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The Journal of Immunology, 2007, 179, 7244 -7253
Copyright © 2007 by The American Association of Immunologists, Inc.
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Defects in the Acquisition of CD8 T Cell Effector Function after Priming with Tumor or Soluble Antigen Can Be Overcome by the Addition of an OX40 Agonist1

William L. Redmond*, Michael J. Gough*, Bridget Charbonneau{dagger},{ddagger}, Timothy L. Ratliff2,{dagger} and Andrew D. Weinberg3,*

* Earle A. Chiles Research Institute, Robert W. Franz Cancer Research Center, Providence Portland Medical Center, Portland, OR 97213; and {dagger} Department of Microbiology and {ddagger} Department of Urology, University of Iowa, Iowa City, IA 52242


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Several members of the TNFR superfamily, including OX40 (CD134), 4-1BB (CD137), and CD27 provide critical costimulatory signals that promote T cell survival and differentiation in vivo. Although several studies have demonstrated that OX40 engagement can enhance CD4 T cell responses, the mechanisms by which OX40-mediated signals augment CD8 T cell responses are still unclear. Previously, we and others have shown that OX40 engagement on Ag-specific CD8 T cells led to increased CD8 T cell expansion, survival, and the generation of greater numbers of long-lived memory cells. Currently, we demonstrate that provision of an OX40 agonist during the activation of naive CD8 T cells primed in vivo with either soluble or tumor-associated Ag significantly augments granzyme B expression and CD8 T cell cytolytic function through an IL-2-dependent mechanism. Furthermore, augmented CTL function required direct engagement of OX40 on the responding CD8 T cells and was associated with increased antitumor activity against established prostate tumors and enhanced the survival of tumor-bearing hosts. Thus, in the absence of danger signals, as is often the case in a tumor-bearing host, provision of an OX40 agonist can overcome defective CD8 T cell priming and lead to a functional antitumor response in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The generation of optimal T cell responses requires TCR-mediated recognition of cognate peptide-MHC complexes on activated APCs that express high levels of costimulatory molecules. In addition to B7-mediated signaling through CD28 expressed on T cells, several members of the TNFR superfamily, such as 4-1BB (CD137), CD27, and OX40 (CD134), have been shown to provide critical signals that enhance and sustain both CD4 and CD8 T cell responses in vivo (1, 2, 3). In particular, costimulation through the OX40 receptor has been shown to play a key role in driving the differentiation of effector and memory CD4 T cells (4, 5, 6, 7, 8, 9, 10, 11). However, the effects of OX40-mediated signaling on CD8 T cell differentiation are less well understood.

Several groups have demonstrated that engagement of OX40 through the endogenous OX40 ligand (OX40L)4 is not required to drive the differentiation of naive CD8 T cells into effector CTL following priming under proinflammatory conditions, including with lymphocytic choriomeningitis virus, influenza virus, or a MHC class I-restricted peptide in adjuvant (9, 12). Under these conditions, the most striking effect of OX40-mediated signaling on CD8 T cell responses appears to be in the determination of the extent of T cell survival, rather than affecting their differentiation into effector CTL (9, 12, 13). However, under certain circumstances, specifically adenovirus-specific priming, endogenous OX40L-mediated signaling was important for the promotion of CD8 T cell differentiation (14). The reasons for these differences are not known, but may be related to the extent to which various pathogens induce various costimulatory molecules, including OX40L, on APCs. There may also be some functional redundancy among the many TNFR family members (1, 15), which may account for the observation that the majority of proinflammatory stimuli induce normal CD8 T cell differentiation in OX40-deficient hosts (12, 16).

In contrast to a proinflammatory environment, the extent of endogenous OX40L expression is limited during the generation of antitumor responses due to the potentially immunosuppressive tumor microenvironment, which often lacks the required danger signals (17, 18). Indeed, engagement of OX40 through endogenously expressed OX40L was recently shown to promote tumor-specific CD8 T cell expansion and survival, but was not required to promote the acquisition of CD8 T cell effector function (13, 19). However, we and others have shown that treatment with an agonist anti-OX40 mAb, or immunization with cells (tumor or APCs) modified to express high levels of OX40L, can promote robust T cell-mediated antitumor responses (3, 20, 21, 22, 23, 24, 25, 26). Although OX40-mediated treatment clearly can lead to the generation of potent antitumor responses, the mechanisms by which OX40 signaling augments tumor-specific CD8 T cell responses are still unknown.

In the current study, we examined the ability of anti-OX40 treatment to augment CD8 T cell differentiation following activation with either soluble or tumor-associated Ag under noninflammatory conditions. Our data demonstrate that OX40 engagement significantly enhanced the differentiation of CD8 T cells in vivo via an IL-2- and granzyme B-dependent mechanism, which ultimately led to the regression of established tumors. Importantly, the generation of optimal tumor-specific effector CTL required direct engagement of OX40 on the responding CD8 T cells. Taken together, these data demonstrate that provision of an OX40 agonist to CD8 T cells stimulated in an environment that lacks danger signals leads to productive CD8 T cell differentiation and the acquisition of effector function in vivo.


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

Wild-type or CD25+/– C57BL/6 mice were purchased from The Jackson Laboratory. OT-I Thy1.1 TCR transgenic (Tg; prostate OVA-expressing Tg) POET-1 Tg (27), and OX40–/– OT-I TCR Tg mice were provided by Dr. C. Surh (The Scripps Research Institute, La Jolla, CA), Dr. T. Ratliff (University of Iowa, Iowa City, IA), and Dr. M. Croft (La Jolla Institute for Allergy and Immunology, La Jolla, CA), respectively. All mice were bred and maintained under specific pathogen-free conditions in the Providence Portland Medical Center animal facility. Experimental procedures were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Adoptive transfer and activation of OT-I T cells

Single-cell suspensions were prepared from the lymph nodes (LN) and spleens of OT-I Thy1.1 TCR Tg mice. Cell suspensions were depleted of CD4+, CD11b+, CD45R+, DX5+, and Ter-119+ cells using the MACS CD8{alpha}+ T cell isolation kit (Miltenyi Biotec). OT-I T cells were purified by negative selection per the manufacturer’s instructions and had a naive phenotype (CD25–CD44lowCD62LhighCD69–) as indicated by flow cytometry (data not shown). 3Three to 4 x 106 OT-I T cells were injected i.v. in 200 µl of PBS into recipient mice.

One day after the adoptive transfer of OT-I T cells (day 0), 500 µg of soluble chicken egg OVA (Sigma-Aldrich) or 6 x 106 TRAMP-C1-membrane-bound OVA (mOVA) tumor cells were injected s.c. in 200 µl of PBS into recipient mice. In addition, mice received either 50 µg of anti-OX40 (OX86) or isotype control rat IgG Ab (Sigma-Aldrich) s.c. on days 0 and 1. Where indicated, recipient mice received 250 µg of anti-CD4 (GK1.5) mAb i.p. (day–2, 0) and the extent of CD4 T cell depletion (>99%) was confirmed by flow cytometry.

Generation of TRAMP-C1-OVA tumor cells

Plasmid DNA vector cloning were as follows: TfRmOVA fusion construct (pWPT-TfRmOVA) was amplified by PCR with Vent polymerase (the template used for PCR is pBlueRIP-TfRmOVA plasmid, provided by Dr. C. Kurts, University of Bonn, Bonn, Germany) and cloned into the lentiviral vector pWPT. The pWPT vector was provided by Dr. D. Trono (Swiss Institute of Technology, Lausanne, Switzerland) and modified to enable the convenient cloning method with the NEB USER Enzyme (New England Biolabs). For the generation of recombinant lentiviruses, HEK 293 cells were transiently transfected with vector plasmid pWPT-TfRmOVA, virus-packaging plasmid pPAX2, envelope plasmid VSV-G MD2, and helper plasmid pAdvantage (Promega). Viral supernatant was used to infect tumor cells and tumor cells were sorted based on surface expression of OVA protein by flow cytometry.

Gene array analysis

Naive OT-I T cells were adoptively transferred into wild-type mice and then activated with soluble OVA and anti-OX40 or rat IgG Ab. Four days later, LN were harvested and donor OT-I T cells were purified by magnetic bead separation using an AutoMACS (Miltenyi Biotec). Total RNA from the donor OT-I T cells was harvested using RNeasy (Qiagen) according to the manufacturer’s instructions. The RNA samples were then sent to the Oregon Health and Science University microarray core facility (Beaverton, OR) for analysis. The core facility produced cRNA from the samples and then probed the Affymetrix MOE 2.0A chip. The probed arrays were scanned and then analyzed using Affymetrix software.

Flow cytometry

LN or spleens were harvested and processed to obtain single-cell suspensions. Ammonium chloride-based RBC lysis buffer (ACK lysing buffer, Cambrex) was added for 5 min at room temperature to lyse RBC. Cells were then rinsed with RPMI 1640 medium (Cambrex) containing 10% FCS (10% cRPMI; Gemini Bio-Products) supplemented with 1 M HEPES, nonessential amino acids, sodium pyruvate, and penicillin-streptomycin-glutamine (Cambrex). Cells were incubated for 30 min on ice with a combination of the following Abs: Thy1.1 FITC, Thy1.1-PE, V{alpha}2-FITC, CD8{alpha}-PE-Cy7, CD25-PE, CD62L-allophycocyanin (Ebioscience), and anti-human/mouse-granzyme B-PE (Caltag Laboratories/Invitrogen Life Technologies). After washing three times with PBS containing 0.1% w/v BSA (Sigma-Aldrich) and 0.02% w/v sodium azide, cells were resuspended in FACS buffer. For intracellular granzyme B staining, cells were fixed in 1% paraformaldehyde for 20 min on ice, rinsed one time with FACS buffer, and then permeabilized with 1x Permwash (BD Pharmingen) for 20 min on ice. Cells were analyzed with a FACSCalibur and CellQuest software (BD Biosciences).

To measure Ag-specific cytokine production, lymphocytes were incubated in 10% cRPMI with 5 µg/ml of the OVA (SIINFEKL) peptide (Anaspec) and 1 µl/ml brefeldin A containing Golgi-Plug solution (BD Pharmingen) for 5 h at 37°C. After washing, cells were stained to detect CD8 and Thy1.1 as described above. Cells were then permeabilized and stained with anti-IL-2-PE and anti-IFN-{gamma}-allophycocyanin mAb using the Cytofix/Cytoperm Plus kit (BD Pharmingen) according to the manufacturer’s instructions.

In vivo and in vitro cytolytic assays

In vivo cytolytic assay. In brief, 3 x 106 nonlabeled, purified OT-I Thy1.1 T cells were injected into recipient mice and activated as described above. Four days later, syngeneic spleen cells were labeled by incubation for 10 min at 37°C with either 5 µM CFSE in PBS (CFSEhigh cells) or 0.5 µM CFSE in PBS (CFSElow cells) and washed twice with HBSS. CFSEhigh cells were pulsed with OVA peptide at 5 µg/ml for 1 h at 37°C. CFSElow cells were not pulsed with peptide and served as an internal control. A mixture of 5 x 106 CFSEhigh peptide-pulsed cells plus 5 x 106 CFSElow nonpulsed cells were injected i.v. into recipient mice. Splenocytes were harvested 4–16 h later and single-cell suspensions were analyzed for detection and quantification of CFSE-labeled cells by flow cytometry.

In vitro cytolytic assay. OT-I T cells were harvested as described above and then stained with V{alpha}2-FITC, CD8, and Thy1.1 to calculate the total number of OT-I T cells per animal. Syngeneic splenocytes were pulsed with either 5 µg/ml OVA-peptide (SIINFEKL) or no peptide for 60 min at 37°C. Cells were then rinsed one time each with 10% cRPMI followed by PBS. Next, peptide-pulsed and nonpeptide-pulsed targets were labeled with 5 µM CFSE (CFSEhigh) or 0.5 µM CFSE (CFSElow), respectively, for 10 min at 37°C. After washing two times with 10% cRPMI, targets were counted and cultured with OT-I T cells at the indicated E:T ratio for 4–7 h. The percent specific lysis was calculated as 100 – (100 x ((CFSElow/CFSEhigh control group)/(CFSElow/CFSEhigh experimental group)).

Tumor challenge and TIL isolation

In brief, 2.5 x 106 TRAMP-C1-mOVA prostate tumor cells were injected s.c. (day 0) into male POET Tg mice. When tumors reached ~25 mm2 (~21–24 days after tumor inoculation), mice received either 3 x 106 (for CD8 T cell analysis) or 5 x 104 (for long-term tumor regression analysis) naive wild-type, OX40–/–, or CD25–/– OT-I T cells. Anti-OX40 or rat IgG Ab (50 µg i.p.) was given the day of OT-I T cell transfer along with an additional dose 1 day later. Four days after adoptive transfer, tumor-infiltrating lymphocytes (TIL) were harvested by dissection of tumors into small fragments followed by digestion in 1 mg/ml collagenase, 100 µg/ml hyaluronidase, and 20 mg/ml DNase in PBS. Following filtration through nylon mesh, donor OT-I T cells were analyzed by flow cytometry. Tumor growth (area) was assessed every 2–3 days with microcalipers and mice were sacrificed when tumors reached >150 mm2.

Statistical analysis

Statistical significance was determined by the unpaired Student t test (for comparison between two groups), one-way ANOVA (for comparison among three or more groups), or Kaplan-Meier survival (for tumor survival studies) using GraphPad InStat or Prism software (GraphPad); a p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Anti-OX40 treatment augments IL-2R{alpha} (CD25) and granzyme B expression in Ag-specific CD8 T cells

Recent studies from our laboratory and others have demonstrated the importance of OX40-mediated signals in enhancing CD8 T cell expansion and survival (13, 23, 26, 28). To elucidate the mechanisms by which anti-OX40 treatment augments the CD8 T cell response, we used DNA microarray analysis to compare global changes in gene expression of Ag-specific CD8 T cells stimulated with an agonist anti-OX40 or a rat IgG control Ab. Purified OT-I Thy1.1 CD8 T cells were adoptively transferred into wild-type recipients, activated with soluble OVA, and treated with anti-OX40 or rat IgG Ab. Four days later, donor OT-I T cells were isolated from the LN of recipient mice and mRNA was extracted for DNA microarray analysis. Anti-OX40 treatment led to the significant (>2.5-fold) up- or down-regulation of ~200 genes. OX40 engagement led to the potent up-regulation of CD25, granzyme A, and granzyme B mRNAs (Table I). Interestingly, the expression of several other effector molecules including Fas ligand, IFN-{gamma}, and perforin was not significantly altered between anti-OX40 and control Ab-treated groups (Table I).


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  		Table I. Expression of effector genes in OX40 agonist-treated CD8 T cellsa

 
To confirm the results obtained from the gene array analysis at the protein level, naive OT-I T cells were adoptively transferred into wild-type recipients and then activated with soluble OVA along with anti-OX40 or control Ab as described above. Four to 12 days later, LN and spleens were harvested and the phenotype of the donor OT-I T cells was determined by flow cytometry. Studies have shown that granzyme B has a nonredundant role in the initiation of target cell apoptosis and is the most potent effector molecule among the granzyme family (29). Therefore, we focused our analysis on the expression of granzyme B rather than granzyme A. Four days after activation, anti-OX40 treatment led to a striking increase in both the percentage and total number of granzyme B+ or CD25+ OT-I T cells as compared with rat IgG-treated controls (Fig. 1, A and B). Although no difference was observed in the overall percentage of IFN-{gamma}+ OT-I T cells upon in vitro restimulation (Fig. 1A), anti-OX40 treatment did lead to an increase in the total number of IFN-{gamma}+ OT-I T cells (Fig. 1B), which is reflective of the ability of OX40 engagement to augment CD8 T cell expansion and survival in vivo (Fig. 1D) (26). Further analysis revealed that the OX40-mediated increase in CD25 and granzyme B expression was transient since the expression of both molecules peaked 4–6 days after activation and returned to baseline by day 12 (Fig. 1C). Additional analysis revealed little or no difference in the extent of CD62L down-regulation following anti-OX40 vs rat IgG Ab treatment (data not shown). Taken together, these data confirm the results obtained from previous studies and the microarray analysis that an OX40 agonist delivered in vivo can enhance CD8 T cell expansion and lead to increased expression of CD25 and the effector molecule granzyme B (13, 26, 28).


Figure 1
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  		FIGURE 1. OX40 agonist treatment augments the expression of granzyme B and IL-2R{alpha} (CD25) in CD8 T cells. Naive OT-I Thy1.1 T cells were adoptively transferred into syngeneic recipients (day–1). Soluble OVA was injected into recipient mice (day 0) along with anti-OX40 or isotype control Ab (rat IgG, days 0 and 1). A, Four days later, spleens (granzyme B and IFN-{gamma}) and LN (CD25) were harvested and donor OT-I Thy1.1 T cells were analyzed by flow cytometry (graphs are gated on total CD8 T cells). B, The total number of donor OT-I Thy1.1 T cells (from A expressing the indicated markers was determined by flow cytometry. C and D, At the indicated time points, spleens and LN were harvested and the activation status (C), percentage of CD8 T cells (D), and total number (D) of the donor OT-I T cells were examined by flow cytometry. Graphs depict the mean ± SD of three to four mice per group from one of three independent experiments with similar results (*, p < 0.05).

 
Treatment with an OX40 agonist enhances CD8 T cell cytolytic function

Several studies (9, 12, 13) have demonstrated that signaling through OX40 is not required for the differentiation of naive CD8 T cells into effector CTL following priming with a variety of pathogens, MHC class-I restricted peptide, or tumor. Since our data demonstrated that OX40 ligation increased the number of granzyme B+ CD8 T cells, we sought to determine whether OX40-mediated signals also promoted CD8 T cell differentiation into cytolytic effector cells. To this end, OT-I T cells were adoptively transferred into syngeneic mice and then activated with soluble OVA along with anti-OX40 or rat IgG Ab. Four or 7 days later, the extent of CD8 T cell effector function was determined by an in vivo CTL assay. Treatment with anti-OX40 significantly augmented the level of CD8 T cell-mediated lysis 4 and 7 days after priming (Fig. 2, A and B). Since OX40 engagement also boosted the expansion and survival of activated CD8 T cells (Fig. 1B) (26), we sought to determine whether OX40-mediated signals could augment CTL function on a per cell basis using an in vitro CTL assay. Following activation in vivo, the total number of activated OT-I T cells (per spleen) was quantitated and then equal numbers of effector cells were cultured with the indicated amount of peptide-loaded targets in vitro. As shown in Fig. 2C, anti-OX40-stimulated CD8 T cells exhibited more potent cytolytic function on a per cell basis when compared with rat IgG-treated cells. These data demonstrate that, following exposure to soluble Ag, treatment with an OX40 agonist in vivo enhances CD8 T cell differentiation and the acquisition of effector function.


Figure 2
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  		FIGURE 2. Anti-OX40 treatment enhances the acquisition of CD8 T cell cytolytic function. Naive OT-I T cells were adoptively transferred into wild-type mice and then activated with soluble OVA along with anti-OX40 or rat IgG Ab (as described in Fig. 1). A, In vivo CTL assay: peptide-pulsed (CFSEhigh) and nonpulsed (CFSElow) target cells were mixed at a 1:1 ratio and then injected into recipient mice at the indicated time points. Data represent the ratio (r) of CFSElow:CFSEhigh target cells harvested from individual mice. B, Graphs represent the ratio (mean ± SD) of CFSElow:CFSEhigh target cells harvested from three to four mice per group. C, In vitro CTL assay: four days after adoptive transfer and activation, the total number of OT-I T cells per mouse was quantified by FACS. Transferred OT-I T cells represented 10.1 ± 2.5% and 4.8 ± 0.7% (mean ± SD) of the total splenocytes for anti-OX40 or rat IgG-treated mice, respectively. Next, the indicated ratio of effector OT-I T cells was cultured with a 1:1 ratio of peptide-pulsed (CFSEhigh) and nonpulsed (CFSElow) target cells. Seven hours later, the extent of specific lysis was assessed by flow cytometry. All data are representative of one of two independent experiments with similar results (*, p < 0.05).

 
OX40 engagement augments CD8 T cell differentiation through both CD8 T cell-dependent and -independent mechanisms

Since the enhancement of CD8 T cell differentiation could result from improved help provided by OX40-stimulated CD4 T cells (3), we sought to determine the extent to which both direct and indirect OX40-specific signaling affected CD8 T cell differentiation. If the CD8 T cell-specific effects of OX40 engagement occur through a CD8 T cell-indirect mechanism, then OX40-deficient CD8 T cells should respond fully to anti-OX40 treatment. To test this hypothesis, wild-type or OX40–/– OT-I T cells were adoptively transferred into wild-type recipients and then activated with soluble OVA along with anti-OX40 or rat IgG Ab. Four days later, spleens and LN were harvested and the activation status of the donor OT-I T cells was determined. Following anti-OX40 treatment, OX40–/– OT-I T cells expressed significantly less granzyme B and CD25 as compared with wild-type controls (Fig. 3A). However, anti-OX40 treatment did increase the level of these molecules in both wild-type and OX40-deficient CD8 T cells over rat IgG-treated controls (Fig. 3A), suggesting that indirect stimulation provides some, but not all of the OX40-mediated effects on CD8 T cells. In addition, we observed little or no difference in the percentage of IFN-{gamma}+ OT-I T cells among the various groups, suggesting that anti-OX40 treatment preferentially enhanced granzyme B and CD25 expression in vivo (Fig. 3A). It should be noted that wild-type OT-I T cells treated with the OX40 agonist underwent the greatest level of expansion (Fig. 3A, total cell numbers), indicating that direct OX40 engagement on the responding CD8 T cells was also critical for driving their optimal expansion and survival in vivo.


Figure 3
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  		FIGURE 3. OX40 engagement augments CD8 T cell differentiation through both CD8 T cell-dependent and -independent mechanisms. A, Wild-type (WT) or OX40–/– OT-I T cells were adoptively transferred into syngeneic recipients and then activated with soluble OVA along with anti-OX40 or rat IgG Ab (as in Fig. 1). Four days later, spleens (granzyme B and IFN-{gamma}) and LN (CD25) were harvested and donor cells were analyzed by flow cytometry. B, OT-I T cells were adoptively transferred into CD4-depleted or wild-type recipients and activated as described above. Four days later, the extent of OT-I T cell activation was assessed. C, Wild-type or CD25–/– OT-I T cells were adoptively transferred into syngeneic recipients and then activated as described above. Four days later, the extent of OT-I T cell activation was assessed. Data represent the percent or total number (per organ) of OT-I T cells expressing the indicated molecules. D, In vitro CTL assay: the total number of OT-I T cells per mouse was quantified by FACS and then the indicated ratio of effector OT-I T cells was cultured with a 1:1 ratio of peptide-pulsed (CFSEhigh) and nonpulsed (CFSElow) target cells for. Seven hours later, the extent of specific lysis was assessed by flow cytometry. Graphs depict the mean ± SD of three to four mice per group. All data are representative of one of two independent experiments with similar results (*, p < 0.05).

 
To assess directly the contribution of CD4 T cell-specific OX40 costimulation to the CD8 T cell response, OT-I T cells were adoptively transferred into CD4 T cell-depleted or intact hosts and then activated with soluble OVA along with anti-OX40 mAb. Four days later, LN and spleens were harvested from the recipient mice and the extent of OT-I T cell activation was determined. In the absence of CD4 T cells, the extent of CD8 T cell-specific granzyme B+ and CD25+ up-regulation as well as total T cell numbers was reduced (Fig. 3B), while no differences were observed in the level of IFN-{gamma}-expression or CD62L down-regulation (Fig. 3B and data not shown). These results complement the data obtained using OX40–/– OT-I T cells (Fig. 3A), suggesting that OX40 engagement on both CD4 and CD8 T cells is required to drive the optimal expansion and differentiation of naive CD8 T cells in vivo.

Optimal OX40-mediated augmentation of CD8 T cell differentiation occurs through an IL-2-dependent mechanism

To understand the mechanism by which anti-OX40 treatment augments CD8 T cell differentiation, we examined the role of IL-2. Although IL-2 is not required for CTL differentiation under all circumstances (30), IL-2-mediated signals are one factor that can induce the up-regulation of granzyme B expression in CD8 T cells (31). The importance of IL-2-mediated signaling is underscored by our observation that OX40 engagement can lead to a substantial increase in level and duration of expression of the high-affinity IL-2R{alpha} (CD25) on Ag-specific CD8 T cells (26) (Table I and Fig. 1). Thus, we compared the differentiation of wild-type vs CD25–/– OT-I T cells following activation with soluble OVA along with anti-OX40 or rat IgG Ab. Interestingly, both maximal OX40 agonist-induced granzyme B expression and CD8 T cell expansion were strongly dependent upon IL-2 signaling, while the percentage of IFN-{gamma} production was equivalent among all of the groups (Fig. 3C). No differences were observed in the level of IL-2 production among the various groups (data not shown). Thus, although activated CD8 T cells acquired the ability to produce IFN-{gamma} in the absence of IL-2, anti-OX40-induced CD8 T cell differentiation into granzyme B+ effector cells and Ag-driven expansion were both IL-2 dependent.

To assess the contribution of direct OX40 engagement and IL-2-mediated signaling to the acquisition of CD8 T cell cytolytic function, wild-type, OX40–/–, or CD25–/– OT-I T cells were adoptively transferred into recipient mice and then activated with soluble OVA along with anti-OX40 or rat IgG Ab. Four days later, spleens were harvested and the extent of CTL lysis was determined in an in vitro CTL assay. Anti-OX40-treated wild-type OT-I T cells exhibited maximal CTL killing at the highest E:T ratio, while OX40–/– OT-I T cells demonstrated significantly lower killing (Fig. 3D). The CTL activity of anti-OX40-treated OX40–/– OT-I T cells was still increased over rat IgG-treated controls (Fig. 3D), demonstrating that both CD8 T cell-direct and -indirect OX40-mediated signals were necessary to generate optimal CTL effector function. In addition, the differentiation of effector CTL following anti-OX40 treatment occurred in an IL-2-dependent manner, because anti-OX40-treated CD25–/– OT-I T cells were unable to lyse peptide-loaded target cells in vitro (Fig. 3D). Taken together, these data demonstrate that anti-OX40 treatment can enhance the acquisition of CD8 T cell effector function through both CD8 T cell-direct and -indirect mechanisms, which require both direct OX40 and IL-2-mediated signaling in vivo.

The addition of an OX40 agonist, but not endogenous OX40-OX40L-mediated signaling, augments tumor-specific CD8 T cell priming

The ability of anti-OX40 treatment to augment CD8 T cell differentiation under noninflammatory conditions is particularly relevant to tumor immunotherapy since tumor growth can create an immunosuppressive environment that provides few, if any, proinflammatory signals (17, 18). Thus, we sought to determine whether anti-OX40 treatment could also augment the acquisition of CD8 T cell effector function following tumor-specific priming in vivo. For these experiments, we modified a poorly immunogenic murine prostate tumor cell line (TRAMP-C1) (32) to express mOVA as a surrogate tumor-associated Ag, which enabled us to track the Ag-specific CD8 T cell response in vivo. Wild-type OT-I T cells were adoptively transferred into syngeneic mice and then activated with TRAMP-C1-mOVA prostate tumor cells along with anti-OX40 or rat IgG Ab. Four days later, the draining LN were harvested and the extent of OT-I T cell activation was determined. Anti-OX40 treatment given during tumor-specific priming led to a substantial increase in granzyme B and CD25 expression as well as augmented CTL function on a per cell basis as compared with controls (Fig. 4, A and B). In contrast, CD8 T cell-specific ligation of OX40 via the endogenous OX40L did not promote the up-regulation of granzyme B or CD25, although optimal IFN-{gamma} production was dependent upon OX40L-mediated signaling (wild-type vs OX40–/– OT-I T cells, rat IgG only; Fig. 4A). Other studies have also shown that, under certain circumstances, signaling via the endogenous OX40L is necessary for inducing optimal IFN-{gamma} production by CD8 T cells (14), although the mechanisms by which OX40 signals regulate this process are unknown. Furthermore, no differences were observed in the response of OT-I T cells activated in wild-type vs OX40L–/– hosts (rat IgG-treated only; data not shown), which indicates that the extent of signaling provided by the endogenous OX40L was not sufficient to augment the acquisition of CD8 T cell effector function.


Figure 4
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  		FIGURE 4. OX40 engagement augments granzyme B and CD25 expression in tumor-primed CD8 T cells. A, Wild-type or OX40–/– OT-I T cells were adoptively transferred into syngeneic recipients and activated with TRAMP-C1-mOVA prostate tumor cells (day 0) along with anti-OX40 or rat IgG Ab (days 0 and 1). Four days later, spleens (granzyme B and IFN-{gamma}) and LN (CD25) were harvested and donor cells were analyzed by flow cytometry. B, Donor OT-I T cells (from A) were cultured with CFSE-labeled SIINFEKL-pulsed target cells at the indicated E:T ratio. Seven hours later, the extent of specific lysis was determined by flow cytometry. Each symbol represents data obtained from an individual animal. C and D, Naive OT-I T cells were transferred into TRAMP-C1-mOVA tumor-bearing (~25- to 40-mm2 tumors) POET Tg mice. Four days later, LN (C) and TILs (D) were harvested and the differentiation status of the donor OT-I T cells was determined. Graphs (A–C) depict the mean ± SD of two to three mice per group from one of two independent experiments with similar results. Each circle (D) represents the results obtained from an individual animal (*, p < 0.05).

 
Thus far, we have demonstrated that OX40-mediated signaling is required for inducing optimal CD8 T cell priming against tumors. Next, we determined whether anti-OX40 treatment augmented the CD8 T cell response against established tumors in a model in which the host was tolerant to the surrogate tumor-associated Ag (OVA). To this end, we implanted TRAMP-C1-mOVA prostate tumors into prostate OVA-expressing transgenic (POET) mice. POET mice express mOVA in an androgen-dependent manner under the control of the rat probasin promoter and become immunologically tolerant to this Ag (27). Approximately 24 days after tumor implantation (tumor size, ~25–40 mm2), OT-I T cells were transferred into tumor-bearing mice along with anti-OX40 or rat IgG Ab. Four days later, the tumor-draining LN and TIL were harvested and the extent of CD8 T cell activation was assessed. Anti-OX40 treatment led to a statistically significant increase in the total number of OT-I T cells recovered from the LN (anti-OX40 = 11.7 x 106 ± 7.3 x 106; rat IgG = 6.6 x 106 ± 2.9 x 106 (mean ± SD), p < 0.05), but not the TIL (anti-OX40 = 9.5 x 105 ± 14.4 x 105; rat IgG = 7.2 x 103 ± 5.2 x 103, p > 0.05) of tumor-bearing animals. Anti-OX40 treatment also significantly enhanced CD25 (LN) and granzyme B (LN and TIL) expression (Fig. 4, C and D) as compared with control-treated mice, while IFN-{gamma} expression was not significantly different in either tissue (Fig. 4, C and D). These results demonstrate that OX40 engagement can augment CD8 T cell effector differentiation to an established tumor in vivo.

Anti-OX40 treatment augments CD8 T cell-mediated tumor regression

Since anti-OX40 treatment enhanced CD8 T cell differentiation following primary tumor challenge (on day 0) or against established tumors (Fig. 4), we examined whether this translated to improved therapy against established prostate tumors in vivo. TRAMP-C1-mOVA tumor-bearing POET mice received 5 x 104 tumor-specific OT-I T cells along with anti-OX40 or rat IgG Ab and tumor progression and long-term survival were monitored.

In the absence of adoptively transferred OT-I T cells, OX40-mediated therapy had no effect on tumor growth, indicating that endogenous T cells were not sufficient to cause tumor regression (Fig. 5A). In contrast, transfer of 5 x 104 wild-type OT-I T cells along with anti-OX40 mAb led to complete tumor regression in 100% of tumor-bearing recipients (Fig. 5, A and B) and greatly enhanced long-term survival of the treated mice (Fig. 5C). The ability of anti-OX40 treatment to enhance tumor regression required direct ligation of OX40 on the CD8 T cell because OX40–/– OT-I T cells treated with an OX40 agonist did not promote enhanced tumor regression (Fig. 5, A and B) or long-term survival (Fig. 5C) compared with rat IgG-treated controls. It is also important to note that signaling via the endogenous OX40L (wild-type vs OX40–/– OT-I T cells, rat IgG treatment) was not sufficient to enhance tumor regression or prolong the survival of tumor-bearing mice (Fig. 5). Taken together, these data demonstrate that the administration of an OX40 agonist directly impacts tumor-specific CD8 T cell differentiation and expansion, which leads to enhanced tumor regression in vivo.


Figure 5
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  		FIGURE 5. Anti-OX40 treatment augments CD8 T cell-mediated tumor regression. TRAMP-C1-mOVA tumor cells were injected into male POET Tg mice (day 0). A, Tumor-bearing (~25- to 40-mm2 tumors, day 24) mice were given 250 µg of anti-OX40 or rat IgG Ab (days 27 and 31 after tumor implantation) along with no additional cells. Alternatively, tumor-bearing mice (day 24) received 5 x 104 wild-type or OX40–/– OT-I T cells along with 50 µg of anti-OX40 or rat IgG Ab (days 24 and 25). B, The percentage of tumor regression (posttreatment) was calculated as follows: [1 – (minimum tumor area/maximum tumor area) x 100)]. C, The survival of tumor-bearing mice treated in A was assessed *, p < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
There are conflicting data regarding the role of OX40-mediated signaling in the generation of effector CD8 T cells. Several studies have suggested that OX40 signaling is not required for the acquisition of CTL function (9, 12, 13), but one study has shown that priming with adenovirus required OX40 engagement for the generation of cytolytic effector cells (14). Thus, although OX40 engagement has been shown to enhance CD4 T cell differentiation (6, 33, 34, 35), whether it similarly augments CD8 T cell differentiation into cytolytic effector cells is not well understood. We have previously shown that exogenous anti-OX40 treatment can augment the expansion, survival, and generation of memory CD8 T cells (26). In the current study, we sought to determine whether treatment with an OX40 agonist could also promote the differentiation of naive CD8 T cells into effector CTL in vivo. We examined the effects of anti-OX40 treatment on CD8 T cell activation following immunization with either soluble or tumor-associated Ag under noninflammatory conditions. Tumors induce immunosuppression through a variety of mechanisms (17, 18) and the tumor microenvironment is therefore unlikely to provide a sufficient proinflammatory milieu to promote extensive endogenous OX40L expression on APCs (36, 37, 38). Therefore, we studied how an OX40 agonist works to promote functional CD8 T cell responses in tumor-bearing animals.

Anti-OX40 treatment greatly enhanced CD8 T cell cytolytic function (Fig. 2), which was associated with increased expression of both CD25 and granzyme B (Table I and Fig. 1). In addition, direct engagement of OX40 on the responding CD8 T cells was required for their optimal differentiation into cytolytic effector cells (Figs. 3, A and D). It should be noted that anti-OX40 treatment was still able to augment partially the acquisition of effector function of OX40–/– CD8 T cells (Fig. 3, A and D), suggesting that OX40 engagement enhances CD8 T cell differentiation in vivo through both direct and indirect mechanisms. Indeed, the presence of CD4 T cells was critical for promoting CD8 T cell differentiation following OX40 engagement (Fig. 3B). This indicates that the generation of an optimal CD8 T cell response to soluble Ag requires OX40-mediated signaling on both CD8 and CD4 T cells (Fig. 3, A and B). In contrast, a recent study demonstrated that anti-OX40 treatment enhanced CD8 T cell expansion and IFN-{gamma} production through a CD4 T cell-dependent mechanism that does not require direct OX40-mediated ligation of CD8 T cells (39). One key difference with this study is that Song et al. (39) stimulated CD8 T cells in vitro for several days before adoptive transfer and anti-OX40 treatment, whereas we provided OX40 agonist treatment during the initial stimulation of naive CD8 T cells in vivo (Fig. 1). Thus, it may be that expression of the OX40 costimulatory receptor is differentially expressed on naive in vivo-stimulated vs in vitro-activated CD8 T cells, which may account for the discrepancy between these two models in terms of the ability of anti-OX40 treatment to augment directly the acquisition of CD8 T cell effector function.

Based upon our observation that anti-OX40 treatment greatly enhanced CD25 expression on CD8 T cells (Fig. 1 and Table I) and the up-regulation of CD4 T cell-specific IL-2 production (40, 41), we sought to determine whether OX40 engagement boosted the acquisition of CD8 T effector function through an IL-2-dependent mechanism. In the absence of IL-2 signaling, anti-OX40 treatment did not augment CD8 T cell expansion or enhance the acquisition of effector function (Fig. 3, C and D). In contrast, IFN-{gamma} production was not affected by the absence of IL-2 signaling, which is consistent with the ability of CD8 T cells to develop the capacity to produce IFN-{gamma} in an IL-2-independent manner (42). Interestingly, recent work from Williams et al. (43) has demonstrated that anti-OX40 treatment can drive CD4 T cells into Th1 effector cells via an IL-2-dependent mechanism. In contrast to the results obtained with CD8 T cells, the OX40-mediated increase in CD4 T cell-specific IFN-{gamma} production was dependent upon IL-2 signaling. The reasons for these differences are unclear, but may reflect distinct mechanisms by which CD4 and CD8 T cells acquire the ability to produce IFN-{gamma}. For example, it is known that the transcription factors STAT4 and T-bet are required for CD4, but not CD8, T cell-specific IFN-{gamma} production (44). Taken together, our data demonstrate that anti-OX40 treatment augments CD8 T cell-specific granzyme B expression and cytolytic function, but not IFN-{gamma} production, through an IL-2-dependent mechanism in vivo.

The mechanisms underlying the striking dependence upon IL-2 signaling for augmenting the acquisition of CD8 T cell effector function following anti-OX40 treatment are still unclear. One recent study demonstrated that IL-2 blockade was associated with a reduction in OX40 expression on CD8 T cells activated in vitro (45), suggesting that the presence of IL-2 may be important for enhancing or sustaining OX40 expression. Thus, in the absence of IL-2-mediated signals, anti-OX40 treatment may be less effective due to diminished expression of the OX40 receptor. Additionally, the contribution of autocrine vs paracrine IL-2 production following OX40 engagement is not known, although treatment with the OX40 agonist did not affect IL-2 production by the responding CD8 T cells (data not shown). Further studies will be needed to determine the extent of cross-regulation among IL-2 production, CD25 expression, and OX40-mediated signaling in regulating CD8 T cell responses.

Next, we examined whether anti-OX40 treatment could enhance tumor-specific CD8 T cell differentiation and antitumor responses. Anti-OX40 treatment greatly enhanced granzyme B and CD25 expression and the cytolytic function of tumor-primed CD8 T cells as compared with rat IgG-treated controls (Fig. 4, A and B). This occurred following a primary tumor challenge or in response to an established tumor (Fig. 4, A and C). Importantly, anti-OX40 treatment augmented the accumulation of granzyme B+ tumor-infiltrating CD8 T cells (Fig. 4D) and helped to promote the regression of established tumors (Fig. 5, A and B). Direct OX40 engagement on the tumor-specific CD8 T cells was required to promote both enhanced tumor regression and long-term survival of tumor-bearing mice (Fig. 5). However, in the absence of exogenous anti-OX40 treatment, the extent of endogenous OX40L-mediated signaling was not sufficient to augment tumor-specific CD8 T cell differentiation (Figs. 4A and 5, A and B, wild-type vs OX40–/–, rat IgG only). Indeed, no increased OX40L expression on APCs was observed following activation with soluble or tumor-associated Ag (data not shown), suggesting that these stimuli were not sufficient to augment OX40L expression in vivo. These data demonstrate the ability of anti-OX40 mAb, but not the endogenous OX40L, to significantly boost the cytolytic function of tumor-specific CD8 T cells in vivo.

One of the potential benefits of OX40-specific treatment is that both tumor-specific CD4 and CD8 T cell responses can be targeted simultaneously, since OX40 expression has been detected on both CD4 and CD8 TILs (21, 46). This is of particular interest given the importance of CD4 T cell help in the generation of optimal CD8 T cell responses (47, 48, 49, 50). As we are currently exploring the safety and efficacy of an anti-OX40 mAb for treatment of human cancer in a phase I clinical trial, it will be of great interest to determine whether OX40-mediated signaling also augments the expansion and differentiation of human CD8 T cells.

In conclusion, our data demonstrate that treatment with an OX40 agonist can overcome defects in CD8 T cell priming to both soluble and tumor-associated Ag in vivo. Anti-OX40 treatment induced CD8 T cell differentiation into cytolytic effector cells through an IL-2-dependent mechanism that also required direct OX40 engagement on the responding CD8 T cells. Furthermore, anti-OX40 treatment, but not engagement through the endogenous OX40L, led to the CD8 T cell-mediated regression of established prostate tumors and enhanced the long-term survival of tumor-bearing mice. Taken together, these data indicate that in the absence of a sufficient proinflammatory milieu, such as in tumor-bearing hosts, OX40 agonists can greatly augment CD8 T cell cytolytic function and promote a potent antitumor response.


   Acknowledgments
 
We thank Kevin Ankrum for providing excellent technical assistance and Drs. Hong-Ming Hu, Carl Ruby, and Walter Urba for critical reading of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
Dr. Weinberg has a patent pending to use anti-OX40 in cancer patients.


   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 This work was supported by grants from the National Institutes of Health (CA122701-01 and CA102577-05 to A.D.W.), an American Cancer Society–Sam E. and Kathleen Henry Postdoctoral Fellowship (to W.L.R.), and funding from the MJ Murdoch Charitable Trust. Back

2 Current address: Department of Comparative Pathobiology, School of Veterinary Medicine, Purdue Cancer Center, Hansen Life Sciences Research Building, Room 145, 201 South University Street, West Lafayette, IN 47907-2064. Back

3 Address correspondence and reprint requests to Dr. Andrew D. Weinberg, Earle A. Chiles Research Institute, Robert W. Franz Cancer Research Center, Providence Portland Medical Center, 4805 NE Glisan Street 5F40, Portland, OR 97213. E-mail address: andrew.weinberg{at}providence.org Back

4 Abbreviations used in this paper: OX40L, OX40 ligand; Tg, transgenic; TIL, tumor-infiltrating lymphocyte; mOVA, membrane-bound OVA; LN, lymph node. Back

Received for publication July 13, 2007. Accepted for publication September 16, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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