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The Journal of Immunology, 2006, 176: 5975-5987.
Copyright © 2006 by The American Association of Immunologists

OX40-OX40 Ligand Interaction through T Cell-T Cell Contact Contributes to CD4 T Cell Longevity1

Pejman Soroosh, Shouji Ine, Kazuo Sugamura and Naoto Ishii2

Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Signals through the OX40 costimulatory receptor on naive CD4 T cells are essential for full-fledged CD4 T cell activation and the generation of CD4 memory T cells. Because the ligand for OX40 is mainly expressed by APCs, including activated B cells, dendritic cells, and Langerhans cells, the OX40-OX40 ligand (OX40L) interaction has been thought to participate in T cell-APC interactions. Although several reports have revealed the expression of OX40L on T cells, the functional significance of its expression on them is still unclear. In this study, we demonstrate that Ag stimulation induced an increase in the surface expression and transcript levels of OX40L in CD4 T cells. Upon contact with OX40-expressing T cells, the cell surface expression of OX40L on CD4 T cells was markedly down-regulated, suggesting that OX40-OX40L binding occurs through a novel T cell-T cell interaction. To investigate the function of this phenomenon, we examined the proliferative response and survival of OX40L-deficient CD4 T cells when challenged with Ag. In vitro studies demonstrated markedly less CD3-induced proliferation of OX40L-deficient CD4 T cells compared with wild-type CD4 T cells. When using TCR transgenic CD4 T cells upon Ag stimulation, survival of OX40L-deficient T cells was impaired. Furthermore, we show that upon antigenic stimulation, fewer OX40L-deficient CD4 T cells than wild-type cells survived following transfer into wild-type and sublethally irradiated recipient mice. Taken together, our findings indicate that OX40L-expressing T cells have an autonomous machinery that provides OX40 signals through a T cell-T cell circuit, creating an additional mechanism for sustaining CD4 T cell longevity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Optimal T cell activation requires not only signals delivered by Ag stimulation, but also costimulatory signals provided by APCs (1, 2). Although the interaction of CD28 with CD80/CD86 provides the best known costimulatory signals, other costimulatory molecules, including TNF superfamily molecules such as OX40 (CD134), CD27, and 4-1BB (CD137), can potently augment the full-fledged activation of T cells (3, 4, 5). Among these costimulatory molecules, OX40, which is transiently expressed by activated T cells, is not only essential for providing optimal CD4 T cell functioning (6, 7, 8, 9), but also critically contributes to the generation of memory T cells by promoting the survival of effector T cells (10, 11, 12). The ligand for OX40 (OX40L)3 is mainly expressed by APCs, such as activated B cells, dendritic cells (DCs), and Langerhans cells, as well as endothelial cells and T cells (13, 14, 15, 16, 17, 18, 19, 20, 21).

In several studies using OX40L-deficient mice, we and others have revealed that the absence of OX40L on APCs leads to a marked reduction in the proliferation and cytokine production of CD4 T cells (8, 9). We further showed that the effector function of encephalitogenic T cells transferred into OX40L-deficient mice could not be sustained in an experimental autoimmune encephalomyelitis system (22). Similarly, pathogenic T cells from mouse models of inflammatory bowel disease and graft-vs-host disease (GVHD) did not cause disease when they were transferred into OX40L-deficient recipient mice (23, 24), primarily because the donor T cells could not produce cytokines normally. These observations imply that OX40L expression, possibly by the APCs of recipient mice, is essential for donor CD4 T cell function.

Accumulating evidence, however, has shown that Ag-experienced T cells do express APC accessory molecules such as CD80, CD86, CD70, and CD40 (25, 26, 27, 28, 29, 30, 31, 32). Bourgeois et al. (31) revealed that, upon activation, CD8 T cells endogenously express CD40, which can bind to CD40L on CD4 T cells through T cell-T cell interactions. The direct cross talk between CD4 and CD8 T cells consequently supports the generation of memory CD8 T cells in vivo. Little is known about whether other accessory molecules on T cells can provide efficient costimulatory signals during T cell-T cell interactions.

Our initial studies of OX40L revealed its up-regulation on HTLV-1-infected T cells (21). Several groups have shown that OX40L is also expressed on activated T cells (16, 18, 33, 34). More recently, in vitro studies demonstrated that T cells can acquire OX40L that apparently originates from OX40L-expressing cells in a non-Ag-specific manner (35, 36). In the present study, we set out to evaluate the expression and biological roles for the OX40L expressed on T cells. In this study, we document the endogenous expression of OX40L by activated CD4 T cells and reveal its costimulatory function in promoting CD4 T cell proliferation and survival through a novel CD4 T cell-T cell interaction.


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

The OX40-deficient (OX40-knockout (KO)) and OX40L-deficient (OX40L-KO) mice were described previously (9, 16). Six- to 8-wk-old female C57BL/6 mice were purchased from Japan SLC. Ly-5.1+ (CD45.1)-C57BL/6 mice were described previously (24). OT-II TCR transgenic mice were a gift from W. Heath (Walter and Eliza Hall Institute, Melbourne, Australia) and used as a source of V{alpha}2+Vbeta5.1-2+ CD4 T cells responsive to peptide 323–339 of OVA (37). OX40-KO, OX40L-KO, and Ly-5.1+ OT-II TCR transgenic mice were generated in-house by intercrossing OT-II mice with OX40-KO, OX40L-KO, and Ly-5.1 mice, respectively. All of the mice used were on a C57BL/6 background, and they were bred and maintained under specific pathogen-free conditions in the Institute for Animal Experimentation, Tohoku University Graduate School of Medicine.

Cell lines

BW5147, a mouse thymoma cell line, was stably transfected with pCXN2-mOX40 expression plasmid, which carries the chicken beta-actin promoter, followed by mouse OX40 cDNA, yielding the BW-mOX40 line. BW-mOX40 cells and their parental BW5147 cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Abs and flow cytometry

The following Abs were purchased from BD Biosciences: anti-CD3 FITC, anti-CD4 allophycocyanin, anti-CD8 allophycocyanin, anti-V{alpha}2 PE, anti-Vbeta5 FITC, anti-Ly-5.1 allophycocyanin, and anti-Ly-5.2 allophycocyanin. Anti-CD28 (eBioscience), control rat IgG (Cappel), and anti-CD3{epsilon} (2C11) were used for cell culture. Agonistic anti-mouse OX40 mAb (OX86) and inhibitory anti-OX40L mAb (MGP34) were used to stimulate and inhibit OX40 signals, respectively (24). Surface expression of OX40 and OX40L was detected with biotin-conjugated anti-OX40 and anti-OX40L mAbs, as briefly previously described (38). In brief, the cells were preincubated with excess rat Ig to block nonspecific binding of the labeled mAb and incubated with the labeled mAbs for 30 min at 4°C. Control staining for OX40 or OX40L was conducted in the presence of excess unlabeled anti-OX40 or anti-OX40L mAb, respectively. Streptavidin-allophycocyanin (BD Biosciences) was used in the second step of the staining. All of the samples were analyzed on a FACSCalibur flow cytometer (BD Immunocytometry Systems). The analyses were conducted using the CellQuest program (BD Immunocytometry Systems).

In vitro cell stimulation and recovery

A single-cell suspension was prepared from mouse spleens and incubated with mouse CD4 MicroBeads (Miltenyi Biotec) for 20 min at 4°C. The suspension was then purified by autoMACS. The purified CD4+ T cells (purity >98%) were stimulated with 25 ng/ml PMA plus 1 µg/ml ionomycin (Sigma-Aldrich) or 10 µg/ml soluble anti-CD3 in the presence of irradiated (30 Gy) splenocytes, which were used as APCs, at 37°C for the indicated times. In some experiments, CD4+ T cells were stimulated with plate-bound anti-CD3 (5 µg/ml) in the absence of APCs. In the case of OT-II T cell stimulation, irradiated (3000 rad) splenocytes that were pulsed with various concentrations of OVA peptide (323–339) were used as the APCs. Proliferation was measured in triplicate by the incorporation of [3H]thymidine (1 µCi/well; Valeant Pharmaceuticals) during the last 8 or 12 h of each culture. In other proliferation studies, purified CD4+ T cells were labeled with CFSE (Molecular Probes) by incubation with 2.5 µM CFSE in protein-free PBS for 10 min at 37°C; they were then diluted with a 10-fold volume of RPMI 1640 medium containing 10% FCS and incubated for 1 min. Cells were then washed twice with chilled PBS. The amount of cell division of CFSE-labeled cells was estimated by the reduction in fluorescence intensity, measured with a flow cytometer.

In vitro long-term T cell culture

Purified CD4+ T cells from OT-II and OX40-KO OT-II mice were stimulated with the OVA peptide in the presence of irradiated (30 Gy) OX40L-KO splenocytes (APCs) in six-well plates. Every five days, one-half of the culture medium was exchanged. At the indicated time, cells were harvested and subjected to cell count, FACS analysis, and real-time PCR analysis for OX40 and OX40L transcript. In vitro T cell survival was determined by trypan blue exclusion, and the calculation for percentage of survival was based on the input number of cells.

Coculture and Transwell experiments

CD4+ T cells from OT-II OX40-KO mice were stimulated with OVA peptide in the presence of irradiated (3000 rad) OX40L-KO splenocytes (APCs) for 48 h. BW5147 cells, a mouse thymoma cell line, or the BW-mOX40 cells were added directly to the cell culture. Alternatively, to inhibit cell-cell contact, BW5147 or BW-mOX40 cells were placed in a 0.2-µm Anopore Membrane Nunc Tissue Culture Insert (Nalge Nunc International) and cultured in the presence of OX40L-expressing OT-II T cells. Twenty-four hours after the beginning of the coculture, OX40L expression on V{alpha}2+Vbeta5.1-2+ cells was examined with a flow cytometer. To inhibit receptor-mediated ligand internalization, 10 mM NaN3 was added into the coculture. OX40-expressing primary T cells were also used instead of cell lines. To prepare OX40-expressing primary T cells, Ly-5.2+ OX40L-KO OT-II T CD4+ cells were stimulated with OVA peptide in the presence of irradiated (3000 rad) OX40L-KO splenocytes (APCs) for 48 h. Activated Ly-5.1+ OX40-KO OT-II CD4+ T cells and Ly-5.2+ OX40L-KO OT-II T CD4+, which can express OX40L and OX40, respectively, were cocultured in medium alone for 24 h. OX40L expression on Ly-5.1+V{alpha}2+ cells was estimated by flow cytometry. To separate OX40L-expressing activated OX40-KO OT-II cells from OX40-expressing activated OX40L-KO OT-II cells, the latter cells were placed into a 0.2-µm Anopore Membrane Nunc Tissue Culture Insert.

Adoptive transfer experiments

Purified naive CD4+ T cells (2.5–10 x 106) from the spleen and lymph nodes of OT-II or OT-II OX40L KO mice were injected i.v. into the tail vein of untreated or sublethally irradiated (500 cGy, 137Cs source) congenic wild-type or OX40L-deficient recipient mice. In some cases, purified CD4+ T cells were labeled with CFSE and then transferred to recipient mice. One day later, the mice were immunized with 2 mg of OVA protein (Worthington Biochemical) plus 50 µg of LPS (Sigma-Aldrich) or 50 µg of OVA323–339 emulsified in CFA. The mice were sacrificed at the indicated times. To examine the endogenous expression of OX40L transcripts in vivo, OT-II T cells (1 x 107) were adoptively transferred into congenic wild-type recipient mice. One day later, the recipient mice were immunized, as described above. After the indicated time points, activated donor T cells were isolated magnetically from the spleen and peripheral lymph nodes of recipient mice using biotinylated anti-Ly-5.2 mAb, followed by anti-biotin MicroBeads (Miltenyi Biotec), and analyzed for OX40L expression. To show ex vivo expression of OX40L on T cells, OT-II and OT-II OX40-KO T cells (5 x 106) were adoptively transferred into congenic wild-type and OX40-deficient recipient mice, which then were immunized the next day, as described above. After the indicated time point, surface expression of OX40 and OX40L was examined on activated donor T cells.

Quantitative real-time PCR

A single-cell suspension of purified CD4+ T cells was prepared and stimulated, as described above. At different time points, T cells were collected from the culture, and their total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies). In a T cell transfer experiment, activated donor T cells were purified from recipient mice using a congenic marker (Ly-5). Total RNA was extracted from the purified donor T cells using TRIzol reagent. Single-strand cDNA was prepared by reverse transcribing 5 µg of total RNA using the SuperScript III kit (Invitrogen Life Technologies). Quantitative real-time PCR was conducted using TaqMan gene expression assays (Applied Biosystems; OX40; Mm00442039, OX40L; Mm00437214, beta-actin; Mm00607939), according to the manufacturer’s instructions. PCR thermal cycling conditions were 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min in a total volume of 25 µl/reaction. Data were collected using an 7500 Real Time PCR System and 7500 System Software (Applied Biosystems). All samples were run in triplicate, and the mean values were used for quantification. The expression level of each gene was normalized to the copies of beta-actin mRNA from the same sample. Standard curves for OX40 and OX40L were generated using seven serial dilutions (1/10, 1/102, 1/103, 1/104, 1/105, 1/106, and 1/107) of cDNA from stimulated CD4 T cells and cDNA from whole splenocyte of OX40L transgenic mice, respectively (linear regression R > 0.99). Standard curves for beta-actin were created from same cDNA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Induction of endogenous OX40L expression by T cells upon Ag stimulation

To address whether the expression of OX40L on activated T cells is endogenous or derived from APCs, wild-type T cells were stimulated with PMA/ionomycin or anti-CD3. In a parallel experiment, OT-II CD4+ T cells were stimulated with the OVA peptide in the presence of OX40L-deficient APCs, which excluded the potential for the T cells’ acquisition of OX40L molecules from the APCs. Three days after stimulation, the up-regulation of OX40L expression as well as of OX40 expression was observed, implying that the OX40L expressed on the activated T cells was not derived from the APCs (Fig. 1, A and B, upper panels). We next analyzed the endogenous expression of the OX40L transcript by RT-PCR. Significant induction of OX40 and OX40L transcripts was observed 24 h after stimulation, and the expression of both molecules was down-regulated after 72 h (Fig. 1, A and B, lower panels). The induction of OX40L gene expression in activated wild-type and OT-II T cells also confirmed that the OX40L that was up-regulated upon activation is endogenous.


Figure 1
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  		FIGURE 1. Expression of OX40 and OX40L on activated CD4 T cells. A and B, upper panels, Purified CD4+ T cells (5 x 104) from wild-type mice were cultured in medium alone, and stimulated with either anti-CD3 (10 µg/ml) or PMA/ionomycin in the presence of irradiated OX40L-KO splenocytes (APCs) (2 x 105). In a parallel study, purified CD4+ T cells (5 x 104) from OT-II TCR transgenic mice were cultured in medium alone or stimulated with OVA peptide (0.1 µM) in the presence of OX40L-KO APCs. Three days after stimulation, the OX40 and OX40L expression on CD4+ cells was examined by flow cytometry (in the case of OT-II T cells, the gated CD4+V{alpha}2+ T cells are shown). Filled histograms represent control staining. A and B, lower panels, Kinetics of OX40 and OX40L mRNA expression. Total RNA was extracted from purified wild-type (A) or OT-II (B) T cells, which had been stimulated with either PMA/ionomycin or OVA peptide at the indicated times. The relative OX40 and OX40L expression was quantified using real-time PCR, and the OX40 and OX40L transcripts were normalized to the copies of beta-actin mRNA from the same sample. The relative expression is expressed as the mean (±SD) of triplicate PCRs. Similar results were obtained in several independent experiments.

 
Down-regulation of surface OX40L expression by OX40 through cell-cell contact

Pippig et al. (16) observed a higher surface expression of OX40L on activated OX40-KO T cells than on wild-type T cells. We found a similar increase in the expression of the OX40L protein on activated OX40-KO T cells (Fig. 2A). Upon evaluating the transcription levels of the OX40L gene, however, we found no significant difference between the OX40-KO and wild-type T cells (Fig. 2B). These results suggest that the surface expression of OX40L on T cells may normally be down-regulated by OX40 via a posttranscriptional mechanism. To determine whether the presence of OX40 can suppress the expression of OX40L on adjacent cells, OX40L-expressing CD4 T cells (OX40-KO OT-II cells previously stimulated with OVA) were cocultured with either an OX40-expressing T cell line (BW-mOX40) or the control parental cell line (BW5147) (Fig. 2C), and the surface expression of OX40L on the OX40-KO OT-II cells was monitored. Coculture with the BW-mOX40 cells markedly reduced the OX40L expression on activated OX40-KO OT-II cells, and the BW5147 parental cell lines did not affect the surface expression of OX40L on the OX40-KO OT-II cells (Fig. 2C). The reduced OX40L expression was not inhibited by azide treatment, which can inhibit ATP-dependent receptor internalization. Therefore, receptor internalization machinery may not be involved in the down-regulation of surface OX40L expression. Separating the OX40L-expressing T cells and OX40-expressing cells (BW-mOX40) with a Transwell culture system completely abrogated the reduction in OX40L expression, suggesting that cell-cell contact is required for the OX40 molecule to block OX40L expression (Fig. 2C). To further investigate whether OX40 expression by primary T cells could also suppress the OX40L expression on adjacent T cells, we cocultured Ly-5.1+ OX40-KO OT-II T cells with Ly-5.2+ OX40L-KO OT-II T cells. In this culture, OX40L was expressed by the OX40-KO T cells only, and OX40 was expressed by the OX40L-KO T cells only. Twenty-four hours after beginning the coculture, the expression level of OX40L on the activated OX40-KO T cells was markedly reduced (Fig. 2D). Separation of the two T cell populations abolished the suppression of OX40L expression by the OX40-expressing T cells (Fig. 2D). Furthermore, the reduction in OX40L expression in the presence of the OX40 receptor raises the possibility that the binding of OX40 to OX40L may reduce staining by the anti-OX40L mAb, which is inhibitory for the OX40-OX40L interaction. To address this question, we washed out cell surface of activated OT-II cells with an acid buffer (pH 3.0), which can abolish the binding between receptor and ligand (35), and then stained the cells with anti-OX40L mAb. However, the acid treatment did not increase the OX40L staining (data not shown). Collectively, these results provide clear evidence that the presence of the OX40 molecule can down-regulate OX40L expression through T cell-T cell contact after Ag stimulation.


Figure 2
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  		FIGURE 2. Suppression of OX40L surface expression on T cells in the presence of the OX40 receptor. A, Purified CD4+ T cells (5 x 104) from wild-type or OX40-KO mice were stimulated with anti-CD3 (10 µg/ml) in the presence of irradiated OX40L-KO splenocytes (APCs) (2 x 105). Alternatively, purified CD4+ T cells (5 x 104) from OT-II mice or OX40-KO OT-II mice were stimulated with OVA peptide (0.1 µM) in the presence of irradiated OX40L-KO splenocytes (APCs) (2 x 105). Filled histograms represent control staining. B, CD4+ T cells from OT-II ({blacksquare}) and OT-II OX40-KO ({cjs2108}) mice express similar levels of OX40L mRNA upon in vitro Ag stimulation. Total RNA was extracted from purified CD4+ T cells, which had been stimulated with OVA peptide for the indicated time, and the relative OX40L expression was quantified, as described in Fig. 1B. The relative expression of OX40L transcript is expressed as the mean (±SD) of triplicate PCRs. Similar results were obtained in three independent experiments. C and D, The presence of OX40 suppresses OX40L expression through cell-to-cell contact. C, Purified OT-II OX40-KO CD4+ T cells (5 x 104) were stimulated with OVA peptide in the presence of OX40L-deficient APCs for 48 h. OX40L-expressing T cells (OX40-KO OT-II, 5 x 104) were collected and cultured alone, in the presence of parental BW5147 (BW) cell line (5 x 103), with the BW5147 cells transfected with mouse OX40 (BW-mOX40) (5 x 103), for 24 h. OX40L expression on V{alpha}2+Vbeta5.1-2+ gated T cells was examined using flow cytometry. To block receptor-mediated ligand internalization, 10 mM azide was added into the coculture. To inhibit cell-cell contact, BW-mOX40- and OX40L-expressing T cells were cultured separately in a Transwell plate. Filled histograms represent control staining. Solid lines represent OX40 and OX40L expression on BW and OX40-KO T cells, respectively. D, OX40-KO Ly-5.1+ OT-II T cells and OX40L-KO Ly-5.2+ OT-II T cells were independently stimulated with OVA peptide in the presence of OX40L-deficient APCs for 48 h. OX40L-expressing cells (OX40-KO Ly-5.1+ OT-II, 5 x 104) were collected and cultured either alone or in the presence of OX40-expressing cells (OX40L-KO Ly-5.2+ OT-II, 5 x 104) for 24 h. OX40L expression on Ly-5.1+V{alpha}2+ gated T cells was examined by flow cytometry. To inhibit cell-cell contact, OT-II OX40-KO Ly-5.1+ and OT-II OX40L-KO Ly-5.2+ were separated using a Transwell plate. Solid lines represent the OX40 and OX40L expression on activated OX40L-KO and OX40-KO T cells, respectively. Filled histograms represent control staining.

 
Sustained OX40 and OX40L expression on long-lived T cells

Previous studies showed that, several days after Ag stimulation, CD4 T cells and activated APCs down-regulate the expression of OX40 and OX40L, respectively (6, 9, 39). To determine whether the expression levels of OX40 and OX40L were maintained on long-lived T cells, OT-II and OX40-KO OT-II T cells were stimulated with OVA in the presence of OX40L-deficient APCs, and the OX40 and OX40L expression levels on T cells were evaluated. For 14 days, OT-II T cells expressed significant amounts of OX40 and OX40L mRNA (Fig. 3A). Although flow cytometric analysis showed quite low expression of OX40L on the OT-II T cells, OX40L expression on 30% of long-lived OT-II OX40-KO T cells was clearly seen (Fig. 3B). Because OT-II and OT-II OX40-KO T cells expressed comparable levels of OX40L transcripts (Fig. 3A, lower panel), the reduced surface OX40L expression on wild-type OT-II T cells may be due to the presence of the OX40 molecule. In addition, during the long-term culture, live OX40L-expressing cell number was also increased in OX40-deficient T cells (Fig. 3C). These data suggest that CD4 effector T cells can maintain the expression of both OX40 and OX40L after Ag priming.


Figure 3
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  		FIGURE 3. Sustained expression of OX40 and OX40L by activated T cells. A, upper panel, Relative expression of the OX40 transcript in activated OT-II cells. Purified CD4+ T cells from OT-II mice were stimulated with the OVA peptide (0.1 µM) in the presence of irradiated OX40L-KO splenocytes (APCs) for the indicated time. Total RNA from the stimulated OT-II CD4+ T cells was subjected to a quantitative real-time PCR for the OX40 transcript, as described in Fig. 1B. The relative expression is expressed as the mean (±SD) of triplicate PCRs. A, lower panel, Relative expression of the OX40L transcript in activated OT-II ({blacksquare}) and OT-II OX40-KO T cells ({cjs2108}). Purified CD4+ T cells from OT-II or OX40-KO OT-II mice were stimulated with the OVA (0.1 µM) peptide in the presence of irradiated OX40L-KO splenocytes (APCs) for the indicated time, and total RNA from the stimulated T cells was subjected to a quantitative real-time PCR to amplify the OX40L transcript, as described in Fig. 1B. The relative expression is expressed as the mean (±SD) of triplicate PCRs. Similar results were obtained in three independent experiments. B, Purified CD4+ T cells (2 x 106) from OT-II and OX40-KO OT-II mice were stimulated with the OVA peptide (0.1 µM) in the presence of irradiated OX40L-KO splenocytes (APCs) (1 x 107) in six-well plates. Fifteen days after the stimulation, OX40 and OX40L expression on CD4+V{alpha}2+ T cells was examined. Solid lines represent the OX40 (upper) and OX40L (lower) expression on OT-II (left) or OX40-KO OT-II (right) T cells. Filled histograms represent control staining. C, Absolute number of total CD4 T cells (upper) and OX40 (middle)- and OX40L (lower)-expressing CD4 T cells, 15 days after Ag stimulation. In these figures, data are the average (± SD) of triplicate cultures, and similar results were obtained in at least three independent experiments.

 
OX40L expression on T cells contributes to T cell proliferation after antigenic stimulation

We next determined whether the expression of OX40L on T cells plays a functional role in immune responses. For this purpose, purified CD4+ T cells from wild-type and OX40L-KO mice were stimulated with anti-CD3 in the presence of wild-type or OX40L-deficient APCs, and T cell proliferation was monitored. As shown in Fig. 4A, the absence of OX40L on either the T cells or APCs resulted in a significant reduction in T cell proliferation. Indeed, the presence of OX40L on both T cells and APCs is crucial for the optimal proliferation of CD4 T cells. Moreover, during the stimulation of CD4 T cells with plate-bound anti-CD3 (in the absence of APCs), the addition of an inhibitory anti-OX40L mAb suppressed the proliferation of wild-type T cells to the same level as OX40L-KO T cells (Fig. 4B). Because addition of anti-OX40L mAb (MGP34) to OX40-deficient T cells, which can express OX40L, showed no effect on T cell proliferation (Fig. 4B), cross-linking of OX40L on T cells cannot deliver any costimulatory or suppressive signals. Furthermore, the suppressive effect by the anti-OX40L mAb was abrogated by adding an agonistic anti-OX40 mAb (Fig. 4C), again suggesting that OX40 signals, but not OX40L signals, in T cells are responsible for CD4 T cell proliferation.


Figure 4
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  		FIGURE 4. OX40L expression on T cells is required for the optimal proliferation of Ag-specific T cells. A, Purified CD4+ T cells (5 x 104) from wild-type or OX40L-KO mice were stimulated with soluble anti-CD3 mAb (10 µg/ml) in the presence of irradiated wild-type or OX40L-KO splenocytes (APCs) (2 x 105). At the indicated days, [3H]thymidine incorporation during the last 8 h of cultures was measured as an indicator of cell proliferation and is expressed as the mean (±SD) of triplicate cultures. Similar results were obtained in at least three independent experiments. B and C, Purified CD4+ T cells (1 x 105) from wild-type ({blacksquare}), OX40L-KO ({square}), and OX40-KO ({cjs2108}) mice were stimulated with plate-bound anti-CD3 mAb (5 µg/ml) in the absence of APCs. An inhibitory anti-OX40L mAb (MGP34; 20 µg/ml), control rat IgG (cont IgG; 20 µg/ml), or anti-OX40L mAb, plus an agonistic anti-OX40 mAb (MGP34 and OX86; 10 µg/ml each) were added, as indicated. [3H]Thymidine incorporation during the last 8 h (Figure legend continues) of the 4-day cultures was measured as an indicator of cell proliferation and is expressed as the mean (±SD) of triplicate cultures. Similar results were obtained in at least four independent experiments. D, Purified CD4+ T cells (5 x 104) from OT-II ({blacksquare}), OT-II OX40L-KO ({triangleup}), and OT-II OX40-KO ({square}) mice were stimulated by irradiated OX40L-sufficient (wild-type) splenocytes (2 x 105) that were pulsed with 0.5 µM OVA peptide. At the different time points, [3H]thymidine incorporation during the last 8 h of cultures was measured as an indicator of cell proliferation and is expressed as the mean (±SD) of triplicate cultures. Similar results were observed in three independent experiments. E and F, Purified CD4+ T cells (5 x 104) from OT-II ({blacksquare}), OT-II OX40-KO ({square}), and OT-II OX40L-KO ({triangleup}) mice, stimulated by either irradiated OX40L-deficient (E) or OX40L-sufficient (wild-type) (F) splenocytes (2 x 105) that were pulsed with various concentrations of OVA peptide [3H]thymidine incorporation during the last 8 h of the 5-day cultures, were measured as an indicator of cell proliferation and expressed as the mean (±SD) of triplicate cultures. Similar results were observed in three independent experiments. G, Purified OT-II, OT-II OX40L-KO, and OT-II OX40-KO CD4+ T cells were labeled with CFSE and stimulated by OVA-pulsed wild-type APCs. Ninety-six hours after stimulation, CFSE intensity of V{alpha}2+ CD4+ T cells was estimated by flow cytometry. Upper panels, Show representatives of cell division patterns in three independent experiments. Lower panels, Demonstrate the mean ± SD of three independent experiments.

 
Previous reports demonstrated that in contrast to stimulation with anti-CD3, when TCR transgenic T cells are used, OX40 signals combined with Ag stimulation promote late cell division, rather than the induction of early cell proliferation (11, 40). Therefore, in addition to CD3 stimulation, we examined whether OX40L expression by T cells may also contribute to the late proliferation of OT-II T cells after Ag priming. OT-II OX40L-KO T cells that had received OX40 signals from OX40L-sufficient APCs proliferated similarly to OT-II T cells at day 3 of stimulation (Fig. 4D); however, they still showed less count of [3H]TdR compared with OT-II T cells at the later phase as well as OT-II OX40-KO T cells (Fig. 4D). This implies that OX40L on T cells contributes to late T cell proliferation or survival of T cells. Indeed, 5 days after stimulation in the presence of OX40L-deficient APCs (Fig. 4E), OT-II OX40-KO and OT-II OX40L-KO T cells showed a similar reduction in their proliferation, and this was most prominent when a low dose of OVA peptide was used. However, OT-II OX40L-KO T cells that received OX40 signals from OX40L-sufficient APCs proliferated more efficiently than OT-II OX40-KO T cells, but still showed impaired proliferation compared with wild-type T cells (Fig. 4F). Analysis of cell division using CFSE also showed slight impairment in cell proliferation of OT-II OX40-KO and OT-II OX40L-KO T cells (Fig. 4G). These results support the conclusion that OX40L on T cells, as well as OX40L on APCs, can provide potent costimulatory signals to regulate the proliferative response of CD4 T cells.

Optimal T cell survival is dependent on the OX40L expression on T cells after Ag priming

The reduction in late T cell proliferation seen in OX40L-deficient T cells (Fig. 4D) may reflect impaired T cell survival due to decreased OX40 signals. To address whether long-term T cell survival is directly controlled by OX40L on T cells, OT-II, OT-II OX40-KO, and OT-II OX40L-KO T cells were stimulated by OVA-loaded OX40L-deficient or OX40L-sufficient wild-type APCs, and the cells’ viability was monitored. OT-II OX40L-KO and OT-II OX40-KO T cells stimulated in the presence of OX40L-deficient APCs, which could not provide any OX40 signals, showed a marked reduction in survival during the 15 days of Ag stimulation (Fig. 5A). In the presence of OX40L-expressing APCs, OT-II OX40L-KO T cells, which are capable of receiving the OX40 signal only through APCs, showed a 85% reduction in their ability to survive compared with OT-II T cells 15 days after Ag stimulation (Fig. 5B, right panel). Similarly to the proliferation assay, survival analysis reveals that OX40L on T cell has a great impact on accumulation of T cells during the contraction phase. Furthermore, adding an inhibitory anti-OX40L mAb, which can bind to OX40L on T cells only when OX40L-KO APCs are used, dramatically suppressed the T cell survival during the 15 days of culture (Fig. 5C). Similarly, the suppressive effect of the OX40L-blocking mAb was rescued by cross-linking the OX40 receptor on T cells when agonistic anti-OX40 mAb was added (Fig. 5C). The reduction in T cell survival due to the absence of functional OX40L on T cells raises the question of whether the OX40-OX40L interaction on T cells imprints the cells for survival during early Ag priming or directly promotes cell survival during the late effector phase. To address this question, OT-II T cells were stimulated by OVA-loaded OX40L-deficient or OX40L-sufficient APCs, and T cell survival was analyzed 10 days poststimulation. To block the OX40-OX40L interaction, the anti-OX40L mAb was added for the initial 3 days, the last 7 days, or for all 10 days. As shown in Fig. 5D, blocking the OX40-OX40L interaction on T cells during either the early or later phase significantly diminished the survival of the CD4 T cells, while adding it for the entire 10 days had the most suppressive effect on T cell survival. Thus, OX40-OX40L interactions on T cells during both early and later phases of Ag priming contribute to the long-term survival of Ag-specific CD4 T cells. Collectively, these findings emphasize the functional importance of the OX40-OX40L interaction during T cell-T cell contact, in that this interaction supports long-term CD4 T cell survival.


Figure 5
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  		FIGURE 5. OX40L expressed by T cells contributes to the long-term survival of CD4 T cells. A and B, Purified CD4+ T cells (2 x 106) from wild-type OT-II ({blacksquare}), OX40-KO OT-II ({square}), and OX40L-KO-OT-II ({triangleup}) mice were activated with OVA-pulsed OX40L-deficient (A) or OX40L-sufficient (wild-type) (B) APCs (1 x 107) in a six-well plate over 15 days. T cell recovery (left panels) and T cell survival (right panels) at the indicated days were assessed by excluding trypan blue-positive cells, and the calculation of the percentage of recovery was based on the input number of cells. Percentage of survival rate was calculated based on ratio (live cell number at the indicated day)/((peak) live cell number at day 3) x 100. In these figures, data are the average (±SD) of triplicate cultures, and similar results were obtained in at least three independent experiments. C, Purified wild-type OT-II CD4+ T cells (2 x 106) were stimulated with OVA-pulsed irradiated OX40L-KO APCs (1 x 107) in the presence of control rat IgG ({blacksquare}; Cont IgG, 20 µg/ml), inhibitory anti-OX40L mAb ({square}; MGP34, 20 µg/ml), or MGP34 plus agonistic anti-OX40 mAb (OX86) ({triangleup}; 10 µg/ml each), and cultured for 15 days. T cell recovery and survival at the indicated days were examined, as described. D, OT-II CD4+ T cells (1 x 105) were stimulated by OVA-loaded OX40L-deficient ({square}) or OX40L-sufficient (wild-type) ({blacksquare}) APCs (4 x 105) over 10 days. Inhibitory anti-OX40L mAb (MGP34; 20 µg/ml) or control rat IgG (20 µg/ml) was added for 3 days starting at the beginning of the culture period. After being washed with PBS, cells were resuspended in complete medium and cultured an additional 7 days in the presence of either control IgG or MGP 34. Ten days later, the percentage of survival was calculated. Similar results were obtained in two independent experiments.

 
OX40L expression on T cells promotes long-term survival of CD4 T cells in vivo

To evaluate the in vivo role of OX40L expression on T cells, we first examined the expression of OX40L on activated OT-II T cells in vivo. To achieve this, Ly-5.2+ OT-II cells were transferred into wild-type non-OT-II recipient mice (Ly-5.1+), followed by immunization of the recipient mice with OVA/LPS. One and 2 days postimmunization, donor T cells were sorted magnetically from the spleen and lymph nodes of the recipient mice, and the expression level of the OX40L transcript in the donor T cells was determined by real-time PCR. Although the OX40L transcript was not detected in naive OT-II T cells, a significant induction of OX40L was observed following the OVA immunization (Fig. 6A). Although the induction of OX40L mRNA was readily detectable, we did not detect any surface expression of OX40L on the activated donor T cells by flow cytometry. We suspect that, as in our in vitro experiments (Fig. 2), the absence of OX40L on the surface of donor T cells may be due to the presence of OX40 expression on donor T cells and the adjacent cells of the recipient mice. To address this, Ly-5.1+ OT-II and OT-II OX40-KO CD4 T cells were transferred into congenic Ly-5.2+ wild-type and OX40-KO recipient mice, followed by Ag immunization. Three days after immunization, in contrast to negligible OX40L expression on OT-II cells that had been transferred to wild-type recipient mice, OX40L expression on OT-II OX40-KO donor T cells in OX40-KO recipient mice was clearly seen (Fig. 6B). These results indicate that the presence of OX40 down-regulates OX40L expression on T cells in vivo.


Figure 6
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  		FIGURE 6. OX40L on donor T cells supports their survival after Ag stimulation in vivo. A, Purified CD4+ T cells (1 x 107) from Ly-5.2+ OT-II TCR transgenic mice were transferred into wild-type (non-OT-II) Ly-5.1+ mice. Twenty-four hours later, each recipient mouse was immunized i.v. with 2 mg of OVA plus 50 µg of LPS. At the indicated time after immunization, the donor Ly-5.2+ T cells were magnetically isolated (Figure legend continues) (purity, >90%) from the spleen of recipient mice, and the total RNA was extracted from the purified donor T cells. Relative OX40L expression was quantified using real-time PCR, as described in Fig. 1B. B, Purified CD4+ T cells (1 x 107) from Ly-5.1+ OT-II and OT-II OX40-KO mice were transferred into congenic (Ly-5.2+) wild-type or OX40-KO mice. Twenty-four hours later, each recipient mouse was immunized i.p. with 50 µg of OVA peptide emulsified in CFA. Three days after immunization, the OX40 and OX40L expression on donor Ly-5.1+ T cells was examined by flow cytometry. Filled histograms represent control staining. C, Purified CD4+ T cells (5 x 106) from Ly-5.2+ OT-II or Ly-5.2+ OX40L-KO OT-II mice were labeled with CFSE and transferred into wild-type Ly-5.1+ mice, and recipient mice were then immunized with OVA plus LPS. Splenocytes were harvested 4 days after immunization, and dilution of CFSE fluorescence intensity of donor T cells was analyzed by FACS. Representative results of two independent experiments were shown. D, Purified CD4+ T cells (1 x 107) from Ly-5.2+ OT-II or Ly-5.2+ OX40L-KO OT-II mice were transferred into wild-type Ly-5.1+ mice, and recipient mice were then immunized with OVA plus LPS. Spleen cells of the recipient mice were harvested 1, 4, and 10 days after Ag administration; stained for Ly-5.2, TCR-V{alpha}2, and TCR-Vbeta5.1; and analyzed by flow cytometry. The absolute number of donor T cells in the spleen of recipient mice (upper) and survival rate of donor T cells (lower) are shown. Percentage of survival rate was calculated based on ratio (donor T cell number at day 10)/(donor T cell number at day 4) x 100. Results represent the mean ± SD from three mice per group, and are representative of two independent experiments. The significance of the data was evaluated by Student’s t test (*, represents p < 0.01). E, Purified CD4+ T cells (2.5 x 106) from Ly-5.1+ OT-II ({blacksquare}) or OX40L-KO OT-II ({square}) mice were transferred to sublethally irradiated (500 cGy) congenic (Ly-5.2) wild-type or OX40L-deficient mice. One day later, recipient mice were immunized with OVA peptide emulsified in CFA. Spleens (upper panel) and peripheral (inguinal and mesenteric) lymph nodes (pLNs, lower panel) were extracted 4 wk after immunization, and the cells were stained for Ly-5.1+, TCR-V{alpha}2, and TCR-Vbeta5.1. The number of surviving donor (Ly-5.1+ TCR-V{alpha}2+ TCR-Vbeta5.1+) T cells was calculated by microscopic cell count and flow cytometric analysis. Results represent the mean ± SD from five mice per group. The significance of the data was evaluated by Student’s t test (* and **, represent p < 0.01 and p < 0.001, respectively).

 
Next, to investigate the in vivo role of OX40L on T cell expansion, OT-II or OX40L-KO OT-II cells (Ly-5.2+) were adoptively transferred into wild-type recipient mice (Ly-5.1+), followed by the immunization of recipient mice with OVA/LPS. In a CFSE analysis, 4 days after transfer, in vivo cell division was similar in OT-II OX40L-KO T cells and OT-II T cells (Fig. 6C). Again, similarly to our in vitro data (Fig. 5B), OX40L-KO OT-II T cells, which can receive OX40 signals from OX40L expressed by recipient APCs, showed an impaired ability to survive for 10 days after stimulation (Fig. 6D). Collectively, OX40L on T cells contributes to T cell survival rather than early T cell expansion.

Finally, we performed an additional adoptive transfer experiment, in which OT-II or OX40L-KO OT-II T cells were transferred into sublethally irradiated congenic wild-type and OX40L-deficient mice. We then immunized the recipient mice with OVA emulsified in CFA. Four weeks after the immunization, OT-II OX40L-KO donor T cells showed significantly reduced numbers compared with OT-II donor T cells in both spleens and peripheral lymph nodes of recipient mice (Fig. 6E). Similarly, the absence of OX40L in recipient mice also appeared to suppress the survival of transferred T cells (Fig. 6E). Indeed, the absence of OX40L both on the donor T cells and in the recipient mice had the most suppressive effect on donor T cell survival. All together, these results provide conclusive evidence that the OX40L on T cells, as well as the OX40L on APCs, contributes to the longevity of Ag-specific CD4 T cells.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we document a precise analysis of the expression and biological function of OX40L expressed on T cells. Using several conventional approaches, we show for the first time that activated CD4 T cells can intrinsically express OX40L both in vitro and in vivo. Although OX40L can also be transferred from OX40L-expressing cells to CD4 T cells (35), our results using OX40L-deficient APCs and recipient mice verified the endogenous expression of OX40L by T cells. We also demonstrated that the presence of the OX40 receptor on the same cell or adjacent cells suppresses the surface expression of OX40L on T cells. Several previous studies have described the expression of OX40L on activated T cells, such as Th1 cells and several CTL clones (18, 34). These studies, however, did not confirm whether the OX40L transcript was present in these T cells. Flow cytometric analysis by Kim et al. (34) demonstrated OX40L expression on Th1, but not Th2 cells. However, the OX40L-expressing Th1 cells had much lower OX40 expression than the Th2 cells, which had barely detectable levels of OX40L. Similarly, Fig. 1A demonstrates that PMA/ionomycin-treated T cells with higher OX40 expression level expressed less OX40L expression as compared with anti-CD3-treated T cells, which had lower level of OX40 expression. Based on our observations, we suggest that the insufficient expression of OX40 on Th1 cells and several CTL clones may have failed to suppress the surface OX40L expression, thus making it possible to detect OX40L on the Th1 cells. Further studies will be required to address the biological significance of the preferential OX40L expression on Th1 cells and CTL clones.

Regarding roles of OX40-OX40L interaction in CD4 T cell activation, when using polyclonal stimulation with anti-CD3 mAb (Fig. 4A), blockade of OX40 signals strongly suppressed early T cell proliferation (Fig. 4A). In contrast, upon stimulation with Ag peptide to naive OT-II T cells, OX40 signals are dispensable for early proliferation of T cells, but essential for T cell survival, as shown in Fig. 4D and previous reports (11, 34, 40, 41). We speculate that stronger TCR signals that may occur in TCR transgenic T cells upon Ag peptide stimulation might be enough for optimal early T cell proliferation even in the absence of OX40 signals. To clarify the precise mechanisms for these observations, further studies will be required.

The presence of OX40L on T cells encouraged us to undertake a more comprehensive analysis of its role in T cell responses. Accumulating evidence has demonstrated that OX40 signals control the frequency of effector CD4 T cells in late primary immune responses by promoting the survival and clonal expansion of T cells (7, 10, 11, 41). The present study has revealed that OX40L on T cells plays a major role in the survival of CD4 T cells that is associated with a significant increase in CD4 T cell proliferation. Given the findings of two previous papers that showed signals through OX40L induce c-fos expression and RANTES production in epithelial cell lines and endothelial cells, respectively (42, 43), it is tempting to speculate that downstream OX40L signals independently promote the proliferation and survival of T cells. However, our results indicated that the OX40 signals rather than the OX40L signals are critical for T cell survival (Fig. 4, B and C). In addition, we found that cross-linking OX40L on T cells with a recombinant soluble OX40 or anti-OX40L mAb did not promote T cell proliferation, but rather suppressed it (data not shown). These results strongly suggest that signals through OX40, but not OX40L, are essential for T cell proliferation and survival. Collectively, these data indicate that OX40L on T cells can initiate OX40 signals in adjacent T cells through direct T cell-T cell contact.

Analyzing CD4 T cell survival in vivo by transferring CD4+ T cells into sublethally irradiated OX40L-sufficient or -deficient hosts clearly showed that OX40L expression is required on donor T cells for full-fledged T cell longevity (Fig. 6E). One previous report, however, demonstrated that adoptively transferred allogeneic OX40L-deficient T cells can successfully induce GVHD in OX40L-sufficient recipient mice in a pattern similar to that of wild-type T cells. By contrast, OX40L-KO recipient mice significantly suppressed the lethality of the GVHD following transfer of allogeneic wild-type T cells (23). These findings indicate that OX40 signals provided only by recipient APCs may be sufficient to induce graft-vs-host responses. Therefore, we speculate that the OX40L on T cells and APCs is used selectively in different types of immune responses. In contrast to little effect of OX40L deficiency on T cell in the GVHD model, deliberate OX40L expression on T cells in OX40L transgenic mice, which were constructed by using the mouse lck proximal promoter, demonstrated significant immunological abnormalities, such as production of autoantibodies, a marked increase in CD4+ effector memory (CD44+CD62Llow) T cell population, and spontaneous development of interstitial pneumonia and inflammatory bowel disease (44). Because excessive OX40 signals by using agonistic anti-OX40 mAb can break CD4 T cell tolerance (24, 45), deliberate OX40L expression on T cells in OX40L transgenic mice may be enough to induce autoimmunity.

We and others have demonstrated that OX40L expressed by APCs, such as DCs and activated B cells, can initiate OX40 signals in activated T cells (8, 9). Thus, the OX40-OX40L interaction has been thought to occur mainly during T cell-APC interaction. However, OX40 expression on naive CD4 T cells peaks 2–3 days after Ag stimulation (6) and continues at least up to 15 days after the stimulation (Fig. 3B). Recently, intravital two-photon imaging of CD4 T cell behavior in the lymph nodes showed that the naive CD4 T cell-DC interaction lasts 24–48 h after Ag priming (46, 47), and about one-half of the Ag-specific T cells leave DCs near high endothelial venules and move into the T cell area in the deep paracortex of lymph nodes (48). These findings support the idea that OX40L expressed by cells different from DCs may also be involved in OX40-OX40L interactions. Our in vitro results (Fig. 2D) demonstrate that OX40L on T cells binds to OX40 on T cells through T cell-T cell contact. In addition, the in vivo results shown in Fig. 6 suggest functional roles for OX40-OX40L binding during T cell-T cell interactions in T cell survival. Although previous studies suggest that OX40L on CD4+CD3– accessory cells (49) and B cells (50) can costimulate OX40+ CD4 T cells, the in vivo roles for the OX40L expression on these cells in supporting CD4 T cell survival are unclear. We have hypothesized that the accumulation of Ag-experienced T cells in the T cell zone of the secondary lymphoid organs may be conducted by OX40-OX40L interactions through direct T cell-T cell cross talk. Moreover, the sustained expression levels of OX40 and OX40L on activated T cells (Fig. 3B) suggest that, after the T cells and APCs (DCs) separate, T cells may still support OX40 signals in an autocrine or paracrine manner. Considering all of the available evidence, we conclude that OX40L expressed by CD4 T cells can provide autonomous OX40 signals through T cell-T cell interactions that contribute to the longevity of Ag-specific CD4 T cells and lead to the generation and survival of CD4 memory T cells.


   Acknowledgments
 
We thank Dr. Ndhlovu for the critical reading and Dr. Murata for the excellent technique.


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


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

1 This work was supported in part by a grant-in-aid for scientific research on priority areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and a grant-in-aid for scientific research on priority areas from the Japan Society for the Promotion of Science. Back

2 Address correspondence and reprint requests to Dr. Naoto Ishii, Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 Japan. E-mail address: ishiin{at}mail.tains.tohoku.ac.jp Back

3 Abbreviations used in this paper: OX40L, OX40 ligand; DC, dendritic cell; GVHD, graft-vs-host disease; KO, knockout. Back

Received for publication September 14, 2005. Accepted for publication February 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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