Although melanoma tumors usually express antigens that can be recognized by T cells, immune-mediated tumor rejection is rare. In many cases this is despite the presence of high frequencies of circulating tumor antigen-specific T cells, suggesting that tumor resistance downstream from T cell priming represents a critical barrier. Analyzing T cells directly from the melanoma tumor microenvironment, as well as the nature of the microenvironment itself, is central for understanding the key downstream mechanisms of tumor escape. In the current report we have studied tumor-associated lymphocytes from a patient with metastatic melanoma and large volume malignant ascites. The ascites fluid showed abundant tumor cells that expressed common melanoma antigens and retained expression of class I MHC and antigen processing machinery. The ascites fluid contained the chemokines CCL10, CCL15, and CCL18 which was associated with a large influx of activated T cells, including CD8(+) T cells recognizing HLA-A2 tetramer complexes with peptides from Melan-A and NA17-A. However, several functional defects of these tumor antigen-specific T cells were seen, including poor production of IFN-gamma in response to peptide-pulsed APC or autologous tumor cells, and lack of expression of perforin. Although these defects were T cell intrinsic, we also observed abundant CD4(+)CD25(+)FoxP3(+) T cells, as well as transcripts for FoxP3, IL-10, PD-L1/B7-H1, and indoleamine-2,3-dioxygenase (IDO). Our observations suggest that, despite recruitment of large numbers of activated CD8(+) T cells into the tumor microenvironment, T cell hyporesponsiveness and additional negative regulatory mechanisms can limit the effector phase of the anti-tumor immune response.

IntroductionSmall GTPases of the Ras family such as Ras, Rho, and Rac are important regulators of growth factor receptor-induced activation events in a variety of cellular systems. A hallmark of GTPases like N-, K-, and H-Ras is that several posttranslational modifications of the synthesized protein must occur before localization to distinct cell membranes is achieved, a necessary prerequisite for functional activity. 1 Prenylation catalyzed by 1 of 3 intracellular enzymes, farnesyltransferase (FTase) or geranylgeranyltransferases (GGTase I and II), is the first critical modification step. Physiologically, prenylation in posttranslational processing of Ras proteins in mammalian cells is predominantly achieved by FTase. However, in cells where FTase activity is blocked, alternative prenylation of K-Ras, N-Ras, and RhoB by GGTase I has been described. 2 The FTase substrate in all Ras family proteins is the common COOHterminal CAAX tetrapeptide sequence. FTase catalyzes the transfer of a 15-carbon farnesyl group from farnesyldiphosphate, a product of the cholesterol biosynthesis pathway, to the CAAX cysteine residue. 3 Besides members of the Ras family and a variety of additional molecules such as HDJ-2 and Lamin A and B, several retinal and centromere-associated proteins are also known substrates of cellular FTase. [4][5][6] Further posttranslational modification for membrane targeting of Ras proteins after prenylation includes proteolysis of the AAX motif followed by alpha-carboxymethylation of the farnesylated cysteine residue. In addition, H-Ras and N-Ras are subsequently palmitoylated. 7 Since posttranslational isoprenoid modification is regarded as the critical event in localization of Ras proteins to cellular membranes, preventing the synthesis of the farnesyl precursor mevalonate by blocking of HMG-CoA reductase results in the depletion of intracellular farnesyl and accumulation of nonprocessed cytosolic Ras. 8 Furthermore, statins, which are HMG-CoAinhibitors widely used as cholesterollowering agents, have recently been attributed clinically relevant immunomodulatory properties. 9 Farnesyltransferase inhibitors (FTIs) are a class of drugs initially generated to interfere with the farnesylation of oncogenic Ras, thereby retaining it in the cytosol and preventing its activity. The development of several structurally different FTIs as anticancer agents was based on the initial observation that the phenotype of oncogenic Ras-transformed fibroblasts could be reversed by FTI treatment 10 followed by a variety of data regarding the antineoplastic activity of FTIs in several in vitro and in vivo tumor models. 5 Cancer cell lines treated with FTI show inhibition of proliferation, 11 induction of apoptosis, 12 or disturbed cell-cycle progression 13 in vitro. In particular, pediatric T-cell acute lymphoblastic leukemia (ALL) and French-American-British (FAB) M5 acute myeloid leukemia (AML) have been proven very sensitive to FTI-mediated cytotoxicity. 14 Inhibition of malignant cell growth could also be demonstrated ...