Viktor Umansky

Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population expanded in cancer and other chronic inflammatory conditions. Here the authors identify the challenges and propose a set of minimal reporting guidelines for mouse and human MDSC.

Bone marrow as a priming site for T-cell responses to blood-borne antigen

Although bone marrow is known as a primary lymphoid organ, its potential to serve as a secondary immune organ has hardly been explored. Here we demonstrate that naive, antigen-specific T cells home to bone marrow, where they can be primed. Antigen presentation to T cells in bone marrow is mediated via resident CD11c+ dendritic cells. They are highly efficient in taking up exogenous blood-borne antigen and processing it via major histocompatibility complex class I and class II pathways. T-cell activation correlates with dendritic cell–T cell clustering in bone marrow stroma. Primary CD4+ and CD8+ T-cell responses generated in bone marrow occur in the absence of secondary lymphoid organs. The responses are not tolerogenic and result in generation of cytotoxic T cells, protective anti-tumor immunity and immunological memory. These findings highlight the uniqueness of bone marrow as an organ important for hemato- and lymphopoiesis and for systemic T cell–mediated immunity.

NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo

NK cells use a variety of receptors to detect abnormal cells, including tumors and their metastases. However, in the case of melanoma, it remains to be determined what specific molecular interactions are involved and whether NK cells control metastatic progression and/or the route of dissemination. Here we show that human melanoma cell lines derived from LN metastases express ligands for natural cytotoxicity receptors (NCRs) and DNAX accessory molecule-1 (DNAM-1), two emerging NK cell receptors key for cancer cell recognition, but not NK group 2 member D (NKG2D). Compared with cell lines derived from metastases taken from other anatomical sites, LN metastases were more susceptible to NK cell lysis and preferentially targeted by adoptively transferred NK cells in a xenogeneic model of cell therapy. In mice, DNAM-1 and NCR ligands were also found on spontaneous melanomas and melanoma cell lines. Interference with DNAM-1 and NCRs by antibody blockade or genetic disruption reduced killing of melanoma cells. Taken together, these results show that DNAM-1 and NCRs are critical for NK cell-mediated innate immunity to melanoma cells and provide a background to design NK cell-based immunotherapeutic strategies against melanoma and possibly other tumors.

Tumor-Infiltrating Monocytic Myeloid-Derived Suppressor Cells Mediate CCR5-Dependent Recruitment of Regulatory T Cells Favoring Tumor Growth

Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of myeloid cells in cancer patients and tumor-bearing mice that potently inhibits T cell responses. During tumor progression, MDSCs accumulate in several organs, including the tumor tissue. So far, tumor-infiltrating MDSC subpopulations remain poorly explored. In this study, we performed global gene expression profiling of mouse tumor-infiltrating granulocytic and monocytic (MO-MDSC) subsets compared with MDSCs from peripheral blood. RMA-S lymphoma–infiltrating MO-MDSCs not only produced high levels of NO and arginase-1, but also greatly increased levels of chemokines comprising the CCR5 ligands CCL3, CCL4, and CCL5. MO-MDSCs isolated from B16 melanoma and from skin tumor–bearing ret transgenic mice also expressed high levels of CCL3, CCL4, and CCL5. Expression of CCR5 was preferentially detected on regulatory T cells (Tregs). Accordingly, tumor-infiltrating MO-MDSCs directly attracted high numbers of Tregs via CCR5 in vitro. Intratumoral injection of CCL4 or CCL5 increased tumor-infiltrating Tregs, and deficiency of CCR5 led to their profound decrease. Moreover, in CCR5-deficient mice, RMA-S and B16 tumor growth was delayed emphasizing the importance of CCR5 in the control of antitumor immune responses. Overall, our data demonstrate that chemokines secreted by tumor-infiltrating MO-MDSCs recruit high numbers of Tregs revealing a novel suppressive role of MDSCs with potential clinical implications for the development of cancer immunotherapies.

CTLA-4 and PD-L1 Checkpoint Blockade Enhances Oncolytic Measles Virus Therapy

We hypothesized that the combination of oncolytic virotherapy with immune checkpoint modulators would reduce tumor burden by direct cell lysis and stimulate antitumor immunity. In this study, we have generated attenuated Measles virus (MV) vectors encoding antibodies against CTLA-4 and PD-L1 (MV-aCTLA-4 and MV-aPD-L1). We characterized the vectors in terms of growth kinetics, antibody expression, and cytotoxicity in vitro. Immunotherapeutic effects were assessed in a newly established, fully immunocompetent murine model of malignant melanoma, B16-CD20. Analyses of tumor-infiltrating lymphocytes and restimulation experiments indicated a favorable immune profile after MV-mediated checkpoint modulation. Therapeutic benefits in terms of delayed tumor progression and prolonged median overall survival were observed for animals treated with vectors encoding anti-CTLA-4 and anti-PD-L1, respectively. Combining systemic administration of antibodies with MV treatment also improved therapeutic outcome. In vivo oncolytic efficacy against human tumors was studied in melanoma xenografts. MV-aCTLA-4 and MV-aPD-L1 were equally efficient as parental MV in this model, with high rates of complete tumor remission (> 80%). Furthermore, we could demonstrate lysis of tumor cells and transgene expression in primary tissue from melanoma patients. The current results suggest rapid translation of combining immune checkpoint modulation with oncolytic viruses into clinical application.

Antitumor Effect of Paclitaxel Is Mediated by Inhibition of Myeloid-Derived Suppressor Cells and Chronic Inflammation in the Spontaneous Melanoma Model

The antitumor effects of paclitaxel are generally attributed to the suppression of microtubule dynamics resulting in defects in cell division. New data demonstrated that in ultralow noncytotoxic concentrations, paclitaxel modulated in immune cells in vitro the activity of small Rho GTPases, the key regulators of intracellular actin dynamics. However, the immunomodulatory properties of paclitaxel in vivo have not been evaluated. In this study, using the ret transgenic murine melanoma model, which mimics human cutaneous melanoma, we tested effects of ultralow noncytotoxic dose paclitaxel on functions of myeloid-derived suppressor cells (MDSCs), chronic inflammatory mediators, and T cell activities in the tumor microenvironment in vivo. Administration of paclitaxel significantly decreased accumulation and immunosuppressive activities of tumor-infiltrating MDSCs without alterations of the bone marrow hematopoiesis. This was associated with the inhibition of p38 MAPK activity, TNF-α and production, and S100A9 expression in MDSCs. The production of mediators of chronic inflammation in the tumor milieu also was diminished. Importantly, reduced tumor burden and increased animal survival upon paclitaxel application was mediated by the restoration of CD8 T cell effector functions. We suggest that the ability of paclitaxel in a noncytotoxic dose to block the immunosuppressive potential of MDSCs in vivo represents a new therapeutic strategy to downregulate immunosuppression and chronic inflammation in the tumor microenvironment for enhancing the efficacy of concomitant anticancer therapies.

Body Fluid Exosomes Promote Secretion of Inflammatory Cytokines in Monocytic Cells via Toll-like Receptor Signaling

Background: Exosomes, secreted from cells, have immunomodulatory capacities. Results: NFκB- and STAT3-mediated cytokine release is triggered by various types of ex vivo exosomes in a TLR-dependent fashion. Conclusion: Exosomes have inherent signaling capacities important for global inflammatory responses. Significance: Detailed knowledge about intercellular communication in cancer and inflammatory diseases is crucial for development of new therapeutic approaches. Tumor-derived exosomes have been shown to induce various immunomodulatory effects. However, the underlying signaling pathways are poorly understood. Here, we analyzed the effects of ex vivo-derived exosomes on monocytic cell differentiation/activation using THP-1 cells as model. We isolated exosomes from various body fluids such as amniotic fluid, liver cirrhosis ascites, and malignant ascites of ovarian cancer patients. We observed that exosomes were internalized by THP-1 cells and induced the production of IL-1β, TNF-α, and IL-6. Analysis of the signaling pathways revealed a fast triggering of NFκB and a delayed activation of STAT3. Pharmacologic and antibody-blocking experiments showed that the initial production of IL-6 was instrumental for subsequent activation of STAT3. Importantly, triggering of cell signaling was not a unique property of tumor exosomes but was also observed with exosomes of noncancerous origin. Exosomal signaling was TLR-dependent as the knockdown of Toll-like receptor 2 (TLR2) and TLR4 blocked NFκB and STAT3 activation. Similar results were obtained with TLR-neutralizing antibodies. Exosomes also triggered the release of cytokines from mouse bone marrow-derived dendritic cells or macrophages. This process was MyD88-dependent, further supporting a role of TLR signaling. Our results suggest that exosomes trigger TLR-dependent signaling pathways in monocytic precursor cells but possibly also in other immune cells. This process could be important for the induction of immunosuppressive mechanisms during cancer progression and inflammatory diseases.

Tumor-Expressed Inducible Nitric Oxide Synthase Controls Induction of Functional Myeloid-Derived Suppressor Cells through Modulation of Vascular Endothelial Growth Factor Release

Inducible NO synthase (iNOS) is a hallmark of chronic inflammation that is also overexpressed in melanoma and other cancers. Whereas iNOS is a known effector of myeloid-derived suppressor cell (MDSC)-mediated immunosuppression, its pivotal position at the interface of inflammation and cancer also makes it an attractive candidate regulator of MDSC recruitment. We hypothesized that tumor-expressed iNOS controls MDSC accumulation and acquisition of suppressive activity in melanoma. CD11b+GR1+ MDSC derived from mouse bone marrow cells cultured in the presence of MT-RET-1 mouse melanoma cells or conditioned supernatants expressed STAT3 and reactive oxygen species (ROS) and efficiently suppressed T cell proliferation. Inhibition of tumor-expressed iNOS with the small molecule inhibitor L-NIL blocked accumulation of STAT3/ROS-expressing MDSC, and abolished their suppressive function. Experiments with vascular endothelial growth factor (VEGF)-depleting Ab and recombinant VEGF identified a key role for VEGF in the iNOS-dependent induction of MDSC. These findings were further validated in mice bearing transplantable MT-RET-1 melanoma, in which L-NIL normalized elevated serum VEGF levels; downregulated activated STAT3 and ROS production in MDSC; and reversed tumor-mediated immunosuppression. These beneficial effects were not observed in iNOS knockout mice, suggesting L-NIL acts primarily on tumor- rather than host-expressed iNOS to regulate MDSC function. A significant decrease in tumor growth and a trend toward increased tumor-infiltrating CD8+ T cells were also observed in MT-RET transgenic mice bearing spontaneous tumors. These data suggest a critical role for tumor-expressed iNOS in the recruitment and induction of functional MDSC by modulation of tumor VEGF secretion and upregulation of STAT3 and ROS in MDSC.

Low-dose gemcitabine depletes regulatory T cells and improves survival in the orthotopic Panc02 model of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human neoplasms with extremely poor prognosis and a low survival rate. Immunosuppressive cell populations, e.g. regulatory T cells (Treg), appear to be important in PDAC, contributing to patients poor prognosis. Therefore, we investigated the PDAC microenvironment with a focus on conventional and regulatory T cells in view of their potential therapeutic importance. We found that tumors from the murine Panc02 orthotopic model of PDAC were infiltrated with high numbers of Treg. Remarkably, these cells exhibited the effector/memory phenotype, suggesting their enhanced suppressive activity and higher proliferation capacity. Although we observed a steady increase in transforming growth factor‐β (TGF‐β) levels in the tumors, treatment with a specific inhibitor of TGF‐β receptor I kinase failed to abrogate Treg accumulation. A CCR4 antagonist did not affect Treg percentage in the tumor either. However, intense Treg cell division in the tumor microenvironment was demonstrated, suggesting local proliferation as a major mechanism of Treg accumulation in PDAC. Notably, this accumulation was reduced by low‐dose gemcitabine administration, resulting in a modestly increased survival of PDAC mice. Our results provide an insight into mechanisms of immunosuppression in PDAC, suggesting an important role for proliferative expansion of effector/memory Treg. Low‐dose gemcitabine therapy selectively depletes Treg, providing a basis for new modalities of PDAC therapy.

Extracellular vesicle-mediated transfer of functional RNA in the tumor microenvironment.

Extracellular vesicles (EVs) have been shown to transfer various molecules, including functional RNA between cells and this process has been suggested to be particularly relevant in tumor-host interactions. However, data on EV-mediated RNA transfer has been obtained primarily by in vitro experiments or involving ex vivo manipulations likely affecting its biology, leaving their physiological relevance unclear. We engineered glioma and carcinoma tumor cells to express Cre recombinase showing their release of EVs containing Cre mRNA in various EV subfractions including exosomes. Transplantation of these genetically modified tumor cells into mice with a Cre reporter background leads to frequent recombination events at the tumor site. In both tumor models the majority of recombined cells are CD45+ leukocytes, predominantly Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs). In addition, multiple lineages of recombined cells can be observed in the glioma model. In the lung carcinoma model, recombined MDSCs display an enhanced immunosuppressive phenotype and an altered miRNA profile compared to their non-recombined counterparts. Cre-lox based tracing of tumor EV RNA transfer in vivo can therefore be used to identify individual target cells in the tumor microenvironment for further mechanistical or functional analysis.