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The Journal of Immunology, 2008, 180, 2140-2148
Copyright © 2008 by The American Association of Immunologists, Inc.
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IL-12 Is Required for Anti-OX40-Mediated CD4 T Cell Survival

Carl E. Ruby*, Ryan Montler*, Rongxui Zheng{dagger}, Suyu Shu{dagger} and Andrew D. Weinberg1,*

* Earle A. Chiles Research Institute, Providence Portland Medical Center, Portland, OR 97213; and {dagger} Center for Surgery Research, Cleveland Clinic, Cleveland, OH 14495


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Engagement of OX40 greatly improves CD4 T cell function and survival. Previously, we showed that both OX40 engagement and CTLA-4 blockade led to enhanced CD4 T cell expansion, but only OX40 signaling increased survival. To identify pathways associated with OX40-mediated survival, the gene expression of Ag-activated CD4 T cells isolated from mice treated with anti-OX40 and -CTLA-4 was compared. This comparison revealed a potential role for IL-12 through increased expression of the IL-12R-signaling subunit (IL-12Rβ2) on T cells activated 3 days previously with Ag and anti-OX40. The temporal expression of IL-12Rβ2 on OX40-stimulated CD4 T cells was tightly regulated and peaked ~4–6 days after initial activation/expansion, but before the beginning of T cell contraction. IL-12 signaling, during this window of IL-12Rβ2 expression, was required for enhanced T cell survival and survival was associated with STAT4-specific signaling. The findings from these observations were exploited in several different mouse tumor models where we found that the combination of anti-OX40 and IL-12 showed synergistic therapeutic efficacy. These results may lead to the elucidation of the molecular pathways involved with CD4 T cell survival that contribute to improved memory, and understanding of these pathways could lead to greater efficacy of immune stimulatory Abs in tumor-bearing individuals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
A member of the TNFR superfamily, OX40 (CD134) profoundly influences CD4 T cell activation and survival. The biologic function of OX40 on CD4 T cells is dependent on its expression and ligation. OX40 expression is induced 16–24 h after Ag and CD28 stimulation of T cells and maintained for up to 120 h (1). OX40 interaction with OX40L, its cognate ligand found on mature DCs and other activated APCs, increases proliferation and differentiation of effector cells, induces greater migration of T cells, and enhances their survival (2, 3, 4, 5, 6, 7). It is the ability of OX40 to enhance survival of CD4 T cells, in some cases 34-fold, that is of particular interest, as this property could aid in the development of effective immunotherapeutic strategies against tumors and/or chronic pathogens (8, 9, 10). Thus, understanding the underlying principles involved in OX40-mediated T cell survival should lead to improved immunotherapy.

Analysis of the pathways responsible for OX40-enhanced CD4 T cell survival has identified several proteins shown to affect apoptosis, yet much remains unknown. A handful of studies have revealed the role of known survival factors, including Akt, Bcl-2, and Bcl-xL, in OX40-mediated survival of CD4 T cells (11, 12). These studies suggest that OX40 can activate and sustain Akt signaling, which may lead to increased expression of the antiapoptotic Bcl-2 family of proteins and boost overall survival of CD4 T cells (11, 12, 13). However, this picture of OX40-mediated survival remains unclear, as the roles of Bcl-2 and Bcl-xL in CD4 T cells, in some cases, have been shown to be dispensable for CD4 T cell survival (6, 14).

Engagement of the CD28 family member CTLA-4 limits CD4 T cell expansion; therefore, CTLA-4 blockade, via Abs, has been shown to enhance CD4 T cell activation and expansion similar to OX40 agonists (15, 16). However, unlike OX40 agonists, systemic administration of anti-CTLA-4 does not enhance CD4 T cell survival (7, 17). It is this critical difference in survival between anti-OX40 and -CTLA-4-treated CD4 T cells that led to the gene array experiments reported here, the results of which demonstrate that the proinflammatory cytokine IL-12 is involved in OX40-enhanced T cell survival.

IL-12 is produced by innate immune cells in response to viral and bacterial products or CD40 stimulation, and enhances CD4 T cell differentiation, cytokine production, and survival. This heterodimeric cytokine, composed of the p35 and p40 proteins, primarily signals through the tightly regulated β2 subunit of the IL-12R, containing the intracellular signaling domains necessary for downstream transcription factor activation (18, 19). Although IL-12 is best known to induce differentiation of activated naive T cells to Th1 effectors, its influence on T cell survival has also been described (20, 21).

In the studies presented here, we investigated the signals responsible for anti-OX40-mediated survival of Ag-activated CD4 T cells. To accomplish this, TCR-transgenic T cell adoptive transfer models were used to track and isolate Ag-specific CD4 T cells primed in vivo with soluble Ag and treated with an agonist anti-OX40 Ab or a CLTA-4 antagonist Ab. OX40 engagement, but not CTLA-4 blockade, led to the expression of IL-12Rβ2 on Ag-activated CD4 T cells and the increase in cell surface expression of the IL-12Rβ2 protein was directly linked to OX40-mediated enhancement of T cell survival. This newly identified function for IL-12 in the survival of OX40-stimulated CD4 T cells appeared to be mediated in part by intracellular signaling of STAT4. Finally, the biologic relationship between OX40 and IL-12 was exploited in three different tumor models in which combined treatment exhibited CD4-dependent therapeutic synergy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Four- to 6-wk-old male and female C57BL/6 and BALB/c mice were purchased from Charles River Laboratories and used at 6–10 wk of age. BALB/c and C57BL/6 IL-12-deficient (p35–/–), DO11.10 (OVA323–339), OT-II (OVA323–339), OT-I (OVA357–364), and OT-II IL-12Rβ2-deficient mice were bred and maintained at the Earle A. Chiles Research Institute Animal Facility (Portland, OR). The DO11.10 STAT4-deficient mice were a gift from Dr. K. Murphy (Washington University, St. Louis, MO). All animal studies were approved by an internal animal care committee.

Adoptive transfer and immunization

Spleens and lymph nodes (LNs)2 from DO11.10, OT-I, OT-II, OT-II IL-12Rβ2-deficient, or DO11.10 STAT4-deficient mice were harvested and processed by crushing between two frosted glass microscope slides and RBC lysed with ACK (Invitrogen Life Technologies). The percentage of DO11.10 T cells or OT-I/II T cells were identified by FACS using KJ-126 or V{alpha}2-FITC and Vβ5-PE Abs, respectively, before transfer (BD Biosciences/BD Pharmingen). A total of 2–3 x 106 transgenic TCR T cells were adoptively transferred i.v. into BALB/c, C57BL/6, or IL-12-deficient recipients. One or 2 days later, mice were immunized s.c. with 500 µg of OVA (Sigma-Aldrich) and 50 µg of anti-OX40 (OX86) or 100 µg of anti-CTLA-4 (9H10), or IgG control (Sigma-Aldrich). The following day, mice were given a second injection of anti-OX40 or CTLA-4 or rat IgG. The blocking CTLA-4 Ab does not signal through CTLA-4, but rather interferes with the ligation of CD80/86 to CTLA-4, increasing the duration of CD28 stimulation, and also blocks potential downstream intra-T cell signaling by CTLA-4 (via Src homology region 2 domain-containing phosphatase 2 acting on the TCR/CD3/protein tyrosine kinase complex) (22).

Gene array analysis

Lateral axillary LNs were collected 4 days after immunization and processed into a single-cell suspension. Cells were stained for 0.5 h on ice with biotinylated KJ-126 Ab (0.5 mg/106 cell) and then washed. Cells were harvested using anti-biotin microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. Total RNA from the enriched KJ-126+ cells (>90%) was collected using RNeasy (Qiagen), according to the manufacturer’s instructions. The RNA samples were then sent to the Oregon Health and Sciences Cancer Institute Microarray Core Facility (Portland, OR) for further analysis. The core facility produced cRNA from the samples and probed the Affymetrix MOE 2.0A chip. The probed arrays were scanned and then analyzed by Affymetrix software.

FACS analysis of cells from peripheral blood, LNs, and spleens

Mice were bled via the tail vein into 50 µl of heparin and 750 µl of RPMI 1640 was added. The blood was underlaid with 500 µl of Lympholyte M (Cedarlane Laboratories) and centrifuged. The cells at the interface were collected and washed with FACS buffer (1% FBS, 0.1% sodium azide in PBS). Spleens and LNs were processed as previously described by Evans et al. (7) and stained with the following Abs: FITC-V{alpha}2, PE-Vβ5, allophycocyanin-CD62L, FITC-CD4, CyChrome-CD8, CyChrome-CD4, PE-CD95, PE-CD25, purified-IL-12Rβ2, or biotinylated-CD25, (BD Biosciences/BD Pharmingen), and PE-Cy5-CD127 (eBioscience). Harvested samples, isotype controls, and single stain controls were run on the FACSCalibur (BD Biosciences).

In vitro T cell cultures

Splenocytes (1–5 x 106) from wild-type, OX40–/–, or t-bet–/– mice were harvested and cultured with 1 µg/ml anti-CD3 for 24 h. Cultures were then treated with 20 µg/ml anti-OX40 (chicken-anti-mouse OX40 IgY) or Ig control (IgY) for an additional 48 h and analyzed.

Intracellular cytokine staining

Splenic T cells were obtained as described above and stimulated for 6 h in vitro with 2.5 or 5 µg/ml OVA peptide 323–339 or control peptide, SIINFEKL, in RPMI 1640 containing 10% FBS and 1 mg/ml Golgi Stop (BD Biosciences/BD Pharmingen). The cells were harvested and stained with FITC-CD4 and KJ-126. Cells were made permeable with CytoPerm/PermWash buffers and stained with allophycocyanin-IFN-{gamma} (BD Biosciences/BD Pharmingen).

Determination of annexin V+ cells

Spleens from adoptively transferred and immunized mice were harvested 4 days after immunization and a total of 1–2 x 106 CD4+ cells were plated in wells of a 24-well plate in 1 ml of medium and incubated overnight at 37°C. Cells were harvested 24 h later and stained according to the manufacturer’s instructions with FITC-CD4, KJ-126, and PE-annexin V (BD Biosciences/BD Pharmingen; Calbiochem).

Immunoblot analysis

LNs were isolated 4, 6, 7, and 8 days after immunization and cells were incubated with biotinylated anti-KJ-126 on ice. DO11.10 T cells were purified (>90%) with antibiotin microbeads via the AutoMacs cell sorter (Miltenyi Biotec). Lysates were prepared, run on polyacrylamide gels (1.5 x 106 cell equivalents/lane or equivalent microgram quantities of protein), and transferred onto nitrocellulose membranes. Abs for phospho-Akt, Akt, Bcl-xL (Cell Signaling Technology) phospho-STAT4, and STAT4 (Santa Cruz Biotechnology) were used to detect these proteins. NIH ImageJ software (National Institutes of Health, Bethesda, MD) was used to analyze the phospho-Akt/Akt data. Data were presented as percent of a ratio of phosphoprotein vs total protein.

Tumor models

The MCA205 sarcoma tumor cell line H12, a stable clone from the 3-methylcholanthrene-induced parental tumor, was suspended in HBSS (1–3 x 105) and injected i.v. into 6- to 8-wk-old female C57BL/6 mice to establish pulmonary metastases. Eighteen days after tumor inoculation, mice were sacrificed and metastatic tumor nodules on the surface of the lung were counted following counterstaining with India ink.

The TRAMP C1 prostate cancer line was derived from the transgenic adenocarcinoma of the mouse prostate (TRAMP) (23) and was shown to be MHC class II negative. Male C57BL/6 mice were challenged with 7.5 x 105 TRAMP C1 cells suspended in HBSS by s.c injection on the right flank. Following tumor challenge, mice were monitored for tumor growth and sacrificed if tumors were ulcerated or if tumor growth reached 150 mm2.

Electrofusion of dendritic cells (DCs) and tumor cells

DCs were generated from the spleens of mice injected with eight daily i.p. injections of Flt-3 ligand, as previously described (24), and were enriched by CD11c microbeads following the manufacturer’s instructions (Miltenyi Biotec). Cells were cultured for 24 h in medium containing 10 µg/ml of both GM-CSF and IL-4. DCs and irradiated MCA205 (H12) tumor cells were electrofused as previously described (25, 26).

Statistical analysis

For all experiments, a Student t test (two-tailed) was used to compare means of selected groups. For analysis, values of p ≤ 0.05 were considered significant and were expressed as follows: *, p ≤ 0.05; **, p ≤ 0.001; and ***, p ≤ 0.0001, if not specifically stated. Experimental replication is indicated in the figure legends.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
OX40 engagement induced the expression of IL-12Rβ2

Previous studies have shown that both OX40 engagement and CTLA-4 blockade effectively boosted the early expansion of Ag-specific CD4 T cells; however, only OX40 engagement led to a substantial increase in long-term T cell survival (7, 17). The molecular events responsible for the differences between these two immune-enhancing strategies could provide insights into the pathways mediating OX40-enhanced survival of CD4 T cells. Ag-activated T cells (DO11.10) were purified directly ex vivo from mice stimulated with anti-OX40 or -CTLA-4. RNA was isolated from the purified cells to determine differences in gene expression between the two groups via Affymetrix gene array analysis. At least a 3-fold increase or decrease in 163 gene products from Ag-specific CD4 T cells was observed in anti-OX40 vs -CTLA-4-treated mice. No significant differences were observed in the level of transcription for Bcl-2, Bcl-xL, or survivin, previously shown to be involved in OX40-mediated T cell survival (11, 27); however, IL-12Rβ2 mRNA was increased 17-fold upon OX40 engagement (Table I). Further analysis of the raw hybridization numbers revealed that IL-12Rβ2 transcript levels were "present" at low levels in the OX40 agonist group vs completely "absent" in the CTLA-4 blockade group.


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  		Table I. Differential gene expression between anti-OX40 stimulation and anti-CTLA-4 blockade in Ag-specific CD4 T cellsa

 
To verify the gene array data, IL-12Rβ2 protein levels were measured on Ag-activated transgenic CD4 T cells stimulated in vivo with Ag, in combination with anti-OX40, anti-CTLA-4, or rat IgG. Four days following immunization, expression of IL-12Rβ2 was measured on Ag-activated transgenic T cells from peripheral blood, draining LNs, and spleens. T cells isolated from anti-OX40-treated mice expressed the IL-12Rβ2 protein, with the greatest levels found in the Ag-draining LNs (Fig. 1A). In contrast, IL-12Rβ2 was not detected on Ag-specific CD4 T cells from either the rat IgG or anti-CTLA-4-treated mice at any time in either immune compartment (Fig. 1A and data not shown). Although the levels detected in the OX40-treated group appear low, they are in agreement with our gene array data where low—but detectable—levels of IL-12Rβ2 mRNA were observed. The kinetics of IL-12Rβ2 cell surface expression peaked 4 days after Ag stimulation and then decline thereafter until undetectable by day 7 (Fig. 1B). These results demonstrate that OX40 engagement produced a window of IL-12Rβ2 expression that occurred after initial expansion, but before contraction.


Figure 1
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  		FIGURE 1. Anti-OX40 induces the expression of IL-12Rβ2 on activated CD4 T cells in vivo and in vitro. A, A total of 3 x 106 OVA-specific DO11.10 CD4 T cells were adoptively transferred into recipient mice and 1 day later mice were immunized s.c. with 500 µg of OVA and 50 µg of anti-OX40, IgG, or 100 µg of anti-CTLA-4. A second s.c. injection of these Abs was given 24 h after the first injection. Four days after immunization, spleens and LNs were harvested and the cells were gated on the OVA-specific CD4 T cells (KJ-126+) and analyzed for the expression of IL-12Rβ2 (open histograms) or isotype control (gray histograms) by FACS. B, LNs from anti-OX40-treated mice were harvested 3, 4, 5, 6, and 7 days following OVA immunization and the kinetics of IL-12Rβ2 expression were analyzed; IL-12Rβ2 (open histograms) or isotype control (gray histograms). C, Cultured T cell blasts (see Materials and Methods) from wild-type, OX40-deficient (OX40–/–), or t-bet-deficient (t-bet–/–) mice were treated with 20 µg/ml anti-OX40 or IgG for 24–48 h, and CD4+ T cells were analyzed for the expression of IL-12Rβ2 and CD25. Data are representative of at least three separate experiments.

 
The specific requirements for IL-12Rβ2 expression on OX40-activated CD4 T cells were also investigated. Wild-type CD4 T cells activated with anti-CD3 and incubated with a control Ig failed to express IL-12Rβ2, but these cells when treated with anti-OX40 displayed a marked increase in IL-12Rβ2 expression (Fig. 1C). T cells deficient in OX40 or t-bet (required for IL-12Rβ2 expression (28)) did not express IL-12Rβ2 upon anti-OX40 stimulation. These data—as well as results from a CD4 T cell peripheral tolerance model that showed OX40 stimulation enabled T cells to become sensitive to IL-12 stimulation (29)—suggest a potential role for IL-12Rβ2 in some of the OX40-mediated effects on T cells.

IL-12 signaling is critical for OX40-enhanced CD4 T cell survival

The kinetics of IL-12Rβ2 expression following OX40 engagement suggested that IL-12 might play a role in OX40-mediated CD4 T cell survival. To determine whether IL-12R signaling directly affected CD4 T cell survival, OVA-specific TCR-transgenic CD4 T cells (OT-II) were backcrossed to IL-12Rβ2-deficient mice. The initial expansion of the IL-12Rβ2-deficient OT-II T cells following Ag and anti-OX40 administration was indistinguishable from wild-type OT-II; however, as the population of Ag-specific T cells contracted, significantly more wild-type OT-II T cells survived compared with the IL-12Rβ2-deficient OT-II T cells (Fig. 2A). Additional studies were conducted using IL-12 (p35)-deficient mice as recipients of wild-type DO11.10 T cells as a second approach. This approach allowed us to verify the OX40/IL-12 survival hypothesis in a different strain of mice with a different genetic mutation that has disrupted IL-12 signaling. In the absence of IL-12, survival of DO11.10 T cells in anti-OX40-immunized mice was severely diminished compared with wild-type mice (Fig. 2B) and similar to what was observed with IL-12Rβ2-deficient OT-II T cells. These differences in survival were evident 6 days after immunization, which was manifested as accelerated CD4 T cell contraction in IL-12-deficient hosts (Fig. 2C), and persisted for 60 days after immunization (Fig. 2D and data not shown). In these experiments, we saw similar levels of the Ag-specific CD4 T cells in the three compartments that were analyzed: PBL, LN, and spleen; however, this probably does not account for all the trafficking events involved. Finally, we have also shown anti-OX40 can enhance the survival of Ag-activated CD8 T cells (30); yet, the IL-12-specific survival, shown here, appeared to be specific for CD4 T cells, as survival of OX40-stimulated Ag-specific CD8 T cells was unaffected in the absence of IL-12 (data not shown).


Figure 2
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  		FIGURE 2. IL-12 is required for anti-OX40-enhanced survival of Ag-specific CD4 T cells, but not for IFN-{gamma} production. A, A total of 3 x 106 OVA-specific OT-II wild-type (Wt) or OT-II IL-12Rβ2-deficient CD4 T cells were adoptively transferred into wild-type recipient mice. One day later, mice were immunized with OVA and anti-OX40 or rat IgG as previously described. After immunization, PBL were collected on various days and the frequency of OVA-specific (KJ-126+) CD4 T cells was determined by FACS (n = 5). B, A total of 3 x 106 OVA-specific DO11.10 CD4 T cells were adoptively transferred into Wt or IL-12 (p35)-deficient mice (IL-12ko) and immunized with OVA and anti-OX40 or rat IgG. Peripheral blood was harvested and the frequency of DO11.10 CD4 T cells was determined for up to 28 days following immunization (n = 4–5). C, Frequency of DO11.10 CD4 T cells from peripheral blood between 4 and 10 days following immunization. D, DO11.10 CD4 T cells were enumerated from the spleens of OVA and anti-OX40 immunized Wt and IL-12-deficient (IL-12ko) mice (n = 3). E, Intracellular production of IFN-{gamma} from DO11.10 CD4 T cells harvested from the spleens of OVA and anti-OX40-immunized Wt or IL-12ko mice (day 4) was measured following ex vivo restimulation with peptide. Data are representative of at least two to four independent experiments.

 
The effector function, assessed by IFN-{gamma} production, of anti-OX40-stimulated Ag-specific CD4 T cells was unaffected by the absence of IL-12. DO11.10 T cells isolated 4 days following immunization and anti-OX40 from IL-12-deficient mice produced similar levels of IFN-{gamma} when compared with wild-type controls (Fig. 2E). Although this data seems to be at odds with the premise that IL-12 drives a Th1 response, previous studies have shown that IL-12 (p35)-deficient mice infected with lymphocytic choriomeningitis virus produce equivalent levels of IFN-{gamma} when compared with wild-type mice (31). Furthermore, a recent study demonstrated IL-12Rβ2 did not play a significant role in the differentiation of Ag-specific CD4 T cells following OX40 engagement (32). Therefore, in anti-OX40-stimulated CD4 T cells, IL-12 appears to be more important for survival than Th1 differentiation.

The experiments described above have demonstrated that anti-OX40-stimulated Ag-specific CD4 T cells primed in the absence of IL-12 signaling contract rapidly and fail to survive. Thus, to determine whether the CD4 T cells were irreversibly affected by the absence of IL-12, they were adoptively transferred into IL-12-sufficient hosts to test whether their survival could be rescued. DO11.10 T cells from OVA- and anti-OX40-immunized IL-12-deficient hosts were harvested 4 days after immunization, and equal numbers of OVA-specific T cells were transferred into wild-type or IL-12-deficient recipients. Fourteen days after immunization, LNs were harvested and the survival of OVA-specific T cells was measured. If IL-12 was required during Ag priming (days 0–4), we would expect that T cells would fail to survive in both recipients. This was not the case, as the number of DO11.10 T cells was significantly greater in IL-12-sufficient mice compared with IL-12-deficient mice, demonstrating that IL-12 signaling several days after Ag priming can rescue T cells primed in the absence of IL-12 (Fig. 3A). The converse was observed when OVA-specific T cells primed for 4 days in IL-12 intact mice were transferred into IL-12-deficient hosts. The numbers of OVA-specific T cells were significantly decreased compared with OVA-specific T cells transferred to intact hosts (Fig. 3B). Furthermore, data in Fig. 1A showed that IL-12Rβ2 expression was undetectable 7 days following immunization and OX40 stimulation, suggesting that the biological effect of IL-12 had to occur after priming but before day 7. To test this hypothesis, OVA-specific T cells were primed in wild-type mice and, 8 days later, were transferred into IL-12-deficient and -sufficient hosts. The number of DO11.10 T cells in LNs harvested 10 days later was the same in IL-12-deficient and -sufficient hosts (Fig. 3C). These results suggest that OX40-enhanced CD4 T cell survival is dependent on the timeframe/kinetics determined by IL-12Rβ2 expression on Ag-specific CD4 T cells.


Figure 3
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  		FIGURE 3. IL-12 signals provided after priming, but before contraction, improved the survival of Ag-specific CD4 T cells stimulated with anti-OX40. A total of 3 x 106 OVA-specific DO11.10 CD4 T cells were adoptively transferred into wild-type (wt) or IL-12 (p35)-deficient mice (IL-12ko) and immunized s.c. with 500 µg of OVA and 50 µg of anti-OX40. A second s.c. injection of 50 µg of anti-OX40 was given 48 h after immunization. A, DO11.10 CD4 T cells from immunized IL-12-deficient mice were harvested from the spleen and LNs 4 days following immunization and 1–2 x 106 DO11.10 T cells were transferred to wild-type (+/+) or IL-12-deficient (–/–) mice immunized with 500 µg of OVA and 25 µg of LPS 4 days before transfer. LNs were harvested 10 days after transfer and DO11.10 CD4 T cells measured (n = 3–6). B, DO11.10 CD4 T cells from immunized wild-type mice were harvested from the spleen and LNs 4 days following immunization and 1–2 x 106 DO11.10 T cells transferred to wild-type (+/+) or IL-12-deficient (–/–) mice immunized with 500 µg of OVA and 25 µg of LPS 4 days before transfer. LNs were harvested 8 or 10 days after transfer and DO11.10 CD4 T cells measured (n = 3). C, DO11.10 CD4 T cells from immunized wild-type mice were harvested from the spleen and LNs 8 days following immunization and 1 x 106 DO11.10 T cells transferred to wild-type (+/+) or IL-12-deficient (–/–) mice immunized with 500 µg of OVA and 25 µg of LPS 8 days before transfer. LNs were harvested 10 days after transfer and DO11.10 CD4 T cells measured (n = 3). The data are representative of two to three independent experiments.

 
STAT4 signaling is involved in OX40-mediated survival

To determine the molecular signaling events by which IL-12 mediates anti-OX40 survival, we investigated two known IL-12-mediated signaling pathways, Akt and STAT4. The Akt-signaling pathway has been shown to be activated by IL-12 and has been linked to OX40-mediated survival (12, 20); STAT4 signaling, mediated by IL-12, is involved with Th1 differentiation (18, 19). Phosphorylation of both proteins is required for downstream signaling and was analyzed in Ag-specific T cells isolated from Ag and anti-OX40 immunized wild-type or IL-12-deficient recipients. There was no difference in the level of phospho-Akt between wild-type and IL-12-deficient mice over the course of 4–7 days after immunization (Fig. 4A). In contrast, levels of phospho-STAT4 in Ag-specific T cells from IL-12-sufficient mice were greater than in IL-12-deficient mice (phospho-STAT4/total STAT4). The greatest difference occurred 7–8 days postimmunization, when Ag-specific CD4 T cells in the absence of IL-12 experienced accelerated contraction (Fig. 4, B and C). Interestingly, substantial levels of phosopho-STAT4 were still detected in OVA-specific CD4 T cells isolated from IL-12-deficient mice, possibly due to other inflammatory cytokines signaling through STAT4 (e.g., IFN-{alpha}/IFN-β and/or IL-23) (31, 33, 34).


Figure 4
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  		FIGURE 4. The effect of IL-12 on Akt and STAT4 signaling in anti-OX40-stimulated Ag-specific CD4 T cells. LNs were harvested from wild-type (+/+) or IL-12-deficient (–/–) mice various days after OVA and anti-OX40 immunization. DO11.10 CD4 T cells were enriched (>90%) and lysates from the DO11.10 CD4 T cells were analyzed by immunoblot. A, Phospho-Akt (PO4-Akt) and total Akt (Akt). B, Phospho-STAT4 (PO4-STAT4) and total STAT4 (STAT4). C, The ratio of phosph-STAT4 to total STAT4 was measured by image analysis (NIH Image J software) in three independent experiments.

 
Differences in STAT4 phosphorylation in OVA-specific T cells from anti-OX40-treated IL-12-deficient mice suggest IL-12-mediated STAT4 activation could be important in OX40-mediated survival. To address the role of STAT4 in OX40-mediated T cell survival, we transferred STAT4-deficient DO11.10 T cells into wild-type mice and compared their lifespan to STAT4-sufficient DO11.10 T cells following OVA and anti-OX40 immunization. Early expansion of both STAT4-deficient and wild-type DO11.10 T cells was similar; however, 7 days following immunization, STAT4-deficient DO11.10 T cells were dramatically decreased when compared with STAT4-sufficient DO11.10 T cells transferred into either wild-type or IL-12-deficient hosts (Fig. 5A). There was a decline in both the frequency and numbers of OVA-specific T cells in these experiments (data not shown). STAT4-deficient T cells were then assessed for an apoptotic phenotype by ex vivo examination of annexin V. The STAT4-deficient and wild-type DO11.10 T cells for these experiments were harvested from mice (wild-type and IL-12–/–) 4 days after immunization. The frequency and number of the transferred transgenic T cells harvested from these mice at this time were not overtly different (Fig. 5B) and reflected previous results (Fig. 5A). The STAT4-deficient DO11.10 T cells displayed increased annexin V staining compared with STAT4-sufficient OVA-specific T cells harvested from wild-type or IL-12-deficient hosts (Fig. 5B).


Figure 5
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  		FIGURE 5. The effect of STAT4 on anti-OX40-stimulated Ag-specific CD4 T cell survival and function. A total of 3 x 106 OVA-specific DO11.10-STAT4-deficient (STAT4–/–) CD4 T cells were adoptively transferred into wild-type (Wt) mice. In addition, 3 x 106 Wt DO11.10 CD4 T cells were adoptively transferred into Wt or IL-12-deficient mice (IL-12–/–). All mice were then immunized with OVA and anti-OX40 as previously described. A, Four and 7 days after immunization, LNs were harvested and the frequency of DO11.10 CD4 T cells determined (n = 3–4). B, Four days after immunization, spleens were harvested and the frequency and number of DO11.10 CD4 T cells determined. Then, 1 x 106 cells were cultured overnight in medium, and 24 h later the DO11.10 CD4 T cells were analyzed for the expression of annexin V. C, Intracellular production of IFN-{gamma} and IL-4 by splenocytes from Wt and STAT4-deficient DO11.10 CD4 T cells harvested 4 days after immunization. Data representative of two to three independent experiments.

 
Finally, STAT4 also plays a role in the differentiation of CD4 T cells by binding to promoter regions acting on IFN-{gamma} expression and IFN-{gamma}-activated genes (35, 36). Analysis of IFN-{gamma} and IL-4 production from STAT4-deficient OVA-specific effector T cells harvested 4 days following immunization revealed impaired IFN-{gamma} production, which is in contrast to wild-type T cells transferred into IL-12-deficient hosts (Figs. 5C and 2E). Therefore, STAT4 signaling appears to be important for both Th1 differentiation and OX40-meditated survival.

IL-12 synergizes with anti-OX40 to enhance antitumor immunity

The critical role for IL-12 in OX40-mediated survival of Ag-specific CD4 T cells described above suggests that the combination of OX40 and IL-12 signaling could drive enhanced immunity in tumor-bearing hosts. We therefore tested the combination of anti-OX40 and IL-12 in several tumor models. First, in a pulmonary metastases model (MCA205), mice were vaccinated with DC:tumor fusion hybrid cells (25, 26) with anti-OX40, IL-12, or the combination 10 days after tumor inoculation. Treatment with DC:tumor fusion cells alone showed no antitumor efficacy (data not shown). Moreover, the combination of fusion cell vaccination with anti-OX40 or IL-12 alone did not reduce lung metastases; however, vaccination combined with both anti-OX40 and IL-12 led to a significant reduction in pulmonary metastases (Fig. 6A).


Figure 6
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  		FIGURE 6. Combined treatment with anti-OX40 and IL-12 enhanced antitumor responses. A, C57BL/6 mice were injected i.v. with 1–3 x 105 MCA205 tumor cells to establish pulmonary metastases. Ten days later, 1 x 106 DC-tumor fusions and/or 300 µg of anti-OX40 and/or 100 ng of rIL-12 were injected i.p. Mice were sacrificed 18 days after tumor challenge and the number of metastases was counted. B, Male C57BL/6 mice were challenged with 7.5 x 105 TRAMP C1 tumors injected s.c. Anti-OX40 or rat IgG (250 µg) was administered i.p. days 3 and 7 after tumor transplantation and IL-12 (100–200 ng) was administered i.p. days 4–9 after tumor transplantation. In addition, mice were injected i.p. with depleting anti-CD4 (GK1.5) 1 day before being challenged with 7.5 x 105 TRAMP C1 tumors and treated with anti-OX40 and IL-12 (n = 5/group). Data representative of two to three independent experiments.

 
The efficacy of the combined treatment of anti-OX40 and IL-12 was also examined in the s.c. tumor model, TRAMP-C1 prostate tumor (23). In this model, anti-OX40 was injected 3 and 7 days posttumor inoculation, as previously described (9). The TRAMP C1 tumor was resistant to anti-OX40 monotherapy and to therapies that combined anti-OX40 with androgen ablation, rIL-2, or irradiated GM-CSF-secreting tumor as a vaccine (data not shown). In addition, IL-12 monotherapy, six daily injections starting 4 days after tumor transplantation, failed to mediate any tumor-free survival (Fig. 6B). The combination of IL-12 treatment with anti-OX40 treatment showed a synergistic therapeutic effect in mice challenged with s.c. TRAMP-C1 tumors (Fig. 6B). To determine whether the protective tumor immunity induced by anti-OX40 and IL-12 was dependent on CD4 T cells, we depleted CD4 T cells 1 day before tumor inoculation and treatment. The ability of the combined treatment to protect against TRAMP-C1 tumors was completely ablated following CD4 T cell depletion (Fig. 6B). The data from these two tumor models—as well as results from a third tumor model, CT26 colon carcinoma (data not shown)—demonstrate the ability of IL-12 to dramatically enhance anti-OX40-mediated tumor therapy.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our results demonstrate that the improved survival of Ag-activated CD4 T cells observed following OX40 engagement requires signaling of the proinflammatory cytokine IL-12. Survival via an IL-12-specific signal is due to the up-regulation of the signaling chain of the IL-12R, IL-12Rβ2, on CD4 T cells 4–6 days following OX40 engagement, though the exact mechanism behind the up-regulation of IL-12Rβ2 by OX40 has yet to be determined. The cooperation of OX40 and IL-12 signaling appears to rely in part on STAT4 activation, which suggests STAT4-mediated transcription not only accounts for the differentiation of Ag-specific effector CD4 T cells, but is also critical for survival. These results may help to explain the enhanced immunotherapeutic effects of combined OX40 stimulation and IL-12 treatment in several tumor models.

The results presented here are the first to describe the involvement of IL-12 signaling in the mechanism of OX40-mediated survival of CD4 T cells. Initial OX40 engagement has been shown to activate the transcription factor NF-{kappa}B, which is capable of inducing the expression of a number of survival proteins (37, 38). The OX40-mediated NF-{kappa}B activation most likely occurs during cell surface expression of OX40, between 24 and 72 h after TCR priming. This "early" survival protein induction could then be maintained by other signals and/or potentially shift to other "long-term" survival proteins and pathways. These proteins may include several antiapoptotic proteins such as Bcl-2, Bcl-xL, and pAkt that have been previously linked to this survival phenomenon (11, 12, 13). Our results suggest that events involved in OX40-mediated survival may require other signals before or in addition to these antiapoptotic proteins (6). For example, our studies show similar levels of pAkt from anti-OX40-treated T cells from IL-12-deficient and -sufficient mice, yet the T cells in the absence of IL-12 signaling failed to survive. Thus, it appears IL-12 facilitates a critical OX40-mediated survival signal that is upstream or independent of some of the antiapoptotic proteins previously described (11, 12, 13).

The survival signal mediated by IL-12 in OX40-stimulated T cells coincided with the temporary expression of IL-12Rβ2 on the Ag-specific CD4 T cells. The surface expression of IL-12Rβ2 on effector T cells occurred near the start of contraction (4–6 days postimmunization), thereby placing the timing of IL-12-mediated survival between a first set of survival signals primarily mediated by CD28 and IL-2 during the Ag priming and a final set of signals that maintains long-term memory likely through IL-7 signaling (39, 40). The limited window for IL-12-mediated effector T cell survival demonstrate that IL-12 is not involved in the maintenance of long-lived memory CD4 T cells, which was also observed in a study measuring fungal Ag-specific T cells in IL-12-deficient hosts (41).

A role for IL-12 survival signaling may not be specific for OX40 and may include other members of the TNFR family. Recently, CD27 stimulation of CD4 T cells was also shown to induce the IL-12Rβ2 protein (42). CD27, a costimulatory member of the TNFR family, has the ability to increase the survival of Ag-primed CD4 T cells (43), like OX40, and IL-12 signaling may play a role in CD27-mediated survival. In contrast, it could be convenient to also speculate that the IL-12Rβ2 induced by CD27 and OX40 may have a different function, due to the differential expression of these two TNFRs, as CD27 expression occurs within 24 h of initial TCR priming, and OX40 24–72 h after TCR priming. Additional studies would be needed to clarify the role of IL-12 in CD27-stimulated T cells and if other members of the TNFR family also induce IL-12Rβ2.

IL-12 signaling in OX40-stimulated CD4 T cells promotes T cell survival that appears to be mediated in part by STAT4. Indeed, decreased levels of phospho-STAT4 were found in anti-OX40-treated T cells isolated from IL-12-deficient hosts, and STAT4-deficient T cells were rapidly deleted following Ag and anti-OX40 priming. STAT4-deficient effector T cells also failed to produce IFN-{gamma}, which suggest that STAT4 also plays a role in effector cell differentiation. Our data point to two possible divergent roles for STAT4 in OX40-stimulated CD4 T cells: 1) IL-12-mediated activation of STAT4 drives survival and 2) early activation of STAT4 during the priming phase by other cytokines (e.g., IFN-{alpha}/IFN-β) may be required for effector cell differentiation (31, 44). The STAT4-specific differentiation signals delivered to OX40-stimulated T cells appear to preclude the STAT4-survival signals initiated by IL-12. Potential survival signals mediated by IL-12/STAT4, may include IL-18R and the short-lived kinase pim-1. IL-12 and STAT4 have been directly linked to expression of IL-18R, which has been shown to "strengthen/extend" T cell responses and directly signals through the transcription factor NF-{kappa}B for a possible second round of survival signaling (45, 46). The survival kinase pim-1 is also induced by IL-12/STAT4 signaling and pim-1 can directly inactivate the proapoptotic protein Bad (47, 48). Additional experiments are required to fully understand the timing, survival proteins induced and roles played by innate cytokines in STAT4 activation, survival, and differentiation.

OX40 signals have been demonstrated to be critical in the generation of both Th1 and Th2 responses (3, 49), yet a role for IL-12 in Th2 survival seems unlikely. The CD4 T cell responses shown here appear to be primarily skewed toward a Th1 response, as assessed by a greater IFN-{gamma}:IL-4 ratio (Fig. 5C). Therefore, this study demonstrated the mutual dependence of OX40 and IL-12 for optimal survival of CD4 T cells that differentiate into Th1 cells, but IL-12 most likely would not influence the survival of CD4 T cells under conditions that favor Th2 differentiation. OX40-mediated Th2 survival may then originate from OX40L-expressing thymic stromal lymphopoietin-activated DCs and/or CD4+CD3– accessory cells found in B cell areas (50, 51, 52).

Successful tumor regression using the combination of OX40 engagement and IL-12 suggest effective antitumor responses in vivo may require critical "danger" signals (e.g., CpG, dsRNA, or LPS). A theory as to why some tumors do not elicit potent immune priming may be the lack of these "danger" signals, which would result in the lack of proinflammatory cytokines and costimulatory molecules, such as IL-12 and OX40 ligand. Restoration of these signals in tumor-bearing mice may help boost adaptive and innate immunity, which are likely missing in the nonimmunogenic tumor environment. The effects of IL-12 and OX40 signaling, observed in animal studies, could translate to increased success of immunotherapy in patients with cancer by increasing the longevity of tumor Ag-specific T cell responses. In fact, a recent clinical study in cancer patients identified a correlation between the persistence of tumor-specific T cells and clinical responses in patients with melanoma (10). The studies presented here also highlight differences in the modes of action of anti-CTLA-4 and anti-OX40 which are both currently in clinical trials for tumor immunotherapy (53) (A. D. Weinberg, unpublished observation).

In conclusion, the data described here identify a novel pathway showing that IL-12 is essential for OX40-mediated CD4 T cell survival in vivo. The dependence of OX40 on IL-12 suggest that this interaction is an important link between the innate and adaptive arms of immunity. Ultimately, these findings could be exploited to enhance immunotherapy in patients with cancer or chronic pathogens and could help to elucidate the exact molecular pathways involved with CD4 T cell survival that lead to improved memory T cell survival.


   Acknowledgments
 
We thank Drs. Walter Urba and Anthony Vella for their critical review of this manuscript, and Dr. Kenneth Murphy for providing the STAT4–/– DO11.10 mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 Address correspondence and reprint requests to Dr. Andrew D. Weinberg at the current address: Laboratory of Basic Immunology, Robert W. Franz Cancer Research Center, 4805 NE Glisan Street, Portland, OR 97213. E-mail address: andrew.weinberg{at}providence.org Back

2 Abbreviations used in this paper: LN, lymph node; TRAMP, transgenic adenocarcinoma of the mouse prostate; DC, dendritic cell. Back

Received for publication September 18, 2007. Accepted for publication December 10, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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