Claudia Hernandez

Arginase-Producing Myeloid Suppressor Cells in Renal Cell Carcinoma Patients: A Mechanism of Tumor Evasion

Myeloid suppressor cells with high arginase activity are found in tumors and spleen of mice with colon and lung cancer. These cells, described as macrophages or immature dendritic cells, deplete arginine and impair T cell proliferation and cytokine production. Although arginase activity has been described in cancer patients, it is thought to originate from tumor cells metabolizing arginine to ornithine needed to sustain rapid cell proliferation. The goal of this study was to determine whether myeloid suppressor cells producing high arginase existed in renal cell carcinoma patients. Peripheral blood mononuclear cells from 123 patients with metastatic renal cell carcinoma, prior to treatment, were found to have a significantly increased arginase activity. These patients had a markedly decreased cytokine production and expressed low levels of T cell receptor CD3zeta chain. Cell separation studies showed that the increased arginase activity was limited to a specific subset of CD11b+, CD14-, CD15+ cells with a polymorphonuclear granulocyte morphology and markers, instead of macrophages or dendritic cells described in mouse models. Furthermore, these patients had low levels of arginine and high levels of ornithine in plasma. Depletion of the CD11b+, CD14- myeloid suppressor cells reestablished T cell proliferation and CD3zeta chain expression. These results showed, for the first time, the existence of suppressor myeloid cells producing arginase in human cancer patients. In addition, it supports the concept that blocking arginase may be an important step in the success of immunotherapy.

Arginase I―Producing Myeloid-Derived Suppressor Cells in Renal Cell Carcinoma Are a Subpopulation of Activated Granulocytes

Myeloid-derived suppressor cells (MDSC) producing arginase I are increased in the peripheral blood of patients with renal cell carcinoma (RCC). MDSC inhibit T-cell function by reducing the availability of L-arginine and are therefore considered an important tumor escape mechanism. We aimed to determine the origin of arginase I-producing MDSC in RCC patients and to identify the mechanisms used to deplete extracellular L-arginine. The results show that human MDSC are a subpopulation of activated polymorphonuclear (PMN) cells expressing high levels of CD66b, CD11b, and VEGFR1 and low levels of CD62L and CD16. In contrast to murine MDSC, human MDSC do not deplete L-arginine by increasing its uptake but instead release arginase I into the circulation. Activation of normal PMN induces phenotypic and functional changes similar to MDSC and also promotes the release of arginase I from intracellular granules. Interestingly, although activation of normal PMN usually ends with apoptosis, MDSC showed no increase in apoptosis compared with autologous PMN or PMN obtained from normal controls. High levels of VEGF have been shown to increase suppressor immature myeloid dendritic cells in cancer patients. Treatment of RCC patients with anti-VEGF antibody bevacizumab, however, did not reduce the accumulation of MDSC in peripheral blood. In contrast, the addition of interleukin-2 to the treatment increased the number of MDSC in peripheral blood and the plasma levels of arginase I. These results may provide new insights on the mechanisms of tumor-induced anergy/tolerance and may help explain why some immunotherapies fail to induce an antitumor response.

Arginase I in myeloid suppressor cells is induced by COX-2 in lung carcinoma

Myeloid suppressor cells (MSCs) producing high levels of arginase I block T cell function by depleting l-arginine in cancer, chronic infections, and trauma patients. In cancer, MSCs infiltrating tumors and in circulation are an important mechanism for tumor evasion and impair the therapeutic potential of cancer immunotherapies. However, the mechanisms that induce arginase I in MSCs in cancer are unknown. Using the 3LL mouse lung carcinoma, we aimed to characterize these mechanisms. Arginase I expression was independent of T cell–produced cytokines. Instead, tumor-derived soluble factors resistant to proteases induced and maintained arginase I expression in MSCs. 3LL tumor cells constitutively express cyclooxygenase (COX)-1 and COX-2 and produce high levels of PGE2. Genetic and pharmacological inhibition of COX-2, but not COX-1, blocked arginase I induction in vitro and in vivo. Signaling through the PGE2 receptor E-prostanoid 4 expressed in MSCs induced arginase I. Furthermore, blocking arginase I expression using COX-2 inhibitors elicited a lymphocyte-mediated antitumor response. These results demonstrate a new pathway of prostaglandin-induced immune dysfunction and provide a novel mechanism that can help explain the cancer prevention effects of COX-2 inhibitors. Furthermore, an addition of arginase I represents a clinical approach to enhance the therapeutic potential of cancer immunotherapies.

Arginase, prostaglandins, and myeloid-derived suppressor cells in renal cell carcinoma.

Tumor-induced tolerance is a well-established phenomenon in cancer patients that can severely impair the therapeutic efficacy of immunotherapy. One mechanism leading to T-cell tolerance is the generation of myeloid-derived suppressor cells (MDSC) by soluble factors produced by the tumor. MDSC express CD11b+ as a common marker but may vary in their stage of maturation, depending on the tumor factors being produced. Arginase production by MDSC depletes arginine from the tumor microenvironment and impairs T-cell signal transduction and function. We studied whether an increase in MDSC could explain the molecular alterations and dysfunction found in T cells of patients with renal cell carcinoma (RCC). Arginase activity in the peripheral blood mononuclear cells of 117 RCC patients was increased between 6- to 8-fold compared with normal controls. The increased arginase activity was limited to the CD11b+CD14− myeloid cells and resulted in significantly decreased serum levels of arginine and increased ornithine in patients. Depletion of MDSC restored IFN-γ production and T-cell proliferation. Preliminary data suggest that prostaglandin E2 produced by the tumor induces arginase I expression in MDSC. Therefore, blocking MDSC activity may enhance the therapeutic efficacy of immunotherapy in RCC.

Helicobacter pylori Arginase Inhibits T Cell Proliferation and Reduces the Expression of the TCR ζ-Chain (CD3ζ)

Helicobacter pylori infects approximately half the human population. The outcomes of the infection range from gastritis to gastric cancer and appear to be associated with the immunity to H. pylori. Patients developing nonatrophic gastritis present a Th1 response without developing protective immunity, suggesting that this bacterium may have mechanisms to evade the immune response of the host. Several H. pylori proteins can impair macrophage and T cell function in vitro through mechanisms that are poorly understood. We tested the effect of H. pylori extracts and live H. pylori on Jurkat cells and freshly isolated human normal T lymphocytes to identify possible mechanisms by which the bacteria might impair T cell function. Jurkat cells or activated T lymphocytes cultured with an H. pylori sonicate had a reduced proliferation that was not caused by T cell apoptosis or impairment in the early T cell signaling events. Instead, both the H. pylori sonicate and live H. pylori induced a decreased expression of the CD3ζ-chain of the TCR. Coculture of live H. pylori with T cells demonstrated that the wild-type strain, but not the arginase mutant rocF(−), depleted l-arginine and caused a decrease in CD3ζ expression. Furthermore, arginase inhibitors reversed these events. These results suggest that H. pylori arginase is not only important for urea production, but may also impair T cell function during infection.

Inhibition of Fatty Acid Oxidation Modulates Immunosuppressive Functions of Myeloid-Derived Suppressor Cells and Enhances Cancer Therapies

Myeloid-derived suppressor cells in tumors, but not in the spleen, activated fatty acid uptake and oxidation (FAO) and increased their immunosuppressive pathways. Blocking FAO with inhibitors induced T-cell–mediated antitumor activity, which provides a novel approach for treatment. Myeloid-derived suppressor cells (MDSC) promote tumor growth by inhibiting T-cell immunity and promoting malignant cell proliferation and migration. The therapeutic potential of blocking MDSC in tumors has been limited by their heterogeneity, plasticity, and resistance to various chemotherapy agents. Recent studies have highlighted the role of energy metabolic pathways in the differentiation and function of immune cells; however, the metabolic characteristics regulating MDSC remain unclear. We aimed to determine the energy metabolic pathway(s) used by MDSC, establish its impact on their immunosuppressive function, and test whether its inhibition blocks MDSC and enhances antitumor therapies. Using several murine tumor models, we found that tumor-infiltrating MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation (FAO). This was accompanied by an increased mitochondrial mass, upregulation of key FAO enzymes, and increased oxygen consumption rate. Pharmacologic inhibition of FAO blocked immune inhibitory pathways and functions in T-MDSC and decreased their production of inhibitory cytokines. FAO inhibition alone significantly delayed tumor growth in a T-cell–dependent manner and enhanced the antitumor effect of adoptive T-cell therapy. Furthermore, FAO inhibition combined with low-dose chemotherapy completely inhibited T-MDSC immunosuppressive effects and induced a significant antitumor effect. Interestingly, a similar increase in fatty acid uptake and expression of FAO-related enzymes was found in human MDSC in peripheral blood and tumors. These results support the possibility of testing FAO inhibition as a novel approach to block MDSC and enhance various cancer therapies. Cancer Immunol Res; 3(11); 1236–47. ©2015 AACR.

l-Arginine Depletion Blunts Antitumor T-cell Responses by Inducing Myeloid-Derived Suppressor Cells

Enzymatic depletion of the nonessential amino acid l-Arginine (l-Arg) in patients with cancer by the administration of a pegylated form of the catabolic enzyme arginase I (peg-Arg I) has shown some promise as a therapeutic approach. However, l-Arg deprivation also suppresses T-cell responses in tumors. In this study, we sought to reconcile these observations by conducting a detailed analysis of the effects of peg-Arg I on normal T cells. Strikingly, we found that peg-Arg I blocked proliferation and cell-cycle progression in normal activated T cells without triggering apoptosis or blunting T-cell activation. These effects were associated with an inhibition of aerobic glycolysis in activated T cells, but not with significant alterations in mitochondrial oxidative respiration, which thereby regulated survival of T cells exposed to peg-Arg I. Further mechanistic investigations showed that the addition of citrulline, a metabolic precursor for l-Arg, rescued the antiproliferative effects of peg-Arg I on T cells in vitro. Moreover, serum levels of citrulline increased after in vivo administration of peg-Arg I. In support of the hypothesis that peg-Arg I acted indirectly to block T-cell responses in vivo, peg-Arg I inhibited T-cell proliferation in mice by inducing accumulation of myeloid-derived suppressor cells (MDSC). MDSC induction by peg-Arg I occurred through the general control nonrepressed-2 eIF2α kinase. Moreover, we found that peg-Arg I enhanced the growth of tumors in mice in a manner that correlated with higher MDSC numbers. Taken together, our results highlight the risks of the l-Arg-depleting therapy for cancer treatment and suggest a need for cotargeting MDSC in such therapeutic settings.

Pegylated arginase I: a potential therapeutic approach in T-ALL

Adult patients with acute lymphoblastic T cell leukemia (T-ALL) have a very poor prognosis and few effective therapeutic options. Therefore, novel therapies that increase the efficacy of the treatments and that prolong T-ALL patient survival are needed. Malignant T cells require high concentrations of nutrients to sustain their increased rate of proliferation. In this study, we determined whether L-Arginine depletion by the pegylated form of the L-Arginine-metabolizing enzyme arginase I (peg-Arg I) impairs the proliferation of malignant T cells. Our results show that peg-Arg I depleted L-Arginine levels in vitro and in vivo. In addition, treatment of malignant T-cell lines with peg-Arg I significantly impaired their proliferation, which correlated with a decreased progression into the cell cycle, followed by the induction of apoptosis. Furthermore, peg-Arg I impaired the expression of cyclin D3, a fundamental protein in T-ALL proliferation, through a global arrest in protein synthesis. Injection of peg-Arg I plus chemotherapy agent Cytarabine prolonged survival in mice bearing T-ALL tumors. This antitumoral effect correlated with an inhibition of T-ALL proliferation in vivo, a decreased expression of cyclin D3, and T-ALL apoptosis. The results suggest the potential benefit of L-Arginine depletion by peg-Arg I in the treatment of T-cell malignancies.

l-Arginine Deprivation Regulates Cyclin D3 mRNA Stability in Human T Cells by Controlling HuR Expression

Myeloid-derived suppressor cells are a major mechanism of tumor-induced immune suppression in cancer. Arginase I-producing myeloid-derived suppressor cells deplete l-arginine (L-Arg) from the microenvironment, which arrests T cells in the G0–G1 phase of the cell cycle. This cell cycle arrest correlated with an inability to increase cyclin D3 expression resulting from a decreased mRNA stability and an impaired translation. We sought to determine the mechanisms leading to a decreased cyclin D3 mRNA stability in activated T cells cultured in medium deprived of L-Arg. Results show that cyclin D3 mRNA instability induced by L-Arg deprivation is dependent on response elements found in its 3′-untranslated region (UTR). RNA-binding protein HuR was found to be increased in T cells cultured in medium with L-Arg and bound to the 3′-untranslated region of cyclin D3 mRNA in vitro and endogenously in activated T cells. Silencing of HuR expression significantly impaired cyclin D3 mRNA stability. L-Arg deprivation inhibited the expression of HuR through a global arrest in de novo protein synthesis, but it did not affect its mRNA expression. This alteration is dependent on the expression of the amino acid starvation sensor general control nonderepressible 2 kinase. These data contribute to an understanding of a central mechanism by which diseases characterized by increased arginase I production may cause T cell dysfunction.

Anti-leukemic mechanisms of pegylated arginase I in acute lymphoblastic T-cell leukemia

New treatments for adults with acute lymphoblastic T-cell leukemia (T-ALL) are urgently needed, as the current rate of overall remission in these patients is only about 40 percent. We recently showed the potential therapeutic benefit of the pegylated-human-arginase I (peg-Arg I) in T-ALL. However, the mechanisms by which peg-Arg I induces an anti-T-ALL effect remained unknown. Our results show the induction of T-ALL cell apoptosis by peg-Arg I, which associated with a global arrest in protein synthesis and with the phosphorylation of the eukaryotic-translation-initiation factor 2 alpha (eIF2α). Inhibition of eIF2α phosphorylation in T-ALL cells prevented the apoptosis induced by peg-Arg I, whereas the expression of a phosphomimetic eIF2α form increased the sensibility of T-ALL cells to peg-Arg I. Phosphorylation of eIF2α by peg-Arg I was mediated through kinases PERK and GCN2 and down-regulation of phosphatase GADD34. GCN2 and decreased GADD34 promoted T-ALL cell apoptosis after treatment with peg-Arg I, whereas PERK had an unexpected anti-apoptotic role. Additional results showed that phospho-eIF2α signaling further increased the anti-leukemic effects induced by peg-Arg I in T-ALL-bearing mice. These results suggest the central role of phospho-eIF2α in the anti-T-ALL effects induced by peg-Arg I and support its study as a therapeutic target.