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

Abstract

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 1 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-cellmediated 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. 3615The tumor microenvironment is characterized by an immunosuppressive cytokine milieu, which promotes immune tolerance and tumor growth. Treg cells secrete interleukin 10 (IL-10), which plays a critical role in suppressing immune responses and in particular the maturation of fully competent DCs [13][14][15]. Among tumor-infiltrating Teff cells, the subpopulation of effector memory T (Tem) cells is the most abundant. Tem cells are CD4 1 CD44 high CD62L low lymphocytes excluded from resting lymph nodes and mainly localized in peripheral tissues; upon stimulation they rapidly activate and secrete effector cytokines like IFN-g, but they have limited proliferative capacity [16]. In experimental models of immune activation, Tem cells constitutively express CD40L at levels sufficient to induce DC activation in an antigen-independent manner [17]. The CD40/CD40L axis is crucial for DC maturation and the subsequent T-cell priming. However in the tumor microenvironment this costimulatory pathway is often dampened, thus impairing the generation of an efficient anti-tumor immune response [18,19].In this study we have investigated the mechanisms by which OX86 modulates Treg-and Teff-cell functions and their reciprocal interactions with DCs at the tumor site. We propose a model of the tumor microenvironment in which, after OX86 treatment, DCs receive a lower IL-10-mediated inhibition by Treg cells on the one hand, and a stronger stimulation from Tem cells, via the CD40/CD40L axis, on the other. In this favorable condition, DCs acquire a stronger migratory ability toward the draining LNs (dLNs), thus inducing a specific anti-tumor immune response. ResultsIntratumoral ...

Divergent effects of type-I interferons on regulatory T cells

“…Indeed, IRF-1 can directly bind to the Il10 promoter and induce IL-10 transcription in response to IFN-a [31]. In a mouse model of cancer, it has been demonstrated that tumorinfiltrating Treg locally release high amounts of IL-10, in line with high expression of IRF-1 [17]. Of note, the engagement of the OX40 receptor on Treg surface, an approach known to reduce the suppressive function of murine Treg, resulted in the downregulation of IRF-1 in vitro, and significantly reduced IL-10 release in vivo in tumor-infiltrating Treg [17].…”

“…In a mouse model of cancer, it has been demonstrated that tumorinfiltrating Treg locally release high amounts of IL-10, in line with high expression of IRF-1 [17]. Of note, the engagement of the OX40 receptor on Treg surface, an approach known to reduce the suppressive function of murine Treg, resulted in the downregulation of IRF-1 in vitro, and significantly reduced IL-10 release in vivo in tumor-infiltrating Treg [17]. These data indicate that IRF-1 may sustain IL-10 production in Treg, even though the impact of type-I IFNs in inducing such response in certain contexts, such as cancer, remains to be clarified.…”

“…It is now well established that Treg accumulate within tumor beds and locally suppress a variety of immune cells and functions, thus inhibiting possible immunosurveillance mechanisms and contributing to tumor escape and progression [16]. Treg exert such a wide and efficacious immune suppression through several mechanisms, among which the release of the immunomodulatory cytokine IL-10 may play a central role [17,18]. Type-I IFNs may represent the driving force for establishing an IL-10-mediated suppression program in tumor-infiltrating Treg, as recently highlighted in experimental models: indeed, mice harboring deficiencies in the IFNAR/STAT1 pathway also showed strikingly reduced frequencies of tumor-infiltrating IL-10-producing Treg, which in turn unleashed the expansion of Th17 cells that exhibited pro-tumoral activities [18].…”

Agonists of Co-stimulation in Cancer Immunotherapy Directed Against CD137, OX40, GITR, CD27, CD28, and ICOS

“…It is believed that one of the main advantages of targeting OX40 is the abrogation of Treg-mediated immunosuppression by inhibition of the activity of Treg cells (114) or by direct depletion of these cells (91,115,116). Thus, combination of anti-OX40 mAbs with cyclophosphamide (chemotherapy that is capable of partially depleting Treg cells) showed a synergistic effect in a poorly immunogenic B16 murine melanoma tumor and was associated with a reduction of Treg at the tumor site (111).…”

OX40 triggering concomitant to IL12-engineered cell vaccine hampers the immunoprevention of HER2/neu-driven mammary carcinogenesis

“…16 PE-Cy7 anti-CD4 (RM4–5), APC anti-Foxp3 (FJK-16s), PE anti-CD103 (2E7) and FITC anti-GITR were purchased from eBioscience. Surface staining was performed on cells obtained from each organ by incubating antibodies at 5 μg/mL on ice for 30 min in PBS containing 2% FBS.…”