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The Journal of Immunology, 2001, 167: 6804-6811.
Copyright © 2001 by The American Association of Immunologists

Engagement of OX40 Enhances Antigen-Specific CD4+ T Cell Mobilization/Memory Development and Humoral Immunity: Comparison of {alpha}OX-40 with {alpha}CTLA-41

Dean E. Evans*, Rodney A. Prell*, Colin J. Thalhofer*, Arthur A. Hurwitz{dagger} and Andrew D. Weinberg2,*

* Earle A. Chiles Research Institute, Robert W. Franz Cancer Research Center, Providence Portland Medical Center, Portland, OR 97213; and {dagger} Department of Microbiology and Immunology, State University of New York Upstate Medical University, Syracuse, NY 13210


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Increasing the long-term survival of memory T cells after immunization is key to a successful vaccine. In the past, the generation of large numbers of memory T cells in vivo has been difficult because Ag-stimulated T cells are susceptible to activation-induced cell death. Previously, we reported that OX40 engagement resulted in a 60-fold increase in the number of Ag-specific CD4+ memory T cells that persisted 60 days postimmunization. In this report, we used the D011.10 adoptive transfer model to examine the kinetics of Ag-specific T cell entry into the peripheral blood, the optimal route of administration of Ag and {alpha}OX40, and the Ag-specific Ab response after immunization with soluble OVA and {alpha}OX40. Finally, we compared the adjuvant properties of {alpha}OX40 to those of {alpha}CTLA-4. Engagement of OX-40 in vivo was most effective when the Ag was administered s.c. Time course studies revealed that it was crucial for {alpha}OX40 to be delivered within 24–48 h after Ag exposure. Examination of anti-OVA Ab titers revealed a 10-fold increase in mice that received {alpha}OX40 compared with mice that received OVA alone. Both {alpha}OX40 and {alpha}CTLA-4 increased the percentage of OVA-specific CD4+ T cells early after immunization (day 4), but {alpha}OX40-treated mice had much higher percentages of OVA-specific memory CD4+ T cells from days 11 to 29. These studies demonstrate that OX40 engagement early after immunization with soluble Ag enhances long-term T cell and humoral immunity in a manner distinct from that provided by blocking CTLA-4.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
During the course of an immune response, Ag-specific CD4+ T cells undergo multiple rounds of division. After this initial stage of activation and expansion, most of the Ag-specific T cells disappear (1, 2). However, a few cells persist as long-lived memory T cells (1). Ideally, an optimized vaccine would not only lead to acute activation and expansion of Ag-specific T cells, but it would also maximize the number of Ag-specific T cells that differentiate and persist as memory T cells. The costimulatory effects of the interaction of CD80 and CD86 with CD28 are critical for the initial stages of naive T cell activation. Blocking the B7/CD28 interaction with soluble CTLA4-Ig prevents T cell activation and proliferation (3). A number of other costimulatory molecules have the potential to increase activation of naive T cells, but few show promise as candidates to save effector T cells from activation-induced cell death. Recently, we demonstrated that engagement of the costimulatory molecule OX40 with an agonist Ab leads not only to enhancement of short-term activation of Ag-specific effector T cells, but also to an increased memory T cell pool (4).

OX40, a 50-kDa transmembrane protein of the TNFR family, is expressed primarily on activated CD4+ T lymphocytes (5, 6, 7). Engagement of OX40 enhances proliferation and cytokine production by CD4+ T cells in vitro (8, 9, 10, 11, 12) as well as survival of Ag-specific CD4+ T cells in vivo (12). Expression of OX40 has been detected on T cells at the site of inflammation during clinical signs of experimental allergic encephalomyelitis (EAE),3 graft-vs-host disease, and on tumor-infiltrating lymphocytes (13, 14, 15, 16, 17). OX40+ T cells isolated from inflammatory lesions in EAE were shown to be the autoantigen-specific cells. The ligand for OX40, OX40L, is a type II transmembrane protein and a member of the TNF family. It is expressed on activated endothelium (18, 19), B cells (6, 20), macrophages/microglia isolated from the central nervous system of mice with EAE (21), and on CD40-activated dendritic cells (22). Blocking OX40/OX40L can ameliorate EAE (21, 23), whereas engagement of OX40 with a soluble OX40L:Ig fusion protein or an agonist Ab enhances anti-tumor immunity (24).

In this study, we investigate the adjuvant properties associated with an agonistic {alpha}OX40 Ab. OVA-specific CD4+ T cells were used to examine the effect of OX40 engagement on long-term survival and responsiveness (up to 95 days) of Ag-specific T cells and on OVA-specific humoral immunity. Administration of {alpha}OX40 within 24–48 h after Ag resulted in increased numbers of Ag-specific T cells in the periphery capable of producing Th1 cytokines upon Ag restimulation. In addition, engagement of OX40 greatly enhanced an anti-OVA humoral immune response. A comparison of {alpha}OX40 to {alpha}CTLA-4 showed that {alpha}OX40 was more effective at maintaining high numbers of long-lived Ag-specific T cells and at enhancing an Ag-specific humoral immune response. Thus, engagement of OX40 during immunization is a potent adjuvant for enhancing long-term T cell and humoral immunity.


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

Four- to 6-wk-old female BALB/cJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and used at 6–10 wk of age. D011.10 TCR-transgenic mice, which recognize the OVA peptide residues 323–339 (OVA323–339) in the context of MHC class II I-Ad (25), were maintained on a BALB/c background and used at 8–12 wk of age. Mice were housed at the Earle A. Chiles Research Institute animal care facility (Providence Portland Medical Center, Portland, OR) and cared for under the National Institutes of Health guidelines.

Adoptive transfer and immunization

The protocol for adoptive transfer of D011.10 cells was slightly modified from a previously published report (1). Spleens were removed from D011.10 mice, and erythrocytes were lysed in ammonium chloride potassium lysis buffer and suspended in Dulbecco’s PBS (DPBS; BioWhittaker, Walkersville, MD). The percentage of cells in the spleen expressing the transgenic TCR specific for OVA was determined before adoptive transfer by flow cytometry using biotinylated KJ1-26 and Cy-chrome {alpha}CD4 Abs in combination with PE-SAV. A total of 1–5 x 106 KJ1-26+CD4+ spleen cells in 0.2 ml of DPBS were adoptively transferred i.v. into the tail vein of unirradiated female BALB/c recipients. Two to 3 days after adoptive transfer, mice were immunized with 500 µg OVA (Sigma-Aldrich, St. Louis, MO) in 0.2 ml DPBS, by the indicated route. They also received 50 µg of {alpha}OX40 or a rat IgG control (Sigma-Aldrich) in DPBS by the indicated route. The next day, mice received an additional injection of the respective Ab. The adoptively transferred OVA-specific DO11.10 cells were detected by dual staining with a fluorochrome-conjugated CD4 Ab and a biotinylated KJ1-26 Ab (26, 27) and then fluorochrome-conjugated streptavidin.

Antibodies

FITC {alpha}CD25 (7D4), FITC {alpha}CD44 (IM7), CyChrome {alpha}CD4, PE {alpha}IL-4 (11B11), PE {alpha}IL-2 (JES6-5H4), PE {alpha}IL-10 (JES5-16E3), PE {alpha}TNF-{alpha} (MP6-XT22), PE {alpha}IFN-{gamma} (XMG1.2), PE {alpha}IL-12 (C15.6), and purified {alpha}CD28 (37.51) were purchased from BD PharMingen (La Jolla, CA). PE-conjugated streptavidin was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Biotinylated KJ1-26 was kindly provided by N. Kerkvliet (Oregon State University, Corvallis, OR). Anti-CTLA-4 (9H10) was grown in a bio-reactor and purified on protein G. The hybridoma that produces the rat-{alpha}murine OX40 mAb (OX86) was obtained from the European Cell Culture Collection (Porton Down, U.K.) and was produced and purified by UniSyn (Hopkinton, MA).

Staining of peripheral blood

Mice were bled by the tail vein into 5-ml heparin-treated polystyrene tubes (Falcon; BD Biosciences, Franklin Lakes, NJ). One milliliter of RPMI 10 was added, and the blood was underlayed with 0.7 ml of Lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) and spun at 2500 rpm for 12 min at room temperature. The cells at the medium/lympholyte M interface were collected, washed, and stained with the indicated Abs in FACS buffer (1% FBS, 0.1% sodium azide in PBS). The samples were run on a FACScan flow cytometer, and analysis was performed using CellQuest analysis software (BD Biosciences, Mountain View, CA).

Intracellular cytokine staining

Two days after adoptive transfer of 5 x 106 KJ1-26+CD4+ T cells (day 0), mice were injected with 500 µg of OVA (s.c.) and 50 µg of {alpha}OX40 or rat IgG (i.v.). The next day, mice received an additional 50 µg of Ab; one-half of the mice also received 50 µg of LPS (i.v.) on day 1. Spleens were removed at the indicated times and were stimulated for 5 h in vitro with 10 µM OVA323–339 peptide in RPMI 10 growth medium containing 10% FBS and 1 µg/ml brefeldin A (Sigma-Aldrich). The cells were harvested and surface stained with bio-KJ1-26, CyChrome-SAV, and FITC {alpha}CD4. The cells were subsequently fixed overnight with 1% formaldehyde (Polysciences, Warrington, PA) in FACS buffer. The following morning the cells were permeabilized in Perm/Wash buffer (BD PharMingen) and stained with the indicated PE-labeled cytokine Ab.

Detection of an Ab response to OVA

OVA-specific Abs were detected by ELISA (28). Standard 96-well ELISA plates were incubated overnight with 100 µl of 1 mg/ml OVA in PBS. The plates were washed in PBS Tween-20 and blocked for 4 h at room temperature with 3% BSA in PBS. The plates were washed and incubated overnight at 4°C with dilutions of the appropriate serum. The plates were washed four times and incubated for 1 h at room temperature with a biotinylated goat anti-mouse IgG1 or IgG2a Ab (Southern Biotechnology Associates, Birmingham, AL) in PBS containing 3% BSA. The plates were washed and incubated for 30 min at room temperature with streptavidin-peroxidase. Tetramethylbenzidine substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added. The reaction was stopped with 10% phosphoric acid and the absorbance was read at 450 nm. The Ab titer was defined as the reciprocal of the serum dilution with an OD at least three times the OD of serum from unimmunized mice (lowest dilution).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Large numbers of Ag-specific T cells migrate into the peripheral blood 4 days after Ag and {alpha}OX40 administration

Two different TCR transgenic systems have shown that engagement of OX40 can dramatically enhance the number of Ag-specific T cells in the spleen and draining lymph nodes after s.c. administration of moth cytochrome c in CFA (12) or i.p. administration of soluble OVA (4). Activation and expansion of T cells in the secondary lymphoid tissues are important for mounting an effective immune response against a potential pathogen. Equally important is the migration of effector T cells into the peripheral circulation and ultimately to the site of infection. Thus, we explored whether {alpha}OX40 treatment would increase the number of Ag-specific T cells entering the periphery (blood) after immunization with a soluble Ag. The DO11.10 transfer model (1) was used to track Ag-specific T cells after immunization. In this model, TCR transgenic T cells specific for OVA peptide (OVA323–339) in the context of class II I-Ad were transferred to normal BALB/c recipients and detected by flow cytometry with the TCR clonotype-specific Ab KJ1-26 (27). The kinetics of migration of KJ1-26+CD4+ T cells into the peripheral circulation after immunization with OVA and treatment with {alpha}OX40 and/or LPS was examined (Fig. 1Goa). The percentage of KJ1-26+CD4+ T cells in the blood 3 days after administration of Ag was low in all groups (<3% of the CD4+ T cells). However, on day 4 the percentage of KJ1-26+CD4+ T cells in the blood increased dramatically in mice that received {alpha}OX40 (20% of CD4+) or {alpha}OX40/LPS (38% of CD4+) compared with the rat IgG control (5% of CD4+). On day 7, the percentage of KJ1-26+CD4+ T cells in the blood was lower in all groups, but remained higher in mice that received {alpha}OX40 compared with control rat IgG. Thus, engagement of OX40 during soluble Ag immunization results in a dramatic increase in the percentage of Ag-specific T cells detected in the peripheral blood between days 3 and 4 after immunization. OVA-specific T cells in the peripheral blood have a memory phenotype, as assessed by increased CD44 expression after immunization with OVA and {alpha}OX40 (Fig. 1Gob).



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  		FIGURE 1. Large numbers of Ag-specific T cells migrate into the peripheral blood 4 days after receiving Ag and {alpha}OX40. a, A total of 1 x 106 KJ1-26+CD4+ spleen cells were transferred (i.v.) into BALB/c mice. Three days later (day 0) 500 µg of OVA and 50 µg of either anti-OX40 or a rat IgG control were injected s.c. Mice received an additional 50 µg of Ab with or without 50 µg of LPS (i.p.) 1 day later (day 1). Peripheral blood lymphocytes obtained on days 3, 4, and 7 were analyzed by flow cytometry for the presence of OVA-specific KJ1-26+CD4+ T cells. The mean of four mice/group ± SE is shown. b, A total of 2 x 106 KJ1-26+CD4+ spleen cells were transferred into BALB/c mice (day -2). Mice were injected s.c. on day 0 with 500 µg of OVA and with 50 µg of either anti-OX40 or a rat IgG control on days 0 and 1. On day 35, peripheral blood KJ1-26+CD4+ T cells were analyzed for CD44 expression. The dotted line represents CD44 expression on KJ1-26+CD4+ T cells from unimmunized mice, and the solid line represents CD44 expression on KJ1-26+CD4+ T cells from mice immunized s.c. with OVA and anti-OX40. The histogram is representative of one of five mice.

 
Route of Ag administration influences the percentage of Ag-specific T cells found in the peripheral blood after OX40 stimulation

The effect the route of administration of Ag has on the percentage of Ag-specific T cells found in the peripheral blood after OX40 engagement was examined. After adoptive transfer of naive KJ1-26+CD4+ T cells, mice were immunized with 500 µg of OVA by one of the indicated routes and treated with {alpha}OX40 or rat IgG. Five days after immunization, KJ1-26+ T cells comprised 20%, 6.1%, and 4.8% of the CD4+ population in the peripheral blood when OVA was administered s.c., i.p., or i.v., respectively (Fig. 2Go). When we followed the percentage of OVA-specific T cells in the peripheral blood over time, the percentage of KJ1-26+ T cells declined in all groups between days 12 and 26 (data not shown). However, the percentage of KJ1-26+ T cells was always highest when OVA was administered s.c. In addition, s.c. and i.v. administration of {alpha}OX40 during OVA immunization were equally effective at increasing the number of OVA-specific T cells in the peripheral blood (data not shown). Based on these data, it appeared that s.c. administration of Ag was the most effective way to increase the percentage of Ag-specific T cells in the peripheral blood (Fig. 2Go) and in the secondary lymphoid organs (data not shown) after OX40 ligation in vivo.



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  		FIGURE 2. The route of Ag administration affects expansion of peripheral blood Ag-specific T cells. A total of 1 x 106 KJ1-26+CD4+ spleen cells were transferred into BALB/c mice (day -2). Mice were injected on day 0 with 500 µg of OVA by different routes (s.c., i.v., or i.p.) and with 50 µg of either anti-OX40 or a rat IgG control on days 0 and 1. Mice were bled on day 5 and the blood was analyzed by flow cytometry for the presence of OVA-specific KJ1-26+CD4+ T cells. The bar graph represents the percentage of CD4+ T cells that were KJ1-26+. The mean of seven mice/group + SE is shown.

 
Anti-OX40 adjuvant effects require early treatment

We investigated the kinetics of the {alpha}OX40-accentuated immune response by providing {alpha}OX40 at different times after Ag priming. After receiving DO11.10 spleen cells (as previously described), mice were injected with OVA on day 0 and 50 µg of LPS on day 1. In two separate experiments, two doses of anti-OX40 were administered as follows: days 0 and 1 or 1 and 2 (Fig. 3Goa); days 1 and 2, 2 and 3, or 3 and 4 (Fig. 3Gob). When {alpha}OX40 was administered on days 0 and 1 or 1 and 2, a 10-fold increase in the percentage of KJ1-26+CD4+ T cells was observed in the peripheral blood. Surprisingly, when {alpha}OX40 was delayed as little as 48 h, the anticipated expansion of KJ1-26+CD4+ T cells never occurred (Fig. 3Gob). Thus, {alpha}OX40 is effective at enhancing expansion and survival of naive T cells when administered within 24 h of Ag encounter, but not 48 h or later.



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  		FIGURE 3. Anti-OX40 adjuvant properties require early treatment. A total of 1 x 106 KJ1-26+CD4+ spleen cells were transferred (i.v.) into BALB/c mice (day -2). Mice were injected s.c. with 500 µg of OVA on day 0 and with 50 µg of LPS (i.p.) on day 1. At the indicated days, the mice were also injected with 50 µg of anti-OX40 or a rat IgG control. Peripheral blood was examined on day 11 by flow cytometry for the presence of KJ1-26+CD4+ T cells. The number in the upper right quadrant is the percentage of CD4+ T cells that are KJ1-26+ (mean of six mice per group). The data represent two independent experiments (a and b).

 
Anti-OX40-stimulated naive T cells give rise to Ag-responsive long-lived memory T cells

Engagement of OX40 in vivo in the presence of Ag led to increased numbers of KJ1-26+CD4+ T cells. However, whether memory T cells generated with soluble Ag and {alpha}OX40 remained responsive to Ag months after immunization had not been investigated. To address this issue, we examined cytokine production by splenic T cells isolated 95 days after immunization with OVA and {alpha}OX40, {alpha}OX40/LPS, or rat IgG/LPS. On day 95, a significantly higher percentage of the CD4+ T cells from mice treated with {alpha}OX40 or {alpha}OX40/LPS were KJ1-26+ (4% and 20%, respectively) compared with CD4+ T cells from mice that received rat IgG/LPS (0.3%) (data not shown). CD4+ T cells from mice immunized with OVA and LPS did not produce detectable levels of cytokines when stimulated in vitro with OVA peptide (Fig. 4Go). In contrast, CD4+ T cells from OVA/{alpha}OX40-treated mice produced TNF-{alpha}, (4.9%), IFN-{gamma} (3.08%), and IL-2 (3.6%) when stimulated in vitro with OVA peptide. Mice immunized with OVA in combination with {alpha}OX40 and LPS had even higher percentages of CD4+ T cells that produced TNF-{alpha} (17%), IFN-{gamma} (13%), and IL-2 (14%) when stimulated with OVA peptide in vitro. Th2 cytokine (IL-4 and IL-10) producing cells were not detected in any group (data not shown). It was not possible to examine directly the percentage of KJ1-26+ T cells that were producing cytokines, because the TCR was down-regulated after in vitro stimulation with OVA peptide (data not shown). However, the percentage of CD4+ T cells that were KJ1-26+ in the absence of restimulation was similar to the percentage of CD4+ T cells that were producing cytokines. Thus, the majority of KJ1-26+CD4+ T cells present 95 days after administration of Ag and {alpha}OX40 remained Ag responsive.



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  		FIGURE 4. Long-lived memory T cells induced by anti-OX40 remain responsive to Ag. A total of 5 x 106 KJ1-26+CD4+ spleen cells were transferred into BALB/c mice (day -2). Mice were injected s.c. with 500 µg of OVA on day 0 and i.v. with 50 µg of either anti-OX40 or a rat IgG control on days 0 and 1. One-half of the mice also received 50 µg of LPS (i.v.) on day 1. The mice were sacrificed on day 95, and spleen cells were stimulated for 5 h in vitro with either OVA peptide or a PCC peptide control. Cells were examined by flow cytometry for expression of CD4 and the intracellular production of TNF-{alpha}, IFN-{gamma}, and IL-2. The number in the upper right quadrant is the percentage of CD4+ cells that stain positive for the indicated intracellular cytokine (mean of three mice). PCCP (TNF-{alpha}) shows TNF-{alpha} production in response to an irrelevant PCC peptide.

 
Anti-OX40 also enhances an Ag-specific humoral immune response

Although it has been shown that engagement of OX40 during Ag priming increases the survival of Ag-specific T cells, the effect on the humoral immune response had not been examined. Therefore, we examined whether engagement of OX40 would affect the Ab response to OVA. Engagement of OX40 during priming with OVA led to anti-OVA IgG1 Ab titers that were 20-fold higher compared with OVA plus rat IgG (Fig. 5Goa). When LPS was administered with {alpha}OX40, the IgG1 Ab response decreased, with only a 6- to 9-fold increase compared with rat IgG and rat IgG/LPS controls. Anti-OVA IgG2a Ab titers were also greatly enhanced by {alpha}OX40; 37-fold higher anti-OVA IgG2a Ab titers were observed in mice treated with {alpha}OX40 vs rat IgG. Furthermore, the combination of {alpha}OX40 and LPS resulted in 14-fold higher IgG2a titers compared with rat IgG plus LPS (Fig. 5Gob). Thus, it appears that engagement of OX40 during priming with a soluble Ag enhances a TH2 (IgG1) Ab response. Our data suggest that LPS skews the {alpha}OX40-mediated Ab response to a TH1 (IgG2a) isotype-biased Ab response. The data support the hypothesis that OX40 engagement can facilitate both TH1 and TH2 responses, depending on the stimuli present within the local microenvironment.



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  		FIGURE 5. Anti-OX40 also enhances anti-OVA humoral immune responses. A total of 5 x 106 KJ1-26+CD4+ spleen cells were transferred into BALB/c mice (day -2). Mice were injected s.c. with 500 µg of OVA on day 0 and i.v. with 50 µg of either anti-OX40 or a rat IgG control on days 0 and 1. One-half of the mice also received 50 µg of LPS (i.v.) on day 1. Mice were bled via the tail vein on day 26, and the serum was analyzed for OVA-specific IgG1 (a) (*, p = 0.0007 compared with rat IgG; p = 0.01 compared with {alpha}OX40, LPS; #, p = 0.03 compared with rat IgG, LPS) and IgG2a Abs (b) (*, p < 0.04 compared with rat and rat, LPS). The mean of five mice/group + SE is shown.

 
Anti-OX40 and {alpha}CTLA-4 have different effects on T cell activation and survival

It has been demonstrated in other systems that blocking a negative signal through CTLA-4 can enhance T cell activation (29, 30, 31, 32, 33). Thus, we were interested in how blocking CTLA-4 and OX40 engagement compared in their ability to enhance T cell expansion and survival. We examined expansion and survival of OVA-specific T cells after immunization with OVA and treatment with either a blocking CTLA-4 or an agonist OX40 Ab. We chose a dose of {alpha}CTLA-4 that had previously been shown to enhance Ag-specific T cell responses in vivo (32). Mice injected with OVA and {alpha}OX40 or with OVA, {alpha}OX40, and LPS had increased percentages of KJ1-26+CD4+ T cells in the blood at all days examined compared with control groups (Fig. 6Goa). The percentage of KJ1-26+CD4+ T cells in the blood on day 4 was also enhanced in mice that received {alpha}CTLA-4 (Fig. 6Goa). However, although {alpha}OX40- and {alpha}OX40/LPS-treated mice exhibited greater expansion on day 11, the percentage of KJ1-26+CD4+ T cells in the blood of {alpha}CTLA-4- and {alpha}CTLA-4/LPS-treated mice had already fallen to one-half the level observed on day 4 (Fig. 6Goa). Whereas on day 29 the percentage of KJ1-26+CD4+ T cells in the blood was lower in mice that received either {alpha}OX40 or {alpha}CTLA-4 (±LPS), they remained higher in {alpha}OX40-treated compared with {alpha}CTLA-4-treated mice (±LPS), and the {alpha}CTLA-4-treated mice were not different from controls (Fig. 6Goa). The mice were sacrificed and the KJ1-26+CD4+ T cells in the spleen, lymph nodes, and lungs were examined. The percentages of KJ1-26+CD4+ T cells were 6-fold and 4-fold higher in the spleens and lymph nodes (Fig. 6Gob) of mice treated with {alpha}OX40/LPS vs {alpha}CTLA-4/LPS, which were at levels observed with the controls. Compared with {alpha}CTLA-4/LPS, mice that received {alpha}OX40 and LPS also had 4- to 6-fold higher numbers of KJ1-26+CD4+ T cells in the spleen and lymph nodes (data not shown). Similar percentages were observed with the spleen, nodes, and lungs in mice treated with {alpha}OX40 or {alpha}CTLA-4 in the absence of LPS (Fig. 6Gob). We also examined CD25 expression on KJ1-26+CD4+ T cells from mice immunized with OVA and treated with {alpha}OX40/LPS or {alpha}CTLA-4/LPS. Interestingly, there were increased levels of CD25 on the cell surface of KJ1-26+CD4+ T cells on day 4 after treatment with {alpha}OX40/LPS but not {alpha}CTLA-4/LPS or rat IgG/LPS (Fig. 6Goc). By day 11, however, CD25 expression was at background levels in all groups (data not shown).



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  		FIGURE 6. Anti-OX40 and anti-CTLA-4 exert different effects on T cell activation and survival. A total of 1 x 106 KJ1-26+CD4+ spleen cells were transferred (i.v.) into BALB/c mice. Three days later (day 0), mice were injected s.c. with 500 µg of OVA and 50 µg of anti-OX40, 100 µg of anti-CTLA-4, or the appropriate IgG control. The next day, the mice received an additional dose of Ab; a subgroup of mice also received 50 µg of LPS (i.p.). a, Mice were bled via the tail vein on the indicated day, and the blood was analyzed by flow cytometry for the presence of OVA-specific KJ1-26+CD4+ T cells. b, The percentage of OVA-specific KJ1-26+CD4+ T cells in the spleen, lymph nodes, and lungs at day 43 in mice that did not receive LPS, or at day 29 in mice that received LPS. c, CD25 and KJ1-26 expression on peripheral blood lymphocytes isolated on day 4. The LPS and no LPS groups are from two independent experiments. The serum from day 29 was analyzed for OVA-specific IgG2a Abs (d) (*, p < 0.005 compared with all other groups). The mean of nine mice per group (LPS) or five mice per group (no LPS) ± SE is shown.

 
Next, we examined whether blocking CTLA-4 during immunization with soluble Ag would alter the humoral immune response. OVA-specific Ab titers were examined on day 29 in the mice immunized with OVA/LPS and treated with either {alpha}OX40 or {alpha}CTLA-4. The anti-OVA IgG2a titers were 7-fold higher in the {alpha}OX40/LPS group compared with the rat IgG/LPS control group (Fig. 6God). The anti-OVA IgG2a titers in the {alpha}CTLA-4/LPS group were not different from those of the hamster IgG/LPS control group (Fig. 6God). There were no significant differences in the OVA-specific IgG1 titers (data not shown). Thus, unlike {alpha}OX40, {alpha}CTL-4 had no effect on the anti-OVA IgG1 or IgG2a response under the conditions examined.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report we demonstrate that: 1) the number of Ag-specific T cells entering the peripheral blood after Ag immunization is greatly enhanced by engagement of OX40, 2) OX40 has to be engaged within 24–48 h of exposure to soluble Ag to exert its adjuvant effect, 3) memory T cells generated by engagement of OX40 during Ag priming remained responsive to Ag stimulation 95 days after primary immunization, 4) engagement of OX40 also enhanced an Ag-specific Ab response, and 5) early engagement of OX40 during Ag-specific priming is more effective at enhancing Ag-specific T cell expansion and humoral immunity than blocking CTLA-4. The data presented clearly show that accentuating OX40 signaling during Ag priming results in enhanced T cell function and humoral immunity distinct from that observed by blocking CTLA-4.

When a potential pathogen gains entry, it usually elicits a robust innate and adaptive immune response that ultimately results in the elimination of the organism. The initial activation of Ag-specific T and B cells does not occur at the site of infection, but instead in secondary lymphoid organs such as the spleen and lymph nodes, where they proliferate and mature into effector and memory T cells. The effector and memory T cells then travel through the blood and into peripheral tissues, where they are important for immune surveillance and clearance of the pathogen (34, 35). Memory and effector T cells that reside in peripheral tissues are phenotypically and functionally distinct from those in the secondary lymphoid organs. They may express chemokine receptors that direct them into a specific tissue (36) and they may be functionally more active. Masopust et al. (35) demonstrated that vesicular stomatitis virus-specific CD8 memory T cells from the liver, lung, and lamina propria, but not from the spleen, were cytolytic when assayed directly ex vivo. Previously, we have shown that engagement of OX40 with an Ab during Ag immunization could increase the number of Ag-specific T cells that persist long-term in the spleen and lymph nodes (4). Here we demonstrate that OX40 engagement during immunization also results in increased numbers of Ag-specific T cells in the peripheral blood (Figs. 1Goa, 2, and 6a) and lungs (Fig. 6Gob). Increased "mobilization" of Ag-specific T cells into the periphery after Ag immunization and OX40 engagement may be the result of increased numbers of activation-induced, cell death-resistant, Ag-specific T cells in the secondary lymphoid organs simply spilling out into the periphery or from up-regulation of chemokine or homing receptors on the T cell surface. We have examined for CD25, CD69, CD62L, and CD44 expression on peripheral blood Ag-specific T cells after Ag immunization and OX40 engagement. Of the molecules we examined, CD44 (Fig. 1Gob) and CD25 (Fig. 6Goc) were expressed at differential levels on KJ1-26+CD4+ T cells in the peripheral blood after Ag and engagement of OX40. Increased CD44 expression after engagement of OX40 is consistent with a memory phenotype (37). Increased CD25 expression was short lived (only observed on day 4) and is consistent with increased expansion of Ag-activated T cells after engagement of OX40. However, it may be an indirect effect of OX40-enhanced IL-2 production. It is possible that up-regulation of CD44 (Fig. 1Gob) or a yet identified adhesion or chemokine receptor is responsible for enhanced mobilization of recently activated T cells into the peripheral tissues after OX40 engagement.

It is interesting that engagement of OX40 during Ag immunization resulted in an increased pool of memory T cells. Potentially more important is whether the T cells remained functional. We demonstrate that memory T cells generated during Ag immunization and OX40 engagement remain functional and produce Th1 cytokines when restimulated in vitro with Ag (Fig. 4Go). Although in vivo the combination of {alpha}OX40 and LPS vs OX40 alone increased the percentage of T cells that survived and produced Th1 cytokines, it did not alter the cytokine secretion profile; e.g., the T cells did not produce Th2 cytokines under either condition (data not shown). Although we did not observe Th2 cytokine production by memory T cells 95 days after Ag and {alpha}OX40 immunization, Th2 cytokines may have been produced early during the immune response, as evidenced by the Th2-like Ab response (IgG1) with {alpha}OX40 alone (Fig. 5Goa). In support, Gramaglia et al. (12) observed that OX40 engagement enhanced both Th1 and Th2 cytokine secretion after in vivo immunization with peptide in CFA. Similarly, mice deficient for OX40 exhibited reduced secretion of both Th1 and Th2 cytokines (12).

In this report, we demonstrated that an agonist Ab to OX40 enhances an IgG1 (Th2) Ab response when OVA is administered in the absence of a "danger" signal (LPS) (Fig. 5Goa). In a model for leishmaniasis, Akiba et al. (38) found that blocking the OX40/OX40L interaction prevented progressive disease in BALB/c mice, which was associated with reduced levels of Leishmania major-specific IgG1 and IgE Ab production. Thus, OX40/OX40L appeared to play a role in the development of a detrimental Th2 response to L. major infection. OX40/OX40L probably also plays a role in development of Th1 Ab responses. We found that soluble OVA in combination with {alpha}OX40 elicited an enhanced IgG2a (Th1) response in the presence of LPS (Fig. 5Gob). In addition, OX40L knockout mice have a reduced capacity to produce both Th1- and Th2-mediated Abs when immunized with keyhole limpet hemocyanin in CFA (39). Thus, a signal through OX40 appeared to augment both Th1 and Th2 humoral responses, depending on what additional signals were received by the T cell.

Expansion, deletion, and tolerance induction are all controlled by a number of positive and negative signals. T cell expansion after TCR engagement can be augmented by costimulatory signals through molecules such as CD28, 4-1BB, and OX40 and can be inhibited by a negative signal through CTLA-4. Induction of tolerance can be inhibited by providing costimulatory signals (40, 41) or by blocking an inhibitory signal through CTLA-4 (42, 43). In support of the hypothesis that CTLA-4 is important for modulating early T cell activation (29), we observed an increase in the percentage of KJ1-26+CD4+ T cells in the blood 4 days after immunization with OVA in the presence of a blocking CTLA-4 Ab (Fig. 6Goa). However, blockade of CTLA-4 did not prevent the percentage of KJ1-26+CD4+ T cells in the peripheral blood from dramatically decreasing from days 11–26 (Fig. 6Goa). In addition, the numbers of KJ1-26+CD4+ T cells in the {alpha}CTLA-4-stimulated group were at background levels in the spleen, lymph nodes, and lungs at time points later than 26 days (Fig. 6Gob). These data indicate that, although blockade of CTLA-4 augments T cell activation and expansion, it does not appear to enhance CD4+ T cell memory. In contrast to CTLA-4 blockade, signaling through OX40 during naive T cell priming enhanced both short-term expansion (Figs. 1Goa and 6a) and long-term persistence (Figs. 4Go and 6Goa) of KJ1-26+CD4+ T cells in the peripheral blood and secondary lymphoid organs. OX40 engagement and CTLA-4 blockade also appeared to have different effects on humoral immunity. Although OX40 engagement in the presence of LPS enhanced an anti-OVA IgG2a response (Fig. 5Gob), blockade of CTLA-4 had little if any effect on humoral immunity (Fig. 6God). This difference might be due to differential trafficking of T cells to germinal centers or to enhanced survival of OVA-specific T cells after OX40 engagement vs CTLA-4 blockade. In support of differential trafficking, Walker et al. (44) showed that germinal center formation was inhibited when OX40 interactions were blocked with an OX40-Ig fusion protein. OX40-mediated T cell homing to B cell areas of secondary lymphoid organs might be due to OX40 up-regulation of chemokine receptor CXCR5 mRNA (45). Although we favor that OX40 delivers a signal that is qualitatively different from that of inhibiting CTLA-4, it is possible that the observed differences are due to {alpha}CTLA-4 having a shorter half-life in vivo. If so, giving additional doses of {alpha}CTLA-4 may further enhance T cell expansion and survival. In addition, because the two Abs are probably enhancing T cell expansion and survival by different mechanisms, they may be synergistic when delivered together. We are currently performing combination experiments to assess whether together they are synergistic for T cell expansion, survival, and Ab production.

It has been proposed that the number of memory T cells specific for a particular pathogen or tumor Ag must reach a certain threshold to provide protection against potential pathogens or malignant cells (46). Because OX40 engagement dramatically enhances the numbers of memory T cells and Ab titers to the immunizing Ag, engagement of OX40 may be a helpful adjuvant for future vaccines to various pathogens or tumors. Recently, we have shown in several tumor models derived from distinct tissues (sarcoma, melanoma, glioma, colon cancer, mammary cancer) that engagement of OX40 with either {alpha}OX40 or soluble OX40L:Ig after administration of tumor s.c. prevented a significant percentage of mice from developing tumor (24, 47). These mice remained resistant to tumor development upon rechallenge with the same tumor, but not to a tumor of different tissue origin. The data suggested that generation of tumor-specific memory T cells was enhanced by engagement of OX40. We also demonstrated that tumor immunity generated by OX40 engagement could be conferred to naive recipients by the adoptive transfer of CD4+ splenic T cells from the OX40-cured mice (24). The data suggest that increasing the number tumor-specific T cells above a certain threshold allowed for protective tumor immunity in these mice.

We demonstrate that in vivo injection of {alpha}OX40 within 24–48 h of administration of soluble Ag enhances both cellular and humoral immune responses to the immunizing Ag. In addition, the adjuvant effects associated with {alpha}OX40 appeared to be distinct from those of {alpha}CTLA-4. Although both Abs are quite effective at increasing the number of Ag-specific T cells in the peripheral blood early after immunization, only {alpha}OX40 enhanced the number of memory T cells detected in the peripheral blood and lymphoid organs on day 26. Anti-OX40 also enhanced an Ag-specific Ab response, whereas {alpha}CTLA-4 did not. In conclusion, the administration of {alpha}OX40 during immunization could serve as a potent adjuvant by enhancing long-term immunity.


   Acknowledgments
 
We thank Walter Urba, Hong Ming Hu, and Jim Allison for their critical review of this manuscript.


   Footnotes
 
1 This work was supported by the National Institutes of Health (Grant R01 CA81383-03), the Multiple Sclerosis Society (Grant RG 3193-A-2), and the Murdoch Trust Foundation. A.A.H. is a CAP CURE young investigator and is supported in part by the Department of Defense Prostate Cancer Research Program (DAMD17-01-1-0085). Back

2 Address correspondence and reprint requests to Dr. Andrew D. Weinberg, Earle A. Chiles Research Institute, Robert W. Franz Cancer Research Center, 4805 NE Glisan, Providence Portland Medical Center, Portland, OR 97213. E-mail address: weinbera{at}ohsu.edu Back

3 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; OX40L, OX40 ligand; DPBS, Dulbecco’s PBS; PCC, pigeon cytochrome c. Back

Received for publication April 26, 2001. Accepted for publication October 9, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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