Intracellular signaling of gp34, the OX40 ligand: induction of c-jun and c-fos mRNA expression through gp34 upon binding of its receptor, OX40.

We investigated the intracellular signaling events of OX40 ligand (gp34), a member of the TNF family. To elucidate the intracellular signaling via gp34, we prepared a model system in which a human gp34-transfected mouse epithelial cell line was stimulated with a recombinant soluble form of OX40. We demonstrated that OX40 binding resulted in increase in c-jun and c-fos mRNA levels in this transfectant by Northern blot analysis, which was blocked by the pretreatment with anti-gp34 Ab. The studies with various gp34 deletion mutants showed that the cytoplasmic portion including the amino acid sequence 16-21 (RPRFER) was required for the induction of c-jun and c-fos mRNA expression. Furthermore, OX40 binding induced c-jun mRNA expression also in HUVECs, which in our previous study have been shown to express gp34 and interact with activated T cells through the OX40/gp34 pathway. On the other hand, c-fos mRNA was detectable neither in unstimulated HUVECs nor in gp34-stimulated HUVECs. These results indicate that the OX40/gp34 system generates two-way signals and may elicit biological effects on vascular endothelial cells.     

Molecular characterization of murine and human OX40/OX40 ligand systems: identification of a human OX40 ligand as the HTLV-1-regulated protein gp34.

A ligand was cloned for murine OX40, a member of the TNF receptor family, using a T cell lymphoma cDNA library. The ligand (muOX40L) is a type II membrane protein with significant identity to human gp34 (gp34), a protein whose expression on HTLV-1-infected human leukemic T cells is regulated by the tax gene. The predicted structures of muOX40L and gp34 are similar to, but more compact than, those of other ligands of the TNF family. Mapping of the muOX40L gene revealed tight linkage to gld, the FasL gene, on chromosome 1. gp34 maps to a homologous region in the human genome, 1q25. cDNAs for human OX40 receptor were cloned by cross-hybridization with muOX40, and gp34 was found to bind the expressed human receptor. Lymphoid expression of muOX40L was detected on activated T cells, with higher levels found on CD4+ rather than CD8+ cells. The cell-bound recombinant ligands are biologically active, co-stimulating T cell proliferation and cytokine production. Strong induction of IL-4 secretion by muOX40L suggests that this ligand may play a role in regulating immune responses. In addition, the HTLV-1 regulation of gp34 suggests a possible connection between virally induced pathogenesis and the OX40 system.     

OX40 ligation of CD4+ T cells enhances virus-specific CD8+ T cell memory responses independently of IL-2 and CD4+ T regulatory cell inhibition

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.     

Characterization of rat OX40 ligand by monoclonal antibody

OX40 (CD134) is a member of the tumor necrosis factor (TNF) receptor superfamily first identified as a rat T cell activation marker. We previously identified the rat ligand for OX40 (OX40L) by molecular cloning. In the present study, we newly generated an anti-rat OX40L mAb (ATM-2) that can inhibit the binding of OX40 to rat OX40L and thus efficiently inhibits the T cell costimulatory activity of rat OX40L. Flow cytometric analyses using ATM-2 and an anti-rat OX40 mAb (MRC OX40) indicated that OX40 was inducible on splenic CD4(+) T cells by stimulation with immobilized anti-CD3 mAb, while OX40L was not expressed on resting or activated T cells. OX40L was expressed on splenic B cells after stimulation with lipopolysaccharide (LPS), but not on peritoneal macrophages. Interestingly, splenic dendritic cells (DC) expressed OX40L constitutively, which was further upregulated by LPS stimulation. The potent costimulatory activities of splenic DC for anti-CD3-stimulated rat CD4(+) T cell proliferation and cytokine (IL-2, IFN-gamma, IL-10, and IL-13) production were substantially inhibited by ATM-2. These results indicated that OX40L is expressed on professional antigen-presenting cells (APC), and may be involved in humoral immune responses via T-B interaction and in cellular immune responses via T-DC interaction in the rat system.     

Constitutive OX40/OX40 ligand interaction induces autoimmune-like diseases

The interaction between OX40 and OX40 ligand (OX40L) is suggested to provide T cells with an effective costimulatory signals during T cell-APC interaction. To examine the in vivo effect of constitutive OX40/OX40L interaction during immune regulation, we report the establishment of OX40L-transgenic (OX40L-Tg) mice that constitutively express OX40L on T cells. Markedly elevated numbers of effector memory CD4(+) T cells, but not CD8(+) T cells, were observed in the secondary lymphoid organs of OX40L-Tg mice. Upon immunization with keyhole limpet hemocyanin in the absence of adjuvant, profound T cell proliferative responses and cytokine productions were seen in the OX40L-Tg mice as compared with wild-type mice. Furthermore, in OX40L-Tg mice administrated with superantigen, this constitutive OX40/OX40L interaction on CD4(+) T cells completely prevented normal in vivo clonal T cell deletion. Interestingly, OX40L-Tg mice on the C57BL/6 background spontaneously developed interstitial pneumonia and inflammatory bowel disease that was accompanied with a significant production of anti-DNA Ab in the sera. Surprisingly, these diseases were not evident on the OX40L-Tg mice on the BALB/c strain. However, such inflammatory diseases were successfully reproducible in recombination-activating gene (RAG)2-deficient mice upon transfer of OX40L-Tg CD4(+) T cells. Blockade of OX40/OX40L interaction in the recipient RAG2-deficient mice completely prevented disease development. The present results orchestrated in this study indicate that OX40/OX40L interaction may be a vital link in our understanding of T cell-mediated organ-specific autoimmunity.     

HIV-1 envelope triggers polyclonal Ig class switch recombination through a CD40-independent mechanism involving BAFF and C-type lectin receptors

Switching from IgM to IgG and IgA is essential for antiviral immunity and requires engagement of CD40 on B cells by CD40L on CD4(+) T cells. HIV-1 is thought to impair CD40-dependent production of protective IgG and IgA by inducing progressive loss of CD4(+) T cells. Paradoxically, this humoral immunodeficiency is associated with B cell hyperactivation and increased production of nonprotective IgG and IgA that are either nonspecific or specific for HIV-1 envelope glycoproteins, including gp120. Nonspecific and gp120-specific IgG and IgA are sensitive to antiretroviral therapy and remain sustained in infected individuals with very few CD4(+) T cells. One interpretation is that some HIV-1 Ags elicit IgG and IgA class switch DNA recombination (CSR) in a CD40-independent fashion. We show that a subset of B cells binds gp120 through mannose C-type lectin receptors (MCLRs). In the presence of gp120, MCLR-expressing B cells up-regulate the CSR-inducing enzyme, activation-induced cytidine deaminase, and undergo CSR from IgM to IgG and IgA. CSR is further enhanced by IL-4 or IL-10, whereas Ab secretion requires a B cell-activating factor of the TNF family. This CD40L-related molecule is produced by monocytes upon CD4, CCR5, and CXCR4 engagement by gp120 and cooperates with IL-4 and IL-10 to up-regulate MCLRs on B cells. Thus, gp120 may elicit polyclonal IgG and IgA responses by linking the innate and adaptive immune systems through the B cell-activating factor of the TNF family. Chronic activation of B cells through this CD40-independent pathway could impair protective T cell-dependent Ab responses by inducing immune exhaustion.     

Multiple levels of activation of murine CD8(+) intraepithelial lymphocytes defined by OX40 (CD134) expression: effects on cell-mediated cytotoxicity, IFN-gamma, and IL-10 regulation.

The involvement of OX40 (CD134) in the activation of CD8(+) intestinal intraepithelial lymphocytes (IELs) has been studied using freshly isolated IELs and in vitro CD3-stimulated IELs. Although freshly isolated CD8(+) IELs exhibited properties of activated T cells (CD69 expression and ex vivo cytotoxicity), virtually all CD8(+) IELs from normal mice were devoid of other activation-associated properties, including a lack of expression of OX40 and the ligand for OX40 (OX40L) and an absence of intracellular IFN-gamma staining. However, OX40 and OX40L expression were rapidly up-regulated on CD8 IELs following CD3 stimulation, indicating that both markers on IELs reflect activation-dependent events. Unlike IELs, activated lymph node T cells did not express OX40L, thus indicating that OX40-OX40L communication in the intestinal epithelium is part of a novel CD8 network. Functionally, OX40 expression was exclusively associated with IELs with active intracellular IFN-gamma synthesis and markedly enhanced cell-mediated cytotoxicity. However, OX40 costimulation during CD3-mediated activation significantly suppressed IL-10 synthesis by IELs, whereas blockade of OX40-OX40L by anti-OX40L mAb markedly increased IL-10 production. These findings indicate that: 1) resident CD69(+)OX40(-) IELs constitute a population of partially activated T cells poised for rapid delivery of effector activity, 2) OX40 and OX40L expression defines IELs that have undergone recent immune activation, 3) OX40(+) IELs are significantly more efficient CTL than are OX40(-) IELs, and 4) the local OX40/OX40L system plays a critical role in regulating the magnitude of cytokine responses in the gut epithelium.     

OX40/OX40L costimulation affects induction of Foxp3+ regulatory T cells in part by expanding memory T cells in vivo

OX40 is a member of the TNFR superfamily and has potent T cell costimulatory activities. OX40 also inhibits the induction of Foxp3(+) regulatory T cells (Tregs) from T effector cells, but the precise mechanism of such inhibition remains unknown. In the present study, we found that CD4(+) T effector cells from OX40 ligand-transgenic (OX40Ltg) mice are highly resistant to TGF-beta mediated induction of Foxp3(+) Tregs, whereas wild-type B6 and OX40 knockout CD4(+) T effector cells can be readily converted to Foxp3(+) T cells. We also found that CD4(+) T effector cells from OX40Ltg mice are heterogeneous and contain a large population of CD44(high)CD62L(-) memory T cells. Analysis of purified OX40Ltg naive and memory CD4(+) T effector cells showed that memory CD4(+) T cells not only resist the induction of Foxp3(+) T cells but also actively suppress the conversion of naive CD4(+) T effector cells to Foxp3(+) Tregs. This suppression is mediated by the production of IFN-gamma by memory T cells but not by cell-cell contact and also involves the induction of T-bet. Importantly, memory CD4(+) T cells have a broad impact on the induction of Foxp3(+) Tregs regardless of their origins and Ag specificities. Our data suggest that one of the mechanisms by which OX40 inhibits the induction of Foxp3(+) Tregs is by inducing memory T cells in vivo. This finding may have important clinical implications in tolerance induction to transplanted tissues.     

Critical involvement of OX40 ligand signals in the T cell priming events during experimental autoimmune encephalomyelitis

OX40 ligand (OX40L) expressed on APCs, and its receptor, OX40 present on activated T cells, are members of the TNF/TNFR family, respectively, and have been located at the sites of inflammatory conditions. We have observed in OX40L-deficient mice (OX40L(-/-)) an impaired APC capacity and in our recently constructed transgenic mice expressing OX40L (OX40L-Tg), a markedly enhanced T cell response to protein Ags. Using these mice, we demonstrate here the critical involvement of the OX40L-OX40 interaction during the T cell priming events in the occurrence of experimental autoimmune encephalomyelitis (EAE). In OX40L(-/-) mice, abortive T cell priming greatly reduced the clinical manifestations of actively induced EAE, coupled with a reduction in IFN-gamma, IL-2, and IL-6 production in vitro. Adoptive transfer experiments however revealed an efficient transfer of disease to OX40L(-/-) mice using wild-type donor T cells, indicating an intact capacity of OX40L(-/-) mice to initiate effector responses. On the other hand, OX40L(-/-) donor T cells failed to transfer disease to wild-type recipient mice. Furthermore, OX40L-Tg mice developed a greater severity of EAE despite a delayed onset, while both OX40L-Tg/CD28(-/-) and OX40L-Tg/CD40(-/-) mice failed to develop EAE demonstrating a requisite for these molecules. These findings indicate a pivotal role played by OX40L in the pathogenesis of EAE.     

OX40 ligation on activated T cells enhances the control of Cryptococcus neoformans and reduces pulmonary eosinophilia

Pulmonary eosinophilia induced in C57BL/6 mice after Cryptococcus neoformans infection is driven by CD4(+) Th2 cells. The immunological mechanisms that protect against eosinophilia are not fully understood. Interaction of OX40 (CD134) and its ligand, OX40L, has been implicated in T cell activation and cell migration. Unlike CD28, OX40 is only expressed on T cells 1-2 days after Ag activation. Manipulation of this pathway would therefore target recently activated T cells, leaving the naive repertoire unaffected. In this study, we show that engagement of OX40 by an OX40L:Ig fusion protein drives IFN-gamma production by CD4(+) T cells and reduces eosinophilia and C. neoformans burden in the lung. Using gene-depleted mice, we show that reduction of eosinophilia and pathogen burden requires IL-12 and/or IFN-gamma. C. neoformans infection itself only partially induces OX40L expression by APCs. Provision of exogenous OX40L reveals a critical role of this pathway in the prevention of C. neoformans-induced eosinophilia.     

Mice deficient in OX40 and CD30 signals lack memory antibody responses because of deficient CD4 T cell memory

Recently, we reported that a CD4(+)CD3(-)CD11c(-) accessory cell provided OX40-dependent survival signals to follicular T cells. These accessory cells express both OX40 ligand and CD30 ligand, and the receptors, OX40 and CD30, are both expressed on Th2-primed CD4 T cells. OX40 and CD30 signals share common signaling pathways, suggesting that CD30 signals might substantially compensate in OX40-deficient mice. In this report we have dissected the signaling roles of CD30 alone and in combination with OX40. CD30-deficient mice showed an impaired capacity to sustain follicular germinal center responses, and recall memory Ab responses were substantially reduced. Deficiencies in OX40 and CD30 signals were additive; secondary Ab responses were ablated in double-deficient mice. Although the initial proliferation of OX40/CD30 double-knockout OTII transgenic T cells was comparable to that of their normal counterparts, they failed to survive in vivo, and this was associated with reduced T cell numbers associated with CD4(+)CD3(-) cells in B follicles. Finally, we show that OX40/CD30 double-knockout OTII transgenic T cells fail to survive compared with normal T cells when cocultured with CD4(+)CD3(-) cells in vitro.     

OX40 costimulatory signals potentiate the memory commitment of effector CD8+ T cells

A T cell costimulatory molecule, OX40, contributes to T cell expansion, survival, and cytokine production. Although several roles for OX40 in CD8(+) T cell responses to tumors and viral infection have been shown, the precise function of these signals in the generation of memory CD8(+) T cells remains to be elucidated. To address this, we examined the generation and maintenance of memory CD8(+) T cells during infection with Listeria monocytogenes in the presence and absence of OX40 signaling. We used the expression of killer cell lectin-like receptor G1 (KLRG1), a recently reported marker, to distinguish between short-lived effector and memory precursor effector T cells (MPECs). Although OX40 was dispensable for the generation of effector T cells in general, the lack of OX40 signals significantly reduced the number and proportion of KLRG1(low) MPECs, and, subsequently, markedly impaired the generation of memory CD8(+) T cells. Moreover, memory T cells that were generated in the absence of OX40 signals in a host animal did not show self-renewal in a second host, suggesting that OX40 is important for the maintenance of memory T cells. Additional experiments making use of an inhibitory mAb against the OX40 ligand demonstrated that OX40 signals are essential during priming, not only for the survival of KLRG1(low) MPECs, but also for their self-renewing ability, both of which contribute to the homeostasis of memory CD8(+) T cells.     

Functional CD4 T cells after intercellular molecular transfer of 0X40 ligand.

OX40/OX40 ligand (OX40L) proteins play critical roles in the T cell-B cell and T cell-dendritic cell interactions. Here we describe the intercellular transfer of OX40L molecules by a non-Ag specific manner. After 2-h coculture of activated CD4(+) T cell (OX40L(-), OX40(+)) with FLAG peptide-tagged OX40L (OX40L-flag) protein-expressing COS-1 cells, the OX40L-flag protein was detected on the cell surface of the CD4(+) T cells by both anti-OX40L and anti-FLAG mAbs. The intercellular OX40L transfer was specifically abrogated by pretreatment of the COS-1 cells with anti-OX40L mAb, 5A8. The OX40L transfer to OX40-negative cells was also observed, indicating an OX40-independent pathway of OX40L transfer. HUVECs, allostimulated monocytes, and human T cell leukemia virus type I-infected T cells, which all express OX40L, can potentially act as the donor cells of OX40L. The entire molecule of OX40L was transferred and stabilized on the recipient cell membrane with discrete punctate formation. The transferred OX40L on normal CD4(+) T cells was functionally active as they stimulated latent HIV-1-infected cells to produce viral proteins via OX40 signaling. Therefore, these findings suggest that the intercellular molecular transfer of functional OX40L may be involved in modifying the immune responses.     

OX40 signals during priming on dendritic cells inhibit CD4 T cell proliferation: IL-4 switches off OX40 signals enabling rapid proliferation of Th2 effectors

In this study we examined the role and regulation of OX40 signals during CD4 T cell priming on dendritic cells (DCs). Contrary to expectation, OX40-deficient cells proliferated more rapidly than their normal counterparts, particularly when stimulated with peptide in the absence of added cytokines. This proliferative advantage was not apparent for Th2-differentiated cells. When the reasons for this were investigated, we found that the cytokine IL-4 specifically down-regulated expression of OX40 ligand on T, B, and DCs, but not on the CD4(+)CD3(-) cells linked with selection of Th2 cells into the memory compartment. OX40 ligand expression was also down-regulated on rapidly proliferating Th1 effectors. These data are compatible with OX40 signals acting during priming as a check on naive T cell proliferation while T cells integrate additional DC signals. This would serve to limit inappropriate T cell responses. In contrast, OX40 signals from CD4(+)CD3(-) cells located in the outer T zone select proliferating Th2 effectors into the memory T cell pool.     

A recombinant vector expressing transgenes for four T-cell costimulatory molecules (OX40L,B7-1, ICAM-1, LFA-3) induces sustained CD4+ and CD8+ T-cell activation,protection from apoptosis,and enhanced cytokine production

The role of OX40L on the activation of T cells was investigated using poxvirus vectors expressing OX40L alone or in combination with three other T-cell costimulatory molecules: B7-1, ICAM-1, and LFA-3. Poxvirus vector-infected cells were used to stimulate nai;ve or activated CD4(+) and CD8(+) T cells. These studies demonstrate that (a) OX40L plays a role in sustaining the long-term proliferation of CD8(+) T cells in addition to the known effect on CD4(+) T cells following activation, (b) OX40L enhances the production of Th1 cytokines (IL-2, IFN-gamma, and TNF-alpha) from both CD4(+) and CD8(+) while no change in IL-4 expression was observed, and (c) the anti-apoptotic effect of OX40L on T cells is likely the result of sustained expression of anti-apoptotic genes while genes involved in apoptosis are inhibited. In addition, these are the first studies to demonstrate that the combined use of a vector driving the expression of OX40L with three other costimulatory molecules (B7-1, ICAM-1, and LFA-3) both enhances initial activation and then further potentiates sustained activation of nai;ve and effector T cells.     

The HTLV-I tax protein transcriptionally modulates OX40 antigen expression

OX40 is a member of the TNF receptor family, expressed on activated T cells. It is the only costimulatory T cell molecule known to be specifically up-regulated in human T cell leukemia virus type-I (HTLV-I)-producing cells. In a T cell line, OX40 surface expression was shown to be induced by HTLV-I Tax alone. To understand molecular mechanisms of OX40 gene regulation and modulation by HTLV-I Tax, we have cloned the human OX40 gene and analyzed its 5'-flanking region. By reporter gene analysis with progressive 5' deletions from nucleotides -1259 to -64, we have defined a 157-bp DNA fragment as a minimal promoter for constitutive expression. In addition, we show that in the OX40+ cell line, Co, Tax is able to further increase OX40 surface expression. Up-regulation of OX40 promoter activity by Tax requires two upstream NF-kappaB sites, which are not active in the constitutive OX40 expression. Their deletion abrogates Tax responsiveness in reporter gene analysis. The site-directed mutagenesis of each NF-kappaB site demonstrates that cooperative NF-kappaB binding is a prerequisite for Tax-directed activity as neither site alone is sufficient for a full Tax responsiveness of the OX40 promoter. Upon Tax expression, both sites bind p65 and c-Rel. These data provide new insight into the direct regulation of OX40 by Tax and add to our understanding of the possible role of the OX40/OX40 ligand system in the proliferation of HTLV-I+ T cells.     

Differential requirements for OX40 signals on generation of effector and central memory CD4+ T cells

Memory T cells can be divided into effector memory (T(EM)) and central memory (T(CM)) subsets based on their effector function and homing characteristics. Although previous studies have demonstrated that TCR and cytokine signals mediate the generation of the two memory subsets of CD8(+) T cells, the mechanisms for generation of the CD4(+) T(EM) and T(CM) cell subsets are unknown. We found that OX40-deficient mice showed a marked reduction in the number of CD4(+) T(EM) cells, whereas the number of CD4(+) T(CM) cells was normal. Adoptive transfer experiments using Ag-specific CD4(+) T cells revealed that OX40 signals during the priming phase were indispensable for the optimal generation of the CD4(+) T(EM), but not the CD4(+) T(CM) population. In a different transfer experiment with in vitro established CD4(+)CD44(high)CD62L(low) (T(EM) precursor) and CD4(+)CD44(high)CD62L(high) (T(CM) precursor) subpopulations, OX40-KO T(EM) precursor cells could not survive in the recipient mice, whereas wild-type T(EM) precursor cells differentiated into both T(EM) and T(CM) cells. In contrast, T(CM) precursor cells mainly produced T(CM) cells regardless of OX40 signals, implying the dispensability of OX40 for generation of T(CM) cells. Nevertheless, survival of OX40-KO T(EM) cells was partially rescued in lymphopenic mice. During in vitro recall responses, the OX40-KO T(EM) cells that were generated in lymphopenic recipient mice showed impaired cytokine production, suggesting an essential role for OX40 not only on generation but also on effector function of CD4(+) T(EM) cells. Collectively, the present results indicate differential requirements for OX40 signals on generation of CD4(+) T(EM) and T(CM) cells.     

OX40 controls functionally different T cell subsets and their resistance to depletion therapy

T cell depletion is a widely used approach in clinical transplantation. However, not all T cells are equally sensitive to depletion therapies and a significant fraction of T cells persists even after aggressive treatment. The functional attributes of such T cells and the mechanisms responsible for their resistance to depletion are poorly studied. In the present study, we showed that CD4(+) T cells that are resistant to polyclonal anti-lymphocyte serum (ALS) mediated depletion exhibit phenotypic features of memory cells and uniformly express OX40 on the cell surface. Studies using the foxp3gfp knockin mice revealed that the remaining CD4(+)OX40(+) cells consist of Foxp3(+) Tregs and Foxp3(-) T effector/memory cells. The ALS-resistant CD4(+)OX40(+) cells failed to mediate skin allograft rejection upon adoptive transferring into congenic Rag(-/-) mice, but removal of Foxp3(+) Tregs from the OX40(+) cells resulted in prompt skin allograft rejection. Importantly, OX40 is critical to survival of both Foxp3(+) Tregs and T effector/memory cells. However, OX40 exhibits opposing effects on the functional status of Foxp3(+) Tregs and T effector/memory cells, as stimulation of OX40 on T effector cells induced amplified cell proliferation but stimulation of OX40 on the Foxp3(+) Tregs impaired their suppressor functions. Our study demonstrates that OX40 is a critical molecule in regulating survival and functions of depletion-resistant T cells; and these findings may have important clinical implications.