Transfection of TAP 1 gene restores HLA class I expression in human small-cell lung carcinoma

HLA class I antigens of the human major histocompatibility complex (MHC) play an important role in immune response. Consistent with their role in immune surveillance, these antigens are expressed on most cell types. However, a marked deficiency or lack of expression of these antigens has been observed in a variety of human neoplasms. We have shown that a number of class I-deficient human tumor cell lines, including small-cell lung carcinoma, lacked products of MHC-encoded TAP1 and LMP2 genes. Since a direct evidence for the role of these genes in class I expression in tumor cells is not available, in the present study we transfected class I-deficient human small-cell lung carcinoma cells with cDNAs corresponding to TAP1 gene and to LMP2 gene. Following transfection, tumor cells expressed products of the respective transfected gene. Cell-surface expression of class I molecules was, however, observed in cells transfected with TAP1, but not in tumor cells transfected with LMP2 gene. Our results provide conclusive evidence for a role of TAP1 gene in class I expression and suggest that transfection of TAP genes may be useful to upregulate class I expression in tumor cells. This strategy for restoration of class I expression by transfection of TAP genes is relevant for tumor rejection and/or abrogation of metastases formation. Int. J. Cancer 75:112–116, 1998.© 1998 Wiley-Liss, Inc.     

HLA class I and neuroendocrine antigen expression in multidrug resistant small cell lung carcinoma cell lines

Antigenic marker expression was studied in a series of eight small cell lung carcinoma (SCLC) cell lines, according to their histological subtype, classic or variant. These lines were obtained from human tumors xenografted into nude mice, originally derived from heterotransplanted tumor biopsy samples. We looked at an altered expression of HLA class I antigens, a battery of neuroendocrine antigens and the P-glycoprotein (Pgp) responsible for MDR1 encoded multidrug resistance, as markers of tumor malignancy. Three cell lines out of four of the classic subtype and two cell lines out of four of the variant subtype showed a lack or a low expression of HLA class I antigen. Recombinant interferon gamma (rIFN-gamma) treatment (100 U/ml, for 48 h) increased HLA class I expression of the cell lines differently, but did not induce an imbalance between HLA-A and HLA-B molecules as described in other tumor models. Neuroendocrine antigens were tested in six out of these eight lines, using a family of monoclonal antibodies developed against the cell membrane antigens of low passage cell lines derived from pleural effusions (de Leij et al, Cancer Res 45: 2192-2200, 1985). Globally, these antigens were more highly expressed in classic subtypes of SCLC. Neuroendocrine antigens corresponding to MOC-21 and MOC-32 monoclonal antibodies were weakly expressed in variant forms. Pgp expression was detectable with the JSB1 monoclonal antibody on the three variant SCLCs out of the six lines. Comparing two cell lines originated from the same patient before and after therapy, we showed that neuroendocrine reactivity to MOC-21 and MOC-32 was lost simultaneously with a gain of Pgp expression, and with a classic to variant histological transition. With regard to the clinical evolution, HLA class I expression and stimulation by rIFN-gamma was not related to malignancy. It appears that for variant forms, a low expression of neuroendocrine antigens detected by MOC-21 and MOC-32 monoclonal antibodies and a high level of Pgp predict for a poor prognosis.     

HLA class I antigens expression in renal cell carcinoma: histopathological and clinical correlation

Immunohistochemical analysis of HLA class I antigens expression in 26 renal cell carcinomas (RCCs)--18 clear cell, 6 granular and 2 chromophobe--was performed with indirect immunoperoxidase method. Results were correlated with extent and immunophenotype of tumor infiltrating mononuclear cells, histopathological (histology, cytology, grade, presence of necrosis) and clinical (tumor diameter, TNM classification) characteristics of RCC. 4 (15%) RCCs showed reduced HLA class I presence and this was associated with greater tumor diameter and more frequent T3, T4 and M1 stage. All tumors with altered HLA class I antigens expression were grade 2 or 3 and strong correlation with presence of necrosis (p=0.006) was noticed, while mononuclear cell infiltrates (especially CD8+ T lymphocytes) were less extensive compared to tumors with normal HLA class I level. Our results suggest more aggressive clinical behavior of RCCs with reduced HLA class I antigens expression, probably due to impaired cellular antitumor immune response.     

HLA antigens in colorectal tumours--low expression of HLA class I antigens in mucinous colorectal carcinomas

Expression of HLA antigens and beta 2-microglobulin was studied by immunoperoxidase staining of frozen sections of 9 mucinous and 10 nonmucinous colorectal adenocarcinomas, 1 cloacogenic carcinoma, 12 colorectal adenomas and 4 samples of normal colorectal mucosa using monoclonal antibodies (MAbs). Staining results were related to histopathological features. HLA Class I antigens were strongly expressed in morphologically normal colorectal epithelium, in all adenomas tested and in all non-mucinous carcinomas. In contrast, expression of HLA class I antigens by the majority of tumour cells was present in only 2 of the 9 mucinous carcinomas, whereas 2 of these mucinous carcinomas were completely negative. In the mucinous carcinomas a striking scarcity of mononuclear inflammatory infiltrate, especially around the mucus accumulations, was observed. HLA class II antigen expression was not detected in normal epithelium and was only focally present in 1 of the 12 adenomas. In 6 out of the 20 carcinomas tested between 20% and 90% of the tumour cells were stained by MAbs against HLA class II antigens. Apart from the low expression of HLA class I antigens in mucinous carcinomas no relationship was found between expression of HLA antigens and histological features of the tumours. The relative poor prognosis of mucinous colorectal carcinoma as reported in the literature may be associated with low expression of HLA class I antigens and scant mononuclear inflammatory infiltrate, which may be a reflection of a weak immune response to the tumour cells.     

Gene expression of human squamous cell carcinoma antigens 1 and 2 in human cell lines

The squamous cell carcinoma antigen (SCCA) serves as a serological marker for squamous cell carcinomas. Molecular cloning of the SCCA genomic region has revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2. SCCA1 is a papain-like cysteine proteinase inhibitor, while SCCA2 is a chymotrypsin-like serine proteinase inhibitor. Little is known concerning how expression of the SCCA1 and SCCA2 genes is regulated in human cell lines. The purpose of this study was to determine whether the SCCA1 gene or SCCA2 gene is more strongly expressed in human cell lines. Squamous cell carcinoma cell lines secreted respectively 4 times and 50 times as much SCCA proteins into medium as normal human keratinocyte and non-squamous cell carcinoma cell lines, as measured by enzyme-linked immunosorbent assay. Quantitative RT-PCR ELISA digoxigenin-labeling assay demonstrated that SCCA1 mRNA expression in squamous cell carcinoma cell lines was respectively 2.8 times and 42 times that in keratinocyte and non-squamous cell carcinoma cell lines. The ratio of SCCA1 to SCCA2 mRNA expression differed distinctly among squamous cell carcinoma, keratinocyte and non-squamous cell carcinoma cell lines (2.8, squamous; 24.1, keratinocyte; 11.0, non-squamous). These findings suggest that SCCA1 is mainly expressed in squamous cell carcinoma, keratinocyte and non-squamous cell carcinoma cell lines and that the ratio of SCCA1 to SCCA2 expression might be a novel marker for the detection of squamous cell carcinoma.     

HLA class I antigens are possible prognostic factors in hepatocellular carcinoma

Thirty patients who underwent hepatectomy for the treatment of hepatocellular carcinoma (HCC) were examined for expression of HLA class I antigens on HCC cells by flow cytometry. The expression was found significantly lower in cases of stage IV compared with those of stage I or stage II (p<0.05), and in cases of intrahepatic metastases compared with those without metastases (p<0.001). In cases of non-curative hepatectomy, the expression of HLA class I antigens was lower compared with those treated by curative resection. Postoperative cumulative disease-free survival rates were well correlated with the expression rate of HLA class I antigens (p<0.05). Expression of HLA class I antigens on HCC may indicate low malignancy and better prognosis.     

High frequency of allele-specific down-regulation of HLA class I expression in uveal melanoma cell lines

Uveal melanoma is the most common primary intra-ocular tumor in adults and has a high mortality rate due to liver metastases, for which no effective treatment is available. To investigate whether immunotherapy might be feasible in uveal melanoma, the HLA class I surface expression of 6 uveal melanoma cell lines was analyzed by flow cytometry using a broad panel of allele-specific monoclonal antibodies. To up-regulate HLA expression, cells were also cultured with IFN-alpha or -gamma. In general, expression of HLA-A alleles was high (except for cell line EOM-3) and could be further up-regulated by both IFN-alpha and -gamma. In cell line EOM-3, IFN-gamma treatment resulted in significant HLA-A expression while IFN-alpha treatment did not. Expression of HLA-B alleles was low or even negative. Variable effects were observed after IFN treatment. In 3 cell lines, expression of some HLA-B alleles could not be induced by IFN-alpha or -gamma: HLA-B44 in cell line 92-1, HLA-B15 in cell line OCM-1 and HLA-B5 in cell line MEL-202. The other B alleles of these cell lines showed enhanced expression levels upon IFN stimulation. In OMM-1 cells, IFN-alpha and -gamma increased the expression of HLA-A but did not induce expression of the 2 B alleles, indicating an HLA-B locus-specific loss. We thus found a high frequency of allele-specific and locus-specific down-regulation of HLA expression in uveal melanoma cell lines. Some of these defects were not restored by IFN-alpha or -gamma treatment. The lack of HLA expression may explain why uveal melanoma cells escape immune surveillance by cytotoxic T cells and complicate the development of immunotherapy in uveal melanoma.     

Loss of HLA haplotype in lung cancer cell lines: implications for immunosurveillance of altered HLA class I/II phenotypes in lung cancer.

Loss of expression of HLA class I antigens has been demonstrated in a wide variety of tumors and is considered to be one of the mechanisms whereby tumors escape T-cell surveillance. Genomic DNA of MHC class I/II molecules in seven lung cancer cell lines was investigated and compared with that in peripheral blood mononuclear cells. In three cell lines, OU-LC-A1, OU-LC-A2, and OU-LC-AS1, a loss of HLA haplotype was observed. Aberrations of HLA class I/II in tumor cell lines should be considered when MHC-restricted phenomena in vitro models are assessed and clinical use of tumor vaccination in vivo is considered.     

Prognostic value of HLA class I expression in patients with oral squamous cell carcinoma

Human leukocyte antigen (HLA) class Ⅰ molecules play a central role in anticancer immunity, but their prognostic value in oral squamous cell carcinoma (OSCC) remains unclear. We examined HLA class I expression in 2 distinct tumor compartments, namely, the tumor center and invasive front, and evaluated the association between its expression pattern and histopathological status in 137 cases with OSCC. Human leukocyte antigen class Ⅰ expression was graded semiquantitatively as high, low, and negative. At the invasive front of the tumor, HLA class I expression was high in 72 cases (52.6%), low in 44 cases (32.1%), and negative in 21 cases (15.3%). The HLA class I expression in the tumor center was high in 48 cases (35.0%), low in 58 cases (42.4%), and negative in 31 cases (22.6%). The 5‐year overall survival and disease‐specific survival rates were good in cases with high HLA class I expression at the invasive front; however, there was no significant difference in survival based on HLA class I expression in the tumor center. In addition, high HLA class I expression was correlated with high CD8⁺ T cell density, whereas negative HLA class I expression was correlated with low CD8⁺ T cell density at the invasive front. These results suggest that it is easier for CD8⁺ T cells to recognize presented peptides in the case of high HLA class Ⅰ expression at the tumor invasive front and could be a prognostic factor for OSCC.     

Reduced expression of HLA class I and II antigens in colon cancer

The expression of HLA class I and II antigens was studied by immunohistochemistry in (a) specimens of colon cancer from 25 patients, (b) normal colonic mucosa obtained 5-10 cm away from each tumor, and (c) colonic mucosa from 13 normal individuals. Thirteen of the tumor specimens had normal epithelium adjacent to the cancer, which thus served as an internal control. The expression of HLA class I antigens in colon cancer was dramatically reduced compared to control (P less than 0.0001): undetectable in 28%, diminished in 68%, normal in 4%. The expression of class II antigens was also reduced in cancer (P less than 0.0001 for all when compared to normal), being undetectable in most (HLA-DP 64%, HLA-DQ 72%, HLA-DR 68%). In 44% of the cancers all three HLA class II antigens were undetectable; in 92% at least one class II antigen was undetectable; and in 20% both class I and class II antigens were undetectable. No cancer specimen had a completely normal HLA phenotype. The expression of other surface antigens was preserved in cancer tissues and, therefore, loss of HLA antigens was not due to a nonspecific decline in surface molecules. When glands of normal mucosa immediately adjacent to cancer were compared to those of normal controls, significantly reduced expression of only HLA class I antigens (P = 0.0149) and HLA-DP (P = 0.034) was found. The expression of the HLA antigens in colonic mucosa remote from the cancer was no different from that of normal controls. Our data show extensive and significant reduction in the expression of HLA antigens in colon cancer; its potential relationship to immunosurveillance in cancer is discussed.     

Induction of HLA class-II antigen expression on human carcinoma cell lines by IFN-Gamma

The effects of recombinant interferon gamma (IFN-gamma) on HLA antigen expression were examined in various human carcinoma cell lines: carcinomas of the lung (ChaGo, Oat 75, SK-LC-LL), colon (HT-29, WiDr), larynx (HEp-2), cervix (ME-180) and mammary gland (AlAb). Surface expression of HLA antigens was determined using monoclonal antibodies directed against monomorphic determinants of HLA class II (HD II, 2.06) and HLA class I (W6/32) by several techniques including indirect immunofluorescence, immunoperoxidase assay, radioimmunoassay of cells, and immunoprecipitation of radioiodinated cells. Without previous exposure to IFN-gamma, HLA class-II antigen was surface-expressed on a small subpopulation of HEp-2 only. All cell lines examined, except AlAb, could be induced to cell-surface expression of HLA class-II antigen after 48 hr incubation in the presence of IFN-gamma (10 units/ml). HLA class-I antigen was present on all cell lines even without treatment, and it was increased after IFN-exposure. Incorporation of 3H-thymidine and 3H-leucine and expression of the transferrin receptor were not significantly altered by IFN-gamma application, indicating a specific regulatory effect of IFN-gamma on HLA antigen expression. Our data demonstrate that HLA class-II surface expression is inducible by IFN-gamma in a variety of epithelial tumor lines. This may have implications for the host immune system defense mechanisms against tumors.     

Cell phenotype dependent expression of MHC class I antigens in Burkitt's lymphoma cell lines.

Burkitt's lymphoma (BL) is a highly malignant B cell tumor characterized by three types of chromosomal translocation which constitutively activate the c-myc oncogene by juxtaposing it to Ig coding sequences. Epstein-Barr virus (EBV) infection, hyperendemic malaria and HIV-caused immunosuppression are thought to contribute to the pathogenesis of the tumor. Cell lines derived from EBV carrying and EBV negative BLs often show altered MHC class I antigen expression. The defects include a lower expression of all HLA class I antigens compared to EBV transformed normal B-blasts, and selective down-regulation of certain HLA-A and HLA-C alleles. As a consequence BL cells are often resistant to cytotoxic T lymphocyte (CTL) mediated destruction. Alleles selective down-regulations are found only in cell lines that maintain the tumor cell phenotype while shift towards a more activated 'B-blast like' phenotype is accompanied by HLA class I up-regulation. A similar pattern of HLA class I expression can be found in a subpopulation of germinal center B cells which express a 'BL like' phenotype. Our findings suggest that the HLA class I expression of BL cells reflects the characteristics of the normal B cell precursor and is probably not the result of immune selection.     

Tissue array analysis of the aberrant expression of HLA class I molecules in human non small cell lung cancer

A clinical significance of the aberrant expression of HLA class I molecules including HLA class I and HLA-G was analyzed using tissue array analysis. Our institute has established a two millimeter spot sized tissue array set of 105 clinical cases of resected human non small cell lung cancer tissues. A loss of HLA class I was observed in the 58.3% of cancer tissues. The aberrant expression of HLA G was also observed in the 55.2% of cancer tissues. Statistically significant correlations were observed among HLA class I expression and tumor size, nodal involvement and pathological stage. Survival analyses were shown that the HLA class I loss was correlated to a recurrence free survival time. The HLA-G expression did not correlate with any clinico-pathological parameters. A loss of HLA class I was probably involved due to a cancer progression in human non-small cell lung cancer through the mechanism of immune escape from the host immune system.     

Molecular evidence for the lack of epidermal growth factor receptor gene expression in small cell lung carcinoma cells

It has been shown that none of the small cell lung carcinoma (SCLC) cell lines possess epidermal growth factor (EGF) binding activity on their surface. We have examined several SCLC cell lines for the possibility that they may have EGF receptors but that the receptors are masked by an EGF-like protein factor(s), which may be produced by an autocrine mechanism. No evidence, however, was found for the production of such factors. We then used an EGF receptor complementary DNA to determine the state of the EGF receptor gene by Southern blot analysis. The receptor gene appears to be present in these cells in an intact, unrearranged form. These cells, however, were found to lack detectable levels of EGF receptor mRNA, suggesting a possible reason for the absence of EGF receptors on the cell surface. Furthermore, karyotype analysis revealed that SCLC cell lines Lu134 and H69 contained a morphologically normal chromosome 7, which carries the EGF receptor gene. Also, these SCLC cells contained the apparently normal chromosome 3 and exhibited the presence of c-raf-1 gene in an unrearranged form. Thus, the previously noted partial deletion of chromosome 3 is not necessarily common to the SCLC cells. Instead, the lack of EGF receptor is frequently found in SCLC cell lines and is distinct from the other types of lung cancer. We postulate that SCLC cells have some active regulatory mechanism which prevents the expression of EGF receptor gene.     

Altered HLA class I expression in non-small cell lung cancer is independent of c-myc activation.

We studied the expression of major histocompatibility complex class I antigens in 59 bronchogenic carcinomas, as well as in pneumocytes and epithelial respiratory cells distant from the tumor. We observed in all cases that normal lung tissue expressed major histocompatibility complex class I antigens, while this expression was completely lost in 16 tumors (27%). The defect in HLA gene expression affected both heavy chain and beta 2-microglobulin, as demonstrated by the null reactivity with the monoclonal antibodies GRH1, W6/32, and HC10. Selective underexpression was detected in 1 tumor for HLA-A locus antigens and in 3 tumors for HLA-B locus antigens. Southern blot analyses of major histocompatibility complex class I genes were performed in 20 tumor tissue specimens and 6 cell lines. No class I gene rearrangements were detected using HLA coding and locus specific noncoding probes. We also used the Southern blot method to investigate the possible relationship between c-myc amplification and HLA class I antigens in non-small cell lung cancers and detected no apparent amplification in 20 tumor tissue specimens (5 negative for HLA class I antigens) and 6 cell lines (3 with decreased expression). Northern blot analysis revealed no relationship between c-myc mRNA levels and specific mRNA for HLA-A and HLA-B antigens in cell lines with imbalanced HLA-A or HLA-B expression.