JNK1 has divergent roles in regulating the effector functions of CD4+ and CD8+ T cells. However, the function of JNK1 in tumor immune surveillance is unknown. In this study, we show that similar to IFN-γ−/− mice, JNK1−/− mice are highly susceptible to tumor development after inoculation of both melanoma cell line B16 and lymphoma cell line EL-4. Using T cell depletion and reconstitution approaches, we show that CD8+ T cells, but not CD4+ T cells, from JNK1−/− mice are responsible for tumor susceptibility. JNK1−/− CD8+ T cells have an intrinsic defect in early IFN-γ gene transcription and production after activation by either anti-CD3/anti-CD28 Abs or dendritic cells loaded with specific Ag in vitro. The impaired IFN-γ production in JNK1−/− CD8+ T cells is associated with reduced expression of both T-bet and Eomesodermin, indicating that JNK1 regulates the transcription program of CD8+ T cells. Finally, JNK1−/− CD8+ T cells showed reduced perforin expression and impaired CTL function. Taken together, our results demonstrate that JNK1 plays an important role in tumor immune surveillance through regulating the effector functions of CD8+ T cells.

It has been clearly demonstrated that both IFN-γ and lymphocytes play a critical role in tumor immune surveillance (1, 2, 3). IFN-γ is an essential cytokine involved in the innate and adaptive immune responses against tumor development (1, 4), whereas both αβ and γδ T cells have been shown to be indispensable in tumor immunity, with their actions dependent on IFN-γ synthesis (3, 5, 6). CD8+ T cells are key players in adaptive immune responses, and have been shown to be crucial for protective immune responses against viruses, intracellular bacteria and a wide range of tumors through cytokine expression and cytolytic activity. However, compared with CD4+ T cells, the molecular mechanisms that control the functional differentiation of CD8+ T cells are largely unknown. Until recently, Eomesodermin (Eomes)3 has been identified as a T-bet analog to control the IFN-γ production and the CTL function of CD8+ T cells (7). In addition, T-bet, a dominant Th1-specific transcription factor for CD4+ T cells, has also been demonstrated to control Ag-driven effector CD8+ T cell IFN-γ production and CTL function (8). It is unknown, however, which signaling pathways control the expression of T-bet and Eomes in CD8+ T cells. It also remains to be seen whether other signaling pathways are involved in functional differentiation of CD8+ T cells.

JNK belong to the MAPK family and are involved in a variety of cellular responses including cell activation, proliferation, differentiation, and cell death (9, 10). Three members of the JNK family have been identified in eukaryotic cells (JNK1, JNK2, and JNK3). Although JNK1 and JNK2 are ubiquitously expressed, JNK3 is limited to brain and heart (11). JNK in T cells is activated by phosphorylating MEK4 and MEK7 in response to TCR and costimulation (CD28) signaling (12). It has been shown by several investigators that JNK1 and JNK2 have divergent effects on CD4+ and CD8+ T cells in terms of their activation, cytokine production, and effector function. Using JNK1−/− and JNK2−/− mice, it has been demonstrated that JNK2 is required for CD4+ T cell IFN-γ production and Th1 differentiation (13), whereas JNK1 is a negative regulator of Th2 development (14). In contrast, JNK1 is required for Ag-stimulated expansion of CD8+ T cells in vitro due to impaired IL-2R α-chain (CD25) expression and survival of activated CD8+ T cells in immune response against viral infections in vivo (15, 16), whereas JNK2 negatively regulates CD8+ T cell IL-2 production and proliferation (15, 16).

JNK1 and JNK2 also play distinct roles in skin tumor formation and carcinogenesis. JNK2−/− mice had reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin papillomas while JNK1−/− mice developed more tumors (17, 18). However, it is unknown from these studies whether the tumor phenotypes originated from JNK regulation of immune cell functions and/or from carcinogenesis.

In this report, we demonstrate that compared with wild-type (Wt) mice, JNK1−/− mice were highly susceptible to both melanoma (B16) or lymphoma (EL-4) tumorigenesis. This high susceptibility is due to the functional defects of CD8+ T cells, but not of CD4+ T cells. Moreover, JNK1−/− CD8+ T cells are intrinsically defective in producing IFN-γ and are compromised in perforin synthesis and CTL function. Our results suggest that JNK1 may serve as an important regulator of the effector functions of the CD8+ T cell, and thus play a critical role in tumor immune surveillance.

C57BL/6 (B6) mice and CD45.1 B6 mice were purchased from the National Cancer Institute. C57BL/6-Ifngtm1Ts (B6 IFN-γ−/−) mice and C57BL/6-Cd8atm1Mak (B6 CD8−/−) mice were purchased from The Jackson Laboratory. B6 JNK1−/− mice were described previously (14). JNK1−/−OT-I mice were generated by intercross of B6 JNK1−/− and B6 OT-I mice. B6 OT-1 mice express a transgenic TCR (Vα2+Vβ5+) specific for the SIINFEKL peptide of OVA in the context of MHC class I (H-2-Kb, Ref.19). B6 MKK3−/− mice (Ref.20 ; were provided by Dr. R. A. Flavell) and B6 NFAT-1−/− mice (Ref.21); originally from Dr. A. Rao, Department of Pathology, Harvard University Medical School, Cambridge, MA, were provided by Dr. D. Rothstein, Yale University School of Medicine, New Haven, CT). All mice were maintained under specific pathogen-free conditions at Yale University.

Recombinant murine IL-2, IL-12, and IL-4 were purchased from R&D Systems. Anti-mouse Abs used for phenotypic and cytokine analysis were all purchased from BD Biosciences. Dye CFSE was purchased from Molecular Probes.

B16 F0 melanoma cells (provided by Dr. M. Mamula, Yale University, School of Medicine, New Haven, CT) or EL-4 (purchased from American Type Culture Collection) were injected s.c. and tumor growth was monitored and recorded daily for over 3 wk as described in our previous studies (6).

B6 mice were administered with i.p. injection of anti-CD4 (GK 1.5) (22, 23) Ab (0.4 g/mouse) on day −7, −4, and −1, followed by reconstitution with purified CD4+ T cells (2 × 106 cells/mouse) from B6 Wt and B6 JNK1−/− on day 1. CD4+ T cells were negatively selected from the splenocytes of B6 Wt or B6 JNK1−/− mice by MACS using Abs against CD8, CD11b, CD11c, and B220 as described in our previous studies (24) and the purity was >90% (data not shown). The reconstituted mice were challenged with B16 F0 melanoma cells (1 × 105 cells/mouse) on day 2. Preliminary results confirmed the complete depletion of CD4+ T cells even at 14 days post-Ab treatment and reconstitution of donor CD4+ T cells by FACS (data not shown).

B6 CD8−/− mice were reconstituted with purified CD8 T cells (2 × 106 cells/mouse) isolated from B6 Wt or B6 JNK1−/− or B6 IFN-γ−/− mice, followed by inoculation of B16 F0 melanoma cells on the next day as described above. CD8+ T cells were purified by negative selection using anti-CD4, anti-CD11b, anti-B220, and anti-CD11c by MACS and the purity was >90% (data not shown).

Naive CD8+ T cells were sorted from B6 Wt and B6 JNK1−/− mice (CD62LhighCD44low) and cultured with 10 μg/ml plate-bound anti-CD3 and 1 μg/ml anti-CD28 Abs in the presence of IL-2 (TC0) or IL-12 (5 ng/ml) plus anti-IL-4 (10 μg/ml, TC1). At different time points (0, 3, 6, 9, 12, 24, 48 h), cells were used for cDNA preparation and IFN-γ and hypoxanthine phosphoribosyltransferase (HPRT) transcripts were detected using real-time PCR analysis as described in details below. The culture supernatants were collected for IFN-γ ELISA analysis using the preoptimized Ab package and protocol from BD Biosciences. For Ag-specific CD8+ T cell proliferation and cytokine production in vitro, naive CD8+ T cells (CD8+Vβ5+CD44lowCD62Lhigh) were sorted from OT-I or JNK1−/−OT-I transgenic mice, labeled with CFSE, and cocultured with bone marrow-derived dendritic cells (DCs). Bone marrow from femurs and tibia of B6 wild-type mice were cultured with RPMI 1640 complete medium containing GM-CSF (10 ng/ml) and IL-4 (10 ng/ml). On day 9, cultured DCs were treated with 100 ng/ml LPS for 2 days, followed by irradiation under 3000 rad and used as APC as described previously (25). CD8+ T cells were cultured either in the presence of different concentrations of SIINFEKL peptide from OVA (OVAp) with a fixed DC-T ratio (1:5) or in the presence of different DC-T ratios (DC-T ratios were 1:5 and 1:25) with a fixed concentration of OVAp (10 pM).

Total RNA was extracted from the cell samples using the RNeasy Mini kit (Qiagen) and reverse transcribed using the Strata Script First Strand Synthesis System (Stratagene). The PCR was performed on an iCycler (Bio-Rad). Cycling conditions were 12 min at 95°C followed by 40 repeats of 95°C for 15 s and 60°C for 60 s. Analysis was performed by sequence detection software supplied with the instrument. Each cytokine was analyzed concurrently on the same plate with HPRT, and cytokine transcripts were normalized to HPRT abundance using primers as described previously (26). The primers included: HPRT sense, 5′-CTGGTGAAAAG GACCTCTCG-3′; HPRT antisense, 5′-TGAAGTACTCATTATAGTCA AGGGCA-3′; HPRT probe, VIC-5′-TGTTGGATACAGGCCAGACTTT GTTGGAT-3′-TAMRA; IFN-γ sense, 5′-GGATGCATTCATGAGTATT GC-3′, IFN-γ antisense, 5′-CCTTTTCCGCTTCCTGAGG-3′; IFN-γ probe, FAM-5′-TTTGAGGTCAACAACCCACAGGTCCA-3′-TAMRA; T-bet sense, 5′-CAACAACCCCTTTGCCAAAG-3′; T-bet antisense, 5′-TCCCCCAAGCAGTTGACAGT-3′; T-bet probe, FAM-5′-CCGGGAGA ACTTTGAGTCCATGTACGC-3′-TAMRA; Eomes sense, 5′-CCTTCAC CTTCTCAGAGACACAGTT-3′; Eomes antisense, 5′-TCGATCTTTAG CTGGGTGATATCC-3′; Eomes probe, FAM-5′-TCGCTGTGACGGCCT ACCAAAACA-3′-BHQ.

Sorted naive CD8+ T cells from OT-I or JNK1−/−OT-I mice were labeled with CFSE cultured with DC-loaded OVAp for 48 h. Brefeldin A was added 3 h before harvesting. Cells were then washed, fixed with 2% formaldehyde and permeabilized with 0.5% saponin (w/v) for intracellular IFN-γ staining as described (24). In the parallel culture, the activated cells as described above were restimulated with anti-CD3 and anti-CD28 in the presence of brefeldin A for 6 h, and cells were then used for intracellular IFN-γ staining as described above. For intracellular perforin staining, naive CD8+ T cells were activated with splenocyte-loaded OVA for 72 h. The activated cells were then washed, fixed, and permeabilized for intracellular perforin staining as described (27). PE-conjugated rat IgG2a (BD Pharmingen) was used as an isotype control. Stained cells were analyzed using a FACSCalibur flow cytometer with CellQuest software.

In vitro cytotoxicity of CD8+ T cells was determined in a JAM test essentially according to a standard methodology (28, 29). Briefly, sorted naive CD8+ T cells from OT1 and JNK1−/− OT1 transgenic mice were stimulated with OVAp (10 pM) and born marrow DCs (DC-T ratio as 1:5) for 5 days. EL-4 cells (target cells, 1000 cells/well) were cultured for 6 h with [3H]thymidine (5 μCi/ml) in the presence or absence of 0.1 μg/ml SIINFKEL peptide. Different numbers of the activated cells as above (effector cells) were added in triplicate to the target cells in a 96-well round-bottom plate. Spontaneous 3H retention was determined by adding medium instead of effector cells. After 4 h of culture, cells were harvested onto filters before scintillation counting, which was then used to calculate the percentage of cytotoxicity using the following formula: ((spontaneous cpm − experimental cpm) × 100)/spontaneous cpm.

We adopted the protocol as described previously (30, 31). Briefly, CD8+ T cells were isolated from B6 OT-1 or B6 JNK1−/−OT-1 mice, cultured with syngeneic T cell-depleted splenocytes loaded with OVA (200 μg/ml) for 24 h. These activated CD8+ T cells were then transferred to B6 Wt mice (1 × 106 cells/mouse) i.v. Five days later, the target cells were injected, and the cytotoxicity was analyzed 20 h later. Wt B6 splenocytes were used as the target cells and labeled with the fluorescent dye CFSE at two different densities. The CFSEhigh cells (5 μM CFSE) were pulsed with peptide SIINFEKL (0.5 μg/ml) at 37°C, 5% CO2 for 90 min, whereas the CFSElow cells (0.5 μM CFSE) were pulsed with the same concentration of a control peptide (SSVVGVWYL) to serve as the internal control. These two populations of CFSE-positive cells were mixed at a 1:1 ratio and coinjected i.v. as target cells. The ratios of CFSEhigh/CFSElow cells from the splenocytes of recipients were used to measure in vivo killing as indicated by loss of the CFSEhigh Ag-pulsed population relative to the control CFSElow population (31) (30, 31). The formula is as following: percent killing = [control (CFSEhigh/CFSElow) − experimental (CFSEhigh/CFSElow)]/control (CFSEhigh/CFSElow) × 100%.

Statistical significance was evaluated by two-tailed unpaired Student’s t test or nonparameter analysis if SDs were significantly different between two compared groups using InState version 2.03 software for Macintosh (GraphPad). The incidence of tumor development was compared and analyzed using the log rank test, performed by GraphPad Prism version 3.0a for Macintosh (GraphPad). Throughout the text, figures, and legends, the following terminology was used to denote statistical significance: ∗, p < 0.001 or p < 0.01; ∗∗, p < 0.05.

Based upon the facts that JNK1−/− mice are more susceptible to TPA-induced skin tumor development (17) and that JNK1 is required for CD8+ T cell activation, we hypothesized that JNK1 might play a critical role in antitumor immune response. To define the role of JNK1 in tumor immune surveillance, sex- and age-matched B6 Wt (n = 18), B6 JNK1−/− (n = 15), and B6 IFN-γ−/− (n = 10) mice were inoculated with the B16 F0 melanoma cell line (1 × 105/mouse) as previously described (6), and tumor growth was recorded daily. Compared with Wt mice, both JNK1−/− and IFN-γ−/− mice showed earlier tumor development in both tumor models (Fig. 1,A) as well as larger tumor size. One example of tumor size comparison between Wt and JNK1−/− mice upon inoculation with B16 F0 melanoma cells is shown (Fig. 1,B). Notably, several other signaling-deficient mice, such as B6 MEK3−/− and B6 NFAT-1−/− mice were included in the experiment, and no significant difference was observed in their tumor growth compared with B6 Wt mice (data not shown), indicating a nonredundant role of JNK1 in antitumor immune responses. To define whether the role of JNK1 in tumor immunity is melanoma-specific, B6 Wt, JNK1−/− and IFN-γ−/− mice were inoculated with thymoma EL-4, and the incidence of tumor growth was monitored. Similar to the experiments with B16 melanoma cell line, JNK1−/− mice were highly susceptible to tumor growth (Fig. 1 C). Our results indicate that JNK1 plays a critical role in tumor immune surveillance.

FIGURE 1.

JNK1 is critical for tumor immune surveillance. A, Both JNK1 and IFN-γ are required for antitumor immune responses against melanoma formation. Sex- and age-matched B6 Wt (n = 18), B6 JNK1-deficient mice (JNK1−/−, n = 15) and B6 IFN-γ-deficient mice (IFN-γ−/−, n = 10) were injected s.c. with 1 × 105 B16 F0 melanoma cells, and tumor growth was recorded daily. Tumor size >4 × 4 mm was considered positive. Data represent three independent experiments. ∗, p < 0.01. B, JNK1 is involved in controlling tumor growth. The mean tumor size from Wt and JNK1−/− mice in A is shown. ∗, p < 0.01. C, Both JNK1 and IFN-γ are involved in protective immune responses against EL-4 tumor cells. Sex- and age-matched B6 Wt (n = 12), B6 JNK1-deficient mice (JNK1−/−, n = 12) and B6 IFN-γ-deficient mice (IFN-γ−/−, n = 12) were injected s.c. with 5 × 105 B16 F0 melanoma cells, and tumor growth was recorded daily. Tumor size >4 × 4 mm was considered positive. Data represent three independent experiments. ∗, p < 0.01.

FIGURE 1.

JNK1 is critical for tumor immune surveillance. A, Both JNK1 and IFN-γ are required for antitumor immune responses against melanoma formation. Sex- and age-matched B6 Wt (n = 18), B6 JNK1-deficient mice (JNK1−/−, n = 15) and B6 IFN-γ-deficient mice (IFN-γ−/−, n = 10) were injected s.c. with 1 × 105 B16 F0 melanoma cells, and tumor growth was recorded daily. Tumor size >4 × 4 mm was considered positive. Data represent three independent experiments. ∗, p < 0.01. B, JNK1 is involved in controlling tumor growth. The mean tumor size from Wt and JNK1−/− mice in A is shown. ∗, p < 0.01. C, Both JNK1 and IFN-γ are involved in protective immune responses against EL-4 tumor cells. Sex- and age-matched B6 Wt (n = 12), B6 JNK1-deficient mice (JNK1−/−, n = 12) and B6 IFN-γ-deficient mice (IFN-γ−/−, n = 12) were injected s.c. with 5 × 105 B16 F0 melanoma cells, and tumor growth was recorded daily. Tumor size >4 × 4 mm was considered positive. Data represent three independent experiments. ∗, p < 0.01.

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To define the subset of T cells that is responsible for the phenotype in JNK1−/− mice, we first assessed the requirement of JNK1 for the effector function of CD8+ T cells in antitumor immune response. B6 CD8−/− mice were reconstituted with either Wt or IFN-γ−/− or JNK1−/− CD8+ T cells (2 × 106/mouse), or with PBS (n = 12 per each group), followed by s.c. inoculation of B16 F0 melanoma cells (Fig. 2,A). Tumor growth was monitored and recorded daily as described above. In the absence of CD8+ T cells (PBS injection), mice were highly susceptible to tumor growth, indicating an essential role of CD8+ T cells in antitumor immunity (Fig. 2,A). Mice reconstituted with JNK1−/− CD8+ T cells showed similar susceptibility to tumor growth as those receiving IFN-γ−/− CD8+ T cells. More importantly, a significantly greater number of mice from these two groups developed tumors as compared with mice reconstituted with Wt CD8+ T cells, implying a possible role of IFN-γ in JNK1-mediated protective antitumor immune response by CD8+ T cells (Fig. 2,A, p < 0.01). We next tested the role of JNK1 in mediating the function of CD4+ T cells in tumor immune surveillance using the same B16 melanoma model. B6 Wt mice were injected with depleting anti-CD4 Ab (clone GK1.5; Refs.22 and 23) followed by reconstitution of purified Wt CD4+ and JNK1−/− CD4+ T cells (2 × 106 cells/mouse). The reconstituted mice were then inoculated with B16 tumor cells as described above. No significant differences were observed between these two groups of mice with regard to tumor formation (Fig. 2 B), indicating that CD4+ T cells are not responsible for the phenotype in JNK1−/− mice. Notably, both Wt and JNK1−/− donor CD4+ T cells were survived equally in the CD4+ T cell-depleted host (data not shown).

FIGURE 2.

JNK1 contributes to tumor immunity by regulating the functions of CD8+, but not CD4+, T cells. A, Both JNK1 and IFN-γ are required for CD8+ T cell-mediated protection in B16 transfer tumor model. B6 CD8−/− mice were reconstituted with either B6 Wt, B6 JNK1-deficient (JNK1−/−), or B6 IFN-γ-deficient (IFN-γ−/−), CD8+ T cells (purified by MACS, purity is >90%, data not shown) or PBS by i.v. injection (2 × 106 cells/mouse, n = 10 per each group). On the following day, these reconstituted mice were then inoculated s.c. with B16 F0 tumor cells (0.1 × 105 cells/mouse) and tumor growth was recorded daily. Data represent three independent experiments. ∗, p < 0.01. B, CD4+ T cells are not required for JNK1-mediated protective antitumor immune response. B6 mice were treated with CD4-depletion Ab (GK1.5) at day −7, −4, and −1, and on day 1, mice were reconstituted with purified CD4+ T cells (purified by MACS; the purity is >90%, data not shown), either from B6 Wt or B6 JNK1-deficient (JNK1−/−) mice (n = 10 per each group) by i.v. injection. These reconstituted mice were then inoculated with B16 F0 tumor cells, and tumor growth was monitored and recorded daily as described above. Data represent three independent experiments.

FIGURE 2.

JNK1 contributes to tumor immunity by regulating the functions of CD8+, but not CD4+, T cells. A, Both JNK1 and IFN-γ are required for CD8+ T cell-mediated protection in B16 transfer tumor model. B6 CD8−/− mice were reconstituted with either B6 Wt, B6 JNK1-deficient (JNK1−/−), or B6 IFN-γ-deficient (IFN-γ−/−), CD8+ T cells (purified by MACS, purity is >90%, data not shown) or PBS by i.v. injection (2 × 106 cells/mouse, n = 10 per each group). On the following day, these reconstituted mice were then inoculated s.c. with B16 F0 tumor cells (0.1 × 105 cells/mouse) and tumor growth was recorded daily. Data represent three independent experiments. ∗, p < 0.01. B, CD4+ T cells are not required for JNK1-mediated protective antitumor immune response. B6 mice were treated with CD4-depletion Ab (GK1.5) at day −7, −4, and −1, and on day 1, mice were reconstituted with purified CD4+ T cells (purified by MACS; the purity is >90%, data not shown), either from B6 Wt or B6 JNK1-deficient (JNK1−/−) mice (n = 10 per each group) by i.v. injection. These reconstituted mice were then inoculated with B16 F0 tumor cells, and tumor growth was monitored and recorded daily as described above. Data represent three independent experiments.

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IFN-γ is an essential cytokine for tumor immune surveillance, and its production is a functional hallmark of CD8+ T cells (1, 4, 32). To define the precise role of JNK1 in regulating the early transcription of IFN-γ, naive CD8+ T cells that were sorted from B6 Wt or B6 JNK1−/− mice were cultured with plate-bound anti-CD3 (10 μg/ml) and anti-CD28 (1 μg/ml) in the presence of IL-2 (20 U/ml, cytotoxic T cells (Tc) 0), or IL-2, IL-12 (5 ng/ml), and anti-IL-4 (10 μg/ml, Tc1) as previously described (24). At different time points after stimulation, cells were used for cDNA preparation, and IFN-γ gene transcription was measured by real-time PCR analysis. Normalized IFN-γ levels are shown (Fig. 3, RNA and ELISA). Wt CD8+ T cells initiated IFN-γ gene transcription as early as 3 h, and these levels were significantly higher under Tc1 conditions. In contrast, CD8+ T cells from JNK1−/− mice exhibited a significant delay in IFN-γ gene transcription (after 12 h), and the mRNA levels were reduced at each time point. To quantify the production of IFN-γ from these activated CD8+ T cells, culture supernatants were used in an IFN-γ-specific ELISA (Fig. 3). Consistently, Wt CD8+ T cells produced IFN-γ as early as 9 h upon activation under Tc1 conditions, whereas JNK1−/− CD8+ T cells showed a significant delay in IFN-γ production as well as reduced levels at all time points (Fig. 3, lower right panel). Interestingly, JNK1−/− CD8+ T cells under neutral conditions showed similar patterns of delay in transcription (Fig. 3, upper left panel) and secretion of IFN-γ (Fig. 3, upper right panel). Our results suggest that JNK1 is critical for TCR- and CD28-mediated early IFN-γ gene transcription and may also be involved in IL-12-mediated IFN-γ production.

FIGURE 3.

JNK1 is required for the early IFN-γ gene transcription and secretion by CD8+ T cells. Naive CD8+ T cells (CD62LhighCD44low) were sorted from B6 Wt mice or JNK1-deficient (JNK1−/−) mice, and cultured with plate-bound anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) in the presence of either IL-2 (2 ng/ml, Tc0) or IL-2 plus IL-12 (5 ng/ml) and anti-IL-4 (10 μg/ml, Tc1). At different hours of culture, cells were used for cDNA preparation and real-time PCR analysis for IFN-γ gene transcription. The ratios between IFN-γ vs HPRT control are shown (left panel). The supernatant collected from all culture conditions was used for analysis of the level of IFN-γ production by IFN-γ-specific ELISA (right panel). A representative sample set of three separated experiments is shown.

FIGURE 3.

JNK1 is required for the early IFN-γ gene transcription and secretion by CD8+ T cells. Naive CD8+ T cells (CD62LhighCD44low) were sorted from B6 Wt mice or JNK1-deficient (JNK1−/−) mice, and cultured with plate-bound anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) in the presence of either IL-2 (2 ng/ml, Tc0) or IL-2 plus IL-12 (5 ng/ml) and anti-IL-4 (10 μg/ml, Tc1). At different hours of culture, cells were used for cDNA preparation and real-time PCR analysis for IFN-γ gene transcription. The ratios between IFN-γ vs HPRT control are shown (left panel). The supernatant collected from all culture conditions was used for analysis of the level of IFN-γ production by IFN-γ-specific ELISA (right panel). A representative sample set of three separated experiments is shown.

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To further determine the role of JNK1 in Ag-specific CD8+ T cell cytokine responses, we obtained CD8+ TCR transgenic mice (OT-1) that recognize the SIINFEKL peptide of OVA (19) and crossed OT-1 to B6 JNK1−/− to generate JNK1−/−OT-1 mice. Naive OT-1 or JNK1−/−OT-1 CD8+ T cells (Vβ5+CD62LhighCD44low) were sorted by FACS, labeled with CFSE, and cocultured with bone marrow-derived DCs, either at DC-T cell ratios of 1:5 in the presence of different concentrations of OVAp (Fig. 4,A), or at the fixed concentration of OVAp (10 pM) with different DC-T ratios (1:5 or 1:25, Fig. 4,B). After 48 h of cell culture, a portion of cultured cells was used for intracellular cytokine staining. Both populations of CD8+ T cells included similar fractions of cells that had undergone one or two cell divisions, and OT-1 CD8+ T cells produced a significantly greater amount of IFN-γ than those of JNK1−/−OT-1 CD8+ T cells. The percentage of IFN-γ-producing cells was 4- to 8-fold lower than those of Wt CD8+ T cells at the fixed DC-T ratio (1:5) and at various concentration of OVAp (Fig. 4,A). Similar results were obtained using different DC-T ratios with fixed OVAp concentration (10 pM) (Fig. 4,B). To define whether the role of JNK1 in the priming of naive CD8+ T cells has any effect on their potential to produce IFN-γ upon restimulation after 84 h of culture, the activated cells as described above (Fig. 4, A and B) were further stimulated with anti-CD3 and anti-CD28 Abs for 6 h and brefeldin A was added during the last 3 h of culture. Cells were then fixed and permeabilized for intracellular cytokine staining. Both the percentage and the mean fluorescence intensity of IFN-γ-positive cells were significantly lower in JNK1−/− CD8+ T cells in all primary culture conditions compared with Wt CD8+ T cells (Fig. 4, C and D). These results strongly suggest that JNK1 is essential for the induction of IFN-γ production by naive CD8+ T cells.

FIGURE 4.

JNK1 selectively regulates Ag-specific IFN-γ production by CD8+ T cells. A and B, Naive transgenic CD8+ T cells (Vβ5+CD62LhighCD44low) were sorted from B6 OT-1 or JNK1-deficient OT-1 (JNK1−/−OT-1) mice, and labeled with CFSE and cultured with bone marrow-derived DCs pulsed with different concentrations of OVAp at a DC-T ratio of 1:5 (A), or cultured with different DC-T ratios (1:5 or 1:25) at the fixed concentration of OVAp (10 pM; B). After 48 h of culture, cells were fixed and permeabilized for intracellular cytokine staining. This figure is representative of three separated experiments. C and D, JNK1 regulates the ability of CD8+ T cells to produce IFN-γ. Cultured cells in A and B at 5 days were restimulated with anti-CD3 and anti-CD28 Abs for 6 h in the presence of brefeldin A for the last 3 h, and then washed and fixed for intracellular cytokine staining. The numbers in the upper right quadrant are the percentage/mean fluorescence intensity (MFI) of IFN-γ-producing cells. Results represent one of three repeated experiments.

FIGURE 4.

JNK1 selectively regulates Ag-specific IFN-γ production by CD8+ T cells. A and B, Naive transgenic CD8+ T cells (Vβ5+CD62LhighCD44low) were sorted from B6 OT-1 or JNK1-deficient OT-1 (JNK1−/−OT-1) mice, and labeled with CFSE and cultured with bone marrow-derived DCs pulsed with different concentrations of OVAp at a DC-T ratio of 1:5 (A), or cultured with different DC-T ratios (1:5 or 1:25) at the fixed concentration of OVAp (10 pM; B). After 48 h of culture, cells were fixed and permeabilized for intracellular cytokine staining. This figure is representative of three separated experiments. C and D, JNK1 regulates the ability of CD8+ T cells to produce IFN-γ. Cultured cells in A and B at 5 days were restimulated with anti-CD3 and anti-CD28 Abs for 6 h in the presence of brefeldin A for the last 3 h, and then washed and fixed for intracellular cytokine staining. The numbers in the upper right quadrant are the percentage/mean fluorescence intensity (MFI) of IFN-γ-producing cells. Results represent one of three repeated experiments.

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Both T-bet and Eomes have been defined as the transcription factors that contribute to IFN-γ production in CD8+ T cells (7, 8, 33). To assess the role of JNK1 in regulating the transcription of T-bet and Eomes, naive and activated CD8+ T cells under Tc1 conditions as described in Fig. 3 were used for real-time PCR analysis. The expression of T-bet is low in naive CD8+ T cells, and is up-regulated upon activation. Strikingly, JNK1−/− CD8+ T cells expressed significantly lower level of T-bet at all time points (Fig. 5, upper panel), indicating that JNK1 is required for the synthesis of T-bet. In contrast, Eomes is expressed in naive Wt CD8+ T cells and are only slightly enhanced upon activation. Interestingly, JNK1−/− CD8+ T cells down-regulate Eomes transcription following activation, and this level was significantly lower than that of Wt CD8+ T cells (Fig. 5, lower panel). These results suggest that JNK1 regulates CD8+ T cell IFN-γ production by controlling the synthesis of T-bet and Eomes.

FIGURE 5.

JNK1 regulates both T-bet and Eomes expression in CD8+ T cells. Naive CD8+ T cells were sorted from B6 Wt or JNK1−/− mice and activated with anti-CD3 and anti-CD28 in the presence Tc1 conditions as described in Fig. 3. Activated cells were collected from different time points and used for real-time PCR analysis and cytokine transcripts were normalized to HPRT abundance. One example of three repeated experiments is shown. ∗, p < 0.01; ∗∗, p < 0.05.

FIGURE 5.

JNK1 regulates both T-bet and Eomes expression in CD8+ T cells. Naive CD8+ T cells were sorted from B6 Wt or JNK1−/− mice and activated with anti-CD3 and anti-CD28 in the presence Tc1 conditions as described in Fig. 3. Activated cells were collected from different time points and used for real-time PCR analysis and cytokine transcripts were normalized to HPRT abundance. One example of three repeated experiments is shown. ∗, p < 0.01; ∗∗, p < 0.05.

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Perforin is an important mediator of CTL function of CD8+ T cells and plays a critical role in antitumor immune response (34, 35). To define the role of JNK1 in the synthesis of perforin by CD8+ T cells, naive transgenic CD8+ T cells (OT-1 or JNK1−/−OT-1) were activated with syngeneic T cell-depleted Wt splenocytes loaded with OVA (200 μg/ml) for 72 h, and cells were then used for intracellular perforin staining as described (27). OT-1 CD8+ T cells expressed significantly higher levels of perforin per single cell compared with those of JNK1−/− CD8+ T cells, with mean fluorescence intensity (mean ± SD) as 13 ± 2 for OT-1 and 8.4 ± 0.7 for JNK1−/−OT-1, respectively (p = 0.018), indicating a critical role of JNK1 in the synthesis of perforin by CD8+ T cells (Fig. 6,A). To further define the impact of JNK1 in CD8+ T cell CTL function, sorted naive OT-1 and JNK1−/−OT-1 CD8+ T cells were activated with bone marrow-derived DC loaded with OVAp as described in Fig. 4 for 5 days, and the CTL ability of these activated CD8+ T cells was analyzed using the JAM test as described previously (28, 29). EL-4 cell-pulsed OVAp were used as the target cells. JNK1−/− CD8+ T cells showed significantly impaired CTL activity at both higher E:T ratios (33:1 and 10:1), suggesting that JNK1 regulates the CTL activity of activated CD8+ T cells (Fig. 6,B). Finally, we investigated the role of JNK1 in CTL function of CD8+ T cells in vivo. Naive CD8+ T cells were activated as described in Fig. 6,A, and the activated cells (1 × 106/mouse) were transferred into B6 Wt mice (n = 3 for each type of CD8+ T cells) for in vivo CTL assay as described in Materials and Methods. JNK1−/− CD8+ T cells showed >30% reduction in their specific cytolytic activity (mean ± SD for OT-1 and JNK1−/−OT-1 are 92 ± 8 and 60 ± 4, respectively (Fig. 6,D)). A representative sample set is shown in Fig. 6,C. To exclude the possibility that the impaired cytolytic activity of JNK1−/− CD8+ T cells was due to the impaired cell expansion in vivo, OVA- activated CD8+ T cells (1 × 106/mouse), as described above, were labeled with CFSE and transferred into a naive Wt host. At day 5 posttransfer, both populations were recovered from the spleen, and both types of cells were found to have undergone similar numbers of cell divisions (Fig. 6 E). Our results demonstrate that JNK1 controls the intrinsic cytolytic activity of CD8+ T cells by regulating perforin synthesis.

FIGURE 6.

JNK1 is required for perforin-dependent CTL function of CD8+ T cells. A, Naive CD8+ T cells (Vβ5+CD62LhighCD44low) were sorted from B6 OT-1 or B6 JNK1-deficient OT-1 (JNK1−/−OT-1) mice and then cultured with T cell-depleted splenocytes loaded with OVA (200 mg/ml) for 3 days. The cells were then fixed, permeabilized, and stained with PE-conjugated perforin Ab or isotype controls. One example of perforin vs isotype control is shown. B, Activated CD8+ T cells as described in Fig. 4 were transferred to 96-well round-bottom plate with 3H-pulsed EL-4 cells in the presence or absence of OVAp and CTL activity was analyzed as JAM test. C, Activated CD8+ T cells as described in A were transferred to B6 Wt mice (1 × 106 cells/mouse, n = 3 per each group). Five days later, the mixture (1:1 ratio) of two populations of target cells labeled with different concentrations of CFSE (CFSEhigh population loaded with SIINFEKL peptide and CFSElow population with control peptide) was injected into the hosts reconstituted with activated CD8+ T cells. After 20 h, the ratios of CFSEhigh/CFSElow cells from spleens of the respective hosts were used to determine the percentage of CTL. One example of CFSE-positive cells from splenocytes is shown. D, The percentage of specific lysis (mean ± SD) calculated from the ratios of CFSEhigh/CFSElow cells is shown. ∗∗, p < 0.05. E, Activated CD8+ T cells as in A were labeled with CFSE (5 μM) and transferred to B6 Wt host by i.v. injection. Five days later CFSE-positive cells were recovered from the splenocytes and analyzed for the numbers of cell divisions. One example is shown.

FIGURE 6.

JNK1 is required for perforin-dependent CTL function of CD8+ T cells. A, Naive CD8+ T cells (Vβ5+CD62LhighCD44low) were sorted from B6 OT-1 or B6 JNK1-deficient OT-1 (JNK1−/−OT-1) mice and then cultured with T cell-depleted splenocytes loaded with OVA (200 mg/ml) for 3 days. The cells were then fixed, permeabilized, and stained with PE-conjugated perforin Ab or isotype controls. One example of perforin vs isotype control is shown. B, Activated CD8+ T cells as described in Fig. 4 were transferred to 96-well round-bottom plate with 3H-pulsed EL-4 cells in the presence or absence of OVAp and CTL activity was analyzed as JAM test. C, Activated CD8+ T cells as described in A were transferred to B6 Wt mice (1 × 106 cells/mouse, n = 3 per each group). Five days later, the mixture (1:1 ratio) of two populations of target cells labeled with different concentrations of CFSE (CFSEhigh population loaded with SIINFEKL peptide and CFSElow population with control peptide) was injected into the hosts reconstituted with activated CD8+ T cells. After 20 h, the ratios of CFSEhigh/CFSElow cells from spleens of the respective hosts were used to determine the percentage of CTL. One example of CFSE-positive cells from splenocytes is shown. D, The percentage of specific lysis (mean ± SD) calculated from the ratios of CFSEhigh/CFSElow cells is shown. ∗∗, p < 0.05. E, Activated CD8+ T cells as in A were labeled with CFSE (5 μM) and transferred to B6 Wt host by i.v. injection. Five days later CFSE-positive cells were recovered from the splenocytes and analyzed for the numbers of cell divisions. One example is shown.

Close modal

The JNK signal transduction pathway has been shown to play an essential role in cell activation, differentiation, and survival (9, 10). Studies with JNK1-or JNK2-deficient mice suggest that different isoforms of JNK have distinct effector functions on different subsets of T cells (CD4+ vs CD8+) (13, 14). More recently, JNK1 has been shown to be critical for the proliferation and expansion of CD8+ T cells both in vitro and in vivo (15, 16). We provide herein the first evidence that JNK1 is critical for tumor immune surveillance by regulating the effector functions of CD8+ T cells, including IFN-γ production and CTL function.

It has been well-established that both lymphocytes and IFN-γ are essential components of tumor immune surveillance (2, 3, 4). Different subsets of lymphocytes contribute to antitumor immune responses at different stages. Our previous studies have defined a critical role of γδ T cells to provide the early source of IFN-γ, which in turn regulates the effector function of CD8+ T cells (6). The critical role of CD8+ T cells in prevention and eradication of tumors has been highlighted by many studies (35, 36, 37). However, the controlling mechanisms of differentiation and effector functions of CD8+ T cells are largely unknown. In this report, we first demonstrate that JNK1 is essential for tumor immune surveillance by affecting the effector functions of CD8+ T cells. In the absence of JNK1, mice were highly susceptible to B16 melanoma (Fig. 1,A) and EL-4 thymoma (Fig. 1,C). Moreover, reconstitution of CD8−/− mice with JNK1−/− CD8+ T cells resulted in similar susceptibility to those of CD8-deficient mice (no reconstitution), with significantly higher tumor incidence compared with those reconstituted with Wt CD8+ T cells (Fig. 2 A). These results strongly suggest a critical role of JNK1 in mediating the effector function of CD8+ T cells in antitumor immune response.

IFN-γ is a necessary cytokine in the innate and adaptive immune responses that protect against tumor development (2, 4). The finding that reconstitution of CD8−/− mice with JNK1−/− CD8+ T cells renders the recipients equally susceptible to tumor growth as those with IFN-γ−/− CD8+ T cells (Fig. 2,A), implies a critical role of JNK1 in controlling the production of IFN-γ by CD8+ T cells. To define the role of JNK1 in the regulation of early IFN-γ gene transcription and secretion, we compared two different populations of naive CD8+ T cells from JNK1−/− and Wt mice, either a polyclonal population from nontransgenic mice (Fig. 3, left panel) or a monoclonal population from OT-1 CD8+ TCR transgenic mice (Fig. 4), and we applied two different activation approaches, either by polyclonal activation with anti-CD3 and anti-CD28 Abs (Fig. 3) or by Ag-specific stimulation (DC-loaded peptide, Fig. 4). Regardless of the source of naive CD8+ T cells or the approach of activation, a similar result was obtained indicating that JNK1 was critical for the early transcription and secretion of IFN-γ by CD8+ T cells.

How does JNK1 regulate IFN-γ gene transcription and production? Similar to CD4+ T cells, CD8+ T cells can be differentiated to Tc1 or Tc2 cells (38). However, the molecular mechanisms that control the differentiation of CD8+ T cells are less clear. Until recently, Eomes, a T-bet analog, has been defined to play a critical role in controlling the effector function of CD8+ T cells, including IFN-γ production and CTL function (7). T-bet, a specific transcription factor for CD4+ Th1 cells, has also been determined to regulate Ag-driven IFN-γ production by CD8+ T cells (8, 33). We found that JNK1−/− CD8+ T cells expressed lower levels of T-bet and Eomes following activation (Fig. 5), suggesting a potential role of JNK1 in regulating the IFN-γ production by CD8+ T cells by modulating the expression of both T-bet and Eomes. It has to be emphasized that other mechanisms may also be involved. For example, JNK1−/− CD8+ T cells have lower AP-1 phosphorylation (15), and AP-1 can bind to the promoter region of the IFN-γ gene directly to promote its transcription (39). Additional experiments are needed to further elucidate the underlying mechanisms whereby JNK1 controls IFN-γ production by CD8+ T cells.

CTL function is the second critical effector function element for cancer immunosurveillance (4). It has been well-established that perforin is a critical molecule in the primary antitumor immune responses (34, 35). To define the role of JNK1 in perforin expression in CD8+ T cells, naive CD8+ TCR transgenic T cells (OT-1 or JNK1−/−OT-1) were activated with the cognate Ag for 72 h, and intracellular perforin expression was analyzed. JNK1−/−OT-1 CD8+ T cells showed lower levels of perforin (Fig. 6,A). This result was further confirmed by the observation that JNK1−/− CD8+ T cells showed significantly lower levels of CTL activity both in vitro and in vivo (Fig. 6, B and C). Further studies are needed to define the role of JNK1 in regulating perforin synthesis in CD8+ T cells. It is unclear at this stage whether the impaired CTL function in JNK1−/− CD8+ T cells is solely perforin-dependent or also resulted from impaired IFN-γ production.

CD8+ T cell effector functions require the generation of sufficient numbers of activated cells, which depend on cell proliferation and survival. We demonstrated that JNK1−/− CD8+ T cells had similar potential for proliferation in vivo (Fig. 6,E) as well as in vitro (Fig. 4). These results were somehow at odds with the previous finding that JNK1−/− CD8+ T cells had impaired ability for proliferation due to their low level of CD25 expression (15). Potential reasons for this discrepancy are the use of a homogenous population of transgenic CD8+ T cells in our current study vs a heterogenous population in the previous studies, and also the methods of stimulation (anti-CD3 vs DC/peptide) used in these studies.

It has to be emphasized that JNK1 may also affect functions of other cell types, such as NK, NKT, γδ T cells and innate immunity (DC and macrophages), which in turn contribute to tumor susceptibility. Studies are in progress to further dissect the impact of JNK1 in these cells to fully understand the role of JNK1 in tumor immune surveillance. In addition, we have obtained B6 JNK2−/− mice (13), and this will allow us to compare the different roles of JNK1 and JNK2 in antitumor immune responses.

In summary, we have presented the first evidence that JNK1 is crucial for tumor immune surveillance by regulating the effector function of CD8+ T cells, including their IFN-γ production and CTL function. These findings will not only shed light on the molecular mechanisms involved in tumor immune surveillance, but may also lead to the development of new strategies for tumor immunotherapy.

We thank Dr. Kim Bottomly from Yale Immunobiology for providing C57BL/6 OT-1 transgenic mice and Dr. Paula Kavathas for providing OVAp for our initial studies. We thank Drs. Fotios Koumpouras, Dan Kaplan, Bohdan Harvev, and Mark Mamula for critical review the manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by an Arthritis Foundation Investigator Award, National Institutes of Health (NIH) (National Institute of Arthritis and Musculoskeletal and Skin Diseases) Grant K01 AR 02188 and NIH (National Institute of Allergy and Infectious Diseases) Grant R01 (R01 AI56219) (to Z.Y.).

3

Abbreviations used in this paper: Eomes, Eomesodermin; TPA, 12-O-tetradecanoylphorbol-13-acetate; Wt, wild type; DC, dendritic cell; HPRT, hypoxanthine phosphoribosyltransferase; Tc, cytotoxic T cell.

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