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The Journal of Immunology, 2007, 178, 7694 -7702
Copyright © 2007 by The American Association of Immunologists, Inc.
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OX40-Mediated Differentiation to Effector Function Requires IL-2 Receptor Signaling but Not CD28, CD40, IL-12Rbeta2, or T-bet1

Cortny A. Williams*, Susan E. Murray*, Andrew D. Weinberg{dagger} and David C. Parker2,*

* Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239; and {dagger} Earle A. Chiles Research Institute, Robert W. Franz Cancer Research Center, Providence Portland Medical Center, Portland, OR 97213


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ag-specific CD4 T cells transferred into unirradiated Ag-bearing recipients proliferate, but survival and accumulation of proliferating cells is not extensive and the donor cells do not acquire effector functions. We previously showed that a single costimulatory signal delivered by an agonist Ab to OX40 (CD134) promotes accumulation of proliferating cells and promotes differentiation to effector CD4 T cells capable of secreting IFN-{gamma}. In this study, we determined whether OX40 costimulation requires supporting costimulatory or differentiation signals to drive acquisition of effector T cell function. We report that OX40 engagement drives effector T cell differentiation in the absence of CD28 and CD40 signals. Two important regulators of Th1 differentiation, IL-12R and T-bet, also are not required for acquisition of effector function in CD4 T cells responsive to OX40 stimulation. Finally, we show that CD25-deficient CD4 T cells produce little IFN-{gamma} in the presence of OX40 costimulation compared with wild type, suggesting that IL-2R signaling is required for efficient OX40-mediated differentiation to IFN-{gamma} secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Effective CD4 T cell immunity requires recognition of cognate Ag followed by additional costimulatory signals. The classical costimulatory receptor, CD28, amplifies signals from the TCR, which decreases the threshold for Ag-specific activation, resulting in responsiveness to lower doses of Ag (1). CD28 signals lead to enhanced expression of transcription factors, antiapoptotic genes, cytokines, and cytokine receptors that lead to survival, differentiation, avoidance of anergy, and effector T cell function (2). A variety of other receptors, such as members of the TNFR family, including 4-1BB, CD27, CD30, and OX40, also exhibit costimulatory function (3). Expression of these receptors and their ligands is selective in time and place and tightly controlled by antigenic stimulation and inflammatory or danger signals. Although these costimulation pathways can reinforce outcomes initiated by CD28, they also have unique roles in survival and promote differentiation to effector cells that contribute to a specific immune response. For example, OX40 and OX40L are induced only after activation (4, 5), and OX40 signaling to CD4 T cells has been shown to directly enhance survival (6, 7, 8) and promote effector function by enhancing either Th1 or Th2 differentiation (9, 10, 11, 12, 13).

Adoptive transfer of Ag-specific CD4 T cells into unirradiated Ag-bearing recipients results in robust proliferation of donor cells and infiltration of tissues, but effector function is limited and recipients do not develop clinical signs of disease (10, 12, 14), presumably due to a lack of costimulation. Transfer of transgenic T cells into CD40-deficient Ag-bearing recipients results in similar proliferation of donor cells, suggesting that conventional costimulation is not limiting for T cell proliferation in this system (12). In contrast, a single injection of agonist anti-OX40 provides a signal that induces accumulation of proliferating donor cells that acquire the ability to produce IFN-{gamma} and cause disease (10, 12). In this model, donor T cells are unresponsive upon restimulation through the TCR, but produce IFN-{gamma} upon stimulation distal to the TCR or through cytokine receptors (12). Agonist anti-OX40 treatment results in death of the recipient, presumably due to cytokine-producing effector T cells infiltrating nonlymphoid organs (12). OX40 also promotes differentiation early in T cell priming and produces larger changes in expression of cytokine and cytokine receptor genes than in survival genes (10). In other reports, OX40-driven differentiation results in enhanced secretion of IL-4 and IL-5 (15) and germinal center formation (16) in Th2 responses and IL-2 and IFN-{gamma} production in Th1 responses (12, 17). OX40 signaling has also been reported to reverse previously established peripheral tolerance (18).

OX40 costimulation could be directly orchestrating the acquisition of effector function or could require supportive signaling from other costimulatory and cytokine receptors or other downstream regulators of differentiation to induce the effector T cell functions seen in each of these models. For example, OX40-driven differentiation could synergize with CD28 to promote activation of transcription factors, as suggested by a recent report (19), or OX40 could require interaction with supporting costimulatory pathways in APC such as CD40. Upon ligation with CD40L on T cells, CD40 enhances several costimulatory ligands on APC that interact with T cell costimulatory receptors, and consequently promote a positive feedback loop that drives differentiation (20). In a previous report, we showed that OX40-deficient donor cells gain effector function as bystanders to wild-type Ag-responsive cells stimulated with anti-OX40 (10). In this system, anti-OX40 was the only exogenous adjuvant provided to initiate costimulation, indicating that OX40 engagement led to activation of alternative pathways capable of driving differentiation of OX40-deficient T cells.

In this study, we use two previously published adoptive transfer systems (10, 12) to determine whether OX40 engagement promotes differentiation in the absence of costimulatory or cytokine receptors, or T-bet, a transcription factor promoting Th1 differentiation. We show that OX40 costimulation drives differentiation to IFN-{gamma}-secreting effector T cells in the absence of CD28 or CD40 costimulation. We also show that OX40 induces acquisition of effector function in the absence of the IL-12R and T-bet, two important regulators of Th1 differentiation. Finally, we examined the effect of OX40 costimulation in the absence of the IL-2R {alpha}-chain, CD25, and found that acquisition of effector function in this case requires functional IL-2R signaling. Thus, while OX40 promotes differentiation of CD4 T cells in the absence of other costimulatory and differentiation pathways, IL-2R signaling is essential for the development of effector CD4 T cells in response to OX40 costimulation.


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

Mice were housed under specific pathogen-free conditions at the Oregon Health and Science University Animal Facility. (B6.CD45.1 x bm12)F1 mice were made by crossing female B6.CD45.1 to B6.C-H2bm12/KhEg mice, obtained from The Jackson Laboratory. C57BL/6J, CD28–/– (B6.129S2-CD28), IL-12Rbeta2–/– (B6.129S1-Il12rb1), T-bet–/– (B6.129S6-Tbx21), and CD25–/– (B6.129S4-Il2ra) were also obtained from The Jackson Laboratory. Pigeon cytochrome c (PCC)3 specific AND TCR-transgenic, Rag-1-deficient mice, PCC-specific AD.10 TCR-transgenic, and Ag-transgenic mice expressing I-Ek plus covalently associated antigenic peptide on the C57BL/6 background sufficient or deficient in CD40 have been described previously (12). Donor splenocytes plus lymph nodes were pooled for each experiment and were prepared for i.v. injection as previously described (12). In some experiments, donor cells were labeled with 2 µM CFSE in 0.1% BSA in PBS for 10 min at 37°C and washed in HBSS with 2% serum. Cells were injected i.v. with 50 µg of anti-OX40 or control IgG in HBSS without serum into unirradiated (B6.CD45.1 x bm12)F1 recipients or into Ag-transgenic recipients.

Abs and cytokines

PerCP anti-CD4 (RM4–5), biotin anti-CD25 (7D4), biotin anti-CD69 (FN50), allophycocyanin anti-IL-2 (JES6-5H4), PE anti-IL-17 (TC1-8H4.1), and labeled isotype controls were purchased from BD Pharmingen. Allophycocyanin anti-IFN-{gamma} (XMG1.2), PE or FITC anti-CD45.1 (A20), allophycocyanin streptavidin, PE goat anti-rat IgG, and appropriate isotype controls were purchased from eBioscience. Biotin chicken anti-OX40 and isotype control were produced and purified for staining cells. Anti-OX40 Ab from clone OX86 (European Cell Culture Collection) was produced and purified for i.v. injection. Rat IgG was purchased from Cappel (ICN Pharmaceuticals). Anti-IL-4 (11B11), anti-IFN-{gamma} (XMG1.2), and recombinant mouse IL-2 were produced and purified for in vitro culture. Recombinant mouse IL-12 was purchased from Cell Sciences; recombinant mouse IL-18 and recombinant mouse IL-23 were purchased from R&D Systems.

Cell culture and flow cytometry

Spleen cell suspensions were prepared for intracellular cytokine staining as previously described (12). In some experiments, splenocytes were stimulated with 100 ng/ml IL-18 and 8 ng/ml IL-23 for 5 h before intracellular cytokine staining. Labeled cells were analyzed on a FACSCalibur flow cytometer (BD Immunocytometry Systems) and analyzed using FlowJo (Tree Star). Th1 and Th2 cultures were set up using AD.10 splenocytes as described previously (21).

Quantitative PCR

A total of 106 AND TCR-transgenic T cells were transferred with 50 µg of anti-OX40 or control IgG into Ag-transgenic recipients congenic for CD45.1; 3.5 days later, TCR-transgenic T cells were isolated from recipient splenocytes by sorting CD45.1-negative CD4-positive cells to ≥92% purity on a FACSVantage flow cytometer (BD Immunocytochemistry Systems). Total RNA was purified from Th1, Th2, and sorted T cell populations as previously described (10). cDNA was made using the Stratascript First-Strand Synthesis System (Stratagene). Quantitative real-time PCR was performed on an ABI 7700 using SYBR Green Master Mix (Applied Biosystems) and previously published primer sequences for T-bet and beta-actin to measure DNA amplification (21). T-bet and beta-actin cycle threshold values were converted to standard curve values, and T-bet/beta-actin ratios were analyzed as fold over baseline (Th2 cells). Th1 cells were used as a positive control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD40 signaling to APC is not required for OX40-driven differentiation

To understand how OX40 signaling affects CD4 T cell differentiation, we sought to examine the requirement for other costimulatory signals during an immune response driven by an agonist Ab to OX40. CD40 engagement functions as a dendritic cell (DC) maturation stimulus, enhancing Ag presentation and inducing costimulatory ligand expression, which contributes to a positive feedback loop to promote donor CD4 T cell differentiation (20). Because DC in our model are not exogenously activated, we reasoned that if OX40 engagement increases CD40L expression, allowing CD40 signaling to initiate DC maturation, then the absence of CD40 on DC may prevent or dampen DC maturation, and subsequently affect the acquisition of effector function in CD4 T cells initiated by OX40. To test this hypothesis in vivo, we used our previously published model in which AND Rag1–/– TCR-transgenic T cells specific for a PCC peptide are transferred into Ag-transgenic mice (12). We transferred 106 TCR-transgenic CD4 T cells in a spleen and lymph node suspension with anti-OX40 or control IgG into Ag-transgenic mice that were deficient in CD40 or not, and harvested spleens 5 days later. As seen in Fig. 1A and Table I, all donor cells divide and dilute CFSE to background levels, but accumulation of donor CD4 T cells in spleen is diminished in CD40-deficient hosts compared with CD40+/+ recipients. However, donor T cells from CD40–/– recipients that received OX40 costimulation accumulated 3-fold more donor cells compared with control IgG, and the donor cells had acquired effector function, as measured by CD25 expression and IFN-{gamma} production (Fig. 1 and Table I). Moreover, a higher percentage of donor CD4 T cells from CD40–/– recipients produced IFN-{gamma}, especially directly ex vivo, compared with CD40+/+ recipients (40 vs 15%), and were able to respond more robustly to restimulation in vitro with IL-12 and IL-18, a cytokine combination known to induce robust IFN-{gamma} production (22), and PMA and ionomycin (Fig. 1B). These data show that OX40 costimulation induces differentiation to cytokine-producing effector CD4 T cells in the absence of CD40 signaling to APC. These data also suggest that signals to DC via CD40 may be important in enhancing CD4 T cell survival because in the absence of CD40, donor cell recovery from spleen was reduced.


Figure 1
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  		FIGURE 1. CD40 signaling is not required to support OX40-mediated acquisition of effector function. A total of 106 TCR-transgenic CD4 T cells from AND Rag1–/– spleen and lymph node suspensions were transferred with 50 µg of anti-OX40 or control IgG into CD40+/+ or CD40–/– Ag-transgenic recipients, and spleens were harvested on day 5. A, Top panels, Percent TCR-transgenic T cells of total CD40–/– (left) or CD40+/+ (right) recipient splenocytes. Bottom panels, CD25 expression on donor TCR-transgenic cells. The number shows the percentage of CFSElow divided donor cells that are also CD25+. B, Bar diagrams show mean and SE of the divided donor TCR-transgenic T cells that are IFN-{gamma}+ after restimulation in vitro for 5 h with medium only, IL-12 and IL-18 (IL-12/18), or PMA and ionomycin (PMA/iono) in two separate experiments, six animals total per group.

 

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  		Table I. Donor CD4+ cell recoveries from spleena

 
IL-12R and T-bet, part of the Th1 commitment pathway, are not required for OX40-mediated differentiation to IFN-{gamma} production

IL-12R signaling is important for commitment to Th1 effector cell differentiation (23). In a previous report, we showed that IL-12R gene expression was enhanced in Th1 effector cells upon OX40 costimulation (10). Agonist anti-OX40 drives effector T cell development in a population of alloreactive B6 CD4 T cells transferred into unirradiated (B6.CD45.1 x bm12)F1 recipients, which differ on one allele from MHC class II I-Ab in the peptide-binding domain (10). To test whether the IL-12R influences the ability of anti-OX40-stimulated alloreactive donor cells to differentiate, we transferred 107 IL-12Rbeta2–/– CD4 T cells in a spleen suspension with anti-OX40 or control IgG into (B6.CD45.1 x bm12)F1 recipients. Seven days after donor cell transfer, splenocytes were analyzed for activation markers and cytokine production. As shown in Fig. 2A, IL-12Rbeta2–/– donor cell recovery and CD25 expression were similar to wild type, in the presence or absence of anti-OX40. Also shown in Fig. 2A, OX40 engagement promotes accumulation of CD4high T cells, which also appear to be CD45.1 positive. This observation is not surprising because activated CD4 T cells have been shown to acquire membrane proteins during T:APC interactions (24). IFN-{gamma} production after in vitro restimulation with medium alone, PMA and ionomycin, and IL-18 was also similar between experimental groups, although IL-12Rbeta2–/– donor cells treated with anti-OX40 did not make as much IFN-{gamma} after IL-12 and IL-18 stimulation, as expected when the IL-12R is absent (Fig. 2B). These results show that although OX40 costimulation enhanced IL-12R expression in wild-type CD4 T cells (10), OX40 promoted differentiation of Th1 effector cells independent of IL-12R signaling.


Figure 2
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  		FIGURE 2. OX40 drives differentiation independent of IL-12R signaling. A total of 107 IL-12Rbeta2–/– or B6 CD4+ T cells in a spleen suspension were transferred with 50 µg of anti-OX40 or control IgG into (B6.CD45.1 x bm12)F1 recipients for 5 days. A, Top panels, Percent CD4+CD45.1– donor cells of total splenocytes. Bottom panels, Donor cell size and CD25 expression. B, Bar diagrams show mean and SE of the divided donor TCR-transgenic T cells that are IFN-{gamma}+ after restimulation in vitro for 5 h with medium, 1 µg/ml IL-18, IL-12 and IL-18, or PMA and ionomycin in two separate experiments, six animals total per group.

 
T-bet is a transcription factor that regulates IFN-{gamma} gene transcription and many reports have demonstrated the requirement for T-bet expression in Th1 effector cell differentiation (25, 26). Given that in our models, OX40 costimulation promotes Ag-specific CD4 T cells to acquire a robust Th1 phenotype, we reasoned that OX40 signaling would enhance T-bet expression to drive Th1 differentiation. To test this hypothesis, we transferred 106 AND TCR-transgenic T cells with anti-OX40 or control IgG into Ag-transgenic recipients congenic for CD45.1; 3.5 days later, we purified TCR-transgenic T cells from the spleen and extracted total RNA for quantitative PCR to measure levels of T-bet mRNA in comparison to in vitro-cultured Th2 cells (baseline) and Th1 cells (positive control). As shown in Fig. 3A, OX40 costimulation induced T-bet mRNA expression >10-fold over control IgG-treated donor cells to levels comparable to cultured Th1 cells.


Figure 3
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  		FIGURE 3. OX40 costimulation enhances T-bet expression, but T-bet is not required for OX40-driven acquisition of effector function. A, Quantitative analysis of the fold-change in T-bet expression from day 3.5 TCR-transgenic T cells subjected to anti-OX40 or control IgG are compared with T-bet expression of in vitro-cultured Th1 and Th2 cells. Quantitative PCR analysis and RNA isolation from recipient Ag-transgenic spleens is described in Materials and Methods. B, A total of 7.5 x 106 T-bet–/– or wild-type (WT) donor cells were transferred with 50 µg of anti-OX40 or control IgG into (B6.CD45.1 x bm12)F1 recipients and spleens were harvested 5 days later. Percent CD4+CD45.1– donor cell recovery is shown in the first row of panels. FACS plots show divided donor CD4+CD45.1– cells that are IL-17+, IFN-{gamma}+, and IL-17+IFN-{gamma}+ double positive after restimulation in vitro for 5 h with medium (second row), IL-18 and IL-23 (third row), or PMA and ionomycin (fourth row); bar diagrams below each column show mean and SE of the divided donor cytokine production from two independent experiments, six animals total per group. C, The bar diagram shows the percentage of CFSElow divided donor cells that are also CD25+ or OX40+ from two experiments, six replicates per group.

 
The data in Fig. 3A show that T-bet expression is elevated as a result of OX40 costimulation, but do not address the importance of T-bet function in the OX40-signaling pathway. Therefore, we sought to test whether the effects of OX40 are dependent on this master regulator of Th1 differentiation, or if OX40 costimulation can independently induce differentiation and IFN-{gamma} effector cytokine production. We transferred 7.5 x 106 T-bet–/– CD4 T cells in a spleen and lymph node suspension with anti-OX40 or control IgG into (B6.CD45.1 x bm12)F1 recipients. Five days after transfer, recovery of donor T-bet–/– cells in spleen was similar to that of wild-type donor cells, with control IgG or anti-OX40 (Fig. 3B, first row, and Table I). Compared with control IgG, anti-OX40 promotes an activated phenotype in T-bet–/– divided donor cells, with high expression of CD25 and OX40 (Fig. 3C). Interestingly, anti-OX40 induces greater expression of OX40 on T-bet–/– donor cells compared with T-bet+/+ donors. These data show that despite a deficiency in T-bet, OX40 costimulation supports an activated phenotype in alloreactive CD4 T cells.

We next examined whether OX40 costimulation could induce effector cytokine production in T-bet–/– CD4 T cells. We measured IL-4, IL-10, TNF-{alpha}, IL-2, and IL-17 in addition to IFN-{gamma} production after in vitro restimulation with PMA and ionomycin or medium alone to detect OX40-mediated acquisition of effector cytokine production. Anti-OX40 promoted IFN-{gamma} production in T-bet-deficient alloreactive donor cells, although 4-fold fewer compared with wild-type donors when stimulated with PMA and ionomycin (Fig. 3B, fourth row). Of note, T-bet–/– donor cells made IL-17 with or without OX40 signaling when stimulated with PMA and ionomycin (Fig. 3B). In addition, IL-23 and IL-18, a cytokine combination reported to induce IL-17 production in CD4 T cells (27), enhanced IL-17 production in T-bet–/– CD4 T cells, and particularly in donor cells from anti-OX40-treated mice (Fig. 3B, third row). No T-bet+/+ donor CD4 T cells made IL-17 in response to IL-23 and IL-18 and very few produced IL-17 in response to PMA and ionomycin (Fig. 3B). IL-12 and IL-18 stimulation prompted T-bet+/+ divided donors to make three times as much IFN-{gamma} as with IL-23 and IL-18 (data not shown), while IL-12 and IL-18 promoted equal IFN-{gamma} production from T-bet–/– divided donor cells as from cells stimulated with IL-23 and IL-18. T-bet–/– donor cells subjected to anti-OX40 made very little IL-2 compared with T-bet+/+ donor cells (0.5 vs 13%), while both groups treated with control IgG made equal amounts of IL-2 (20%) when restimulated with PMA and ionomycin (data not shown). All other cytokines tested were not induced above background in any experimental group (data not shown). These data show that in the absence of T-bet, total OX40-mediated effector cytokine production is reduced compared with wild type, indicating that T-bet is required for optimal cytokine production. However, these data also suggest that in the absence of T-bet, OX40 supports the ability to acquire alternative effector functions, as noted by the additional responsiveness to IL-23 and IL-18, and production of IL-17 in addition to IFN-{gamma}.

A signal through OX40 can overcome the defect in differentiation in CD28–/– CD4 T cells

Signals through constitutively expressed CD28 promote initial T cell activation and additional T cell effector function (2), while OX40 expression is delayed in T cell priming. To test the hypothesis that OX40 promotes differentiation in CD4 T cells in the absence of CD28, we transferred 7.5 x 106 CD4 T cells deficient in CD28 into (B6.CD45.1 x bm12)F1 recipients. Five days after T cell plus control IgG transfer, few CD28–/– donor cells were recovered from spleen, but some had proliferated, as measured by CFSE dilution (Fig. 4A). Wild-type donor cells accumulated 15- to 20-fold more donor cells with or without OX40 costimulation, confirming that CD28 is important for progressive cell division and/or accumulation of divided cells (Fig. 4A and Table I). However, in the absence of CD28, OX40 enhanced accumulation of divided donor cells compared with control IgG, suggesting that OX40 signals contribute to optimal accumulation of divided donor cells independent of CD28. We also looked at T cell activation by measuring CD25 and OX40 expression. As seen in previous in vitro studies (28), OX40 is expressed on CD28-deficient divided donor cells, as well as on wild type (Fig. 4A, bottom row), and anti-OX40 enhanced CD25 expression in CD28–/– divided donor cells compared with control IgG (Fig. 4A, middle row). To determine whether OX40 can promote effector cytokine production in the absence of CD28, we examined IFN-{gamma} production from CD28–/– donor CD4 T cells exposed to anti-OX40. Day 5 splenocytes from each animal were restimulated in vitro for 5 h with medium alone, IL-12 and IL-18, or PMA and ionomycin. Although a higher percentage of wild-type than CD28–/– donor cells from anti-OX40-treated mice are able to make IFN-{gamma} upon in vitro restimulation, OX40 costimulation enhanced IFN-{gamma} production by CD28–/– divided donor cells in response to IL-12 and IL-18 or PMA and ionomycin compared with CD28–/– donor cells plus control IgG (Fig. 4B). Although CD28 is required for optimal effector cytokine production, these data show that OX40 signaling supports the necessary machinery to acquire effector cytokine production without CD28 costimulation.


Figure 4
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  		FIGURE 4. OX40 costimulation enhances proliferation and effector function of CD28–/– CD4 T cells. A total of 7.5 x 106 CD4 T cells in a spleen and lymph node suspension from CD28–/– or B6 mice were transferred with 50 µg of agonist anti-OX40 or control IgG into unirradiated (B6.CD45.1 x bm12)F1 recipients and spleens were harvested 5 days later. A, Top panels, Percent CD4+CD45.1– donor CD28–/– (left) or wild-type (right) cells of total splenocytes. Middle and bottom rows, The phenotype of CD4+CD45.1– donor cells. The number shows the percentage of CFSElow divided donor cells that are also CD25+ or OX40+. B, Bar diagrams show mean and SE of the divided donor CD4 T cells that are IFN-{gamma}+ after restimulation in vitro for 5 h with medium alone, IL-12 and IL-18 (IL-12/18), or PMA and ionomycin (PMA/iono) in two separate experiments, six animals total per group.

 
OX40-mediated differentiation is dependent on IL-2R signaling

IL-2 has primarily been characterized as a T cell growth factor (29). However, many recent studies have found that IL-2 also plays a role in driving effector T cell differentiation (30, 31, 32, 33). In our systems, we note that OX40 engagement induces and maintains expression of the IL-2R {alpha}-chain, CD25, but the importance of CD25 expression in acquisition of effector function is unknown (10, 12). To test the importance of IL-2R signaling on OX40-mediated effector cell development, we transferred 7.5 x 106 CD25–/– CD4 T cells from mice <8 wk of age with anti-OX40 or control IgG into (B6.CD45.1 x bm12)F1 recipients, and 5 days later looked at donor CD4 T cell recovery in the spleen. Few CD25–/– donor cells were recovered, but some proliferated, as measured by CFSE dilution (Fig. 5A and Table I). However, 10- to 20-fold more divided wild-type donor cells accumulated with or without costimulation (Fig. 5A and Table I), suggesting that CD25 is important for cell division or accumulation of dividing cells. Importantly, in the absence of CD25, OX40 still induced a 3-fold increase in accumulation of the divided donor cells compared with control IgG (Table I), suggesting that costimulation via OX40 can rescue the accumulation defect to some extent, but not to wild-type levels.


Figure 5
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  		FIGURE 5. OX40-induced acquisition of effector function is dependent on IL-2R signaling. A total of 7.5 x 106 CD25–/– or WT donor cells were transferred with 50 µg of anti-OX40 or control IgG into (CD45.1.B6 x bm12)F1 recipients, and spleens were harvested 5 days later. A, Percent CD4+CD45.1– donor cell recovery is shown in the first row of panels. FACS plots show divided donor CD4+ CD45.1– cells that are IFN-{gamma}+ after restimulation in vitro for 5 h with medium (second row), IL-18 and IL-12 (third row), or PMA and ionomycin (fourth row), and the bar diagram in B shows mean and SE of the divided donor IFN-{gamma} production from two independent experiments, six animals total per group.

 
To determine whether OX40 costimulation induces effector function in CD25-deficient CD4 T cells, we restimulated splenocytes from each group with medium, IL-12 and IL-18, or PMA and ionomycin for 5 h. Surprisingly, we found that although OX40 engagement of CD25-deficient donor T cells causes an increase in the percentage of IFN-{gamma}+ cells in unstimulated cultures and in response to IL-12 and IL-18, the percentage of IFN-{gamma}+ cells is 20-fold less than that of wild-type donor cells from OX40-treated animals (Fig. 5A). CD25–/– donor cells from control IgG and anti-OX40-treated animals were equally able to make IFN-{gamma} upon PMA and ionomycin stimulation and at the same low levels as wild-type donor cells from control IgG-treated animals (Fig. 5A). These data indicate that although OX40 signaling is able to provoke slight accumulation of effector CD4 T cells independent of CD25 expression, IL-2R signaling is essential for efficient differentiation to IFN-{gamma} secretion in OX40-responsive CD4 T cells.

CD25-deficient T cells develop autoimmunity due to a defect in regulatory T cells (34, 35). To ensure that the observed donor CD4 T cell proliferation was Ag specific and not a byproduct of autoimmunity, we transferred CD25–/– cells into syngeneic B6 hosts with anti-OX40 or control IgG and looked at donor CD4 T cell recovery and differentiation 5 days later. As shown in Table I, very few divided donor CD4 T cells accumulated in syngeneic hosts with either anti-OX40 or control IgG, confirming that the transfer of CD25-deficient CD4 T cells into (B6.CD45.1 x bm12)F1 recipients, reported above, resulted in an Ag-specific immune response. In addition, the divided donor cells transferred into syngeneic hosts do not produce IFN-{gamma} upon in vitro restimulation (data not shown). We measured the expression of OX40 on CD25–/– divided donor cells and found that the lack of differentiation reported above was not due to lack of the OX40R on CD25–/– donor cells (data not shown). Taken together, these data show that OX40 signaling greatly enhances effector CD4 T cell effector function in an IL-2R-dependent manner.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Ag-specific CD4 T cells transferred into unirradiated Ag-bearing recipients undergo massive proliferation, but survival of divided cells is limited and acquisition of effector function does not occur (10, 12, 14). Lack of differentiation in this case could be attributed to lack of costimulatory signals, as there is no exogenous adjuvant or danger signal provided in this system to bolster costimulation. The introduction of a single injection of anti-OX40, a costimulatory signal that promotes differentiation (10, 12), provides a model for understanding the mechanism of effector CD4 T cell development. In this study, we asked whether OX40 costimulation required other costimulatory or differentiation signals to drive acquisition of effector function. We found that OX40 costimulation promotes effector cytokine production in the absence of CD28 and CD40. OX40 signals also partially rescue a survival and/or accumulation defect in CD28 mutants, and can promote limited accumulation of donor T cells in CD40–/– recipients. We also found that OX40 drives acquisition of effector function in the absence of two important Th1 differentiation signals, IL-12R and T-bet. Finally, we show that functional IL-2R signaling is required for enhanced differentiation to IFN-{gamma} secretion in donor T cells responsive to OX40 costimulation.

Ag recognition and costimulation have been reported to promote T cell activation and proliferation independent of IL-2, but differentiation to effector function in these models was IL-2 dependent (32, 36, 37). Recently, two reports have used recombinant IL-2/anti-IL-2 immune complexes, which enhanced the potency of IL-2 for T cells in vivo, and showed the importance of IL-2 in promoting differentiation of effector T cells (31, 33). In our model, OX40 costimulation promoted limited accumulation of CD25-deficient donor CD4 T cells, but very few of those cells acquired the ability to secrete IFN-{gamma}. Although CD25 was not necessary for OX40 expression, it is possible that the lack of CD25 expression in CD4 T cells disconnects the OX40-signaling pathway that drives differentiation. OX40 activates NF-{kappa}B (38), which can then interact with the NF-{kappa}B-responsive elements in the IL-2 and CD25 promoters (39). Enhanced IL-2R signaling induces other cytokine and cytokine receptor expression, including CD25, ultimately driving full effector cytokine production (40, 41). In this way, OX40 costimulation could require functional IL-2R signaling to enhance effector cytokine and cytokine receptor expression to promote effector function. Alternatively, IL-2R signaling could be important in establishing development of effector function early in T cell priming, and effects of OX40 engagement could depend upon early signals from the IL-2R. In a previous report, we showed that Ag-responsive CD4 T cells expressed high levels of CD25 within 24 h after T cell transfer, but in the absence of OX40 costimulation, expression waned by 48 h and few cells expressed CD25 at 72 and 120 h. OX40 costimulation induced effector cytokine production beginning at 48 h, and CD25 expression was maintained at high levels at all time points (10). Those experiments did not show that enhanced CD25 expression accounts for OX40 effects on acquisition of effector function, but data in this report clearly show that IL-2R signaling is essential for enhanced effector function in CD4 T cells in response to OX40 engagement.

We found that OX40 ligation induces effector cytokine production in the absence of either CD28 on donor CD4 T cells or CD40 on recipient APC. These data support findings in a related model of skin graft rejection, in which CD28 and CD40L double-deficient naive or memory T cells were able to induce graft rejection, even in the presence of blocking Abs to inducible costimulator:inducible costimulator ligand, 4-1BB:4-1BBL, or CD27:CD70. Furthermore, OX40+ CD4high T cells were found in the target organ, and blocking OX40:OX40L signaling improved acceptance of the graft (42, 43). Our results also show that when CD40 is deficient on recipient APC, OX40 is less effective at enhancing accumulation of donor cells but more effective at promoting IFN-{gamma} secretion. These data suggest that OX40 does not independently enhance accumulation of donor cells in this model, and instead requires additional signals from DC licensing via CD40 engagement to restore accumulation of CD4 T cells. It is also possible that additional signals from endogenous activated T cells capable of responding to anti-OX40 could affect effector cytokine production and accumulation of donor CD4 T cells. However, we did not detect a difference in effector cytokine production owing to anti-OX40 from endogenous CD4 T cells in any experimental group (data not shown). Thus, the primary role of OX40 signaling may be to allow the expression of cytokines and cytokine receptors, which then both lead to further development of effector function and enhance survival.

Although we showed that anti-OX40 enhanced T-bet expression and Th1 effector cell development in a Th1-biased model, when IFN-{gamma} was dampened in the absence of T-bet, OX40 engagement still promoted accumulation of cytokine-producing effector cells. OX40 signaling in the absence of T-bet enhanced IL-23 responsiveness measured by IL-17 production, as compared with the IL-12 responsiveness measured by IFN-{gamma} production when T-bet was functional. Ectopic T-bet expression has been shown to abrogate IL-17 production in Th17-polarized cells (27). This suggests that a lack of IL-17 production from wild-type CD4 T cells in this report could be due to the enhanced T-bet expression in OX40-responsive CD4 T cells. However, a recent report shows that small-interfering RNA targeted toward T-bet suppresses IL-23R expression and subsequent IL-17 production (44). We show that in the genetic absence of T-bet, OX40 enhanced IL-23 responsiveness measured by IL-17 production, suggesting that OX40 signaling promotes additional pathways for effector cell differentiation.

Although the data in Fig. 3 showed a small increase in IL-17 production upon in vitro stimulation, the majority of T-bet–/– divided donor cells did not produce IL-17, IFN-{gamma}, or other measured cytokines, suggesting that T-bet expression is important for optimal effector cytokine production. Interestingly, recipients that received T-bet–/– donor cells plus anti-OX40 developed larger spleens, thickened intestines, and had excessive mucous in their eyes after 5 days, compared with recipients that received T-bet+/+ donor cells and anti-OX40 (C. Williams, unpublished observations). These observations could indicate that in the absence of T-bet, anti-OX40 promoted sufficient IL-17 production in effector CD4 T cells to lead to exacerbated gross pathology compared with wild type. Alternatively, it is possible that OX40 signals up-regulated other proinflammatory cytokines normally suppressed in the presence of T-bet, which may have acted alone or in synergy with the small amount of IFN-{gamma} and IL-17 produced by T-bet–/– CD4 T cells to enhance disease progression.

In summary, we provide evidence that OX40-mediated differentiation to cytokine-producing effector CD4 T cells is not strictly dependent on costimulatory signals such as CD28 and CD40. In addition, although OX40 enhances regulators of Th1 differentiation such as IL-12Rbeta2 and T-bet expression (10), OX40 signals still promote accumulation of cytokine producing effector T cells in their absence. Of the accessory signals tested, the absence of IL-2R has the most severe effect in curtailing OX40-driven differentiation to IFN-{gamma} production by the recovered donor cells. It appears that OX40 signals amplify the original effector cell developmental program and that this effect occurs in the absence of CD28, CD40, IL-12Rbeta2, and T-bet. However, OX40 signals depend on the IL-2R to support effector cell differentiation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
A. D. Weinberg has a patent pending to use anti-Ox40 Ab 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 AI50823 (to D.C.P.) and CA81383 (to A.D.W.) from the National Institutes of Health. C.A.W. was supported as a trainee in the Molecular Hematology Training Program, Grant T32-HL007781. Back

2 Address correspondence and reprint requests to Dr. David C. Parker, Department of Molecular Microbiology and Immunology, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, L220, Portland, OR 97239. E-mail address: parkerd{at}ohsu.edu Back

3 Abbreviations used in this paper: PCC, pigeon cytochrome c; DC, dendritic cell. Back

Received for publication November 27, 2006. Accepted for publication March 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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