Therapeutic effect of anti-OX40L and anti-TNF-alpha MAbs in a murine model of chronic colitis

Interaction of OX40 (CD134) on T cells with its ligand (OX40L) on antigen-presenting cells has been implicated in pathogenic T cell activation. This study was performed to explore the involvement of OX40/OX40L in the development of T cell-mediated chronic colitis. We evaluated both the preventive and therapeutic effects of neutralizing anti-OX40L MAb on the development of chronic colitis in SCID mice induced by adoptive transfer of CD4(+)CD45RB(high) T cells as an animal model of Crohn's disease. We also assessed the combination of anti-OX40L and anti-TNF-alpha MAbs to improve the therapeutic effect. Administration of anti-OX40L MAb markedly ameliorated the clinical and histopathological disease in preventive and therapeutic protocols. In vivo treatment with anti-OX40L MAb decreased CD4(+) T cell infiltration in the colon and suppressed IFN-gamma, IL-2, and TNF-alpha production by lamina propria CD4(+) T cells. The combination with anti-TNF-alpha MAb further improved the therapeutic effect by abolishing IFN-gamma, IL-2, and TNF-alpha production by lamina propria CD4(+) T cells. Our present results suggested a pivotal role of OX40/OX40L in the pathogenesis of T cell-mediated chronic colitis. The OX40L blockade, especially in combination with the TNF-alpha blockade, may be a promising strategy for therapeutic intervention of Crohn's disease.     

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 ligation enhances cell cycle turnover of Ag-activated CD4 T cells in vivo.

OX40 costimulates T cells, increases activated T cell longevity, and promotes memory acquisition. T cells activated in vivo with agonist anti-OX40 and ovalbumin have a unique pattern of survival and cell division compared to control cells, but are able to respond to recall Ag equally well. BrdU incorporation shows that early cellular division rates of the anti-OX40-treated and the control groups are similar. Nevertheless, more BrdU(+) Ag-specific T cells accumulate in lymphoid tissue upon anti-OX40 administration. Thus, OX40 ligation does not necessarily lead to increased cell cycle entry, but promotes the accumulation of dividing cells. However, CFSE staining shows that OX40 ligation allows cells to progress through more cellular division cycles, while control cells stall or die. Moreover, OX40 ligation leads to a proportional decrease in apoptotic Ag-specific T cells. Thus, OX40 ligation boosts immunity by promoting an increase in the number cell cycles completed, thereby increasing the life span of Ag-activated CD4 T cells.     

Amelioration of experimental autoimmune encephalomyelitis with anti-OX40 ligand monoclonal antibody: a critical role for OX40 ligand in migration, but not development, of pathogenic T cells

OX40 (CD134) and its ligand (OX40L) have been implicated in T cell activation and migration. In this study, we examined the contribution of these molecules to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) by administering a neutralizing mAb against murine OX40L (RM134L) to proteolipid protein (139-151) peptide-induced EAE in SJL mice. Administration of RM134L effectively ameliorated the disease in both actively induced and adoptively transferred EAE models. Histological examination showed that the RM134L treatment greatly reduced mononuclear cell infiltration into the spinal cord. The RM134L treatment did not inhibit the development of pathogenic T cells, given that proliferative response and IFN-gamma production by draining lymph node cells were not reduced or rather enhanced upon restimulation with proteolipid protein (139-151) in vitro, and these cells effectively transferred EAE to naive SJL mice. Flow cytometric analyses showed that the RM134L treatment inhibited the accumulation of OX40-expressing CD4(+) T cells and the migration of adoptively transferred CD4(+) T cells in the spinal cord. Immunohistochemical staining showed that OX40L was most prominently expressed on endothelial cells in the inflamed spinal cord. These results suggest that the OX40/OX40L interaction plays a critical role for the migration of pathogenic T cells into the CNS in the pathogenesis of EAE.     

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 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.     

Gastrointestinal dendritic cells promote Th2 skewing via OX40L

Mice can be sensitized to food proteins by oral administration with the adjuvant cholera toxin (CT), such that they undergo anaphylaxis when rechallenged with the sensitizing allergen. In contrast, feeding of Ags alone leads to oral tolerance. Our aim was to define the mechanisms by which gastrointestinal dendritic cells (DCs) participate in the deviation of tolerance to allergic sensitization in the gut in response to CT. BALB/c mice were fed with CT or PBS. The impact of CT on DC subsets in the mesenteric lymph node (MLN) was assessed by flow cytometry. Ag presentation assays were performed with DCs isolated from the MLN of PBS- or CT-fed mice, using OVA-specific CD4(+) T cells as responder cells. Gene expression in MLN DCs was determined by real-time PCR, and neutralizing Abs were used to test the function of OX40 ligand (OX40L) in Th2 skewing. Oral administration of CT induced an increase in the total CD11c(+) population in the MLN. CT induced a selective increase in migration of the CD11c(+)CD11b(-)CD8alpha(-) DC subset and the maturation of all DC subsets. Maturation of DCs in vivo enhanced T cell proliferation and cytokine secretion. Oral CT induced up-regulation of Jagged-2 and OX40L by MLN DCs. Neutralizing anti-OX40L Abs completely abrogated the CT-induced Th2 cytokine response. We show that oral CT induces selective DC migration, maturation, and T cell priming activity in the MLN. Th2 skewing is mediated by OX40L, and we speculate that this molecule may be an important inducer of allergic sensitization to food allergens.     

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.     

Transfer of T cell surface molecules to dendritic cells upon CD4+ T cell priming involves two distinct mechanisms

Activation of CD4(+) T cells by APCs occurs by multiple Ag recognition events including the exchange of costimulatory signals and cytokines. Additionally, the T cells acquire APC-derived surface molecules. Herein, we describe for the first time the transfer of human and murine T cell surface receptors to APCs after Ag-specific interaction. This transfer occurs in two qualitatively different phases. The first group of molecules (e.g., CD2) derived from the T cell surface was transferred rapidly after 2 h of interaction, was strongly bound on the DC surface (acid wash-resistant), was strictly dependent on dendritic cell-T cell contact, and transferred independently of T cell activation. The second group, including the CD3/TCR complex, CD27, and OX40, was of intracellular origin, transferred later after 10-16 h in a cell-cell contact-independent fashion, was noncovalently bound, and was strictly dependent on Ag-specific T cell activation. Functionally, murine dendritic cells that received TCR molecules from OVA-specific CD4(+) T cells after Ag-specific interaction were less efficient in priming naive CD4(+) T cells of the same specificity without losing their ability for CD8(+) T cell stimulation, indicating that the transferred TCR molecules mask the Ag-bearing MHC II molecules, thereby reducing their accessibility to following Ag-specific CD4(+) T cells. While the first group of transferred T cell surface molecules might facilitate the detachment of the CD4(+) T cell from the dendritic cell during the early scanning phases, the second group could play an important immunomodulatory role in intraclonal competition of T cells for APC access, making the physical presence of CD4(+) T cells unnecessary.     

IL-12 is required for anti-OX40-mediated CD4 T cell survival

Engagement of OX40 greatly improves CD4 T cell function and survival. Previously, we showed that both OX40 engagement and CTLA-4 blockade led to enhanced CD4 T cell expansion, but only OX40 signaling increased survival. To identify pathways associated with OX40-mediated survival, the gene expression of Ag-activated CD4 T cells isolated from mice treated with anti-OX40 and -CTLA-4 was compared. This comparison revealed a potential role for IL-12 through increased expression of the IL-12R-signaling subunit (IL-12Rbeta2) on T cells activated 3 days previously with Ag and anti-OX40. The temporal expression of IL-12Rbeta2 on OX40-stimulated CD4 T cells was tightly regulated and peaked approximately 4-6 days after initial activation/expansion, but before the beginning of T cell contraction. IL-12 signaling, during this window of IL-12Rbeta2 expression, was required for enhanced T cell survival and survival was associated with STAT4-specific signaling. The findings from these observations were exploited in several different mouse tumor models where we found that the combination of anti-OX40 and IL-12 showed synergistic therapeutic efficacy. These results may lead to the elucidation of the molecular pathways involved with CD4 T cell survival that contribute to improved memory, and understanding of these pathways could lead to greater efficacy of immune stimulatory Abs in tumor-bearing individuals.     

4-1BB and OX40 act independently to facilitate robust CD8 and CD4 recall responses

Mice deficient in OX40 or 4-1BB costimulatory pathways show defects in T cell recall responses, with predominant effects on CD4 vs CD8 T cells, respectively. However, OX40L can also stimulate CD8 T cells and 4-1BBL can influence CD4 T cells, raising the possibility of redundancy between the two TNFR family costimulators. To test this possibility, we generated mice deficient in both 4-1BBL and OX40L. In an adoptive transfer model, CD4 T cells expressed 4-1BB and OX40 sequentially in response to immunization, with little or no overlap in the timing of their expression. Under the same conditions, CD8 T cells expressed 4-1BB, but no detectable OX40. Thus, in vivo expression of 4-1BB and OX40 can be temporally and spatially segregated. In the absence of OX40L, there were decreased CD4 T cells late in the primary response and no detectable secondary expansion of adoptively transferred CD4 T cells under conditions in which primary expansion was unaffected. The 4-1BBL had a minor effect on the primary response of CD4 T cells in this model, but showed larger effects on the secondary response, although 4-1BBL(-/-) mice show less impairment in CD4 secondary responses than OX40L(-/-) mice. The 4-1BBL(-/-) and double knockout mice were similarly impaired in the CD8 T cell response, whereas OX40L(-/-) and double knockout mice were similarly impaired in the CD4 T cell response to both protein Ag and influenza virus. Thus, 4-1BB and OX40 act independently and nonredundantly to facilitate robust CD4 and CD8 recall responses.     

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.     

OX40 and Bcl-xL promote the persistence of CD8 T cells to recall tumor-associated antigen

The molecular signals that allow primed CD8 T cells to persist and be effective are particularly important during cancer growth. With response to tumor-expressed Ag following adoptive T cell transfer, we show that CD8 effector cells deficient in OX40, a TNFR family member, could not mediate short-term tumor suppression. OX40 was required at two critical stages. The first was during CD8 priming in vitro, in which APC-transmitted OX40 signals endowed the ability to survive when adoptively transferred in vivo before tumor Ag encounter. The second was during the in vivo recall response of primed CD8 T cells, the stage in which OX40 contributed to the further survival and accumulation of T cells at the tumor site. The lack of OX40 costimulation was associated with reduced levels of Bcl-x(L), and retroviral expression of Bcl-x(L) in tumor-reactive CD8 T cells conferred greatly enhanced tumor protection following adoptive transfer. These data demonstrate that OX40 and Bcl-x(L) can control survival of primed CD8 T cells and provide new insights into both regulation of CD8 immunity and control of tumors.     

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.     

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.     

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.     

OX40 ligand regulates splenic CD8 dendritic cell-induced Th2 responses in vivo

In mice, splenic conventional dendritic cells (cDCs) can be separated, based on their expression of CD8α into CD8⁻ and CD8⁺ cDCs. Although previous experiments demonstrated that injection of antigen (Ag)-pulsed CD8⁻ cDCs into mice induced CD4 T cell differentiation toward Th2 cells, the mechanism involved is unclear. In the current study, we investigated whether OX40 ligand (OX40L) on CD8⁻ cDCs contributes to the induction of Th2 responses by Ag-pulsed CD8⁻ cDCs in vivo, because OX40–OX40L interactions may play a preferential role in Th2 cell development. When unseparated Ag-pulsed OX40L-deficient cDCs were injected into syngeneic BALB/c mice, Th2 cytokine (IL-4, IL-5, and IL-10) production in lymph node cells was significantly reduced. Splenic cDCs were separated to CD8⁻ and CD8⁺ cDCs. OX40L expression was not observed on freshly isolated CD8⁻ cDCs, but was induced by anti-CD40 mAb stimulation for 24 h. Administration of neutralizing anti-OX40L mAb significantly inhibited IL-4, IL-5, and IL-10 production induced by Ag-pulsed CD8⁻ cDC injection. Moreover, administration of anti-OX40L mAb with Ag-pulsed CD8⁻ cDCs during a secondary response also significantly inhibited Th2 cytokine production. Thus, OX40L on CD8⁻ cDCs physiologically contributes to the development of Th2 cells and secondary Th2 responses induced by Ag-pulsed CD8⁻ cDCs in vivo.