The matricellular protein osteopontin (OPN, Spp-1) is widely associated with cancer aggressiveness when produced by tumor cells, but its impact is uncertain when produced by leukocytes in the context of the tumor stroma. In a broad study using Spp1 À/À mice along with gene silencing in tumor cells, we obtained evidence of distinct and common activities of OPN when produced by tumor or host cells in a spontaneously metastatic model of breast cancer. Different cellular localization of OPN is associated with its distinct activities, being mainly secreted in tumor cells while intracellular in myeloid cells. OPN produced by tumor cells supported their survival in the blood stream, whereas both tumor-and host-derived OPN, particularly from myeloid cells, rendered the metastatic site more immunosuppressive. Myeloid-derived suppressor cells (MDSC) expanded with tumor progression at both primary and lung metastatic sites. Of the expanded monocytic and granulocytic cell populations of MDSCs, the monocytic subset was the predominant source of OPN. In Spp1 À/À mice, the inhibition of lung metastases correlated with the expansion of granulocyte-oriented MDSCs. Notably, monocytic MDSCs in Spp1 À/À mice were less suppressive than their wild-type counterparts due to lower expression of arginase-1, IL6, and phospho-Stat3. Moreover, fewer regulatory T cells accumulated at the metastatic site in Spp1 À/À mice. Our data find correlation with lung metastases of human mammary carcinomas that are associated with myeloid cells expressing OPN. Overall, our results unveiled novel functions for OPN in shaping local immunosuppression in the lung metastatic niche.

Mast cell (MC)-deficient c-Kit mutant Kit W/W-v mice are protected against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, suggesting a detrimental role for MCs in this disease. To further investigate the role of MCs in EAE, we took advantage of a recently characterized model of MC deficiency, Kit W-sh/W-sh . Surprisingly, we observed that myelin oligodendrocyte glycoprotein (MOG) 35-55 -induced chronic EAE was exacerbated in Kit W-sh/W-sh compared with Kit þ / þ mice. Kit W-sh/W-sh mice showed more inflammatory foci in the central nervous system (CNS) and increased T-cell response against myelin. To understand whether the discrepant results obtained in Kit W-sh/W-sh and in Kit W/W-v mice were because of the different immunization protocols, we induced EAE in these two strains with varying doses of MOG and adjuvants. Although Kit W-sh/W-sh mice exhibited exacerbated EAE under all immunization protocols, Kit W/W-v mice were protected from EAE only when immunized with high, but not low, doses of antigen and adjuvants. Kit W-sh/W-sh mice reconstituted systemically, but not in the CNS, with bone marrow-derived MCs still developed exacerbated EAE, indicating that protection from disease could be exerted by MCs mainly in the CNS, and/or by other cells possibly dysregulated in Kit W-sh/W-sh mice. In summary, these data suggest to reconsider MC contribution to EAE, taking into account the variables of using different experimental models and immunization protocols. MCs are key factors in IgE-associated immediate hypersensitivity reactions, during which they release a wide spectrum of inflammatory mediators in response to IgE and antigen (Ag) stimulation. 1 However, recent findings have pointed out that MCs may exert also important effector and/or immunomodulatory functions in other physiopathological conditions as venom detoxification, pathogen clearance, tumor growth, contact hypersensitivity, allograft acceptance and autoimmunity. 2 Most of these studies have assessed the in vivo role of MCs by the use of mouse models carrying spontaneous mutations in c-Kit receptor (WBB6F 1 -Kit W/W-v or C57BL/6-Kit W-sh/W-sh mice) or c-Kit ligand (WCB6F 1 -Kitl Sl /Kitl Sl-d ), which show severe deficiency of MC populations. 3,4 MCs may either enhance or suppress the inflammation associated to different types of autoimmune diseases. MC-deficient Kit W/W-v and Kitl Sl/Sl-d mice are protected from autoantibodymediated models of rheumatoid arthritis and bullous pemphigoid, inflammatory disorders affecting, respectively, the joint and the skin. Reconstitution of Kit W/W-v mice with bone marrow derived, in vitro cultured MCs (BMMCs) restores complete disease susceptibility in both conditions, thus indicating a proinflammatory role of these cells in such diseases. 5,6 Conversely, MCs seem to exert a protective function in experimental autoimmune glomerulonephritis, by limiting clinical and histological glomerular pathology and mortality. 7,8 However, recent work has shown that, unlike Kit W/W-v , Ki...

OX40 stimulation is known to enhance activation of effector T cells and to inhibit induction and suppressive function of Treg. Here we uncovered a novel role of OX40 in sustaining Treg competitive fitness in vivo, during repopulation of lymphopenic hosts and reconstitution of BM chimeras. Defective expansion of OX40-null Treg diminished their ability to suppress inflammation in a model of lymphopenia-driven colitis. OX40-mediated promotion of Treg fitness spanned beyond lymphopenic environments, as endogenous Treg in OX40-null mice showed decreased accumulation during thymic development, enhanced susceptibility to antibody-mediated depletion and defective turnover following thymectomy. In vitro, OX40-deficient Treg were found to be intrinsically hyporesponsive to IL-2, in terms of Stat5 phosphorylation and proliferation, according to elevated SOCS1 content and reduced miR155 expression. Therefore, OX40 is a key factor in shaping Treg sensitivity to IL-2 and promoting their proliferation and survival, toward accurate immune regulation.Key words: Competitive fitness . Costimulatory molecules . Cytokines . Treg Supporting Information available online IntroductionTreg are a specialized subset of CD4 1 T lymphocytes deputed to maintain self-tolerance through inhibition of a variety of immune responses [1]. Dysregulation in functions or numbers of Treg has been associated to severe autoimmunity and inflammation in both mice and humans. Treg selectively express the transcription factor Foxp3, which determines their regulatory program, and constitutively express the a-chain of the IL-2 receptor CD25, which mediates their peripheral homeostasis.Also, murine Treg constitutively express OX40 [2,3], a member of the Tumor Necrosis Factor-Receptor superfamily, encoded by the Tnfrsf4 gene. OX40-null mice show defective T-cell activation and impaired reactivity in multiple settings [4]. Indeed, this receptor intervenes at different checkpoints in shaping optimal immune responses and in counteracting both recessive and dominant tolerance. OX40, expressed on non-Treg upon activation, conveys survival signals [5], promotes the acquisition of effector functions [6], impedes and reverts T-cell anergy [7] and even prevents the acquisition of a regulatory phenotype under TGF-b exposure [8,9] or under Tr1-promoting conditions [10]. A further level of control is represented by the à These authors contributed equally to this work. 2902ability of OX40 triggering to inhibit Treg suppressive function in vitro and in vivo, as shown in models of graft-versus-host disease [3], colitis [2] and cancer immunotherapy [11,12]. Recently we have found OX40 bridging the bidirectional interaction between Treg and mast cells, leading to blockade of histamine degranulation and anaphylactic response on the mast cell side [13] and to inhibition of suppressive activity on the Treg side [14].Little is known about the role of OX40 in Treg development and homeostasis. Young Tnfrsf4 À/À mice show reduced numbers of CD41 CD25 1 cells in central and peripheral l...

Osteosarcoma, the most common primary bone tumor, is characterized by an aggressive behavior with high tendency to develop lung metastases as well as by multiple genetic aberrations that have hindered the development of targeted therapies. New therapeutic approaches are urgently needed; however, novel combinations with immunotherapies and checkpoint inhibitors require suitable preclinical models with intact immune systems to be properly tested. We have developed immunocompetent osteosarcoma models that grow orthotopically in the bone and spontaneously metastasize to the lungs, mimicking human osteosarcoma. These models have been used to test the efficacy of trabectedin, a chemotherapeutic drug utilized clinically for sarcomas and ovarian cancer. Trabectedin, as monotherapy, significantly inhibited osteosarcoma primary tumor growth and lung metastases by both targeting neoplastic cells and reprogramming the tumor immune microenvironment. Specifically, trabectedin induced a striking differentiation of tumor cells by favoring the recruitment of Runx2, the master genetic regulator of osteoblastogenesis, on the promoter of genes involved in the physiologic process of terminal osteoblast differentiation. Differentiated neoplastic cells, as expected, showed reduced proliferation rate. Concomitantly, trabectedin enhanced the number of tumor-infiltrating T lymphocytes, with local CD8 T cells, however, likely post-activated or exhausted, as suggested by their high expression of the inhibitory checkpoint molecule PD-1. Accordingly, the combination with a PD-1-blocking antibody significantly increased trabectedin efficacy in controlling osteosarcoma progression. These results demonstrate the therapeutic efficacy of trabectedin in osteosarcoma treatment, unveiling its multiple activities and providing a solid rationale for its combination with immune checkpoint inhibitors. .

Systemic immune stimulation has been associated with increased risk of myeloid malignancies, but the pathogenic link is unknown. We demonstrate in animal models that experimental systemic immune activation alters the bone marrow stromal microenvironment, disarranging extracellular matrix (ECM) microarchitecture, with downregulation of secreted protein acidic and rich in cysteine (SPARC) and collagen-I and induction of complement activation. These changes were accompanied by a decrease in Treg frequency and by an increase in activated effector T cells. Under these conditions, hematopoietic precursors harboring nucleophosmin-1 (NPM1) mutation generated myeloid cells unfit for normal hematopoiesis but prone to immunogenic death, leading to neutrophil extracellular trap (NET) formation. NET fostered the progression of the indolent NPM1-driven myeloproliferation toward an exacerbated and proliferative dysplastic phenotype. Enrichment in NET structures was found in the bone marrow of patients with autoimmune disorders and in NPM1-mutated acute myelogenous leukemia (AML) patients. Genes involved in NET formation in the animal model were used to design a NET-related inflammatory gene signature for human myeloid malignancies. This signature identified two AML subsets with different genetic complexity and different enrichment in NPM1 mutation and predicted the response to immunomodulatory drugs. Our results indicate that stromal/ECM changes and priming of bone marrow NETosis by systemic inflammatory conditions can complement genetic and epigenetic events towards the development and progression of myeloid malignancy.