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OX40 Ligation of CD4⁺ T Cells Enhances Virus-Specific CD8⁺ T Cell Memory Responses Independently of IL-2 and CD4⁺ T Regulatory Cell Inhibition¹

Qigui Yu^*, Feng Yun Yue^*, Xiao X. Gu^*, Herbert Schwartz, Colin M. Kovacs and Mario A. Ostrowski^2,*,

^* Clinical Sciences Division, University of Toronto, Toronto, Ontario, Canada; St. Michael’s Hospital, University of Toronto, Toronto, Ontario, Canada; Canadian Immunodeficiency Research Collaborative, Toronto, Ontario, Canada; and Department of Physiology, National University of Singapore, Singapore

	Abstract

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We have previously shown that CD4⁺ T cells are required to optimally expand viral-specific memory CD8⁺ CTL responses using a human dendritic cell-T cell-based coculture system. OX40 (CD134), a 50-kDa transmembrane protein of the TNFR family, is expressed primarily on activated CD4⁺ T cells. In murine models, the OX40/OX40L pathway has been shown to play a critical costimulatory role in dendritic cell/T cell interactions that may be important in promoting long-lived CD4⁺ T cells, which subsequently can help CD8⁺ T cell responses. The current study examined whether OX40 ligation on ex vivo CD4⁺ T cells can enhance their ability to "help" virus-specific CTL responses in HIV-1-infected and -uninfected individuals. OX40 ligation of CD4⁺ T cells by human OX40L-IgG1 enhanced the ex vivo expansion of HIV-1-specific and EBV-specific CTL from HIV-1-infected and -uninfected individuals, respectively. The mechanism whereby OX40 ligation enhanced help of CTL was independent of the induction of cytokines such as IL-2 or any inhibitory effect on CD4⁺ T regulatory cells, but was associated with a direct effect on proliferation of CD4⁺ T cells. Thus, OX40 ligation on CD4⁺ T cells represents a potentially novel immunotherapeutic strategy that should be investigated to treat and prevent persistent virus infections, such as HIV-1 infection.

	Introduction

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In the control of a number of acute and chronic viral infections such as EBV (1, 2), CMV (3, 4), SIV (5, 6), and HIV-1 (7, 8, 9, 10), CD8⁺ CTL play a central role. In viral infections that tend to persist, CD4⁺ T cells have been shown to play a crucial role in the maintenance of an effective ongoing CD8⁺ CTL response (11). CD4⁺ T cells can help CD8⁺ T cells through their interactions with dendritic cells (DC)³ (12, 13, 14, 15, 16). After contact with their cognate Ag, CD4⁺ T cells are activated to express the TNF superfamily molecule CD40L, which then induces a signal through the CD40R on DC, superactivates the DC, and allows it to become a more efficient inducer of CD8⁺ CTL responses (16, 17, 18, 19, 20, 21, 22, 23).

Other members of the TNFR/ligand superfamily have also been proposed to play a role in either enhancing or bypassing CD4⁺ T cell help in eliciting potent antiviral CTL responses (22, 23, 24). The OX40L (CD134L)/OX-40 (CD134) pathway has been shown to play a critical costimulatory role in DC/ T cell interactions. OX40 (CD134) is a 50-kDa transmembrane protein of the TNFR family. OX40 has a unique pattern of expression which is expressed on activated CD4⁺ T cells (25). The expression of OX40 on activated naive T cells peaks within 24–48 h of engagement of the TCR by peptide Ag in the context of MHC class II, and returns to baseline levels 120 h later (26). Effector T cells up-regulate their expression of OX40 more rapidly than naive T cells and the majority of effector cells express OX40 by 4 h after Ag stimulation (26). OX40L, a TNF family member, has been shown to be expressed on mature-activated DCs (for example, CD40L-conditioned DCs). In murine models, OX40 ligation on activated CD4⁺ T cells potentiates and sustains the proliferation of both Th1 and Th2 effector cell populations via induction of IL-2 and IL-2/IL-4, respectively (26, 27). OX40L-deficient mice (OX40L^–/–) demonstrate defects in delayed-type hypersensitivity responses representative of defects in CD4⁺ T cell priming (28). OX40-deficient (OX40^–/–) mice have been reported to also have defective CD4⁺ T cell proliferative responses to influenza and lymphocytic choriomeningitis virus (LCMV), but interestingly have preserved primary antiviral CTL and Ig (i.e. intact class switching) responses (29). It is unclear to what extent these antiviral CTL responses were dependant on CD4⁺ T cell help in this latter report (29) and long-term memory responses were not studied. Weinberg et al. and others (30, 31) were able to dramatically enhance antitumor immunity in a murine tumor model by in vivo engagement of the OX40R with agonist Abs. The responding mice showed enhanced CD4⁺ T cell immune responses in the vicinity of tumors, although the precise mechanisms (i.e., CD4⁺ T cell effects directly on tumor cells or by helping CD8⁺ T cell CTL) were not elucidated. Thus, OX40/OX40L interactions appear to act after initial activation events of CD4⁺ T cell to prolong their clonal expansion and enhance effector cytokine secretion. It has been suggested that the OX40/OX40L pathway may be most important in promoting long-lived CD4⁺ T cell responses in vivo (26).

The role of the OX40L-OX40 pathway in enhancing CD4⁺ T cell help of CTL responses in human systems is not known. HIV-1 infection is characterized by chronic, persistent viral replication in the face of ongoing detectable CD8⁺ CTL responses (32, 33). In addition, early qualitative and later quantitative abnormalities in CD4⁺ T cells are seen typically in HIV-1-infected individuals (34, 35, 36, 37). It is has been suggested that deficient CD4⁺ Th function in HIV-1 infection is responsible for the inability of CD8⁺ T cells to completely contain HIV-1 replication (38, 39). We have previously shown that HIV-1-specific memory responses require CD4⁺ T cell help to expand in in vitro DC/T cocultures. It is currently unknown whether stimulating CD4⁺ T cells through OX40 can further enhance CD4⁺ T cell help of CTL responses. The current study evaluated the role of OX40 signaling to enhance CD4⁺ T cell help of CTL responses against two persistent viral infections, EBV and HIV-1.

	Materials and Methods

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Study subjects

We were interested in studying human memory HIV-1- and EBV-specific antiviral responses. Thus, untreated HIV-1-infected individuals and HIV-1-negative EBV-seropositive individuals were recruited. For CTL assays, four untreated, HIV-1-seropositive individuals (participant nos. 1–4) with varying stages of disease progression were studied whose clinical profiles are depicted in Table I. Three asymptomatic HIV-1-uninfected individuals (participant nos. 5–7) who were HLA-A*0201-positive and had detectable EBV-specific CD8⁺ T cell IFN- responses by ELISPOT assay (data not shown) were also studied. Leukopheresis was performed to obtain large amounts of PBMCs. Prior to the study, individuals were class I HLA-typed and screened for HIV-1- or EBV-specific CTL by culturing PBMC with HLA-restricted HIV-1 or EBV peptides and detecting IFN--producing CD8⁺ T cells by ELISPOT assay as previously described (data not shown and Ref.16). HLA-restricted epitopes to HIV-1 for individual participant nos. 1–4 are shown in Table I. HIV-1-uninfected participant nos. 5–7 responded to the HLA-A *0201-restricted BMLF1 region of EBV (GLCLVAML). Informed consent was obtained from participants in accordance with the guidelines for conduction of clinical research at the University of Toronto and St. Michael’s Hospital. All investigational protocols were approval by the University of Toronto and St. Michael’s Hospital institutional review boards.

A soluble recombinant human OX40L-human IgG1 Fc fusion protein (hOX40L-IgG1) was used at 10 µg/ml final concentration. hOX40L-IgG1 was a gift from Xenova. As a control, soluble human IgG1 (hIgG1) was used at a final concentration of 10 µg/ml (Sigma-Aldrich). Staphylococcal enterotoxin B (SEB) superantigen was used at a final concentration of 1 µg/ml (Sigma-Aldrich). Anti-human CD3 (clone HIT3) and anti-human CD28 (clone L293) were obtained from BD Pharmingen. Recombinant human IL-2 was purchased from PeproTech and used at a final concentration of 100 U/ml for cell proliferation.

Antibodies

The anti-human fluorochrome-conjugated Abs directed against human CD3, CD4, OX40, CD8, CD80, CD83, CD1a, CD14, HLA-DR, CD27, CD28, CD40L, CCR5, CXCR4, IFN-, IL-2, and TNF-, as well as matched isotype Ab controls were obtained from BD Pharmingen. The recombinant -chain of the human IL-2R (rhIL-2 sR), polyclonal anti-human common -chain (IL-2 R (AF284)), and monoclonal anti-human common -chain (IL-2 R (MAB284)) Abs were purchased from R&D Systems. An FITC-conjugated Ab for human FoxP3 was obtained from eBioscience.

Generation of monocyte-derived DCs

Monocyte-derived DCs (MDDCs) were generated by a modification of a method previously described (16, 40). Briefly, PBMCs obtained by the Ficoll-Paque gradient centrifugation (Amersham Pharmacia Biotech) were separated on multistep Percoll gradients (Sigma-Aldrich). The recovered monocytes were depleted of contaminating B cells, T cells, NK cells, and granulocytes using Ab-conjugated magnetic beads from the Monocyte Negative Isolation kit (Dynal). Purified monocytes were cultured at 1 x 10⁶ cells/ml in medium consisting of RPMI 1640 plus 10% FCS, 2 mM glutamine, 25 mM HEPES, and antibiotics in the presence of 50 ng/ml rhGM-CSF and 100 ng/ml rhIL-4 (ProTech). GM-CSF and IL-4 were added again on days 3 and 5 with the fresh complete RPMI 1640 medium. After 7 days of culture, >50% of the cells were CD1a⁺, MHC class II⁺, CD80^low, and CD14^–, which represent an immature DC phenotype.

Expansion of Ag-specific T cells and assessment of effector responses

The protocol for expanding circulating memory CTL ex vivo was described previously (16). Freshly isolated or thawed autologous PBMCs were prepared in both unfractionated (CD4⁺ T cell containing) and CD4⁺ T cell-depleted conditions. Two rounds of magnetic bead depletion of CD4⁺ T cells were used for preparation of CD4⁺ T cell-depleted PBMCs (Dynal) with resulting populations containing <0.1% CD4⁺ T cells (data not shown). Both unfractionated and CD4⁺ T cell-depleted PBMCs were cultured for 24 h at 37° C in the following conditions: 1) medium alone, 2) soluble rhOX40L-human IgG1 Fc fusion protein (hOX40L-IgG1) at 10 µg/ml, 3) as a control, soluble hIgG1 at a final concentration of 10 µg/ml. Cells were harvested (supernatant was saved for cytokine detection) and cocultured with autologous immature MDDCs, either pulsed or nonpulsed with the specific HLA class I-restricted peptide at 40 µg/ml for 1 h at 37° C, at a ratio of 10:1 (2 x 10⁶ CD8⁺ T cells of PBMCs vs 0.2 x 10⁶ MDDCs). The percentage of CD8⁺ T cells within total unfractionated PBMCs and CD4⁺ T cell-depleted PBMCs was determined by flow cytometric analysis so that equal input of CD8⁺ T cells could be plated in both unfractionated (total PBMCs) and CD4⁺ T cell-depleted conditions. On days 3, 5, and 7, the medium was changed. On day 10, duplicate wells were pooled and cells were harvested and tested for effector activity by intracellular IFN- expression against peptide-pulsed target cells (see below) and chromium release assays. The percentages of CD8⁺ T cells in both unfractionated and CD4⁺-depleted conditions were again determined by flow cytometric analysis before CTL assays to assure for equal inputs of CD8⁺ T cells for all conditions.

Flow cytometric analysis of intracellular IFN- to assess CTL response

Cells were stained in PBS/1% FCS/0.02% NaN₃ using fluorochrome-conjugated Abs obtained from BD Pharmingen. Cell fluorescence was analyzed and compared with appropriate isotype-matched controls with a FACSCalibur flow cytometer and CellQuest (BD Biosciences) or FlowJo (Tree Star) software. Intracellular staining was performed using the Cytofix/Cytoperm kit (BD Pharmingen) in accordance with the manufacturer’s recommendations to enumerate the number of IFN--producing cells, as previously described (41). Briefly, for peptide-specific IFN- staining, 0.25 x 10⁶ cells were cultured in U-bottom 96-well plates in the presence of peptide-pulsed (1–10 µM) autologous B-lymphoblastoid cell lines (B-LCL) or autologous T cell-depleted PBMCs as stimulator cells; nonpeptide-pulsed stimulator cells were used as background controls. Positive control cells were stimulated with SEB. Cells were incubated with peptide-pulsed or nonpeptide-pulsed stimulator cells for 6 h at 37 ° C in 6% CO₂. Monensin was added for the duration of the culture period to facilitate intracellular cytokine accumulation.

Cytotoxicity assay

Autologous B-LCL or CD2-depleted PBMCs were labeled by incubating in 100 µCi sodium chromate, ⁵¹Cr, and pulsed with the specific peptide at 10 µM for 1 h at 37°C. Control B-LCL or CD2-depleted PBMCs were either pulsed with an irrelevant peptide or cultured in complete RPMI 1640 alone. Labeled target cells and serial dilutions of effector cells in triplicate were incubated in complete RPMI 1640 medium for 4 h. Supernatants were then collected and analyzed in a microplate scintillation counter (TopCount; Packard Instrument). Background chromium release was always<1%. The percentage of lysis was calculated from the formula: 100% x (E – M)/(T – M), in which E is experimental release, M is the release in the presence of complete RPMI 1640 medium, and T is the release in the 5% Triton X-100 detergent. Specific lysis was determined by subtracting the lysis of control targets from the lysis of peptide-pulsed targets.

Measurement of cytokines

Supernatants from cell cultures were harvested and stored at –80° C until analysis for secreted cytokines. IL-2 and IL-4 production in the supernatants was measured using ChemiKine Human IL-2 and IL-4 Sandwich ELISA kits (Chemicon International) according to the manufacturer’s specifications. The detection limits of these ELISA tests were 1.10 and 0.87 pg/ml for IL-2 and IL-4, respectively.

To determine whether intracellular cytokine production by CD4⁺ T cells could be influenced by OX40/OX40L, unfractionated (CD4⁺ T cell containing) PBMCs were incubated with hOX40L-IgG1 (10 µg/ml), hIgG1 (10 µg/ml), or medium alone for 9 h at 37° C. Monensin was added for the duration of last 8-h culture period to facilitate intracellular cytokine accumulation. Cells were harvested and assessed using four-color cytometric analysis with Abs of CD3^{allophycocyanin} /CD4^PerCP /IL-2^FITC and IFN-^PE, or TNF-^PE. Matched isotype Ab^FITC or Ab^PE was included for staining control. When immobilized anti-human CD3 or CD28 Ab was used, 50 µ l of either anti-CD3 or anti-CD28 at 2 µg/ml in plain RPMI 1640 medium was added to a flat-bottom 96-well-plate and immobilized for 1 h at 37° C. Ab was then aspirated and wells were washed once with plain RPMI 1640 medium.

Proliferation assays

PBMCs were incubated with hOX40L-IgG1 (10 µg/ml), hIgG1 (10 µg/ml), or medium alone for 24 h and then cocultured with autologous immature MDDCs at 37°C, 5% CO₂ for 7 days. [³H]Thymidine (PerkinElmer) was added at 1 µCi/ml for the duration of last 6-h culture period. The cells were harvested onto glass fiber filters using a Tomtec cell harvester (Wallac) and incorporated radioactivity was determined by liquid scintillation counting (Wallac 1205 Betaplate). The results from triplicate wells were averaged and are reported as mean cpm ± the SE of the mean (cpm ± SEM).

Thymidine incorporation can only measure bulk cell division and does not give any information on the division of specific cell types. To more specifically determine the effect of OX40/OX40L interactions on the CD4⁺ T cell proliferation, stable incorporation of the intracellular fluorescent dye CFSE was used. PBMCs at 1 x 10⁷/ml in PBS containing 5% FBS were stained at room temperature for 5 min with the concentrations of 5 µM CFSE. Staining was terminated by adding PBS containing 5% FBS and subsequent washings. Cells were then resuspended in complete RPMI 1640 culture medium and cultured in U-bottom 96-well plates (5 x 10⁵/well, 200 µl) with hOX40L-IgG1 (10 µg/ml), hIgG1 (10 µg/ml), or medium alone at 37°C, 5% CO₂ for 7 days. Cells were harvested and stained with CD3^{allophycocyanin} and CD4^PerCP. Unstained cells were included in all experiments and were used to set the compensations on flow cytometer.

Depletion of CD4⁺ CD25⁺ cells

CD4⁺ CD25⁺ T cells were depleted using the CD25 Microbead Depletion kit according to the manufacturer’s instructions (Miltenyi Biotec) and purity of depletion was determined by flow cytometry.

Statistical analysis

Data were compared using the paired two-tailed Student t test.

	Results

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OX40 expression on ex vivo peripheral blood CD4⁺ T cells

Consistent with previous reports (42, 43), OX40 expression was detected on activated peripheral human CD4⁺ T cells by surface staining with stimulation of the TCR by anti-CD3 inducing the most potent expression (Fig. 1A). To determine whether OX40 is expressed constitutively on ex vivo human CD4⁺ T cells, we performed flow cytometric analysis on ex vivo PBMC from HIV-1-negative and -seropositive individuals. We found that in uninfected individuals, 1.3% (range 0.8–2.0%; n = 6) of CD4⁺ T cells express OX40, whereas in HIV-1-infected individuals, 4.5% (range 1.8–7.5%; n = 14) of CD4⁺ T cells express OX40 (p < 0.05) as defined by three-color flow cytometric analysis (Fig. 1, B and C). In addition, we have measured very high levels (>45%) of OX40 expression on HIV-1-specific CD4⁺ T cell clones obtained from HIV-1-infected individuals (Fig. 1D). Interestingly, in some HIV-1-infected individuals, low levels of OX40 expression were also observed on CD8⁺ T cells (always <2.0%, data not shown). To further define the phenotype of OX40-expressing CD4⁺ T cells, whole blood samples were examined for other markers of T cell function. We find that OX40-expressing CD4⁺ T cells are predominantly CD27⁺/CD28⁺ and CD27^–/CD28⁺, CCR5^–, and HLA-DR⁺ (Fig. 1E). This was observed in both uninfected and HIV-1-infected volunteers. This phenotype is consistent with an early activated memory phenotype prior to full effector function (44). In murine studies (45), it has been shown that CD25⁺CD4⁺ T regulatory cells express OX40 after stimulation. We thus determined the relative expression of typical markers of T regulatory cells (46, 47, 48) with OX40 in peripheral blood samples. We find that only a minority of OX40-expressing CD4⁺ T cells from peripheral blood express typical markers of T regulatory cells, such as CD25 (mean 23 ± 5% SE) and intracellular FoxP3 (19 ± 10% SE) (Fig. 1F). Thus, OX40 expression ex vivo identifies a population of activated CD4⁺ T cells, with an early memory phenotype, the majority of which do not show markers typical of T regulatory cells. Also, HIV-1-infected individuals express higher OX40 on their CD4⁺ T cells compared with HIV-1-uninfected individuals, which likely reflects a higher baseline level of activation of CD4⁺ T cells in HIV-1 infection. These findings also show that ex vivo human CD4⁺ T cells may be manipulated by stimulating them through OX40.

View larger version (32K): [in this window] [in a new window]   FIGURE 1. OX40 expression on ex vivo peripheral blood CD4+ T cells. OX40 expression on CD4+ T cells was assessed in PBMCs (A) that were incubated with immobilized anti-CD3, -CD28 Ab or medium alone at 37°C for 16 h. Cells were analyzed for surface OX40 expression on CD4+ T cells. B, OX40 expression on ex vivo CD4+ T cells isolated from HIV-1-positive and -negative participants. C, Peripheral blood from 6 HIV-1-negative and 14 untreated HIV-1-positive participants were analyzed for OX40 expression on CD4+ T cells. D, OX40 expression on HIV-1-specific CD4+ T cell clones obtained from HIV-1 infected individuals. Very high levels of OX40 expression were observed from two HIV-1-specific CD4+ T cell clones. A representative experiment from one clone is shown. Values indicate percentage of OX40-positive CD4+ T cells of total CD4+ T cells. E, Whole blood from a normal volunteer was stained; the OX40-positive CD4+ T cells were gated and examined for expression of the activation marker HLA-DR and the T cell maturation markers, CD27, CD28, and CCR5. Numbers in the upper right represent the percentage of cells within each quadrant. Similar results were obtained from blood in an HIV-1-infected individual. F, Ex vivo CD4+ T cells from peripheral blood are gated and then examined for expression of OX40, intracellular FoxP3, and CD25. Numbers represent the percentage of OX40 CD4+ T cells expressing FoxP3 and CD25, respectively. A representative of three experiments from three HIV-infected individuals is shown.

After determining that OX40 was expressed on ex vivo CD4⁺ T cells, we then determined whether HIV-1-specific memory CTL responses could be influenced by OX40 ligation. We hypothesized that stimulation of OX40 on CD4⁺ T cells would enhance the ability of CD4⁺ T cells to help virus-specific CTL responses. We have used an in vitro system in which peptide-pulsed MDDCs and T cells are cocultured in the absence of exogenous cytokines to expand memory CTL responses. We have previously used this system to study the ability of CD4⁺ T cells to help expand CD8⁺ T cell responses (16, 22). Four HIV-1-seropositive individuals with detectable memory CTL responses to defined HLA-restricted epitopes against HIV-1 were examined (see Table I). PBMCs prepared in both unfractionated (CD4⁺ T cell containing) and CD4⁺ T cell-depleted conditions were stimulated with an OX40L-IgG1Fc fusion protein to provide signals through OX40 on OX40-expressing cells, a control human IgG1, or medium alone for 24 h, washed, and then cocultured with autologous MDDCs pulsed or nonpulsed with an HIV-1-specific peptide. After 10 days of coculture, CTL effector activity was assessed by intracellular IFN- production and by chromium release killing assays after exposure to peptide-pulsed targets (autologous B-LCL or T cell-depleted PBMCs). A representative experiment measuring effector IFN- CD8⁺ T cell responses against an HLA-B27-restricted epitope (KRWIIGLNK) of HIV-1 gag by intracellular flow cytometric analysis from participant no. 1 is illustrated in Fig. 2A. Summary data of CTL responses in participant nos. 2–4 are shown in Fig. 2B, and a representative chromium release killing assay from participant no. 3 is shown in Fig. 2C. Of note, effector CD8⁺ T cell responses are greater in cocultures containing CD4⁺ T cells confirming the role of CD4⁺ T cell help to expand HIV-1-specific memory CTL responses. In addition, pretreatment of PBMC with OX40L (CD134L) dramatically enhanced the ability of CTL responses to be expanded when compared with hIgG1 (the control protein for the OX40L fusion protein) or medium-treated PBMCs. Although, OX40L also minimally enhanced CTL responses in CD4⁺ T cell-depleted conditions in all HIV-1-infected participants, the most dramatic effects were observed if CD4⁺ T cells were present indicating that ligation of OX40 on CD4⁺ T cells is the predominant mechanism for enhancing the expansion of memory CTL. Low level expression of OX40 on activated CD8⁺ T cells (49, 50) has been previously noted. We have also observed low level OX40 expression on CD8⁺ T cells, particularly in HIV-1-infected individuals, which likely represents enhanced activation of these cells due to HIV-1 infection. Thus, we postulate that the enhancement observed with OX40L treatment in CD4⁺ T cell-depleted conditions (>99.9% depleted) is due to ligation of OX40 directly on CD8⁺ T cells. We also did not observe any enhancement in CTL responses in OX40L pretreated PBMCs if cells were cocultured with MDDCs that were not pulsed with peptide, ruling out a nonspecific induction of CTL (data not shown).

View larger version (31K): [in this window] [in a new window]   FIGURE 2. OX40L ligation on CD4+ T cells in expansion of HIV-1-specific memory CTL in vitro. CD4+ T cell-containing or -depleted PBMCs from HIV-1-positive participants were incubated with hOX40L-IgG1 Fc fusion protein (10 µg/ml), hIgG1 (10 µg/ml), or medium alone for 24 h, then cocultured with autologous MDDCs pulsed or nonpulsed with HIV-1-specific peptide. On day 10, HIV-1-specific CTL activity was assessed by intracellular flow cytometric analysis of IFN--producing cells and chromium release of peptide-pulsed target cells. A, Representative intracellular IFN- flow cytometric data obtained from HIV-1-positive participant no. 1. For intracellular cytokine flow cytometric analysis, cells were gated on CD3 and IFN--producing CD8+ T cells were enumerated. Experiments were repeated in HIV-1-positive participant no. 1 with similar results obtained. B, Summary data from intracellular IFN- flow cytometric analysis of HIV-1-positive participant nos. 2–4. C, A representative chromium release CTL killing assay from participant no. 3 is shown. Similar results were obtained with participant no. 4 (data not shown). The experiment from participant no. 1 was repeated with similar results. DC, MDDC not pulsed with peptide; DCP, MDDC pulsed with peptide; OX40L/DCP, PBMC cultured with OX40L-IgG1 + MDDC pulsed with peptide; hIgG1/DCP, PBMC cultured with control human IgG + MDDC pulsed with peptide.

Although, OX40 expression was lower on CD4⁺ T cells from HIV-1-uninfected individuals, we wanted to determine whether OX40 ligation of ex vivo CD4⁺ T cells could still enhance help of virus-specific memory CTL. Thus, three healthy HIV-1-uninfected individuals who had previously demonstrated an HLA-A*0201-restricted response to the EBV epitope of BMLF1 (lytic cycle Ag) were studied. PBMCs derived from these individuals were prepared in both unfractionated (CD4⁺ T cell containing) and CD4⁺ T cell-depleted conditions, were stimulated with hOX40L-IgG1 fusion protein, control human IgG1, or medium alone for 24 h, and then cocultured with autologous MDDCs pulsed or nonpulsed with EBV-specific peptide. After 10 days of coculture, CTL effector activity was assessed by intracellular IFN- production and chromium release after exposure to peptide-pulsed targets (autologous B-LCL or T cell-depleted PBMCs). Flow cytometric data from all three individuals are graphically illustrated in Fig. 3A and a representative chromium release killing assay is depicted in Fig. 3B.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. OX40L ligation on CD4+ T cells in expansion of EBV-specific memory CTL in vitro. CD4+ T cell-containing or -depleted PBMCs from EBV-positive participants were incubated with human hOX40L-IgG1 Fc fusion protein (10 µg/ml), human IgG1 (10 µg/ml), or medium alone for 24 h, then cocultured with autologous MDDCs pulsed or nonpulsed with EBV-specific peptide. On day 10, EBV-specific CTL activity was assessed by intracellular flow cytometric analysis of IFN--producing cells and chromium release of peptide-pulsed target cells. A, Summary data from intracellular IFN- flow cytometric analysis of EBV-positive participant nos. 5–7 are graphically depicted. The experiment from participant no. 5 was repeated with similar results. Experiments with patient no. 7 were performed only with unfractionated PBMC (CD4-containing conditions). DC, MDDC not pulsed with peptide; DCP, MDDC pulsed with peptide; OX40L/DCP, PBMC cultured with hOX40L-IgG1 + MDDC pulsed with peptide; hIgG1/DCP, PBMC cultured with control human IgG + MDDC pulsed with peptide. B, A representative CTL killing assay from participant no. 5 is shown using unfractionated PBMC (CD4-containing conditions).

Pooled data from experiments from all participants (nos. 1–7) studied are graphically illustrated in Fig. 4. In summary, pretreatment of CD4⁺ T cells with hOX40L-IgG1 significantly expanded virus-specific memory CTL responses compared with control IgG1 treatment (p < 0.05), by about 4-fold on average for HIV-1-specific CTL and 3-fold for EBV-specific CTL in uninfected individuals. Although Ox40L-IgG1 tended to enhance CTL responses in CD4⁺ T cell-depleted conditions from HIV-1-infected individuals, overall, the differences were not statistically significant if CTL responses from HIV-1-uninfected individuals were also included.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. OX40 ligation expands virus-specific CTL in CD4+ T cell-containing conditions, summary data. Pooled data of CD4+ T cell-containing and CD4+ T cell-depleted experiments from all participants (nos. 1–7) are shown. Squares represent HIV-1-specific CTL responses and circles represent EBV-specific CTL responses of individual participants. Horizontal bars represent means of pooled responses.

CD4⁺ T cells help CTL responses by a number of mechanisms, including via expression of CD40L to stimulate DCs and by direct release of IL-2 which directly stimulates CD8⁺ T cells in their vicinity (22). To define the mechanism whereby OX40L-stimulated CD4⁺ T cells help CTL, we examined cytokine production, CD40L expression and apoptosis of CD4⁺ T cells after hOX40L-IgG1 treatment (data not shown). OX40 ligation of CD4⁺ T cells did not increase IL-2, IL-4, IFN-, or TNF- production from CD4⁺ T cells (data not shown). In addition, OX40 ligation did not enhance intracellular production of IL-2, IFN-, or TNF- production in CD4⁺ T cells that were also costimulated with anti-CD3, anti-CD28, or anti-CD3 + anti-CD28 (data not shown). OX40 ligation did not enhance intracellular or extracellular expression of CD40L, nor did it affect apoptosis of CD4⁺ T cells based on activated caspase-3 expression (data not shown). Thus, the enhancement of CTL through OX40-ligated CD4⁺ T cells is not through IL-2, CD40L, or specific inhibition of apoptosis of CD4⁺ T cells.

Ligation of OX40 induces CD4⁺ T cell proliferation in vitro

To determine whether the effect of OX40 ligation on CTL responses was related to proliferation of CD4⁺ T cells, we assessed the proliferative ability of CD4⁺ T cells in our DC-T cell coculture experiments. PBMCs were treated with soluble hOX40L-IgG1 or control protein for 24 h, then cocultured with autologous MDDC and lymphocyte proliferation was assessed by [³H]thymidine incorporation at day 7 (Fig. 5A). Cultures in which PBMCs were treated with hOX40L-IgG1 showed significantly enhanced proliferation by about 2- to 4-fold (p < 0.05).

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Ligation of OX40L induces lymphocyte proliferation in vitro independent of IL-2. Lymphocyte proliferation in response to OX40L stimulation. A, CD4+ T cell-containing PBMCs were incubated with soluble hOX40L-IgG1 (10 µg/ml), hIgG1 (10 µg/ml), or medium alone for 24 h, then cultured with autologous MDDCs at a ratio of 10:1 for 7 days in complete medium. Lymphocyte proliferation was assessed at 7 days by measuring [3H]thymidine incorporation during last 6-h culture period. The results from triplicate wells were averaged and are reported as mean cpm ± the SE of the mean (cpm ± SEM). B, CFSE staining was used to determine the effect of OX40-OX40L interactions on CD4+ T cell proliferation. PBMCs stained with CFSE were incubated with hOX40L-IgG1 (10 µg/ml), hIgG1 (10 µg/ml), or medium alone at 37°C, 5% CO2 for 7 days (upper row) or with immobilized anti-CD3 (lower row). Cells were harvested and stained with CD3allophycocyanin and CD4PerCP. The percentage of proliferating CD4+ T cells is illustrated after gating on viable CD3+CD4+ T cells. When immobilized anti-human CD3 Ab was used, 50 µl of Ab at 2 µg/ml in plain RPMI 1640 medium was coated to a flat-bottom 96-well plate and immobilized for 1 h at 37°C. Ab was then aspirated and wells were washed once with plain RPMI 1640 medium before adding CFSE-stained PBMCs. Results are representative of five experiments from HIV-infected and uninfected individuals. C, PBMCs were CFSE-labeled as above and then stimulated with SEB plus medium, OX40L, or hIgG1 and then assessed for proliferation at day 7. Numbers in the upper plots are the percentage of CD4+ T cells that have maximally diluted CFSE. Results are representative of three experiments. D, PBMCs were CFSE-labeled, stimulated as above, but also cultured in the presence of cytokine inhibitors, including rIL-2R (rhIL-2 sR, 2.5 µg/ml), a polyclonal (IL-2 R, clone AF284, 100 µg/ml), and a monoclonal (IL-2 R, clone AF284, 10 µg/ml) Ab to the common -chain and then assessed at day 7 for proliferation by flow cytometry. rIL-2 was used as a positive control at a final concentration of 100 U/ml. Numbers in upper left of plots are the percentage of CD4+ T cells that are diluting CFSE from the main population of CD4+ T cells. Results are representative of two experiments.

IL-2 has previously been shown to be the major cytokine to induce proliferation of CD4⁺ T cells (51). To assess whether IL-2 production was driving proliferation of OX40-ligated CD4⁺ T cells, we performed IL-2-blocking experiments using the soluble -chain of the IL-2R (IL-2R , as well as Abs to the common - chain of the IL-2/IL-15/IL-7/IL-4R) (Fig. 5D). However, we could not inhibit OX40L-induced CD4⁺ T cell proliferation, although, we could block proliferation induced directly by rhIL-2 with soluble receptors and Abs (Fig. 5D), confirming that OX40L-induced CD4⁺ T cell proliferation is IL-2-independent.

In addition, OX40L-induced enhancement of HIV-1-specific CTL could not be inhibited with the soluble IL-2R (Fig. 6), confirming that the effects of OX40L are independent of IL-2.

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Effect of the soluble IL-2R on OX40L-induced CTL enhancement. Unfractionated PBMCs from HIV-1-positive participant no. 2 were incubated with hOX40L-IgG1 fusion protein (10 µg/ml) with or without soluble IL-2R (10 µg/ml) to neutralize secreted IL-2 and then cocultured with autologous MDDCs pulsed with HIV-1-specific peptide. Soluble IL-2R (10 µg/ml) was replenished throughout cultures to inhibit IL-2. On day 10, HIV-1-specific CTL activity was assessed by intracellular flow cytometric analysis of IFN--producing cells. Shown are intracellular IFN- flow cytometric data obtained against peptide-pulsed targets similar to as described in Fig. 2. Numbers represent the percentage of CD8+ T cells expressing IFN- against targets. No differences in CTL response was observed in the presence of soluble IL-2R. Results are representative of two experiments.

Recent studies in murine cells have shown that triggering OX40 on CD25⁺CD4⁺ T cells can block the inhibitory activity of these T regulatory cells (52). Thus, a possible mechanism for the enhancement of CTL through OX40L may be due to inhibition of T regulatory function. We thus assessed the ability of OX40L to enhance CTL responses in CD25-depleted conditions. We find that OX40 ligation of CD25-depleted CD4⁺ T cells can still enhance CTL responses to similar degrees as in the presence of CD25⁺CD4⁺ T cells (see Fig. 7 and Fig. 4). Thus, the enhancing effect of OX40 ligation is independent of any inhibitory effect on T regulatory cell function.

View larger version (53K): [in this window] [in a new window]   FIGURE 7. Removal of CD25+CD4+ T cells does not abrogate CTL enhancement of OX40 ligation. CD4+ T cell-containing PBMCs from HIV-1-positive participant nos. 4 and 3 were depleted of CD25-positive cells prior to incubation with hOX40L-IgG1 Fc fusion protein (10 µg/ml), human IgG1 (10 µg/ml), or medium alone for 24 h, and then cocultured with autologous MDDCs pulsed or nonpulsed with HIV-1-specific peptide as described in Fig. 2. On day 10, HIV-1-specific CTL activity was assessed by intracellular flow cytometric analysis of IFN--producing cells in response to peptide-pulsed target cells. A, More than 90% CD25 depletion was confirmed in PBMCs by flow cytometry. Bold-lined histogram represents CD25 expression prior to depletion and thin-lined histogram represents CD25 expression postdepletion, and shaded histogram is isotype control. B, Intracellular IFN- flow cytometric data obtained form both participants. For intracellular cytokine flow cytometric analysis, cells were gated for CD3 and CD8 to enumerate IFN--producing CD8+ T cells only. Similar degrees of CTL enhancement are seen as compared with those in the presence of CD25-expressing cells (see Fig. 4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Effective and sustained memory responses are important for virologic control of persistent infections. Because OX40 has been shown to be expressed on memory T cells that have become activated after recontact with Ag (26, 54), signaling through OX40 is likely to be important to maintain potent memory T cell responses to persistent or re-exposed Ag, by enhancing the ability of CD4⁺ T cells to help CTL. In murine models, OX40 ligation has been shown to help CTL through a number of mechanisms. These include 1) an enhanced ability of CD4⁺ T cells to produce IL-2 (26), 2) an enhanced capacity of CD4⁺ T cells to proliferate (55), and 3) inhibition of apoptosis of CD4⁺ T cells via increased Bcl-2/Bcl-x expression (56). In our coculture system, we were unable to show any effect of OX40 ligation on cytokine secretion from CD4⁺ T cells, including IL-2, IL-4, IFN-, or TNF-. In addition, OX40 ligation had no effect on apoptosis in short-term CD4⁺ T cell cultures, nor any effect on CD4⁺ T cell expression of CD40L, a potent inducer of DC maturation. In our coculture system, the effects of OX40 ligation on CTL responses were independent of IL-2. We also explored the hypothesis that OX40 ligation enhances CTL responses by inhibiting the function of OX40-expressing T regulatory cells. First, we noted that only a minor subset of OX40-expressing cells also coexpress markers found on T regulatory cells, such as CD25 and FoxP3. Because CD25 and FoxP3 markers are not entirely specific for T regulatory cells, it is unclear whether this subset truly represents functional T regulatory cells (46, 47, 48). To rule out an effect of OX40 on T regulatory cell function, we repeated our CTL assays in the absence of CD25-expressing cells and still found a similar degree of enhancement through OX40 ligation. We did, however, show that OX40 ligation enhanced the proliferative capacity of CD4⁺ T cells. Thus, the effect of OX40 signaling may simply be to increase the number of Ag-specific CD4⁺ T cells early on in the immune response to interact with DCs and/or CD8⁺ T cells. In this regard, Song et al. (55) recently demonstrated that CD4⁺ T cells stimulated through OX40 can maintain their ability to expand in vitro independently from IL-2 secretion, because CD4⁺ T cells from OX40 knockout are impaired in their ability to expand but produce normal amounts of IL-2 in response to Ag. The mechanisms responsible for a direct CD4⁺ T cell proliferative effect of OX40 ligation are yet to be determined. Similar to other TNFR family members, OX40 ligation recruits TRAF2 adapter proteins which can provide links to PI3K/AKT, MAPK, JNK/AP1, or NF-B signaling pathways leading to activation of cyclins and cyclin-dependent kinases resulting in cell division (24, 54). Further studies dissecting these respective pathways will be needed to identify the specific mechanisms responsible for direct cell proliferation induced by OX40 ligation. In summary, our findings are consistent with OX40 being up-regulated early after activation of memory CD4⁺ T cells, in which signaling through OX40 directly leads to proliferation of these cells. The resulting increased numbers of CD4⁺ T cells will then directly stimulate DC or CD8⁺ T cells to enhance effector CTL responses.

This study has potentially important implications for the use of OX40L in vaccine strategies or as an effective immunotherapy for chronic-persistent virus infections, such as HIV-1. The incorporation of OX40L in the design of antiviral vaccines might enhance their ability to induce CTL immune responses in vivo. Also, the use of OX40L as an immunotherapeutic agent in infectious diseases in which CD4⁺ T responses are weakened due to anergy, such as hepatitis C virus and HIV-1, would be an attractive approach for clinical application. In the setting of HIV-1 infection, such therapies, however, would have to be designed in conjunction with highly active antiretroviral therapy, as OX40 ligation of CD4⁺ T cells has been demonstrated to enhance HIV-1 replication in vitro (57).

	Acknowledgments

 
We thank our patients for their time and commitment and Tania Watts for helpful discussions.

	Disclosures

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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 through a grant supplied by the Canadian Institutes for Health Research (CIHR) and an Aventis-Pasteur-University of Toronto Research Program grant program. M.A.O. is a career scientist of the CIHR.

² Address correspondence and reprint requests to Dr. Mario A. Ostrowski, Clinical Sciences Division, University of Toronto, Room 6271, 1 King’s College Circle, Medical Sciences Building, Toronto, Ontario, Canada M5S 1A8. E-mail address: m.ostrowski{at}utoronto.ca

³ Abbreviations used in this paper: DC, dendritic cell; LCMV, lymphocytic choriomeningitis virus; h, human; SEB, staphylococcal enterotoxin B; rh, recombinant human; MDDC, monocyte-derived DC; B-LCL, B-lymphoblastoid cell line.

Received for publication June 17, 2005. Accepted for publication November 28, 2005.

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