ADAM10 (22,23). Therefore, a similar mechanism may be involved in IL-12 shedding from the cell surface. Thus, the inactivation of the redox activity of Ape1/Ref-1 may inhibit extracellular proteases, such as ADAM family enzymes, resulting in the accumulation of IL-12 on the cell surface. Further studies will be required to clarify the mechanism by which Ape1/Ref-1 controls the release of IL-12 from the cell surface.
Cell surface-expressed IL-12 appears to be active, given that a genetically engineered membrane-bound form of IL-12 is biologically active (16,17), and naïve Th cell activation requires cell-cell interaction with APCs via an immunological synapse. Furthermore, our finding that plate-bound IL-12 functions as a Th1-inducing cytokine strongly supports this possibility. Although we do not know whether the cell surface-expressed IL-12 is more effective for promoting the differentiation of Th1 cells than secreted IL-12, it is likely to be responsible for Th1 cell differentiation under physiological conditions. The Ape1/Ref-1 redox function-specific inhibitor E3330 has therapeutic potential for many tumors (24). Our finding that the inhibition of the redox function of Ape1/Ref-1 enhances the IFN-␥-producing Th1 response may reveal one of the mechanisms behind the E3330-induced anti-tumor activity in vivo.

Ape1/Ref-1 Modulates Helper T Cell Responses
Ape1/Ref-1 redox suppression by small molecule inhibitors, such as E3330, may be a useful therapeutic approach for inducing anti-tumor immunity.

Experimental Procedures
Mice-C57BL/6J mice were purchased from CLEA Japan. OT-II transgenic mice (OT-II mice) (25), which express T cell receptor (TCR) ␣ and ␤ chains that recognize the MHC class II I b -restricted OVA peptide (residues 323-339) in a C57BL/6J background, were kindly provided by Dr. W. Heath (WEHI, Melbourne, Australia). The mice were bred at the animal facilities of Yamagata University, Faculty of Medicine, under specific pathogen-free conditions and were used for experiments at 6 -12 weeks of age. All of the animal experiments were approved by the Animal Experiment Committee of Yamagata University, Faculty of Medicine.
Preparation of Splenocytes and Isolation of CD4 ϩ T Cells-Mouse spleen was homogenized with a glass homogenizer. The cell suspensions were passed through nylon mesh, and the cells were suspended in RPMI 1640 medium containing L-glutamine and 25 mM HEPES and supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 50 M 2-mercaptoethanol, 100 units/ml of penicillin, and 100 g/ml of streptomycin (complete RPMI 1640 medium). CD4 ϩ T cells were isolated using BD TM IMag anti-mouse CD4 particles (BD Biosciences) according to the manufacturer's instructions. To activate CD4 ϩ T cells with Abs, purified CD4 ϩ T cells were treated with anti-CD3⑀ (plate-bound, 10 g/ml; catalog number 100314, lot number B133074, BioLegend) and anti-CD28 (2 g/ml; catalog number 16-0281-85, lot number E06391-1637, eBioscience) Abs.
To investigate whether solid-phase IL-12 functions as a cytokine, we pre-treated culture plates with recombinant murine IL-12 for 2 h at 37°C. The IL-12 was washed out completely, and the plates were further coated with anti-CD3⑀ and anti-CD28 Abs. After rinsing these Abs, purified splenic CD4 ϩ T cells were cultured in the pretreated plates in the presence of IL-2 (10 units/ml).
Preparation of BMDCs in Vitro-BMDCs were prepared as previously described (27). Briefly, bone marrow cells were isolated from the femurs and tibias of 5-10-week-old mice by flushing with PBS. The red blood cells were lysed using ACK buffer, and the cells were cultured in bacterial dishes in complete RPMI 1640 medium containing 10 ng/ml of murine GM-CSF. The medium was replaced every 2 or 3 days, and the

Ape1/Ref-1 Modulates Helper T Cell Responses
BMDCs that were generated after 6 -8 days of culture were used in experiments.
Flow Cytometry Analysis-Intracellular cytokine staining and flow cytometry analysis of T cells were performed as described previously (28). T cells were stimulated with 25 ng/ml of PMA and 1 M ionomycin in the presence of 1 g/ml of brefeldin A for the last 4 h of culture, followed by cell surface CBA Analysis-Cytokines in the cell culture supernatants were analyzed by CBA (BD Biosciences) as described previously (29). A customized set of fluorescent anti-cytokine multiplex microbeads was obtained together with recombinant standards with which to prepare a standard curve for each cytokine. The assays were performed on a FACSCanto II flow cytometer, and the data were analyzed using FCAP Array software (version 1.0, BD Bioscience).
Quantitative Real-time RT-PCR Analysis-Total RNA isolation and quantitative RT-PCR were performed as described previously (30). Thermo-cycling was performed using a Light-Cycler (Roche Molecular Systems). The IL-12 p35 and p40 mRNA levels were expressed relative to the corresponding ␤-actin mRNA level. The primer sets used in the real-time RT-PCR were as follows: mouse IL-12 p35 sense, 5Ј-ATGTGTCA-ATCACGCTACCTCC-3Ј and antisense, 5Ј-GAAGAAGTCT-