Defects in HLA class I antigen processing machinery (APM) component expression often have a negative impact on the clinical course of tumors and on the response to T cell-based immunotherapy. Since only scant information is available about the frequency and clinical significance of HLA class I APM component abnormalities in prostate cancer, the APM component expression pattern was analyzed in 59 primary prostate carcinoma, adjacent normal tissues, as well as in prostate carcinoma cell lines. The IFN-γ inducible proteasome subunits LMP2 and LMP7, TAP1, TAP2, calnexin, calreticulin, ERp57, and tapasin are strongly expressed in the cytoplasm of normal prostate cells, whereas HLA class I heavy chain (HC) and β2-microglobulin are expressed on the cell surface. Most of the APM components were downregulated in a substantial number of prostate cancers. With the exception of HLA class I HC, TAP2 and ERp57 not detectable in about 0.5% of tumor lesions, all other APM components were not detected in at least 21% of lesions analyzed. These APM component defects were associated with a higher Gleason grade of tumors and an early disease recurrence. Prostate carcinoma cell lines also exhibit a heterogeneous, but reduced constitutive APM component expression pattern associated with lack or reduced HLA class I surface antigens, which could be upregulated by IFN-γ. Our results suggest that HLA class I APM component abnormalities are mainly due to regulatory mechanisms, play a role in the clinical course of prostate cancer and on the outcome of T cell-based immunotherapies.

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Defective expression of HLA class I molecules is common in tumor cells and may allow escape from CTL-mediated immunity. We here investigate alterations in expression of HLA class I and their underlying molecular mechanisms in ovarian cancer patients. The HLA class I and HLA-A2 expression levels on noncultured tumor cells of 12 patients diagnosed with ovarian carcinoma were investigated by flow cytometry. Molecular analyses of antigen-processing machinery (APM) components were done in metastatic cancer cells, and the HLA genotype was determined in both these and the primary tumor. HER-2/neu-specific immunity was evaluated by enzyme-linked immunospot assays. The metastatic tumor cells from all patients expressed low levels of HLA class I surface antigens. In six of nine HLA-A2+ patients, HLA-A2 expression was heterogeneous with a subpopulation of tumor cells exhibiting decreased or absent HLA-A2 expression. One patient-derived tumor cell line completely lacked HLA-A2 but exhibited constitutive expression of APM components and high HLA class I expression that was further inducible by IFN-gamma treatment. Genotyping showed a haplotype loss in the metastatic tumor cells, whereas tumor tissue microdissected from the primary tumor exhibited an intact HLA gene complex. Interestingly, HLA-A2-restricted HER-2/neu-specific T-cell responses were evident among the lymphocytes of this patient. Abnormalities in HLA class I antigen expression are common features during the progression of ovarian cancer, and haplotype loss was, for the first time, described as an underlying mechanism.

Under physiological conditions, the non-classical major histocompatibility complex class Ib molecule human leukocyte antigen G (HLA-G) is selectively expressed in placental trophoblasts, thymus and cornea. In pathological situations, HLA-G expression was frequently found in tumour cells of distinct origin, thereby allowing these tumour cells to escape immune surveillance. Although HLA-G expression occurs at a relatively high frequency in renal cell carcinoma (RCC) of the clear cell subtype, the molecular mechanisms of its aberrant expression in RCC has not yet been determined. Therefore, the constitutive and cytokine-mediated HLA-G expression as well as its mode of regulation was investigated. In addition to HLA-G-specific mRNA expression, membrane-bound and soluble/shed HLA-G protein was determined. Eight of 14 RCC cell lines analysed (57%) exhibited HLA-G-specific transcripts, whereas only 6 of 14 RCC cell lines (43%) expressed HLA-G protein, suggesting a post-transcriptional control of HLA-G in some cases. Treatment of RCC cell lines with either interferon-gamma or interleukin-10, respectively, increased HLA-G-specific mRNA and protein in six of eight HLA-G(+) RCC lines (75%), but not in HLA-G(-) RCC cells. A 5'-aza-2-deoxycytidine (5-Aza-dC)-mediated demethylation of the HLA-G promoter DNA resulted in an enhanced HLA-G expression in four of six RCC cell lines, whereas a de novo induction of HLA-G was only observed in one HLA-G(-) RCC cell line on treatment with 5-Aza-dC. Thus, there exist multiple mechanisms controlling HLA-G expression in RCC, which might also have an impact on the development of RCC-specific immunotherapies.

The HER2 oncogene is frequently over-expressed in human cancers and a promising target for immune therapy. Previous studies have shown that over-expression of mouse or rat HER2 leads to markedly reduced levels of major histocompatibility complex (MHC) class I and molecules of the antigen processing and presentation machinery (APM), thus resulting in a phenotype promoting tumor escape from the immune system. Our study focuses on analyzing the effect of HER2 on MHC class I antigen presentation and sensitivity to tumor-antigen specific cytotoxic T lymphocytes (CTLs) in HLA-A2.1 1 melanomacell lines. We demonstrate significant inverse correlations both between the expression of HER2 and total MHC class I surface expression as well as between HER2 and HLA-A2. A significant reduction of HLA-A2 levels was found when melanoma and carcinoma cell lines were transfected with a human HER2 gene. A signaling-competent HER2 molecule was crucial for the observed HLA-A2 down-regulation, as transfectants expressing high levels of HER2 mutated in the tyrosine signaling domain did not show altered HLA-A2 expression. Importantly, the human melanoma cell line EST049 demonstrated reduced HER2 and melanoma antigen-specific recognition by CTLs upon HER2 transfection. In addition, high expression of HER2 prevented both IFN-c mediated HLA-A2 up-regulation and improved recognition by HLA-A2-restricted CTLs in treated cells. Moreover, key APM molecules were down-regulated by HER2. These findings implicate that HER2 over-expressing tumors may be more prone to escape from HLA-A2 restricted CTLs suggesting that immunotherapy approaches inducing an integrated humoral, cellular and innate immune response would be most effective.