[Show abstract][Hide abstract] ABSTRACT: In preclinical tumor models, aOX40 therapy is often successful at treating small tumors, but is less effective once the tumors become large. For a tumor immunotherapy to be successful to cure large tumors, it will most likely require not only an agonist to boost effector T-cell function but also inhibitors of T-cell suppression. In this study, we show that combining aOX40 antibodies with an inhibitor of the TGFb receptor (SM16) synergizes to elicit complete regression of large established MCA205 and CT26 tumors. Evaluation of tumor-infiltrating T cells showed that SM16/aOX40 dual therapy resulted in an increase in proliferating granzyme B þ CD8 T cells, which produced higher levels of IFNg, compared with treatment with either agent alone. We also found that the dual treatment increased pSTAT3 expression in both CD4 and CD8 T cells isolated from tumors. Because others have published that STAT3 signaling is detrimental to T-cell function within the tumor microenvironment, we explored whether deletion of STAT3 in OX40-expressing cells would affect this potent combination therapy. Surprisingly, we found that deletion of STAT3 in OX40-expressing cells decreased the efficacy of this combination therapy, showing that the full therapeutic potential of this treatment depends on STAT3 signaling, most likely in the T cells of tumor-bearing mice. Cancer Immunol Res; 3(5); 1–10. Ó2015 AACR.
[Show abstract][Hide abstract] ABSTRACT: We examined the phenotype and function of lymphocytes collected from the peripheral blood (PBL) and tumor (TIL) of patients with two different solid malignancies: colorectal cancer liver metastases (CRLM) and ovarian cancer (OVC).
Tumor and corresponding peripheral blood were collected from 16 CRLM and 22 OVC patients; immediately following resection they were processed and analyzed using a multi-color flow cytometry panel. Cytokine mRNA from purified PBL and TIL CD4(+) T cells were also analyzed by qPCR.
Overall, we found similar changes in the phenotypic and cytokine profiles when the TIL were compared to PBL from patients with two different malignancies. The percentage of Treg (CD4(+)/CD25(+)/FoxP3(+)) in PBL and TIL was similar: 8.1% versus 10.2%, respectively in CRLM patients. However, the frequency of Treg in primary OVC TIL was higher than PBL: 19.2% versus 4.5% (p <0.0001). A subpopulation of Treg expressing HLA-DR was markedly increased in TIL compared to PBL in both tumor types, CRLM: 69.0% versus 31.7% (p = 0.0002) and OVC 74.6% versus 37.0% (p <0.0001), which suggested preferential Treg activation within the tumor. The cytokine mRNA profile showed that IL-6, a cytokine known for its immunosuppressive properties through STAT3 upregulation, was increased in TIL samples in patients with OVC and CRLM. Both TIL populations also contained a significantly higher proportion of activated CD8(+) T cells (HLA-DR(+)/CD38(+)) compared to PBL (CRLM: 30.2% vs 7.7%, (p = 0.0012), OVC: 57.1% vs 12.0%, (p <0.0001)).
This study demonstrates that multi-color flow cytometry of freshly digested tumor samples reveals phenotypic differences in TIL vs PBL T cell sub-populations. The TIL composition in primary and metastatic tumors from two distinct histologies were remarkably similar, showing a greater proportion of activated/suppressive Treg (HLA-DR(+), CD39(+), CTLA-4(+) and Helios(+)) and activated cytotoxic T cells (CD8(+)/HLA-DR(+)/CD38(+)) when compared to PBL and an increase in IL-6 mRNA from CD4 TIL.
[Show abstract][Hide abstract] ABSTRACT: Decline in CD4 T cell immune responses is associated with aging. Although a number of immunological defects have been identified in elderly mice (>18 months old), a key early-onset immune defect at middle age could be a driver or contributor to defective CD4 T cell responses. Our studies demonstrate that age-related alterations in DC subsets within the priming environment of middle-aged mice (12 months old) correlate with and can directly contribute to decreases in antigen-specific CD4 T cell Th1 differentiation, which measured by T-bet and IFN-γ expression, was decreased significantly in T cells following VSV infection or s.c. immunization with a protein antigen in the context of immune stimulation via OX40. The deficient Th1 phenotype, observed following protein antigen challenge, was found to be the result of an age-related decrease in an inflammatory DC subset (CD11b+ Gr-1/Ly6C+) in the dLN that corresponded with T cell dysfunction. In the virus model, we observed significant changes in two DC subsets: mDCs and pDCs. Thus, different, early age-related changes in the DC profile in the priming environment can significantly contribute to impaired Th1 differentiation, depending on the type of immunological challenge.
[Show abstract][Hide abstract] ABSTRACT: Immune responses wane during aging, posing challenges to the potential effectiveness of cancer immunotherapies. We previously demonstrated that in the context of a promising immunotherapeutic, OX40 agonist (αOX40), older animals exhibited impaired anti-tumor immune responses and diminished CD4 T cell effector differentiation. In this study, we hypothesized that tumor immune responses could be maintained during aging through caloric restriction (CR) or dietary supplementation with resveratrol (RES), a CR mimetic. Mice were placed on either a calorically restricted diet or a RES-formulated diet starting between 4 and 6 months of age and continued until mice reached 12 months of age. Tumor immune responses were assessed after challenging with either sarcoma or breast tumor cells followed by αOX40 treatment. Our results show that CR, but not RES, maintained OX40-mediated anti-tumor immunity. In addition, CR fully sustained antigen-specific CD4 T cell priming in aged hosts (12 months old), whereas tumor-specific CD8 T cell priming was not fully maintained compared to young reference animals (2 months old). Thus, CR appears to maintain immunological fitness of the CD4 T cell priming environment during aging, which is critical for optimal OX40-mediated responses.
Electronic supplementary material
The online version of this article (doi:10.1007/s00262-014-1542-y) contains supplementary material, which is available to authorized users.
Cancer Immunology and Immunotherapy 03/2014; 63(6). DOI:10.1007/s00262-014-1542-y · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OX40 is a potent co-stimulatory receptor that can potentiate T cell receptor signaling on the surface of T lymphocytes, leading to their activation by a specifically recognized antigen. In particular, OX40 engagement by ligands present on dendritic cells dramatically increases the proliferation, effector function and survival of T cells. Preclinical studies have shown that OX40 agonists increase anti-tumor immunity and improve tumor-free survival. In this study, we performed a Phase I clinical trial using a mouse monoclonal antibody (mAb) that agonizes human OX40 signaling in patients with advanced cancer. Patients treated with one course of the anti-OX40 mAb showed an acceptable toxicity profile and regression of at least one metastatic lesion in 12/30 patients. Mechanistically, this treatment increased T and B cell responses to reporter antigen immunizations, led to preferential upregulation of OX40 on CD4+ FoxP3+ regulatory T cells in tumor-infiltrating lymphocytes andincreased the anti-tumor reactivity of T and B cells in patients with melanoma. Our findings clinically validate OX40 as a potent immune-stimulating target for treatment in cancer patients, providing a generalizable tool to favorably influence the antitumor properties of circulating T cells, B cells and intratumoral regulatory T cells.
Cancer Research 10/2013; 73(24). DOI:10.1158/0008-5472.CAN-12-4174 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: T cell-mediated rejection of tumors requires signals from the T cell receptor and co-stimulatory molecules to license effector functions of tumor-antigen specific T cells. There is also an array of immune suppressive mechanisms within the tumor microenvironment that can suppress anti-tumor immunity. The use of monoclonal antibodies to overcome this suppression and/or enhance tumor-antigen specific T cell responses has shown promise in clinical trials. In particular, targeting co-stimulatory members of the tumor necrosis factor receptor (TNFR) family with agonist Abs enhances T cell function, which has led to encouraging therapeutic results in cancer-bearing hosts. These encouraging data establish TNFRs as important targets for enhancing tumor-specific immune responses in mice and man. This review will focus on agonists that target the TNFRs OX40, 4-1BB, and CD40.
Current opinion in immunology 02/2013; 25(2). DOI:10.1016/j.coi.2013.01.004 · 7.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Surface expression of the IL-2 receptor α-chain (CD25) has been used to discriminate between CD4(+) CD25(HI) FOXP3(+) regulatory T (Treg) cells and CD4(+) CD25(NEG) FOXP3(-) non-Treg cells. However, this study reports that the majority of resting human memory CD4(+) FOXP3(-) T cells expresses intermediate levels of CD25 and that CD25 expression can be used to delineate a functionally distinct memory subpopulation. The CD25(NEG) memory T-cell population contains the vast majority of late differentiated cells that respond to antigens associated with chronic immune responses and are increased in patients with systemic lupus erythematosus (SLE). In contrast, the CD25(INT) memory T cells respond to antigens associated with recall responses, produce a greater array of cytokines, and are less dependent on costimulation for effector responses due to their expression of CD25. Lastly, compared to the CD25(NEG) and Treg-cell populations, the CD25(INT) memory population is lost to a greater degree from the blood of cancer patients treated with IL-2. Collectively, these results show that in humans, a large proportion of CD4(+) memory T cells express intermediate levels of CD25, and this CD25(INT) FOXP3(-) subset is a functionally distinct memory population that is uniquely affected by IL-2.
European Journal of Immunology 07/2012; 42(7):1893-905. DOI:10.1002/eji.201242444 · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The treatment of high-grade tumours must consider a tumour environment dominated by cells that support cancer growth. In addition to directing angiogenesis and invasion, alternatively activated macrophages in the tumour provide protection from adaptive immunity and permit tumour growth. Agonist antibodies to the tumour necrosis factor receptor family member OX40 are an effective therapy for cancer in a range of murine models; however, as with many immune therapies, αOX40 therapy is less effective as the tumour grows and develops an immune suppressive environment. We demonstrate that αOX40 directly activates T cells and that this T-cell activation alters macrophage differentiation in the tumour environment. We demonstrate that macrophages in the tumour limit the efficacy of αOX40 therapy, and that combining αOX40 therapy with inhibitors of arginase significantly enhances survival of tumour-bearing mice. These data demonstrate that macrophages in the tumour environment limit the effectiveness of OX40-based immunotherapy, and combination therapies that target both the cell-mediated immune response and the suppressive tumour environment will be required for translation of effective immunotherapies to patients with established tumours.
[Show abstract][Hide abstract] ABSTRACT: The provision of T cell co-stimulation via members of the TNFR super-family, including OX40 (CD134) and 4-1BB (CD137), provides critical signals that promote T cell survival and differentiation. Recent studies have demonstrated that ligation of OX40 can augment T cell-mediated anti-tumor immunity in pre-clinical models and more importantly, OX40 agonists are under clinical development for cancer immunotherapy. OX40 is of particular interest as a therapeutic target as it is not expressed on naïve T cells but rather, is transiently up-regulated following TCR stimulation. Although TCR engagement is necessary for inducing OX40 expression, the downstream signals that regulate OX40 itself remain unclear. In this study, we demonstrate that OX40 expression is regulated through a TCR and common gamma chain cytokine-dependent signaling cascade that requires JAK3-mediated activation of the downstream transcription factors STAT3 and STAT5. Furthermore, combined treatment with an agonist anti-OX40 mAb and IL-2 augmented tumor immunotherapy against multiple tumor types. Dual therapy was also able to restore the function of anergic tumor-reactive CD8 T cells in mice with long-term well-established (>5 wks) tumors, leading to increased survival of the tumor-bearing hosts. Together, these data reveal the ability of TCR/common gamma chain cytokine signaling to regulate OX40 expression and demonstrate a novel means of augmenting cancer immunotherapy by providing dual anti-OX40/common gamma chain cytokine-directed therapy.
PLoS ONE 04/2012; 7(4):e34467. DOI:10.1371/journal.pone.0034467 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OX40 (CD134) is a tumor necrosis factor (TNF) receptor expressed primarily on activated CD4(+) and CD8(+) T cells and transmits a potent costimulatory signal when engaged. OX40 is transiently expressed after T-cell receptor engagement and is upregulated on the most recently antigen-activated T cells within inflammatory lesions (e.g. sites of autoimmune destruction and on tumor-infiltrating lymphocytes). Hence, it is an attractive target to modulate immune responses: OX40 blocking agents to inhibit undesirable inflammation or OX40 agonists to enhance immune responses. In regards to this review, OX40 agonists enhance anti-tumor immunity, which leads to therapeutic effects in mouse tumor models. A team of laboratory and clinical scientists at the Providence Cancer Center has collaborated to bring the preclinical observations in cancer models from the bench to the bedside. This review describes the journey from in vitro experiments through preclinical mouse models to the successful translation of the first OX40 agonist to the clinic for the treatment of patients with cancer.
[Show abstract][Hide abstract] ABSTRACT: Effective tumor immunotherapy may require not only activation of anti-tumor effector cells, but also abrogation of tumor-mediated immunosuppression. The cytokine TGF-β, is frequently elevated in the tumor microenvironment and is a potent immunosuppressive agent and promoter of tumor metastasis. OX40 (CD134) is a member of the TNF-α receptor superfamily and ligation by agonistic antibody (anti-OX40) enhances effector function, expansion, and survival of activated T cells. In this study, we examined the therapeutic efficacy and anti-tumor immune response induced by the combination of a small molecule TGF-β signaling inhibitor, SM16, plus anti-OX40 in the poorly immunogenic, highly metastatic, TGF-β-secreting 4T1 mammary tumor model. Our data show that SM16 and anti-OX40 mutually enhanced each other to elicit a potent anti-tumor effect against established primary tumors, with a 79% reduction in tumor size, a 95% reduction in the number of metastatic lung nodules, and a cure rate of 38%. This positive treatment outcome was associated with a 3.2-fold increase of tumor-infiltrating, activated CD8+ T cells, an overall accumulation of CD4+ and CD8+ T cells, and an increased tumor-specific effector T cell response. Complete abrogation of the therapeutic effect in vivo following depletion of CD4+ and CD8+ T cells suggests that the anti-tumor efficacy of SM16+ anti-OX40 therapy is T cell dependent. Mice that were cured of their tumors were able to reject tumor re-challenge and manifested a significant tumor-specific peripheral memory IFN-γ response. Taken together, these data suggest that combining a TGF-β signaling inhibitor with anti-OX40 is a viable approach for treating metastatic breast cancer.
Cancer Immunology and Immunotherapy 10/2011; 61(4):511-21. DOI:10.1007/s00262-011-1119-y · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
European Journal of Immunology 04/2011; 41(4):1024-34. DOI:10.1002/eji.201040449 · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The initial phase of sepsis is characterized by massive inflammatory cytokine production that contributes to multisystem organ failure and death. Costimulatory molecules are a class of receptors capable of regulating cytokine production in adaptive immunity. Recent studies described their presence on neutrophils and monocytes, suggesting a potential role in the regulation of cytokine production in innate immunity. The purpose of this study was to determine the role for OX40-OX40 ligand (OX40L) interaction in the innate immune response to polymicrobial sepsis. Humans with sepsis demonstrated upregulation of OX40L on monocytes and neutrophils, with mortality and intensive care unit stay correlating with expression levels. In an animal model of polymicrobial sepsis, a direct role for OX40L in regulating inflammation was indicated by improved survival, decreased cytokine production, and a decrease in remote organ damage in OX40L(-/-) mice. The finding of similar results with an OX40L Ab suggests a potential therapeutic role for OX40L blockade in sepsis. The inability of anti-OX40L to provide significant protection in macrophage-depleted mice establishes macrophages as an indispensable cell type within the OX40/OX40L axis that helps to mediate the clinical signs of disease in sepsis. Conversely, the protective effect of anti-OX40L Ab in RAG1(-/-) mice further confirms a T cell-independent role for OX40L stimulation in sepsis. In conclusion, our data provide an in vivo role for the OX40/OX40L system in the innate immune response during polymicrobial sepsis and suggests a potential beneficial role for therapeutic blockade of OX40L in this devastating disorder.
The Journal of Immunology 10/2010; 185(8):4856-62. DOI:10.4049/jimmunol.1000404 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The existence of tumor-specific T cells, as well as their ability to be primed in cancer patients, confirms that the immune response can be deployed to combat cancer. However, there are obstacles that must be overcome to convert the ineffective immune response commonly found in the tumor environment to one that leads to sustained destruction of tumor. Members of the tumor necrosis factor (TNF) superfamily direct diverse immune functions. OX40 and its ligand, OX40L, are key TNF members that augment T-cell expansion, cytokine production, and survival. OX40 signaling also controls regulatory T-cell differentiation and suppressive function. Studies over the past decade have demonstrated that OX40 agonists enhance antitumor immunity in preclinical models using immunogenic tumors; however, treatment of poorly immunogenic tumors has been less successful. Combining strategies that prime tumor-specific T cells together with OX40 signaling could generate and maintain a therapeutic antitumor immune response.
Seminars in Oncology 10/2010; 37(5):524-32. DOI:10.1053/j.seminoncol.2010.09.013 · 3.94 Impact Factor