Immunogenicity of HLA-A1-restricted peptides derived from S100A4 (metastasin 1) in melanoma patients

S100A4 (metastasin 1) belongs to the S100 family of Ca²⁺ binding proteins. While not present in most differentiated adult tissues, S100A4 is upregulated in the micromilieu of tumors. It is primarily expressed by tumor-associated macrophages, fibroblasts, and tumor endothelial cells. Due to its strong induction in tumors S100A4 is a promising target for cancer immunotherapy. By reverse immunology, using epitope prediction programs, we identified 3 HLA-A1-restricted peptide epitopes (S100A4 A1-1, A1-2, and A1-3) which are subject to human T cell responses as detected in peripheral blood of melanoma patients by means of IFN-γ ELISPOT and cytotoxicity assays. In addition, IFN-γ responses to S100A4 A1-2 can not only be induced by stimulation of T cells with peptide-loaded DC but also by stimulation with S100A4 protein-loaded DC, indicating that this epitope is indeed generated by processing of the endogenously expressed protein. In addition, S100A4 A1-2 reactive T cells demonstrate lysis of HLA-A1⁺ fibroblasts in comparison to HLA-A1⁻ fibroblasts. In summary, this HLA-A1-restricted peptide epitope is a candidate for immunotherapeutical approaches targeting S100A4-expressing cells in the tumor stroma.     

Incubation of antigen-sensitized T lymphocytes activated with bryostatin 1 + ionomycin in IL-7 + IL-15 increases yield of cells capable of inducing regression of melanoma metastases compared to culture in IL-2

Regression of established tumors can be induced by adoptive immunotherapy (AIT) with tumor draining lymph node (DLN) lymphocytes activated with bryostatin and ionomycin (B/I). We hypothesized that B/I-activated T cells cultured in IL-7 + IL-15 might proliferate and survive in culture better than cells cultured in IL-2, and that these cells would have equal or greater anti-tumor activity in vivo. Tumor antigen-sensitized DLN lymphocytes from either wild-type or T cell receptor transgenic mice were harvested, activated with B/I, and expanded in culture with either IL-2, IL-7 + IL-15 or a regimen of alternating cytokines. Cell yields, proliferation, apoptosis, phenotypes, and in vitro responses to tumor antigen were compared for cells grown in different cytokines. These T cells were also tested for anti-tumor activity against melanoma lung metastases established by prior i.v. injection of B16 melanoma cells. IL-7 + IL-15 or alternating cytokines resulted in much faster and prolonged proliferation and much less apopotosis of B/I-activated T cells than culturing the same cells in IL-2. This resulted in approximately tenfold greater yields of viable cells. Culture in IL-7 + IL-15 yielded higher proportions of CD8+ T cells and a higher proportion of cells with a central memory phenotype. Despite this, T cells grown in IL-7 + IL-15 had higher IFN-γ release responses to tumor antigen than cells grown in IL-2. Adoptive transfer of B/I-activated T cells grown in IL-7 + IL-15 or the alternating regimen had equal or greater efficacy on a “per-cell” basis against melanoma metastases. Activation of tumor antigen-sensitized T cells with B/I and culture in IL-7 + IL-15 is a promising modification of standard regimens for production of T cells for use in adoptive immunotherapy of cancer.     

Characterization of MHC class-I restricted TCRαβ<Superscript>+</Superscript> CD4<Superscript>−</Superscript> CD8<Superscript>−</Superscript> double negative T cells recognizing the gp100 antigen from a melanoma patient after gp100 vaccination

The immune attack against malignant tumors require the concerted action of CD8⁺ cytotoxic T lymphocytes (CTL) as well as CD4⁺ T helper cells. The contribution of T cell receptor (TCR) αβ⁺ CD4⁻ CD8⁻ double-negative (DN) T cells to anti-tumor immune responses is widely unknown. In previous studies, we have demonstrated that DN T cells with a broad TCR repertoire are present in humans in the peripheral blood and the lymph nodes of healthy individuals. Here, we characterize a human DN T cell clone (T4H2) recognizing an HLA-A2-restricted melanoma-associated antigenic gp100-peptide isolated from the peripheral blood of a melanoma patient. Antigen recognition by the T4H2 DN clone resulted in specific secretion of IFN-γ and TNF. Although lacking the CD8 molecule the gp100-specifc DN T cell clone was able to confer antigen-specific cytotoxicity against gp100-loaded target cells as well as HLA-A2⁺ gp100 expressing melanoma cells. The cytotoxic capacity was found to be perforin/granzymeB-dependent. Together, these data indicate that functionally active antigen-specific DN T cells recognizing MHC class I-restricted tumor-associated antigen (TAA) may contribute to anti-tumor immunity in vivo. A. Mackensen and K. Fischer contributed equally to this work and should be considered joint senior authors. This work was supported by the Deutsche Forschungsgemeinschaft (MA 1351/5-1, KFO 146) and NIH grants CA90873, CA102280, 104947 (MIN). Companion paper: “Relationship between CD8-dependent antigen recognition, T cell functional avidity, and tumor cell recognition” by Tamson V. Moore et al. doi: .     

Modified peptides in anti-cancer vaccines: are we eventually improving anti-tumour immunity?

The discovery of tumour antigens recognized by T cells and the features of immune responses directed against them has paved the way to a multitude of clinical studies aimed at boosting anti-tumour T cell immunity as a therapeutic tool for cancer patients. One of the different strategies explored to ameliorate the immunogenicity of tumour antigens in vaccine protocols is represented by the use of optimized peptides or altered peptide ligands, whose amino acid sequence has been modified for improving HLA binding or TCR interaction with respect to native epitopes. However, despite the promising results achieved with preclinical studies, the clinical efficacy of this approach has not yet met the expectations. Although multiple reasons could explain the relative failure of altered peptide ligands as more effective cancer vaccines, the possibility that T cells primed by modified tumour peptides might may be unable to effectively cross-recognize tumour cells has not been sufficiently addressed. Indeed, the introduction of conservative amino acid substitutions may still produce diverse and unpredictable changes in the HLA/peptide interface, with consequent modifications of the TCR repertoire that can interact with the complex. This could lead to the expansion of a broad array of T cells whose TCRs may not necessarily react with equivalent affinity with the original antigenic epitope. Considering the results presently achieved with this vaccine approach, and the emerging availability of alternative strategies for boosting anti-tumour immunity, the use of modified tumour peptides could be reconsidered. This article is a symposium paper from the conference “Immunotherapy—From Basic Research to Clinical Applications”, Symposium of the Collaborative Research Center (SFB) 685, held in Tübingen, Germany, 6–7 March 2008.     

Induction of apoptosis by Hax-1 siRNA in melanoma cells

HS1-associated protein X-1 (Hax-1) is a novel intracellular protein and recent studies suggested that it is an anti-apoptotic factor in different tumors. Hax-1 expression was upregulated in various metastatic tumors and cancer cell lines, including melanoma. To understand the role of Hax-1 in melanoma development and progression, we constructed Hax-1 short interfering RNA (siRNA) expression vectors to downregulate Hax-1 expression in a human melanoma A375 cell line. One of the two Hax-1 RNA interference (RNAi) constructs significantly reduced melanoma cell viability, which was due to induction of apoptosis in A375 cells. Molecularly, the induced apoptosis through downregulation of Hax-1 expression was mediated by activation of caspase-3 and poly-ADP-ribose polymerase (PARP) enzymatic activity in A375 cells. The data indicate that Hax-1 plays a role in suppression of apoptosis and promotion of melanoma cell growth, suggesting that this Hax-1 siRNA has a therapeutic indication in control of melanoma.     

Expression profile of SIRT2 in human melanoma and implications for sirtuin-based chemotherapy

Melanoma is cancer of melanin-containing melanocyte cells. This neoplasm is one of the most deadly forms of skin cancer, and currently available therapeutic options are insufficient in significantly improve outcomes for many patients. Therefore, novel targets are required to effectively manage this neoplasm. Several sirtuins have previously been found to be upregulated in melanoma, so in this study, the expression profile of SIRT2 was determined. Employing a tissue microarray containing benign nevi, primary melanomas, and lymph node metastases, we have found that the tissue from lymph node metastases appears to have a significant upregulation of SIRT2 relative to primary tumors across the nuclear, cytoplasmic, and whole cell data. Additionally, SIRT2 staining was found to be higher in the nucleus of metastatic melanomas compared to cytoplasmic staining. As SIRT2 is considered to be a predominantly cytoplasmic protein, this is a novel and very interesting finding. This, combined with previous studies that show other sirtuins are increased in melanoma and involved in cellular proliferation and survival, leads to the suggestion that exploring pan-sirtuin inhibitors may be the best target for the next iteration of melanoma chemotherapeutics.     

Ultrasound-mediated interferon β gene transfection inhibits growth of malignant melanoma

We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon β (IFN-β) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-β in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-β genes mixed with microbubbles. Successful sonotransfection with IFN-β gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-β gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.     

Acid ceramidase expression modulates the sensitivity of A375 melanoma cells to dacarbazine

Dacarbazine (DTIC) is the treatment of choice for metastatic melanoma, but its response in patients remains very poor. Ceramide has been shown to be a death effector and to play an important role in regulating cancer cell growth upon chemotherapy. Among ceramidases, the enzymes that catabolize ceramide, acid ceramidase (aCDase) has been implicated in cancer progression. Here we show that DTIC elicits a time- and dose-dependent decrease of aCDase activity and an increase of intracellular ceramide levels in human A375 melanoma cells. The loss of enzyme activity occurred as a consequence of reactive oxygen species-dependent activation of cathepsin B-mediated degradation of aCDase. These events preceded autophagic features and loss of cell viability. Down-regulation of acid but not neutral or alkaline ceramidase 2 resulted in elevated levels of ceramide and sensitization to the toxic effects of DTIC. Conversely, inducible overexpression of acid but not neutral ceramidase reduced ceramide levels and conferred resistance to DTIC. In conclusion, we report that increased levels of ceramide, due to enhanced degradation of aCDase, are in part responsible for the cell death effects of DTIC. These results suggest that down-regulation of aCDase alone or in combination with DTIC may represent a useful tool in the treatment of metastatic melanoma.     

miRNA-205 suppresses melanoma cell proliferation and induces senescence via regulation of E2F1 protein

MicroRNAs (miRNAs) regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-205 is significantly suppressed in melanoma specimens when compared with nevi and is correlated inversely with melanoma progression. miRNA target databases predicted E2F1 and E2F5 as putative targets. The expression levels of E2F1 and E2F5 were correlated inversely with that of miR-205 in melanoma cell lines. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR sequences complementary to either E2F1 or E2F5. Overexpression of miR-205 in melanoma cells reduced E2F1 and E2F5 protein levels. The proliferative capacity of melanoma cells was suppressed by miR-205 and mediated by E2F-regulated AKT phosphorylation. miR-205 overexpression resulted in induction of apoptosis, as evidenced by increased cleaved caspase-3, poly-(ADP-ribose) polymerase, and cytochrome c release. Stable overexpression of miR-205 suppressed melanoma cell proliferation, colony formation, and tumor cell growth in vivo and induced a senescence phenotype accompanied by elevated expression of p16INK4A and other markers for senescence. E2F1 overexpression in miR-205-expressing cells partially reversed the effects on melanoma cell growth and senescence. These results demonstrate a novel role for miR-205 as a tumor suppressor in melanoma.