No abstract

Purpose: Immune dysfunction reported in renal cell carcinoma (RCC) patients may contribute to tumor progression. Myeloid-derived suppressor cells (MDSC) represent one mechanism by which tumors induce T-cell suppression. Several factors pivotal to the accumulation of MDSC are targeted by the tyrosine kinase inhibitor, sunitinib. The effect of sunitinib on MDSC-mediated immunosuppression in RCC patients has been investigated. Experimental Design: Patient peripheral blood levels of MDSC and regulatoryT-cell (Treg) and T-cell production of IFN-g were evaluated before and after sunitinib treatment. Correlations between MDSC and Treg normalization as well asT-cell production of IFN-g were examined. The in vitro effect of sunitinib on patient MDSC was evaluated. Results: Metastatic RCC patients had elevated levels of CD33 + HLA-DR -and CD15 + CD14-MDSC, and these were partially overlapping populations. Treatment with sunitinib resulted in significant reduction in MDSC measured by several criteria. Sunitinib-mediated reduction in MDSC was correlated with reversal of type 1 T-cell suppression, an effect that could be reproduced by the depletion of MDSC in vitro. MDSC reduction in response to sunitinib correlated with a reversal of CD3 + CD4 + CD25 hi Foxp3 + Treg cell elevation. No correlation existed between a change in tumor burden and a change in MDSC, Treg, or T-cell production of IFN-g. In vitro addition of sunitinib reduced MDSC viability and suppressive effect when used at z1.0 Ag/mL. Sunitinib did not induce MDSC maturation in vitro. Conclusions: Sunitinib-based therapy has the potential to modulate antitumor immunity by reversing MDSC-mediated tumor-induced immunosuppression.

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.

l-Arginine plays a central role in the normal function of several organs including the immune system. It is metabolized in macrophages by inducible nitric oxide synthase to produce nitric oxide, important in the cytotoxic mechanisms, and by arginase I (ASE I) and arginase II (ASE II) to synthesize l-ornithine and urea, the first being the precursor for the production of polyamines needed for cell proliferation. l-Arginine availability can modulate T cell function. Human T cells stimulated and cultured in the absence of l-arginine lose the expression of the TCR ζ-chain (CD3ζ) and have an impaired proliferation and a decreased cytokine production. The aim of this work was to test whether activated macrophages could modulate extracellular levels of l-arginine and alter T cell function, and to determine which metabolic pathway was responsible for this event. The results show that macrophages stimulated with IL-4 + IL-13 up-regulate ASE I and cationic amino acid transporter 2B, causing a rapid reduction of extracellular levels of l-arginine and inducing decreased expression of CD3ζ and diminished proliferation in normal T lymphocytes. Competitive inhibitors of ASE I or the addition of excess l-arginine lead to the re-expression of CD3ζ and recovery of T cell proliferation. In contrast, inducible nitric oxide synthase or ASE II failed to significantly reduce the extracellular levels of l-arginine and modulate CD3ζ expression. These results may provide new insights into the mechanisms leading to T cell dysfunction and the down-regulation of CD3ζ in cancer and chronic infectious diseases.

L-Arg plays a central role in the normal function of several organ systems including the immune system. L-Arg can be depleted by arginase I produced by macrophages and hepatocytes in several disease states such as trauma and sepsis and following liver transplantation. The decrease in L-Arg levels induces a profound decrease in T cell function through mechanisms that have remained unclear. The data presented here demonstrate that Jurkat T cells cultured in medium without L-Arg (L-Arg-free RPMI) have a rapid decrease in the expression of the T cell antigen receptor chain (CD3), the principal signal transduction element in this receptor, and a decrease in T cell proliferation. This phenomenon is completely reversed by the replenishment of L-Arg but not other amino acids. These changes are not caused by cell apoptosis; instead, the diminished expression of CD3 protein is paralleled by a decrease in CD3 mRNA. This change in CD3 mRNA expression is not caused by a decrease in the transcription rate but rather by a significantly shorter CD3 mRNA half-life. This mechanism is sensitive to cycloheximide. Therefore, the regulation of L-Arg concentration in the microenvironment could represent an important mechanism to modulate the expression of CD3 and the T cell receptor and consequently of T cell function.L-Arg plays a central role in several functions of the immune system (1-3). It is metabolized in macrophages by two independent enzymatic pathways (4), the inducible nitric-oxide synthetase and arginase I, leading to different effects on the immune system (4 -11). L-Arg is metabolized by inducible nitricoxide synthetase to produce nitric oxide, one of the principal cytotoxic mechanisms in macrophages (12-15). Alternatively, arginase I metabolizes L-Arg to L-ornithine and urea, the first being the precursor for the production of polyamines that are essential for cell proliferation and fibroblast function (5, 6, 11). The depletion of L-Arg by an increased production of arginase I following liver transplantation (16 -18), severe trauma (9, 20), or sepsis (21) coincides with a major decrease in T cell proliferation. Furthermore, the infusion of high doses of L-Arg results in a recovery of T cell function and an increase in the number of CD4 ϩ cells (22-24), suggesting that L-Arg may play an important role in regulating the T cell function by mechanisms that have remained unclear.The T cell receptor chain (CD3) is the principal signal transduction element of the T cell antigen receptor (TCR) (25)(26)(27). A decreased expression of CD3 has been described in T cells from patients with cancer (28 -32), lupus (33), and chronic infectious diseases such as leprosy (34) and tuberculosis (35). The mechanisms mediating the CD3 decrease are poorly understood. We tested the effect of the absence of L-Arg on T cell signal transduction. The results show that Jurkat T cells cultured in tissue culture medium without L-Arg had a rapid decrease in the expression of CD3 but not of other chains of the TCR such as CD3⑀ (36). The absence of L-Arg...

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 À myeloid cells and resulted in significantly decreased serum levels of arginine and increased ornithine in patients. Depletion of MDSC restored IFN-g production and T-cell proliferation. Preliminary data suggest that prostaglandin E 2 produced by the tumor induces arginase I expression in MDSC. Therefore, blocking MDSC activity may enhance the therapeutic efficacy of immunotherapy in RCC. Immunotherapy with cytokines, such as interleukin-2 (IL-2),has become a standard of care for patients with renal cell carcinoma (RCC). However, only 20% to 30% of patients have a partial or complete response (1), which is significantly lower than the therapeutic efficacy suggested by animal models. One possible explanation is that tumor-induced tolerance diminishes the potential therapeutic effect of T cells, the principal effector cells in most forms of immunotherapy. Over the last decade, several mechanisms by which tumors escape the immune response have been described. These can be divided into three major groups: alterations in antigen expression in tumor cells to make them less detectable, the active suppression of dendritic and T-cell function by inhibitory molecules or factors produced by tumors, and the induction of cells that can suppress the immune response, including myeloid-derived suppressor cells (MDSC) and regulatory T cells. We have focused our work on characterizing MDSC and the mechanisms by which they cause T-cell dysfunction in cancer.

No abstract

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.