Yulia Nefedova

Antigen-Specific Inhibition of CD8+ T Cell Response by Immature Myeloid Cells in Cancer Is Mediated by Reactive Oxygen Species

Tumor growth is associated with the accumulation of immature myeloid cells (ImC), which in mice are characterized by the expression of Gr-1 and CD11b markers. These cells suppress Ag-specific CD8+ T cells via direct cell-cell contact. However, the mechanism of immunosuppressive activity of tumor-derived ImC remains unclear. In this study we analyzed the function of ImC isolated from tumor-free control and tumor-bearing mice. Only ImC isolated from tumor-bearing mice, not those from their control counterparts, were able to inhibit the Ag-specific response of CD8+ T cells. ImC obtained from tumor-bearing mice had significantly higher levels of reactive oxygen species (ROS) than ImC isolated from tumor-free animals. Accumulation of H2O2, but not superoxide or NO, was a major contributor to this increased pool of ROS. It appears that arginase activity played an important role in H2O2 accumulation in these cells. Inhibition of ROS in ImC completely abrogated the inhibitory effect of these cells on T cells, indicating that ImC generated in tumor-bearing hosts suppress the CD8+ T cell response via production of ROS. Interaction of ImC with Ag-specific T cells in the presence of specific Ags resulted in a significant increase in ROS production compared with control Ags. That increase was independent of IFN-γ production by T cells, but was mediated by integrins CD11b, CD18, and CD29. Blocking of these integrins with specific Abs abrogated ROS production and ImC-mediated suppression of CD8+ T cell responses. This study demonstrates a new mechanism of Ag-specific T cell inhibition mediated by ROS produced by ImCs in cancer.

Hyperactivation of STAT3 Is Involved in Abnormal Differentiation of Dendritic Cells in Cancer

Abnormal differentiation of myeloid cells is one of the hallmarks of cancer. However, the molecular mechanisms of this process remain elusive. In this study, we investigated the effect of tumor-derived factors on Janus kinase (Jak)/STAT signaling in myeloid cells during their differentiation into dendritic cells. Tumor cell conditioned medium induced activation of Jak2 and STAT3, which was associated with an accumulation of immature myeloid cells. Jak2/STAT3 activity was localized primarily in these myeloid cells, which prevented the differentiation of immature myeloid cells into mature dendritic cells. This differentiation was restored after removal of tumor-derived factors. Inhibition of STAT3 abrogated the negative effects of these factors on myeloid cell differentiation, and overexpression of STAT3 reproduced the effects of tumor-derived factors. Thus, this is a first demonstration that tumor-derived factors may affect myeloid cell differentiation in cancer via constitutive activation of Jak2/STAT3.

Regulation of Dendritic Cell Differentiation and Antitumor Immune Response in Cancer by Pharmacologic-Selective Inhibition of the Janus-Activated Kinase 2/Signal Transducers and Activators of Transcription 3 Pathway

Abnormal dendritic cell differentiation and accumulation of immunosuppressive myeloid cells in cancer is one of the major factors of tumor nonresponsiveness. We have previously shown that hyperactivation of the Janus-activated kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) induced by tumor-derived factors (TDF) is responsible for abnormal dendritic cell differentiation. Here, using a novel selective inhibitor of JAK2/STAT3 JSI-124, we investigated the possibility of pharmacologic regulation of dendritic cell differentiation in cancer. Our experiments in vitro have shown that JSI-124 overcomes the differentiation block induced by TDF and promotes the differentiation of mature dendritic cells and macrophages. JSI-124 significantly reduced the presence of immature myeloid cells in vivo and promoted accumulation of mature dendritic cells. In addition to a direct antitumor effect in several animal models, JSI-124 significantly enhanced the effect of cancer immunotherapy. This indicates that pharmacologic inhibition of the JAK2/STAT3 pathway can be an important new therapeutic strategy to enhance antitumor activity of cancer immunotherapy.

Lipid accumulation and dendritic cell dysfunction in cancer

Dendritic cells (DCs), a type of professional antigen-presenting cells, are responsible for initiation and maintenance of immune responses. Here we report that a substantial proportion of DCs in tumor-bearing mice and people with cancer have high amounts of triglycerides as compared with DCs from tumor-free mice and healthy individuals. In our studies, lipid accumulation in DCs was caused by increased uptake of extracellular lipids due to upregulation of scavenger receptor A. DCs with high lipid content were not able to effectively stimulate allogeneic T cells or present tumor-associated antigens. DCs with high and normal lipid levels did not differ in expression of major histocompatibility complex and co-stimulatory molecules. However, lipid-laden DCs had a reduced capacity to process antigens. Pharmacological normalization of lipid abundance in DCs with an inhibitor of acetyl-CoA carboxylase restored the functional activity of DCs and substantially enhanced the effects of cancer vaccines. These findings suggest that immune responses in cancer can be improved by manipulating the lipid levels in DCs.

Bone marrow stromal-derived soluble factors and direct cell contact contribute to de novo drug resistance of myeloma cells by distinct mechanisms

The tumor microenvironment plays a critical role in determining the fate of tumor cells. We have previously reported that adhesion of human myeloma and leukemia cell lines to the extracellular matrix protein, fibronectin, confers a multidrug-resistant phenotype. Mechanisms associated with this cell adhesion-mediated drug resistance are drug-type specific. In the present study, we examined the influence of bone marrow stromal cells (BMSCs) on myeloma cell response to the topoisomerase II inhibitor, mitoxantrone. Apoptosis was inhibited by more than 50% when cells were adhered to BMSCs as compared to myeloma cells maintained in suspension. To investigate the mechanisms contributing to the resistance of myeloma cells in contact with BMSCs, we examined the protective effects of BMSCs under four separate conditions: (1) direct cell contact; (2) BMSCs conditioned medium; (3) medium conditioned by coculturing myeloma cells in direct contact with BMSCs; and (4) medium conditioned by coculturing myeloma cells and BMSCs without direct physical contact. Conditioned medium from BMSCs alone was not sufficient to protect myeloma cells from drug-induced apoptosis; however, soluble factors produced during the myeloma-BMSCs interaction decreased the sensitivity of myeloma cells to mitoxantrone, suggesting a dynamic interaction between myeloma cells and BMSCs. We also found that myeloma cells in direct contact with BMSCs underwent growth arrest, whereas soluble factors produced by myeloma cells-BMSCs coincubation stimulated the proliferation of myeloma cells. These data show that both cell–cell adhesion of BMSCs with myeloma cells and soluble factors induced by this cell–cell interaction are involved in the protection of myeloma cells from mitoxantrone-induced apoptosis; however, the mechanisms contributing to the drug resistance are different.

Mechanism of All-Trans Retinoic Acid Effect on Tumor-Associated Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSC) play an important role in tumor escape by suppressing T-cell responses. MDSC represent a group of cells of myeloid lineage at different stages of differentiation. Increased arginase activity and production of reactive oxygen species (ROS) are among the main functional characteristics of these cells. Recent studies have shown that all-trans retinoic acid (ATRA) had a potent activity in eliminating MDSC in cancer patients and in tumor-bearing mice. ATRA differentiates these cells into mature myeloid cells. However, the mechanism of this effect is unclear. Here, we have shown that ATRA dramatically and specifically up-regulated gene expression and protein level of glutathione synthase (GSS) in MDSC. This resulted in accumulation of glutathione (GSH) in these cells, observed in both mice and cancer patients. Blockade of GSH synthesis cancelled the effect of ATRA on MDSC. Accumulation of GSH in these cells using N-acetyl-L-cysteine mimicked the effect of ATRA on MDSC differentiation. Analysis of potential mechanisms of ATRA effect on GSS revealed that ATRA regulates its expression not by directly binding to the promoter but primarily via activation of extracellular signal-regulated kinase 1/2. Thus, ATRA induced differentiation of MDSC primarily via neutralization of high ROS production in these cells. This novel mechanism involves specific up-regulation of GSS and accumulation of GSH and could be used in developing and monitoring therapeutic application of ATRA.

Epigenetic silencing of retinoblastoma gene regulates pathologic differentiation of myeloid cells in cancer

Two major populations of myeloid-derived suppressor cells (MDSCs), monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs) regulate immune responses in cancer and other pathologic conditions. Under physiologic conditions, Ly6ChiLy6G− inflammatory monocytes, which are the normal counterpart of M-MDSCs, differentiate into macrophages and dendritic cells. PMN-MDSCs are the predominant group of MDSCs that accumulates in cancer. Here we show that a large proportion of M-MDSCs in tumor-bearing mice acquired phenotypic, morphological and functional features of PMN-MDSCs. Acquisition of this phenotype, but not the functional attributes of PMN-MDSCs, was mediated by transcriptional silencing of the retinoblastoma gene through epigenetic modifications mediated by histone deacetylase 2 (HDAC-2). These data demonstrate a new regulatory mechanism of myeloid cells in cancer.

Activation of Dendritic Cells via Inhibition of Jak2/STAT3 Signaling

Signaling via Jak2/STAT3 is critically important for normal dendritic cell (DC) differentiation. In addition, we have previously demonstrated that hyperactivation of the Jak2/STAT3 pathway induced by tumor-derived factors (TDF) may be responsible for abnormal DC differentiation in cancer. In this study, using a novel selective inhibitor of Jak2/STAT3, JSI-124, we investigated the mechanism of the Jak2/STAT3 effect on DCs and the possibility of pharmacological regulation of DC differentiation in cancer. Our experiments have demonstrated that JSI-124 overcomes the differentiation block induced by TDF and promotes the differentiation of mature DCs and macrophages. Surprisingly, inhibition of Jak2/STAT3 signaling resulted in dramatic activation of immature DCs generated in the presence of TDF as well as in control medium. This activation manifested in up-regulation of MHC class II, costimulatory molecules, and a dramatic increase in the ability to stimulate allogeneic or Ag-specific T cells. Inhibition of Jak2/STAT3 signaling resulted in activation of the transcription factor NF-κB. This up-regulation was not due to a conventional pathway involving IκBα, but was probably due to a block of the dominant negative effect of STAT3. This indicates that Jak2/STAT3 play an important role in negative regulation of DC activation, and pharmacological inhibition of the Jak2/STAT3 pathway can be used to enhance DC function.

Myeloid derived suppressor cells regulate growth of multiple myeloma by inhibiting T cells in bone marrow

Myeloid-derived suppressor cells (MDSC) are one of the major factors limiting the immune response in cancer. However, their role in bone marrow (BM), the site of primary localization of multiple myeloma (MM), is poorly understood. In this study, we found a significant accumulation of CD11b+CD14−CD33+ immunosuppressive MDSC in BM of patients with newly diagnosed MM. To assess the possible role of MDSC in MM, we used immunocompetent mouse models. Immunosuppressive MDSC accumulated in BM of mice as early as 1 wk after tumor inoculation. S100A9 knockout (KO) mice, which are deficient in their ability to accumulate MDSC in tumor-bearing hosts, demonstrated reduced MDSC accumulation in BM after injection of MM cells compared with wild-type mice. Growth of the immunogenic MM cells was significantly reduced in S100A9KO mice. This effect was associated with the accumulation of Ag-specific CD8+ T cells in BM and spleens of S100A9KO mice, but not wild-type mice, and was abrogated by the administration of anti-CD8 Ab or adoptive transfer of MDSC. Thus, the accumulation of MDSC at early stages of MM plays a critical role in MM progression and suggests that MDSC can be considered a possible therapeutic target in this disease.

Lectin-type oxidized LDL receptor-1 distinguishes population of human polymorphonuclear myeloid-derived suppressor cells in cancer patients

PMN-MDSC in cancer patients can be distinguished from neutrophils by a genomic signature and by expression of the LOX-1 receptor. Stressing myeloid-derived suppressor cells in cancer Immunotherapies for cancer have shown promising results in part because they overcome the suppressive effects of the tumor microenvironment on immune cells. Condamine et al. now report that polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) can be distinguished from neutrophils in the same cancer patient by the expression of the lipid metabolism–related molecule lectin-type oxidized LDL receptor-1 (LOX-1). LOX-1–expressing neutrophils were nearly undetectable in healthy individuals but were found prominently in tumor tissues. Moreover, exposing neutrophils from healthy individuals to endoplasmic reticulum stress resulted in up-regulation of LOX-1 and increased suppressive function. These data support the specific targeting of LOX-1–expressing PMN-MDSC for cancer immunotherapy. Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are important regulators of immune responses in cancer and have been directly implicated in the promotion of tumor progression. However, the heterogeneity of these cells and the lack of distinct markers hamper the progress in understanding the biology and clinical importance of these cells. Using partial enrichment of PMN-MDSC with gradient centrifugation, we determined that low-density PMN-MDSC and high-density neutrophils from the same cancer patients had a distinct gene profile. The most prominent changes were observed in the expression of genes associated with endoplasmic reticulum (ER) stress. Unexpectedly, low-density lipoprotein (LDL) was one of the most increased regulators, and its receptor oxidized LDL receptor 1 (OLR1) was one of the most overexpressed genes in PMN-MDSC. Lectin-type oxidized LDL receptor-1 (LOX-1) encoded by OLR1 was practically undetectable in neutrophils in peripheral blood of healthy donors, whereas 5 to 15% of total neutrophils in cancer patients and 15 to 50% of neutrophils in tumor tissues were LOX-1+. In contrast to their LOX-1− counterparts, LOX-1+ neutrophils had gene signature, potent immunosuppressive activity, up-regulation of ER stress, and other biochemical characteristics of PMN-MDSCs. Moreover, induction of ER stress in neutrophils from healthy donors up-regulated LOX-1 expression and converted these cells to suppressive PMN-MDSCs. Thus, we identified a specific marker of human PMN-MDSC associated with ER stress and lipid metabolism, which provides new insights into the biology and potential therapeutic targeting of these cells.