OX40 promotes Bcl-xL and Bcl-2 expression and is essential for long-term survival of CD4 T cells

It is important to understand which molecules are essential for long-lived immunity. We show that OX40 (CD134) is required with CD28 for the survival of CD4 T cells following antigen-driven expansion. In contrast to CD28-/- T cells, which show defects early, OX40-/- T cells are relatively unimpaired in IL-2 production, cell division, and expansion. However, OX40-/- T cells fail to maintain high levels of Bcl-xL and Bcl-2 4-8 days after activation, and undergo apoptosis. Conversely, OX40 stimulation promotes Bcl-xL and Bcl-2 and suppresses apoptosis. Moreover, retroviral transduction of OX40-/- T cells with Bcl-xL or Bcl-2 reverses their survival defect. Thus, a temporal relationship exists between CD28 and OX40, with OX40 being a critical regulator of antigen-driven T cell survival.     

Targeting OX40 and OX40L for the treatment of autoimmunity and cancer

The optimal activation of na?ve T cells requires TCR-mediated recognition of cognate peptide-MHC complexes on antigen presenting cells in the presence of costimulatory signals. Although signals provided via CD28-B7 interactions are important for enhancing the initial T-cell response, other costimulatory signals are required for sustaining the response and promoting both T-cell differentiation and survival. In particular, engagement of OX40 (CD134) by its natural ligand OX40L (CD134L) or OX40 agonists has been shown to provide key signals that can augment CD4 and CD8 T-cell responses. Importantly, numerous studies have highlighted the ability of OX40-specific agonists or antagonists to enhance antitumor immunity or ameliorate autoimmune disease, respectively. On the basis of these studies, the development of OX40- and OX40L-specific reagents has been pursued for clinical use. Given the emerging role of OX40 and OX40L as novel therapeutic targets, this review will focus on the cellular and molecular mechanisms of OX40-mediated T-cell costimulation with a special emphasis on the role of OX40-OX40L interactions in the etiology and treatment of autoimmunity and cancer.     

Cooperation between CD4 and CD8 T cells for anti-tumor activity is enhanced by OX40 signals

The relative contribution of OX40 (CD134) to priming of CD8 T cells in complex systems where CD4 and CD8 cells respond and cooperate together is not clear. We previously found that OX40 expressed on tumor-reactive CD8 T cells controls their initial persistence when adoptively transferred in vivo and is required for delayed tumor growth. We now show that exogenous stimulation of OX40 with agonist antibody augments its ability to suppress the growth of new as well as established tumors, correlating with marked expansion of adoptively transferred CD8 T cells. Concomitantly, anti-OX40 strongly enhanced the number of tumor antigen-reactive CD4 T cells. Moreover, the augmented accumulation of CD8 T cells was prevented in animals lacking MHC class II or depleted of CD4 cells and did not occur in OX40-deficient animals receiving wild-type CD8 cells, demonstrating that non-CD8 cells are the major target of OX40 signals. These results suggest that while OX40 signaling to a CD8 T cell can control its expansion, OX40 expressed on non-CD8 cells strongly influences CD8 priming and in vivo activity. OX40 therefore represents an important signal for allowing effective cooperation between CD4 and CD8 cells and for promoting cell interplay and tumor rejection where CD8 activity is limiting.     

Functional characterization of OX40 expressed on human CD8+ T cells

In the present study, we investigated the expression of OX40 on human CD8(+) T cells with regard to expression induction, costimulatory function and possible involvement in cytotoxicity. Human CD8(+) T cells were purified from peripheral blood mononuclear cell (PBMC) of healthy donors and cocultured with allogeneic monocyte-derived dendritic cells. Flow cytometric analysis showed that expression of OX40 was induced on CD8(+) T cells within 1 day and increased to the maximum levels on day 3. An addition of anti-OX40 ligand (OX40L) mAb suppressed CD25 expression, proliferation and IFN-gamma production of CD8(+) T cells, suggesting that OX40 functions as a costimulatory molecule not only for CD4(+) T cells but also for CD8(+) T cells. In parallel, coculture of pre-activated CD8(+) T cells with OX40L-transfected murine epithelial cells (MMCE-OX40L) resulted in an increase in CD25 expression, proliferation and IFN-gamma producing cells, compared with that with the mock control (MMCE-mock). Finally, non-specific cytotoxic activity of preactivated CD8(+) T cells was examined using OKT3 hybridoma as target cells after coculture with these transfectants. Coculture with MMCE-OX40L induced slightly higher cytotoxicity of CD8(+) T cells than that with MMCE-mock. These results indicate that OX40 is induced transiently on CD8(+) T cells upon activation and its signals contribute to both clonal expansion and functional reinforcement.     

The generation of T cell memory: a review describing the molecular and cellular events following OX40 (CD134) engagement

OX40 (CD134), a membrane-bound member of the tumor necrosis factor-receptor superfamily, is expressed primarily on activated CD4(+) T cells. Following engagement on the cell surface, OX40 delivers a costimulatory signal that leads to potent, proinflammatory effects. Engagement of OX40 during antigen (Ag)-specific stimulation of T cells leads to increased production of memory T cells, increased migration of Ag-specific T cells, enhanced cytokine production by effector T cells, and the ability to break peripheral T cell tolerance in vivo. Therefore, OX40 engagement in vivo could have important ramifications for the enhancement of vaccine strategies and inhibition of unwanted inflammation. This review summarizes the molecular and cellular events that occur following OX40 engagement during Ag-specific T cell activation.     

Anti-OX40 stimulation in vivo enhances CD8+ memory T cell survival and significantly increases recall responses

There is growing evidence that engagement of OX40 (CD134), a member of the TNF receptor superfamily, can directly stimulate antigen-specific CD8+ T cells. It has been shown that CD8+ T cells express OX40 following activation, but the response of antigen-specific CD8+ T cells to OX40 stimulation has not been fully characterized. We utilized an antigen-specific transgenic CD8+ T cell model (OT-I) to determine if OX40 engagement can boost the generation of antigen-specific CD8+ T cell memory. Our results demonstrate that enhanced OX40 costimulation, via an agonist anti-OX40 antibody, increases CD25 and phospho-Akt expression on the antigen-specific CD8+ T cells and significantly increases the generation of long-lived antigen-specific CD8+ memory T cells. The increased numbers of memory CD8+ T cells generated via anti-OX40 treatment still required the presence of CD4+ T cells for their long-term maintenance in vivo. In addition, anti-OX40 costimulation greatly enhanced antigen-specific CD8+ T cell recall responses. These data show that OX40 engagement in vivo increases the number of antigen-specific CD8+ memory T cells surviving after antigen challenge and has implications for the development of more potent vaccines against pathogens and cancer.     

Contribution of OX40/OX40 ligand interaction to the pathogenesis of rheumatoid arthritis

OX40 ligand (OX40L) and OX40 (CD134) are a pair of cell surface molecules belonging to the TNF/TNF receptor family. Interaction of OX40L with its receptor OX40 is thought to be important in T cell activation through T cell/antigen-presenting cell interaction. However, involvement of these molecules in the pathogenesis of rheumatoid arthritis (RA) remains unclear. To explore the contribution of OX40/OX40L interaction to the pathogenesis of RA in vivo, we evaluated the effect of a neutralizing anti-OX40L monoclonal antibody (mAb) on the development of collagen-induced arthritis (CIA) in DBA/1 mice as an animal model for RA. Administration of anti-OX40L mAb into type II collagen (CII) -immunized DBA/1 mice dramatically ameliorated the disease severity. In vivo treatment with anti-OX40L mAb did not inhibit the expansion of CII-reactive T cells, but suppressed IFN-gamma and anti-CII IgG2a production. Therefore, OX40/OX40L interaction appears to play a critical role in the development of CIA by enhancing Th1-type autoimmune response. In addition, T lymphocytes in synovial fluid and synovial tissue from RA patients expressed OX40, while OX40L was expressed on sublining cells in synovial tissue. These results indicate that OX40/OX40L interaction may play a critical role in the development of RA.     

Prevention of diabetes in NOD mice at a late stage by targeting OX40/OX40 ligand interactions

Autoreactive T cells play a major role in the development of insulin-dependent diabetes mellitus, suggesting that costimulatory molecules that regulate T cell responses might be essential for disease progression. In NOD mice, CD28/B7 and CD40/CD40 ligand (L) interactions control the onset of diabetes from 2 to 4 weeks of age, but blocking these molecules has little effect after this time. Hence, it is possible that other ligand/receptor pairs control a later phase of disease. We now show that OX40 is expressed on CD4 and CD8 T cells several weeks prior to islet destruction, which is initiated around weeks 12-14, and that OX40L is present on dendritic cells in both secondary lymphoid organs and the pancreas from 11 to 13 weeks of age. Blocking OX40L at 6, 9, or 15 weeks after birth had little effect on disease; however, inhibiting OX40/OX40L interactions at week 12, or continuous treatment from week 12 onwards, significantly reduced the incidence of diabetes. Histological examination showed that islet destruction was prevented and insulitis reduced by targeting OX40L. These studies show that OX40/OX40L interactions form a late checkpoint in diabetes development and suggest that these molecules are realistic targets for therapeutic intervention.     

Phenotypic characterization of regulatory CD4+CD25+ T cells in rats

CD25 has become widely used as a marker for a subset of regulatory CD4(+) T cells present in the thymus and periphery of mice, rats and humans. However, CD25 is also expressed on conventionally activated T cells that are not regulatory and not all peripheral regulatory T cells express CD25. The identification of a stable and unique marker for regulatory T cells would therefore be valuable. This study provides a detailed account of the phenotype of CD4(+)CD25(+) regulatory T cells in rats. In the thymus, CD4(+)CD8(-)CD25(+) cells were found to have a more mature phenotype than the corresponding CD4(+)CD8(-)CD25(-) cells with respect to expression of Thy1 (CD90), CD53 and CD44, suggesting that CD25 expression, and perhaps commitment to regulatory function, might be a late event in thymocyte development. CD4(+)CD25(+) cells in both the thymus and periphery were found to have enriched and heterogeneous expression of activation markers such as OX40 (CD134) and OX48 (an antibody determined in this study to be specific for CD86). CD4(+)CD25(+) T cells were also found to have enriched expression of CD80, at both the mRNA and protein level. However, functional studies in vitro and in vivo showed that neither OX40 or CD86 were useful markers for the further subdivision of regulatory T cells. Our studies indicate that, at present, CD25 remains the most useful marker to enrich for regulatory CD4(+) T cells in rats and no further subdivision of the regulatory component of CD4(+)CD25(-)CD45RC(low) T cells has yet been achieved.     

Ligation of the OX40 co‐stimulatory receptor reverses self‐Ag and tumor‐induced CD8 T‐cell anergy in vivo

Tumor‐specific CD8 T‐cell peripheral tolerance occurs through clonal deletion, suppression, and the induction of anergy and can limit the generation of anti‐tumor immunity. Several groups have demonstrated that prostate cancer can render tumor‐specific CD8 T cells anergic, suggesting reversing tumor‐induced anergy may greatly augment anti‐tumor immunity. Recent work has demonstrated that signaling through the OX40 (CD134) co‐stimulatory receptor, a member of the TNFR super‐family, can augment CD4 and CD8 T‐cell expansion, differentiation, and the generation of memory cells. However, whether OX40 ligation can reverse CD8 T‐cell anergy, and more specifically, tumor‐induced CD8 T‐cell anergy, remains unclear. In the current study, we demonstrate that OX40 ligation can reverse CD8 T‐cell anergy to a prostate‐specific self‐Ag in non‐tumor‐bearing hosts. Furthermore, OX40 engagement reversed tumor‐specific CD8 T‐cell anergy and restored the proliferative capacity of tumor‐reactive CD8 T cells, which attenuated tumor growth and enhanced the survival of tumor‐bearing hosts. These data demonstrate that OX40 ligation can rescue the function of anergic self‐ or tumor‐reactive CD8 T cells in vivo and suggests that OX40‐mediated therapy may provide a novel means of boosting anti‐tumor immunity by restoring the responsiveness of previously anergic tumor‐specific CD8 T cells.     

Costimulation through OX40 is crucial for induction of an alloreactive human T-cell response

The alloreactive immune response is a series of events initiated by the interaction of T cells with allogeneic dendritic cells (DCs), involving alloantigen recognition and costimulatory signals. In this study, we investigated the role of OX40 in alloreactivity in vitro. We first demonstrated that anti-OX40 ligand (anti-OX40L) monoclonal antibody (mAb) could markedly suppress the mixed leucocyte reaction (MLR) of peripheral blood mononuclear cells (PBMC). To further define the contribution of the OX40/OX40L system to the MLR, we set up a co-culture system of CD4+ T cells and allogeneic monocyte-derived dendritic cells (DCs). After 2 days, OX40 expression was induced on CD4+ T cells and this induction was strongly inhibited by the addition of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-Fc fusion protein, suggesting that the expression of OX40 during alloreaction is dependent on CD28 signalling. Next we examined the effects of anti-OX40L mAb, CTLA-4-Fc fusion protein and anti-human leucocyte antigen (HLA)-DR mAb on the proliferative response of CD4+ T cells to allogeneic DCs. The proliferation of T cells was almost completely suppressed by anti-OX40L mAb, which was comparable with that of CTLA-4-Fc. Measurement of interleukin-2 (IL-2) production in the culture supernatants showed that suppression of a proliferative response was at least in part ascribed to reduced IL-2 production. Furthermore, purified OX40L- allogeneic DCs could induce considerable proliferation of CD4+ T cells, which was suppressed by anti-OX40L mAb. These results suggest that the OX40/OX40L system is crucial for induction of the allogeneic T-cell response and the OX40/OX40L system is subsequent to and dependent on CD28 signalling, but is crucial for the end outcome of the human alloreactive T-cell response.     

OX40: targeted immunotherapy--implications for tempering autoimmunity and enhancing vaccines

OX40 (CD134), a membrane-bound member of the tumor-necrosis-factor-receptor superfamily, is expressed primarily on activated CD4+ T cells. Recently, several groups have reduced clinical signs of autoimmunity in animal models by blocking the OX40-OX40-ligand interaction or depleting OX40+ T cells. By contrast, engagement of OX40 in the setting of active immunization has potent adjuvant properties, leading to enhanced cytokine production and increased numbers of antigen-specific memory T cells. These potent adjuvant effects lead to an enhancement of anti-tumor responses. OX40 has several unique features that make it a clinically relevant target. They include: (1) T cells isolated from a site of inflammation that express OX40 are T cells that have been stimulated recentlythrough the T-cell receptor in vivo; (2) OX40 is only expressed on T cells found at the site of inflammation, therefore, targeting this receptor does not interfere with the peripheral T-cell repertoire; and (3) the biological function of OX40 is limited primarily to effector CD4+ T cells, which are a major source of cytokines to induce and maintain ongoing immune responses.     

Growth and Differentiation Advantages of CD4+OX40+ T Cells from Allogeneic Hematopoietic Stem Cell Transplantation Recipients

OX40 (CD134), an activation-induced costimulatory molecule, is mainly expressed on CD4⁺ T cells. Several reports, including previous reports from our laboratory, suggest that OX40-mediated signaling plays an important role in the development of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (Allo HSCT). Here, we show that peripheral blood CD4⁺OX40⁺ T cells are a unique cell subset as they possess the homing receptors of lymph nodes, and some of them have an exceptional capacity to produce high levels of interleukin-2 (IL-2) upon the stimulation through T cell receptors. Stimulation with IL-7 acts selectively on CD4⁺OX40⁺ T cells not only to induce antigen-independent growth but also to increase the frequency of cells with IL-2-producing potential. Simultaneous, but not sequential, ligation of the T cell receptor and OX40 induces CD4⁺OX40⁺ T cells to produce far more IL-2, which causes them to proliferate abundantly and differentiate readily into Th1- or Th2-biased effector memory T cells, especially in Allo HSCT recipients. Although not all the CD4⁺OX40⁺ T cells had IL-2-producing capacity, Allo HSCT recipients with chronic GVHD (cGVHD) had a significantly higher frequency of IL-2-producing OX40⁺ cells in their peripheral blood CD4⁺ T cell subset than Allo HSCT recipients without cGVHD. Collectively, CD4⁺OX40⁺ T cells with IL-2-producing potential are expected to be privileged for growth and differentiation in lymph nodes upon antigen presentation, suggesting that they might be involved in the process of inducing or maintaining cGVHD.     

CD4 T cell traffic control: in vivo evidence that ligation of OX40 on CD4 T cells by OX40-ligand expressed on dendritic cells leads to the accumulation of CD4 T cells in B follicles.

We report here that CD40- but not lipopolysaccharide (LPS)-activated murine dendritic cells (DC) express OX40-ligand (OX40L) as has been reported in humans. To understand how OX40 ligation affects differentiation of CD4 T cells at the time of priming, we constitutively expressed OX40L on DC using the DC-specific promoter of CD11c. Transgenic mice showed greatly increased numbers of CD4 but not CD8 T cells in their B cell areas. This effect was to a great extent immunization dependent, as spleen and lymphoid tissue with no germinal center reactions from mice which had not been deliberately immunized did not show marked CD4 T cell accumulation. The increased numbers of CD4+ CD62low cells in transgenic mice suggest that it is activated CD4 T cells that accumulate within B cell follicles. These data are consistent with the notion that physiological engagement of OX40 (CD134) on activated CD4 T cells either initiates their migration into or causes them to be retained in B follicles. In contrast, LPS-treated CD did not up-regulate OX40L expression. This dichotomy provides a molecular explanation of how DC might integrate environmental and accessory signals to control cytokine differentiation and migration in CD4 effector cells.     

Identification of a soluble OX40 isoform: development of a specific and quantitative immunoassay

OX40 (CD134) is a member of the TNF receptor family and is expressed selectively on activated T lymphocytes. Through interactions with OX40 ligand, OX40 delivers potent costimulatory signals to T cells. Here, we describe a sandwich ELISA method for the detection and quantification of OX40 using the monoclonal anti-OX40 antibodies ACT35 and L106 as capture and detecting antibodies, respectively. With this ELISA, the existence of naturally occurring soluble forms of OX40 (sOX40) could be demonstrated for the first time. Soluble OX40 is detectable in serum of subpopulations of healthy donors and patients with autoimmune disease and cancer. Among the five diseases that were investigated, chronic lymphocytic leukemia was identified as the one with the highest frequency of sOX40-positive sera and with the highest mean sOX40 serum concentration. The stability of sOX40 is not affected by repeated freeze/thaw cycles nor by prolonged storage at -20 degrees C, allowing studies on the potential diagnostic significance of sOX40 using existing banks of sera.     

Highly active antiretroviral therapy in patients infected with human immunodeficiency virus increases CD40 ligand expression and IL-12 production in cells ex vivo

Highly active anti-retroviral therapy (HAART) restores CD4(+) T-cell numbers in the periphery; however, its efficacy in restoring functional immunity is not fully elucidated. Here we evaluated longitudinal changes in the expression of several key markers of T-cell activation, namely CD40 ligand (CD154), OX40 (CD134), or CD69, after anti-CD3/CD28 activation, as well as levels of IL-12 production after Staphylococcus aureus Cowan stimulation in 28 HIV-infected adult patients. Patients were followed up to 12 mo post-HAART initiation. Viral burdens and CD4 cell counts were measured at the same time points. A control group of 15 HIV-uninfected adult subjects was included for comparison. Significant increases in CD40L and OX40 expression, but not of CD69 expression, were observed over time in the overall patient population, and more particularly in patients with baseline CD4 counts lower than or equal to 200 cells/μL, or those with baseline viral loads lower than or equal to 10(5) RNA copies/mL. Similar increases were seen for IL-12 production. Viral loads were inversely associated with CD40L expression or IL-12 production in a mixed linear model analysis, while CD4 counts were directly associated. CD40L expression and IL-12 production were significantly correlated. In conclusion, HAART-mediated control of viral replication led to partial restoration of CD40L upregulation/expression, and to increased IL-12 production, but the magnitude of the response depended on the baseline characteristics of the treated patients.     

Direct B/B-cell interactions in immunoglobulin synthesis

The principal roles of B/B-cell interactions in immune response have not yet been established. We therefore investigated B/B-cell interactions in immunoglobulin synthesis via direct cell-to-cell contact, particularly in the tumour necrosis factor receptor (TNFR)/tumour necrosis factor (TNF) family. We prepared highly purified peripheral blood B cells and stimulated them with Epstein--Barr virus (EBV)-transformed B lymphoblastoid cell lines (LCLs) as activated human B cells. The IgG production by B cells was increased by the addition of fixed LCLs in a dose-dependent manner in the presence of IL-10 plus IL-2. LCLs strongly expressed CD40 and CD70 on their surface, but marginal or no CD154, CD27, OX40 (CD134) and CD134 ligand. The enhancement of immunoglobulin production by LCLs was completely blocked by the initial addition of anti-CD70 blocking MoAb, but not by anti-CD154 or anti-CD134 ligand MoAb. The addition of LCLs also caused a reduction in CD27 expression on B cells, and this effect was completely blocked by anti-CD70 MoAb, indicating a direct B cell--LCL contact via CD27/CD70. LCLs markedly promoted B-cell differentiation into plasma cells in the presence of IL-10 plus IL-2. These findings demonstrate that direct interactions between B and B cells via CD27/CD70 induce immunoglobulin production by promoting the generation of plasma cells.     

Disruption of CD40/CD40 ligand interaction with cleavage of CD40 on human gingival fibroblasts by human leukocyte elastase resulting in down-regulation of chemokine production

CD40 is a crucial element in the process of fibroblast activation. We demonstrated that treatment of human gingival fibroblast (HGF) with human leukocyte elastase (HLE), a neutrophil serine protease, down-regulated the expression of CD40 and binding to the CD40 ligand (CD40L) using flow cytometry. The other neutrophil serine proteases, cathepsin G and proteinase 3, exhibited markedly less activity for CD40 reduction. The CD40 reduction by HLE was also observed in skin and lung fibroblasts, but not in monocytes, macrophages, and dendritic cells. The reduction resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD40 on fixed HGF and also on cell lysates and membranes. HLE treatment of HGF decreases interleukin (IL)-8 and macrophage chemoattractant protein-1 production by HGF when stimulated by CD40L, but not by IL-1alpha, suggesting that HLE inhibited a CD40-dependent cell activation. These results suggest that HLE possesses an anti-inflammatory effect for the HGF-mediated inflammatory process.     

Requirements for the functional expression of OX40 ligand on human activated CD4+ and CD8+ T cells

Interaction between OX40 expressed on activated T cells and its ligand (OX40L) on antigen presenting cells (APC) provides a co-stimulatory signal for T cells to promote acquired immunity. In the present study, we have examined various culture conditions for optimum OX40L expression on T cells stimulated with immobilized anti-CD3/CD28 monoclonal antibodies (mAbs). Although the day 3 primed T cells expressed minimal OX40L, after repeated stimulations both the CD4+ and CD8+ T cells became OX40L positive as determined by flow cytometry. Interleukin (IL)-12 interfered with the OX40L expression. Among activated T cells, a higher frequency of CD8+ T cells expressed OX40L than CD4+ T cells. By blocking OX40L-OX40 interaction by an anti-OX40 mAb, the number of OX40L+ T cells significantly increased. Screening of various cytokines showed that transforming growth factor (TGF)-beta1 was capable of induction of OX40L on the activated T cells within 3 days. The OX40L expressed on T cells was functional, as they bound soluble OX40 and stimulated human immunodeficiency virus-1 (HIV-1) production from cell lines chronically infected with HIV-1 and expressing OX40. Altogether the present study findings indicate that functional OX40L is inducible on human activated CD4+ and CD8+ T cells, and that the expression is enhanced by TGF-beta1.