OX40 Stimulation Research Articles

Dual Immune Checkpoint Agents in Combination with Rituximab for Patients with Relapsed or Refractory Follicular Lymphoma

Background: Follicular lymphoma (FL) is an inherently immunosensitive cancer. While PD-1 blockade was associated with a low objective response rate (ORR) in FL, some durable responses were seen. Manipulation of other checkpoint pathways, like 4-1BB, have shown promising activity (Gopal, CCR 2020) and OX40 stimulation may enhance activity of other immunotherapies. We therefore conducted a multi-cohort phase 1b trial testing different combinations of immune checkpoint therapy targeting 4-1BB (utomilumab), OX-40 (PF-04518600), and PD-L1 (avelumab) all in combination with rituximab (R) among patients (pts) with relapsed/refractory (R/R) FL. Methods: Adult pts with R/R grade 1-3a FL who had received ≥ 1 prior therapies (including a CD20 monoclonal antibody) were enrolled. All pts had measurable disease and required therapy based on modified GELF criteria. Pts in need of urgent cytoreductive therapy were excluded. Pts were enrolled onto 2 of 3 planned cohorts (A1 - R, utomilumab, avelumab; A2 - PF-04518600 +/- R, utomilumab). The trial was closed early when development of utomilumab and PF-04518600 was halted by Pfizer. Enrollment ended before dose expansion was completed for cohorts A1 and A2 and before enrollment began for a 3rd cohort (R, avelumab, PF-04518600). Each cohort began with a dose escalation stage (using a standard 3+3 design) followed by a dose expansion stage at the recommended phase 2 dose (RP2D) (15 pts). Treatment consisted of six 28-day cycles for both cohorts (see table for dose levels and dosing details). The co-primary endpoints were RP2D and complete response rate (CRR), assessed using 2014 Lugano criteria. Peripheral blood, stool samples, and tissue biopsies were collected for correlative studies. Results: 24 pts were enrolled at 4 centers, including 1 pt who was initially treated on cohort A2 (with R + PF-04518600), ended treatment due to progression, and then enrolled on cohort A1. In total, 16 pts were treated on cohort A1 and 9 on cohort A2. The median age was 58 (42-76). Pts had received a median of 2 (1-9) prior therapies. 11 pts (46%) had progression within 24 months of initial therapy (POD24). At trial entry, 6 pts (25%) were R-refractory, defined as no response to last R-containing regimen or progression ≤6 months after last R dose. The highest planned dose level (dose level 2) was selected as the RP2D for cohorts A1 and A2. There were no treatment discontinuations due to adverse events (AEs) and no treatment-related deaths. 1 dose-limiting toxicity (grade 3 gastric obstruction, unrelated to study treatment) was observed for a pt in cohort A1. In total, 19/25 (76%) pts had at least 1 treatment-related (tr)AE of any grade, including 7 (28%) pts with grade 3 trAEs. Typical immune-related AEs were not observed. The most common trAEs of any grade were fatigue (24%) and infusion reaction (20%). Grade 3 trAEs included neutropenia in 2 pts (8%), and hypertension, hypertriglyceridemia, hypophosphatemia, lymphopenia, COVID infection, and thromboembolic event in 1 pt each (4%). In cohort A1, the ORR and CRR were 44% and 19%, respectively. Among 10 pts treated at the RP2D, the ORR and CRR were 50% and 30%, respectively. ORRs were observed in 2/5 POD24 pts and 1/4 R-refractory pts in cohort A1. In cohort A2, no responses were observed. After a median follow-up of 16 months, the median progression-free survivals in cohorts A1 and A2 were 7.0 and 3.2 months (m), respectively. The median duration of response in A1 was 9.7m. The 18m overall survivals in cohorts A1 and A2 were 81% and 100%, respectively. We performed 16S ribosomal RNA sequencing of pre- and on-treatment stool samples from pts in cohort A1 and found a positive association between abundance of Akkermansia and ORR to study therapy (p<0.001). Other correlative studies including multiplex immunofluorescence and single-cell analyses (assessing serial biopsy samples) are ongoing. Conclusions: We observed clinical activity with the combination of utomilumab, avelumab, and R, supporting a possible role for 4-1BB agonist therapy in FL. In contrast, our limited results do not support a role for OX40 targeting in this disease. Correlatives studies are ongoing which may shed further light on predictors of response. Interestingly, we found an association between Akkermansia and response, which has been reported in checkpoint blockade studies in solid tumors and supports the hypothesis that the microbiome impacts response to immunotherapies in lymphomas. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Explant Modeling of the Immune Environment of Head and Neck Cancer.

Patients exhibit distinct responses to immunotherapies that are thought to be linked to their tumor immune environment. However, wide variations in outcomes are also observed in patients with matched baseline tumor environments, indicating that the biological response to treatment is not currently predictable using a snapshot analysis. To investigate the relationship between the immune environment of tumors and the biological response to immunotherapies, we characterized four murine head and neck squamous cell carcinoma (HNSCC) models on two genetic backgrounds. Using tumor explants from those models, we identified correlations between the composition of infiltrating immune cells and baseline cytokine profiles prior to treatment. Following treatment with PD-1 blockade, CTLA-4 blockade, or OX40 stimulation, we observed inter-individual variability in the response to therapy between genetically identical animals bearing the same tumor. These distinct biological responses to treatment were not linked to the initial tumor immune environment, meaning that outcome would not be predictable from a baseline analysis of the tumor infiltrates. We similarly performed the explant assay on patient HNSCC tumors and found significant variability between the baseline environment of the tumors and their response to therapy. We propose that tumor explants provide a rapid biological assay to assess response to candidate immunotherapies that may allow matching therapies to individual patient tumors. Further development of explant approaches may allow screening and monitoring of treatment responses in HNSCC.

New pathways in immune stimulation: targeting OX40

Immune checkpoint blockers (ICB) reinvigorate the immune system by removing the molecular brakes responsible for the scarce activity of immune phenotypes against malignant cells. After having proven their remarkable role as monotherapy, combinations of anti-Programmed cell death 1 (PD-1)/Programmed death-ligand 1 (PD-L1) agents with cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) antibodies, chemotherapy and/or anti-angiogenic compounds provide unprecedented results and durable responses across a variety of tumour types. Nevertheless, the main drawbacks of ICB are represented by primary and acquired resistance, translating into disease progression, as well as by immune-related toxicities. In this sense, novel strategies to foster the immune system through its direct stimulation are being tested in order to provide additional clinical improvements in patients with cancer. In this scenario, the co-stimulatory molecule OX40 (CD134) belongs to the next generation of immune therapeutic targets. Preliminary results of early clinical trials evaluating OX40 stimulation by means of different agents are encouraging. Here we review the rationale of OX40 targeting, highlighting the combination of OX40-directed therapies with different anticancer agents as a potential strategy to foster the immune system against malignant phenotypes.

From the Editor’s desk…: June 2019

From the Editor’s desk…: June 2019

OX40 stimulation and PD-L1 blockade synergistically augment HBV-specific CD4 T cells in patients with HBeAg-negative infection

Current antiviral therapies lack the potential to eliminate persistent hepatitis B virus (HBV) infection. HBV-specific T cells are crucial for HBV control and have recently been shown to be protective in patients following discontinuation of antiviral therapy. Thus, T cell-based approaches may greatly improve the therapeutic landscape of HBV infection. We aimed to augment HBV-specific CD4 T cells from chronically infected patients by targeting different immunological pathways. Expression of various co-stimulatory and inhibitory receptors on HBV- and influenza-specific CD4 T cells was analyzed directly ex vivo by MHC class II-tetramers. Patients infected with HBV genotype D were screened for CD4 T cell responses by IFN-γ ELISpot and intracellular cytokine staining following stimulation with overlapping peptides (OLPs) spanning the HBV-polyprotein. Stimulation with recombinant IL-7, an agonistic OX40-antibody or blockade of PD-L1 was performed in antigen-specific in vitro cultures. Cytokine secretion and expression of transcription factors were analyzed by flow cytometry. Responses targeting influenza, Epstein-Barr virus and tetanus toxoid served as controls. Tetramer-staining revealed that the IL-7 receptor-alpha (CD127), OX40 and PD-1 constitute possible therapeutic targets as they were all strongly expressed on HBV-specific CD4 T cells ex vivo. The HBV-specific CD4 T cell responses identified by OLP screening targeted predominantly the HBV-polymerase and core proteins. Combined OX40 stimulation and PD-L1 blockade significantly augmented IFN-γ and IL-21 producing HBV-specific CD4 T cells in vitro, suggesting active T helper type 1 cell and follicular T helper cell programs. Indeed, transcription factors T-bet and Bcl6 were strongly expressed in cytokine-producing cells. Combined OX40 stimulation and PD-L1 blockade augmented secretion of the helper T cell signature cytokines IFN-γ and IL-21, suggesting that immunotherapeutic approaches can improve HBV-specific CD4 T cell responses. CD4 T cells are important in controlling viral infections but are impaired in the context of chronic hepatitis B virus (HBV) infection. Therapeutic approaches to cure chronic HBV infection are highly likely to require an immune-stimulatory component. This study demonstrates that HBV-specific CD4 T cells can be functionally augmented by combined stimulation of the co-stimulatory molecule OX40 and blockade of the inhibitory PD-1 pathway.

OX40 Regulates Both Innate and Adaptive Immunity and Promotes Nonalcoholic Steatohepatitis

Both innate and adaptive immune cells are involved in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the crosstalk between innate and adaptive immunity is largely unknown. Here we show that compared with WT mice, OX40-/- mice exhibit decreased liver fat accumulation, lobular inflammation, and focal necrosis after feeding with diets that induce NASH. Mechanistically, OX40 deficiency suppresses Th1 and Th17 differentiation, and OX40 deficiency in Tcells inhibits monocyte migration, antigen presentation, and M1 polarization. Soluble OX40 stimulation alone upregulates antigen presentation, chemokine receptor expression, and proinflammatory cytokine secretion by liver monocytes. Furthermore, plasma soluble OX40 levels are positively associated with NASH in humans, suggesting clinical relevance of the findings. In conclusion, we show a mechanism for Tcell regulation of innate immune cells. OX40 is a key regulator of both intrahepatic innate and adaptive immunity, generates two-way signals, and promotes both proinflammatory monocyte and macrophage and Tcell function, resulting in NASH development.

Radiation Followed by OX40 Stimulation Drives Local and Abscopal Antitumor Effects in an Anti-PD1-Resistant Lung Tumor Model.

Purpose: Radiation is used extensively to treat localized cancer, but improved understanding of its effects on the immune system has increased interest in its potential systemic (abscopal) effects, particularly in combination with checkpoint inhibitors such as anti-PD1. The majority of patients either do not respond or develop resistance to monotherapy over time. Here, we investigated the efficacy of OX40 (CD134) stimulation as an alternative immunotherapeutic approach in combination with radiotherapy (XRT) in a murine model of anti-PD1-resistant lung tumors.Experimental Design: We established a bilateral tumor model in 129Sv/Ev mice using an anti-PD1-resistant lung tumor cell line. Primary tumors were treated with intratumoral injection of an OX40 agonist antibody, given as adjuvant therapy after XRT (36 Gy in three 12-Gy fractions), whereas secondary tumors were left untreated to investigate abscopal outcomes.Results: The combination of XRT followed by OX40 stimulation effectively inhibited local and systemic antitumor growth, limited lung metastases, and improved survival rates. This treatment regimen augmented CD4+ and CD8+ T-cell expansion. XRT induced the expression of OX40 on T cells in tumors and spleens and increased the percentages of splenic CD103+ dendritic cells.Conclusions: Our data extend the benefits of radiation to systemic disease control, especially when combined with anti-OX40 agonist to promote immunologically mediated abscopal effects. Moreover, this study provides a rational treatment approach and sequence to overcome anti-PD1-resistant poorly immunogenic tumors. Clin Cancer Res; 24(22); 5735-43. ©2018 AACR.

OX40 Stimulation Enhances Protective Immune Responses Induced After Vaccination With Attenuated Malaria Parasites.

Protection against a malaria infection can be achieved by immunization with live-attenuated Plasmodium sporozoites and while the precise mechanisms of protection remain unknown, T cell responses are thought to be critical in the elimination of infected liver cells. In cancer immunotherapies, agonistic antibodies that target T cell surface proteins, such as CD27, OX40 (CD134), and 4-1BB (CD137), have been used to enhance T cell function by increasing co-stimulation. In this study, we have analyzed the effect of agonistic OX40 monoclonal antibody treatment on protective immunity induced in mice immunized with genetically attenuated parasites (GAPs). OX40 stimulation enhanced protective immunity after vaccination as shown by an increase in the number of protected mice and delay to blood-stage infection after challenge with wild-type sporozoites. Consistent with the enhanced protective immunity enforced OX40 stimulation resulted in an increased expansion of antigen-experienced effector (CD11ahiCD44hi) CD8+ and CD4+ T cells in the liver and spleen and also increased IFN-γ and TNF producing CD4+ T cells in the liver and spleen. In addition, GAP immunization plus α-OX40 treatment significantly increased sporozoite-specific IgG responses. Thus, we demonstrate that targeting T cell costimulatory receptors can improve sporozoite-based vaccine efficacy.

Neoadjuvant OX40 therapy in patients with head and neck cancer induces profound changes in tumor-infiltrating lymphocytes

Abstract OX40 is a member of the tumor necrosis factor (TNF) receptor family and, as a co-stimulatory molecule, enhances T-cell proliferation, survival and memory formation. We have also shown that it induces robust antitumor responses in patients with metastatic disease. To examine the effect of OX40 stimulation on the immune response in cancer patients, 16 head and neck squamous cell carcinoma (HNSCC) patients were enrolled in a neoadjuvant phase Ib trial and received anti-OX40 infusion 2 days, 1 week and 2 weeks prior to surgical resection. To evaluate changes in the tumor microenvironment (TME), tissue and peripheral blood samples were obtained prior to infusion and at time of surgery. Assessment of changes in tumor infiltrating lymphocyte (TIL) and peripheral blood populations were performed by flow cytometry and fluorescent multiplex immunohistochemistry (IHC). OX40 administration was well tolerated with low toxicity. Activation and proliferation of CD4+ and CD8+ memory T-cell populations in both the TME and periphery peaked between 12 and 19 days after OX40 infusion, as demonstrated by increased levels of Ki67, CD38 and ICOS. In the TIL, expression of CD39, ICOS and PD-1 was increased on CD4+ T cells in most patients and the frequency of tumor-reactive CD39+CD103+ CD8+ T cells was increased in some patients. IHC analysis revealed striking changes in tissue integrity and increased lymphocyte infiltrates in 4/16 patients. With this study, we have found that neoadjuvant OX40 immunotherapy is feasible and safe in patients with HNSCC and results in robust activation and proliferation of T cells within the tumor. Based on these findings, we are now comparing the transcriptome and changes in TIL repertoire and metabolism before and after treatment.

Guidance of super-enhancers in regulation of IL-9 induction and airway inflammation.

Th9 cells are prominently featured in allergic lung inflammation, but the mechanism that regulates IL-9 induction in T helper cells remains poorly defined. Here we demonstrate that formation of super-enhancers (SEs) is critical in robust induction of IL-9 and that assembly of the Il9 SEs in Th cells requires OX40-triggered chromatin acetylation. Mechanistically, we found that OX40 costimulation induces RelB expression, which recruits the histone acetyltransferase p300 to the Il9 locus to catalyze H3K27 acetylation. This allows binding of the SE factor Brd4 to organize assembly of the SE complex, which in turn drives robust IL-9 expression and Th9 cell induction. Thus, Th9 cells are strongly induced upon OX40 stimulation, and disruption of SEs abolished Th9 cell induction in vitro and inhibited Th9 cell-mediated allergic airway inflammation in vivo. Together, our data suggest that formation of SEs is essential in IL-9 expression and Th9 cell induction. These findings may have important clinical implications.

PKC-θ is dispensable for OX40L-induced TCR-independent Treg proliferation

Abstract Previously, we have shown that GM-CSF-induced Bone marrow-derived dendritic cells (G-BMDCs), upon co-culture with splenic CD4+ T cells, have the capability to induce preferential proliferation of regulatory T cells (Tregs) in TCR-independent and OX40L/OX40 dependent manner. In this study we investigated the signaling involved in this Treg proliferation induced by G-BMDCs. Firstly, using splenic CD4+ T cells from OX40 deficient mice and G-BMDCs from WT mice, we confirmed the important role of OX40L/OX40 interaction in Treg expansion. Since OX40 stimulation in the absence of TCR engagement has been reported to induce the assembly of a signalosome involving PKC-θ, we investigated the requirement of PKC-θ in our Treg proliferation mechanism. Interestingly, PKC-θ deficient-CD4+ T cells showed impaired Treg proliferation upon co-culture with WT G-BMDCs. However, this defect in proliferation could be rescued upon IL-2 addition suggesting that PKC-θ was dispensable for Treg proliferation. We further demonstrated that PKC-θ was essential for T-effectors to provide IL-2, which was required for OX40L/OX40-induced Treg proliferation. Furthermore, injecting WT, OX40- deficient, and PKC-θ-deficient mice with soluble OX40L, we established that OX40L/OX40 signaling was sufficient to induce in vivo expansion of Tregs in WT and PKC- deficient mice, but not in OX40-deficient mice. Finally, our data suggested a possible involvement of TRAF1 in Tregs, downstream of OX40 activation.

TNF superfamily receptor OX40 triggers invariant NKT cell pyroptosis and liver injury

Tissue-resident immune cells play a key role in local and systemic immune responses. The liver, in particular, hosts a large number of invariant natural killer T (iNKT) cells, which are involved in diverse immune responses. However, the mechanisms that regulate survival and homeostasis of liver iNKT cells are poorly defined. Here we have found that liver iNKT cells constitutively express the costimulatory TNF superfamily receptor OX40 and that OX40 stimulation results in massive pyroptotic death of iNKT cells, characterized by the release of potent proinflammatory cytokines that induce liver injury. This OX40/NKT pyroptosis pathway also plays a key role in concanavalin A-induced murine hepatitis. Mechanistically, we demonstrated that liver iNKT cells express high levels of caspase 1 and that OX40 stimulation activates caspase 1 via TNF receptor-associated factor 6-mediated recruitment of the paracaspase MALT1. We also found that activation of caspase 1 in iNKT cells results in processing of pro-IL-1β to mature IL-1β as well as cleavage of the pyroptotic protein gasdermin D, which generates a membrane pore-forming fragment to produce pyroptotic cell death. Thus, our study has identified OX40 as a death receptor for iNKT cells and uncovered a molecular mechanism of pyroptotic cell death. These findings may have important clinical implications in the development of OX40-directed therapies.

Enforced OX40 Stimulation Empowers Booster Vaccines to Induce Effective CD4+ and CD8+ T Cell Responses against Mouse Cytomegalovirus Infection.

There is an imperative need for effective preventive vaccines against human cytomegalovirus as it poses a significant threat to the immunologically immature, causing congenital disease, and to the immune compromised including transplant recipients. In this study, we examined the efficacy of synthetic long peptides (SLPs) as a CD4+ and CD8+ T cell-eliciting preventive vaccine approach against mouse CMV (MCMV) infection. In addition, the use of agonistic OX40 antibodies to enhance vaccine efficacy was explored. Immunocompetent C57BL/6 mice were vaccinated in a prime-boost vaccination regiment with SLPs comprising various MHC class I- and II-restricted peptide epitopes of MCMV-encoded antigens. Enforced OX40 stimulation resulted in superior MCMV-specific CD4+ as CD8+ T cell responses when applied during booster SLP vaccination. Vaccination with a mixture of SLPs containing MHC class II epitopes and OX40 agonistic antibodies resulted in a moderate reduction of the viral titers after challenge with lytic MCMV infection. Markedly, the combination of SLP vaccines containing both MHC class I and II epitopes plus OX40 activation during booster vaccination resulted in polyfunctional (i.e., IFN-γ+, TNF+, IL-2+) CD4+ and CD8+ T cell responses that were even higher in magnitude when compared to those induced by the virus, and this resulted in the best containment of virus dissemination. Our results show that the induction of strong T cell responses can be a fundamental component in the design of vaccines against persistent viral infections.

The Costimulatory Receptor OX40 Inhibits Interleukin-17 Expression through Activation of Repressive Chromatin Remodeling Pathways

T helper 17 (Th17) cells are prominently featured in multiple autoimmune diseases, but the regulatory mechanisms that control Th17 cell responses are poorly defined. Here we found that stimulation of OX40 triggered a robust chromatin remodeling response and produced a "closed" chromatin structure at interleukin-17 (IL-17) locus to inhibit Th17 cell function. OX40 activated the NF-κB family member RelB, and RelB recruited the histone methyltransferases G9a and SETDB1 to the Il17 locus to deposit "repressive" chromatin marks at H3K9 sites, and consequently repressing IL-17 expression. Unlike its transcriptional activities, RelB acted independently of both p52 and p50 in the suppression of IL-17. In an experimental autoimmune encephalomyelitis (EAE) disease model, we found that OX40 stimulation inhibited IL-17 and reduced EAE. Conversely, RelB-deficient CD4(+) Tcells showed enhanced IL-17 induction and exacerbated the disease. Our data uncover a mechanism in the control of Th17 cells that might have important clinic implications.

Exogenous OX40 Stimulation during Lymphocytic Choriomeningitis Virus Infection Impairs Follicular Th Cell Differentiation and Diverts CD4 T Cells into the Effector Lineage by Upregulating Blimp-1

T cell costimulation is a key component of adaptive immunity to viral infection but has also been associated with pathology because of excessive or altered T cell activity. We recently demonstrated that the TNFR family costimulatory molecule OX40 (CD134) is critically required to sustain antiviral T cell and Ab responses that enable control of viral replication in the context of chronic lymphocytic choriomeningitis virus (LCMV) infection. In this study, we investigated whether reinforcing OX40 stimulation through an agonist Ab had the potential to prevent LCMV persistence. We observed that anti-OX40 injection early after LCMV clone 13 infection increased CD8 T cell-mediated immunopathology. More strikingly, OX40 stimulation of virus-specific CD4 T cells promoted expression of the transcriptional repressor Blimp-1 and diverted the majority of cells away from follicular Th cell differentiation. This occurred in both acute and chronic infections, and resulted in dramatic reductions in germinal center and Ab responses to the viral infection. The effect of the OX40 agonist was dependent on IL-2 signaling and the timing of OX40 stimulation. Collectively, our data demonstrate that excessive OX40 signaling can result in deleterious consequences in the setting of LCMV infection.

Enhanced Activity of Akt in Teff Cells From Children With Lupus Nephritis Is Associated With Reduced Induction of Tumor Necrosis Factor Receptor–Associated Factor 6 and Increased OX40 Expression

The breakdown of peripheral tolerance mechanisms is central to the pathogenesis of systemic lupus erythematosus (SLE). Although true Treg cells in patients with SLE exhibit intact suppressive activity, Teff cells are resistant to suppression. The underlying mechanisms are incompletely understood. This study was undertaken to examine the Akt signaling pathway and molecules that may alter its activity in T cells in lupus patients. The Akt pathway and its regulators were analyzed in Teff and Treg cells from children with lupus nephritis and controls using flow cytometry and real-time quantitative polymerase chain reaction. T cell proliferation was assessed by analysis of 5,6-carboxyfluorescein succinimidyl ester dilution. CD4+CD45RA-FoxP3(low) and FoxP3- Teff cells from children with lupus nephritis expressed high levels of activated Akt, resulting in the down-regulation of the proapoptotic protein Bim and an enhanced proliferative response. The induction of tumor necrosis factor receptor-associated factor 6 (TRAF6) was impaired, and TRAF6 levels inversely correlated with Akt activity. Although the expression of OX40 was enhanced on Teff cells from children with lupus nephritis compared to controls, OX40 stimulation failed to significantly increase TRAF6 expression in cells from patients, in contrast to those from healthy controls, but resulted in further increased Akt activation that was reversed by blockade of OX40 signaling. Moreover, inhibition of Akt signaling markedly decreased the proliferation of Teff cells from lupus patients. Our findings indicate that hyperactivation of the Akt pathway in Teff cells from children with lupus nephritis is associated with reduced induction of TRAF6 and up-regulation of OX40, which may cause Teff cell resistance to Treg cell-mediated suppression.

Regulation of A1 by OX40 Contributes to CD8+ T Cell Survival and Anti-Tumor Activity

The TNFR family member OX40 (CD134) is critical for optimal clonal expansion and survival of T cells. However, the intracellular targets of OX40 in CD8 T cells are not fully understood. Here we show that A1, a Bcl-2 family protein, is regulated by OX40 in effector CD8 T cells. In contrast to wild-type T cells, OX40-deficient CD8 T cells failed to maintain A1 expression driven by antigen. Conversely, enforced OX40 stimulation promoted A1 expression. In both situations, the expression of A1 directly correlated with CD8 T cell survival. In addition, exogenous expression of A1 in OX40-deficient CD8 T cells reversed their survival defect in vitro and in vivo. Moreover, forced expression of A1 in CD8 T cells from OX40-deficient mice restored the ability of these T cells to suppress tumor growth in a murine model. These results indicate that OX40 signals regulate CD8 T cell survival at least in part through maintaining expression of the anti-apoptotic molecule A1, and provide new insight into the mechanism by which OX40 may impact anti-tumor immunity.

Young T Cells Age During a Redirected Anti-Tumor Attack: Chimeric Antigen Receptor-Provided Dual Costimulation is Half the Battle

Adoptive therapy with chimeric antigen receptor (CAR)-redirected T cells showed spectacular efficacy in the treatment of leukemia in recent early phase trials. Patient’s T cells were ex vivo genetically engineered with a CAR, amplified and re-administered to the patient. While T cells mediating the primary response were predominantly of young effector and central memory phenotype, repetitive antigen engagement irreversible triggers T cell maturation leaving late memory cells with the KLRG1+ CD57+ CD7− CCR7− phenotype in the long-term. These cells preferentially accumulate in the periphery, are hypo-responsive upon TCR engagement and prone to activation-induced cell death. A recent report indicates that those T cells can be rescued by CAR provided CD28 and OX40 (CD134) stimulation. We discuss the strategy with respect to prolong the anti-tumor response and to improve the over-all efficacy of adoptive cell therapy.

Intratumor OX40 stimulation inhibits IRF1 expression and IL‐10 production by Treg cells while enhancing CD40L expression by effector memory T cells

Treg cells maintain the tumor microenvironment in an immunosuppressive state preventing an effective anti-tumor immune response. A possible strategy to overcome Treg-cell suppression focuses on OX40, a costimulatory molecule expressed constitutively by Treg cells while being induced in activated effector T cells. OX40 stimulation, by the agonist mAb OX86, inhibits Treg-cell suppression and boosts effector T-cell activation. Here we uncover the mechanisms underlying the therapeutic activity of OX86 treatment dissecting its distinct effects on Treg and on effector memory T (Tem) cells, the most abundant CD4+ populations strongly expressing OX40 at the tumor site. In response to OX86, tumor-infiltrating Treg cells produced significantly less interleukin 10 (IL-10), possibly in relation to a decrease in the transcription factor interferon regulatory factor 1 (IRF1). Tem cells responded to OX86 by upregulating surface CD40L expression, providing a licensing signal to DCs. The CD40L/CD40 axis was required for Tem-cell-mediated in vitro DC maturation and in vivo DC migration. Accordingly, OX86 treatment was no longer therapeutic in CD40 KO mice. In conclusion, following OX40 stimulation, blockade of Treg-cell suppression and enhancement of the Tem-cell adjuvant effect both concurred to free DCs from immunosuppression and activate the immune response against the tumor.

OX40 engagement stabilizes Mxd4 and Mnt protein levels in antigen‐stimulated T cells leading to an increase in cell survival

OX40 engagement on activated T cells leads to increased proliferation, expansion and survival of Ag-specific T cells. Direct ex vivo examination of Ag-stimulated murine T cells show that the Myc antagonists, Mxd4 and Mnt, are transiently upregulated and translocated to the nucleus following OX40 engagement and may be involved in suppressing cell death. Both Mxd4 and Mnt are upregulated following OX40 stimulation through increased protein stability and we identify a critical phosphorylation site in Mxd4 that controls Mxd4 stability. The upregulation of Mxd4 and Mnt contributes to OX40-mediated T-cell survival because siRNA knockdown of Mxd4 and Mnt led to increased cell death. We hypothesize the upregulation of c-Myc following OX40 engagement drives T-cell proliferation and that upregulation of Mxd4 and Mnt suppresses Myc-dependent cell death. Thus, Mxd4 and Mnt upregulation following OX40 engagement most likely increases T-cell survival.