Expression Of HLA class-I Antigens Research Articles

Up-regulation of HLA class-I antigen expression and antigen-specific CTL response in cervical cancer cells by the demethylating agent hydralazine and the histone deacetylase inhibitor valproic acid

BackgroundDNA hypermethylation and histone deacetylation are epigenetic events that contribute to the absence or downregulated expression of different components of the tumor recognition complex. These events affect the processing and presentation of antigenic peptides to CTLs by HLA class-I molecules. In this work evaluated the effect of the DNA hypomethylating agent hydralazine and the histone deacetylase inhibitor valproic acid, on the expression of HLA class-I molecules and on the antigen-specific immune recognition of cervical cancer cells.MethodsCell lines C33A (HPV-), CaSki (HPV-16+) and MS751 (HPV-18+) were treated with hydralazine and valproic acid to assess the expression of HLA class-I molecules by flow cytometry and RT-PCR. Promoter methylation of HLA class-I -A, -B and C, was also evaluated by Methylation-Specific PCR. Primary cervical tumors of four HLA-A*0201 allele patients were typed for HPV and their CTL's stimulated in vitro with the T2 cell line previously loaded with 50 μM of the HPV peptides. Cytotoxicity of stimulated CTL's was assayed against Caski and MS751 cells pre-treated with hydralazine and valproic acid.ResultsValproic acid and hydralazine/valproic acid up-regulated the constitutive HLA class-I expression as evaluated by flow cytometry and RT-PCR despite constitutive promoter demethylation at these loci. Hydralazine and valproic acid in combination but no IFN-gamma hyperacetylated histone H4 as evaluated by ChiP assay. The antigenic immune recognition of CaSki and MS751 cells by CTLs specific to HPV-16/18 E6 and E7-derived epitopes, was increased by VA and H/VA and the combination of H/VA/IFN-gamma.ConclusionThese results support the potential use of hydralazine and valproic acid as an adjuvant for immune intervention in cervical cancer patients whenever clinical protocols based on tumor antigen recognition is desirable, like in those cases where the application of E6 and E7 based therapeutic vaccines is used.

Antigenic profile of mucosal melanoma lesions

Ten primary and 6 metastatic mucosal melanoma lesions were tested, utilizing the immunoperoxidase reaction with a panel of 26 monoclonal antibodies (MAbs). The latter included MAbs recognizing membrane-bound and cytoplasmic-melanoma-associated antigens, adhesion molecules and their receptors, and HLA Class-I and Class-II antigens. The melanoma lesions displayed differential reactivity with the panel of MAbs. Mucosal melanoma lesions differ from other types of melanoma in their antigenic profile, since they are the only type of melanoma to display a higher expression of p97 MAA, CEA-MAA and NGF-R in primary lesions than in metastatic ones. Furthermore, the pattern of HMW-MAA expression in mucosal melanoma lesions is different from that in nodular and uveal melanoma lesions and only partly resembles that observed in acral lentiginous melanoma lesions. Like other types of melanoma, mucosal melanoma lesions display a heterogeneous expression of HLA antigens, a selective loss of reactivity with some of the anti-HLA Class-I MAbs and a higher frequency of abnormalities in HLA Class-I antigen expression in metastatic than in primary lesions. Expression of HMW-MAA, 110-kDa MAA, p97 MAA, CEA-MAA, NGF-R and GD2 ganglioside in primary melanoma lesions was found to be associated with a poor prognosis. Although preliminary in nature, these results suggest that analysis of mucosal melanoma lesions with MAbs, in conjunction with information about the clinical course of the disease, will contribute to determine the biological and clinical significance of the distinct characteristics of the antigenic profile of mucosal melanoma lesions.

Suppression of basal, PMA- and IFN-alpha-, but not IFN-gamma-induced expression of HLA class I in v-myc-transformed U-937 monoblasts.

Recent studies have suggested that certain oncogenes, in particular members of the myc family, may be involved in the down-regulation of HLA class-I antigen expression observed in many types of tumor. We report that constitutive expression of an OK10 v-myc gene in human monoblastic U-937 cells results in a reduced expression of HLA class-I cell-surface expression and decreased levels of HLA class-I protein and mRNA. All class-I alleles, with the possible exception of HLA A3, were affected, as shown by one-dimensional isoelectric focusing (ID-IEF). Basal expression of the beta 2m chain was also reduced, although to a lesser extent. In addition, we show that the PMA-, and at least partially the IFN-alpha-induced increase in HLA class-I antigen expression, was inhibited in U-937-myc cells both at the protein and the mRNA level. In contrast, the response to IFN-gamma was normal. Another important difference in the response to IFN-gamma and alpha was that, while IFN-gamma abrogated the v-myc block of PMA-induced differentiation of U-937 cells, as previously reported, IFN-alpha did not. Our data show that v-myc negatively affects the regulation of both basal and inducible HLA class-I antigen expression.

Expression of HLA class-I antigens on renal cell carcinoma and non-transformed renal tissue.

To determine the HLA class-I antigen expression in renal cell carcinoma, the expression of beta-2-microglobulin (beta 2-m) was analyzed. 20 renal tumor specimens and 20 normal renal tissues were examined for beta 2-m expression by immunofluorescence. Homogenous staining of all normal renal tissues was observed, as expected. From the panel of 20 tumors, 17 also had a strong expression of beta 2-m. Furthermore, two newly established renal cancer cell lines exhibited a normal distribution of beta 2-m at two different passages. From these results we conclude that in most cases malignant transformation in renal cancer is not associated with a loss of HLA class-I antigens. This could be a favorable explanation for the restricted results of interferon-gamma in renal cell carcinoma, the main function of which is probably to enhance major histocompatibility complex class-I expression.

In vivo induction of HLA molecules in patients with myeloproliferative syndrome during IFNα treatment

Eighteen patients with myeloproliferative syndrome (14 with chronic myeloid leukemia, four with essential thrombocytosis) were investigated for modulation of HLA antigens on peripheral blood lymphocytes, monocytes, and hematopoietic precursors during IFN alpha therapy as a sign of potentially increased immune recognition of malignant cells. After 1 month of IFN alpha therapy, an increased number of monocytes and hematopoietic precursor cells, but not of lymphocytes, expressed HLA-DQ antigens. In addition, a strong induction of HLA class-I antigens was found on both hematopoietic progenitors and normal peripheral blood mononuclear cells. With daily injections of IFN in the first month of therapy stimulation continuously increased, suggested a major effect of IFN alpha on hematopoietic progenitors with sustained enhanced expression of HLA class-I antigens during differentiation of myelomonocytic cells. HLA class-I antigen expression was consistently augmented by IFN alpha in all patients, irrespective of their hematological response.

Aberrant expression of HLA Class‐I antigens in burkitt lymphoma cells

HLA class-I expression has been investigated by biochemical methods in 14 Burkitt lymphoma (BL) cell lines and the corresponding Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) derived from the same individuals. Selective down-regulation of one or more HLA class-I specificities was demonstrated in 9 out of 14 BL lines. The defect was restricted to a single HLA-A allele in 3 of the lines (BL29, BL72, WW-I-BL). Four lines (BL28, BL37, BL41 and Jijoye M13) showed down-regulation of both HLA-A and -C alleles, and one (BL36) failed to express one HLA-C allele. Only one BL line (WW-2-BL) had lost one HLA-A and one HLA-B allele. The allele-specific defects were mainly detected in cell lines that had maintained the phenotypic characteristics of the original tumor. Expression of B-cell activation markers and the EBV-encoded nuclear antigen (EBNA)-2 correlated with up-regulation of the Cw4 allele in the P79 subline of the BL line Jijoye. Treatment with gamma-interferon (IFN) resulted in full or partial reversion of the HLA class-I defects in some of the cases but had no significant effect in others. This was not due to a cell-line-related unresponsiveness to IFN, nor did it reflect an allele-specific mode of regulation because the same allele could respond differently in different cell lines. The data suggest that defective expression of HLA class-I antigens, which appears to be more prevalent for alleles within the HLA-A and -C loci, is a common feature of BL cell lines. Different regulatory mechanisms appear to be involved.

Molecular abnormalities in the expression of HLA class-I antigens by melanoma cells

Testing of a panel of cultured human melanoma cells with radiolabelled anti-HLA-class-I monoclonal antibodies (MAbs) in a binding assay has shown lack of reactivity of FO-I and SK-MEL-33 cells and low reactivity of SK-MEK-19 cells. SDS-PAGE analysis of the components immunoprecipitated from the 3 intrinsically radiolabelled melanoma cell lines by antibodies to the 2 subunits of HLA-class-I antigens has not detected beta 2-mu in the immunoprecipitates from melanoma cells FO-I and SK-MEL-33 and only a low level of HLA-class-I heavy chain in the immunoprecipitate from SK-MEL-19 cells. Northern blotting analysis with probes specific for HLA-class-I heavy chain and for beta 2-mu indicates that the abnormalities in HLA-class-I-antigen expression reflects a defect at the transcriptional level in FO-I cells and at the post-transcriptional level in SK-MEL-19 and in SK-MEL-33 cells. FO-I, SK-MEL-19 and SK-MEL-33 cells represent useful models to analyze the molecular mechanisms underlying the loss of HLA-class-I-antigen expression which is often associated with malignant transformation of melanocytes and to characterize the role of HLA-class-I antigens in the biology of melanoma cells and in their interactions with effector cells.

K‐ras mutations (codon 12) are not involved in down‐regulation of mhc class‐i genes in colon carcinomas

Fifty-eight colorectal carcinomas were studied for HLA class-I antigen expression and for the presence of point mutations in codons 12 and 61 of the K-ras gene. Eight carcinomas were completely negative for class I by the APAAP technique. Analyses using the polymerase chain reaction (PCR) method, together with selective hybridization using mutation-specific synthetic oligonucleotides, demonstrated K-ras mutations in 14 cases (24.1%), all of them in codon 12. None of the mutations corresponded to the negative cases for class-I HLA antigen expression. We did not observe any correlation between K-ras mutations and the extent of tumor differentiation.

Differential modulation by tumor necrosis factor and immune interferon of HLA class‐II antigens expressed by melanoma cells

Tumor necrosis factor (TNF-alpha) was compared to immune interferon (IFN-gamma) for its ability to modulate the expression and shedding of HLA antigens, of intercellular adhesion molecule I (ICAM I) and of high-molecular-weight melanoma-associated antigen (HMW MAA) by a panel of melanoma cell lines. The latter included the melanoma cell line MeWo and its metastatic variant MeM 50-10, which display differential susceptibility to modulation of HLA class-II antigens by IFN-gamma and the cell lines SK-MEL-93-DX-2 and SK-MEL-93-DX-3, which originated from anatomically distinct metastases in patient DX. TNF-alpha enhanced the expression of HLA class-I antigens on all 7 melanoma cell lines tested, although to a lower extent than IFN-gamma and the combination of IFN-gamma and TNF-alpha. TNF-alpha displayed a differential effect on the expression of HLA class-II antigens by the 7 melanoma cell lines: it enhanced it on 3 out of the 4 cell lines with constitutive expression of HLA class-II antigens and induced them on 1 of 3 cell lines without detectable expression of these antigens. The effects of IFN-gamma were different since it enhanced HLA class-II antigens on the 4 cell lines with constitutive expression of these antigens and induced them on 2 out of the remaining 3 lines. Interestingly, both TNF-alpha and IFN-gamma enhanced the expression of HLA class-II antigens by SK-MEL-93-DX-3 cells. On the other hand only TNF-alpha induced the expression of HLA class-II antigens by MeWo cells and only IFN-gamma induced such expression by MeM 50-10 cells and by SK-MEL-93-DX-2 cells. The effect of the combination of TNF-alpha and IFN-gamma was similar to that of the individual cytokines. Both TNF-alpha and IFN-gamma displayed a differential effect on the expression of the gene products of the HLA-D region by the melanoma cell lines. Northern blot analysis with HLA-DR beta-, DQ beta- and DP beta-specific probes suggests that the modulation of HLA class-II antigens by both cytokines reflects transcriptional and post-transcriptional events. TNF-alpha enhanced the expression of ICAM-I on all the melanoma cell lines, although to a lower extent than IFN-gamma and the combination of IFN-gamma and TNF-alpha. Lastly, neither TNF-alpha nor IFN-gamma displayed a marked effect on the expression of HMW-MAA by the melanoma cell lines tested.

Acquisition of tumorigenic potential in the human myoepithelial HBL100 cell line is associated with decreased expression of HLA class I, class II and integrin β3 and increased expression of c‐myc

The human breast cell line HBL100 acquires the capacity to invade normal tissues and to replace them by proliferation in vitro only at high passage levels (HPL). These cells therefore are a useful model for studying tumor progression in vitro. We have analyzed the expression of cell-surface markers supposed to be involved in the control of the neoplastic process. Quantitative flow cytometry has revealed that: (1) spontaneous expression of HLA class-I antigens strongly decreases in HPL HBL100 cells vs. LPL cells, which parallels amplification and over-expression of c-myc oncogene; (2) HLA DR antigens can be induced by IFN-gamma in LPL but not in HPL HBL100 cells; (3) HBL100 cells secrete a soluble protein factor which specifically inhibits HLA DR induction by IFN-gamma even in heterologous cell systems; (4) 50% of LPL HBL100 cells express integrin beta 3, whereas HPL HBL100 cells lose this antigen; (5) this cell line is myoepithelial in origin, since 100% of HBL100 cells exhibit the CD10 antigen. Our data stress a role of HLA antigens, of some integrins and of c-myc in the acquisition of malignant potential by myoepithelial mammary cells of the HBL100 line.

Differential Expression of HLA Class-I Antigens on B and T Lymphocytes Obtained from Human Lymphoid Tissues

The amount of HLA class-I antigens was determined on the surface of enriched populations of B and T lymphocytes obtained from human peripheral blood, lymph nodes and spleens. The assays were performed using monoclonal antibodies that recognize different determinants on HLA class-I antigens and utilizing a simple and sensitive enzyme-linked immunosorbent assay (ELISA). Our results indicate that B lymphocytes obtained from human peripheral blood, lymph nodes and spleens express at least twice as many HLA class-I antigens as T lymphocytes obtained from the same organs.