Interferon-gamma down-regulates NKG2D ligand expression and impairs the NKG2D-mediated cytolysis of MHC class I-deficient melanoma by natural killer cells.

NKG2D operates as an activating receptor on natural killer (NK) cells and costimulates the effector function of alphabeta CD8(+) T cells. Ligands of NKG2D, the MHC class I chain-related (MIC) and UL16 binding protein (ULBP) molecules, are expressed on a variety of human tumors, including melanoma. Recent studies in mice demonstrated that NKG2D mediates tumor immune surveillance, suggesting that antitumor immunity in humans could be enhanced by therapeutic manipulation of NKG2D ligand (NKG2DL) expression. However, signals and mechanisms regulating NKG2DL expression still need to be elucidated. Here, we asked whether the proinflammatory cytokine Interferon-gamma (IFN-gamma) affects NKG2DL expression in melanoma. Cell lines, established from MHC class I-negative and -positive melanoma metastases, predominantly expressed MICA and ULBP2 molecules on their surface. Upon IFN-gamma treatment, expression of MICA, in some cases, also of ULBP2 decreased. Besides melanoma, this observation was made also for glioma cells. Down-regulation of NKG2DL surface expression was dependent on the cytokine dose and the duration of treatment, but was neither due to an intracellular retention of the molecules nor to an increased shedding of ligands from the tumor cell surface. Instead, quantitative RT-PCR revealed a decrease of MICA-specific mRNA levels upon IFN-gamma treatment and siRNA experiments pointed to an involvement of STAT-1 in this process. Importantly, IFN-gamma-treated MHC class I-negative melanoma cells were less susceptible to NKG2D-mediated NK cell cytotoxicity. Our study suggests that IFN-gamma, by down-regulating ligand expression, might facilitate escape of MHC class I-negative melanoma cells from NKG2D-mediated killing by NK cells.

The coincidence of chromosome 15 aberrations and beta2-microglobulin gene mutations is causative for the total loss of human leukocyte antigen class I expression in melanoma.

PURPOSE: Total loss of surface presentation of human leukocyte antigen (HLA) class I molecules, protecting tumor cells from the recognition by cytotoxic host CD8+ T cells, is known to be caused by mutations in the beta2-microglobulin (beta2m) gene. We asked whether abnormalities of chromosome 15, harboring the beta2m gene on 15q21, in addition to beta2m gene mutations, are causative for the HLA class I-negative phenotype of melanoma cells. EXPERIMENTAL DESIGN: To answer this, we established primary cell lines from the beta2m-negative metastatic melanoma tissues of four different patients and analyzed them for beta2m gene mutations and chromosome 15 aberrations, the latter by loss of heterozygosity analysis, fluorescence in situ hybridization (FISH), and multicolor FISH. RESULTS: Mutations at the beta2m gene level were detected in all cell lines. The loss of heterozygosity analysis of microsatellite markers located on chromosome 15 in three of the four cell lines pointed to an extensive loss of chromosome 15 material. Subsequent molecular cytogenetic analysis revealed the coexistence of apparently normal and rearranged versions of chromosome 15 in three cell lines whereas the fourth cell line solely showed rearranged versions. Two of the four cell lines exhibited a special type of intrachromosomal rearrangement characterized by FISH signals specific for the subtelomeric region of 15q at both ends of the chromosome and one centromeric signal in between. CONCLUSIONS: Our data indicate that the complete loss of HLA class I expression in melanoma cells is due to the coincidence of the following mutational events: (a) chromosome 15 instability associated with an extensive loss of genetic material and (b) beta2m gene mutations.

Identification of HLA class I dependent immunogenic peptides from clonotypic TCRbeta expressed in cutaneous T-cell lymphoma.

The clonotypic T-cell receptor (TCR) is a potential target antigen for specific immunotherapy of cutaneous T-cell lymphoma (CTCL). We identified T-cell epitopes from the rearranged TCR beta chain of the malignant T-cell population by the "reverse immunology" approach. Peptide-specific T-cell lines were generated against predicted epitopes and tested for the recognition of tumor cells and cells transfected with the full-length DNA coding for TCRV beta chain. Two peptides derived from the clonotypic TCRVbeta of a HLA-A2 positive patient could induce peptide-specific T cells from peripheral blood mononuclear cells of healthy donors and the patient as assessed by IFN-gamma ELISpot assay. Furthermore, the reactive CTLs efficiently recognized autologous Sézary tumor cells, as well as HLA-A2 positive 293 cells transfected with recombinant plasmid expressing the corresponding TCRVbeta29 protein. Similar results were obtained in a HLA-A3+ patient for TCRVbeta7-Jbeta2.7. In conclusion, our experiments show that the TCR beta chain harbors epitopes suitable as targets for specific vaccination which might be a promising approach for the specific immunotherapy of cutaneous T-cell lymphoma patients.

A vitellogenic-like carboxypeptidase expressed by human macrophages is localized in endoplasmic reticulum and membrane ruffles.

Carboxypeptidase, vitellogenic-like (CPVL) is a serine carboxypeptidase of unknown function that was first characterized in human macrophages. Initial studies suggested that CPVL is largely restricted to the monocytic lineage, although it may also be expressed by cells outside the immune system. Here, we use a new monoclonal antibody to characterize the properties and localization of CPVL in human macrophages to elucidate a possible function for the protease. CPVL is up-regulated during the maturation of monocytes (MO) to macrophages, although the protein can be seen in both. In primary macrophages, CPVL is glycosylated with high mannose residues and colocalizes with markers for endoplasmic reticulum, while in MO it is more disperse and less clearly associated with endoplasmic reticulum. CPVL is highly expressed in lamellipodia and membrane ruffles, which also concentrate markers of the secretory pathway (MIP-1alpha and tumour necrosis factor-alpha) and major histocompatibility complex (MHC) class I and II molecules. CPVL can be seen on early latex bead and Candida albicans phagosomes, but it is not retained in the maturing phagosome, unlike MHC class I/II. CPVL has a mixed cytosolic and membrane-associated localization but is not detectable on the outer plasma membrane. We propose that CPVL may be involved in antigen processing, the secretory pathway and/or in actin remodelling and lamellipodium formation.