Characterization of HLA class I altered phenotypes in a panel of human melanoma cell lines

Abstract

Background: Altered HLA class I cell surface expression is one of the major mechanisms by which tumor cells escape from T lymphocytes. Immunohistochemistry-defined phenotypes of lost HLA class I expression have been described in human solid tumors, nut less information is available on melanoma cell lines.

Objectives: To describe the frequency and distribution of different types of HLA class I antigen alterations in 91 melanoma cell lines from the European Searchable Tumour Cell and Databank (ESTDAB).

Methods: The HLA class I expression was assessed by flow cytometry and HLA genotyping.

Results: We found various types of HLA class I cell surface alterations in about 67% of the melanoma cell lines. These alterations range from total to selective HLA class I loss due to loss of heterozygosity (LOH), haplotype loss, beta2-microglobulin gene mutation, and/or total or selective down-regulation of HLA class I molecules. The most frequently observed phenotype is down-regulation of HLA-B locus that was reversible after treatment with IFN -gamma.

Conclusions: In general, HLA class I alterations in the majority of the cells analyzed were of regulatory nature and could be restored by IFN-gamma. Analysis of the frequency of distinct HLA class I altered phenotypes in these melanoma cell lines revealed specific differences compared to other types of tumors.

Fig. 1

Distribution of HLA class I phenotypes in melanoma cell lines: HLA class I total loss or down-regulation (phenotype I), HLA class I haplotype loss (phenotype II), HLA-B down-regulation (phenotype III), combination of HLA class I haplotype loss and HLA-B down-regulation (phenotype V), and IFN-γ resistance (phenotype VI). Surface expression of HLA class I antigens was determined by indirect immunofluorescence using anti-HLA-ABC/β2m (W6/32) monoclonal antibodies. Results are expressed as mean florescence intensity (MFI)

Fig. 2

Real-time PCR showed lower (statistically significant) expression of LMP-7 and TAP-2 genes in cell lines with HLA class I down-regulation as compared to cell lines with normal HLA class I expression. Total cellular RNA was isolated from melanoma cell lines and was subjected to reverse transcription. Quantitative real time PCR was used to analyze the reverse-transcribed products from melanoma cell lines for the expression of various genes, including HLA class I heavy chain, TAP-1, TAP-2, LMP-2, and LMP-7. To control for variations in amounts of RNA, G6PDH, and β2-m were also tested as housekeeping genes

Fig. 3

a Patterns of polymorphic markers on chromosome 6. Microsatellite analysis was performed on DNA from melanoma cell lines using six short tandem repeats (STRs) that map chromosome 6. Microsatellite analysis plot using DNA isolated from a healthy donor demonstrates an example of retention of heterozygocity (two peaks at each marker). Examples of homo/hemizygous patterns are shown for cell lines E-058 and E-071 (single peak for majority of the markers and strongly reduced peaks at other markers) suggesting LOH at chromosome 6. b Representative images of FISH assay of cells counterstained with DAPI and hybridized with a centromeric probe showing examples of diploidy (E-064), triploidy (E-058), and tetraploidy (E-037) in cell lines. LOH at chromosome 6 along with triploidy according to FISH assay indicates triplication of a single chromosome 6 in cell line E-058