Loss of single HLA class I allospecificities in melanoma cells due to selective genomic abbreviations.
Expression of human leucocyte antigen (HLA) Class I molecules is essential for the recognition of malignant melanoma (MM) cells by CD8(+) T lymphocytes. A complete or partial loss of HLA Class I molecules is a potent strategy for MM cells to escape from immunosurveillance. In 2 out of 55 melanoma cell cultures we identified a complete phenotypic loss of HLA allospecificities. Both patients have been treated unsuccessfully with HLA-A2 peptides. To identify the reasons underlying the loss of single HLA-A allospecificities, we searched for genomic alterations at the locus for HLA Class I alpha-chain on chromosome 6 in melanoma cell cultures established from 2 selected patients with MM in advanced stage. This deficiency was associated with alterations of HLA-A2 gene sequences as determined by polymerase chain reaction-sequence specific primers (PCR-SSP). Karyotyping revealed a chromosomal loss in Patient 1, whereas melanoma cell cultures established from Patient 2 displayed 2 copies of chromosome 6. Loss of heterozygosity (LOH) using markers located around position 6p21 was detected in both cases. By applying group-specific primer-mixes spanning the 5'-flanking region of the HLA-A2 gene locus the relevant region was amplified by PCR and subsequent sequencing allowed alignment with the known HLA Class I reference sequences. Functional assays using HLA-A2-restricted cytotoxic T-cell clones were performed in HLA-A2 deficient MM cultures and revealed a drastically reduced susceptibility to CTL lysis in HLA-A2 negative cells. We could document the occurrence of selective HLA-A2 deficiencies in cultured advanced-stage melanoma metastases and identify their molecular causes as genomic alterations within the HLA-A gene locus.
Geertsen
Ralf
R
Department of Dermatology, University Hospital, Zürich, Switzerland.
Böni
Roland
R
Blasczyk
Rainer
R
Romero
Pedro
P
Betts
David
D
Rimoldi
Donata
D
Hong
Xin
X
Laine
Elisabeth
E
Willers
Jörg
J
Dummer
Reinhard
R
D000483:Alleles; D000951:Antigens, Neoplasm / Q000235:genetics*; D002896:Chromosomes, Human, Pair 6 / Q000235:genetics; D005434:Flow Cytometry; D005805:Genes, MHC Class I / Q000235:genetics; D015789:HLA-A2 Antigen / Q000235:genetics*; D006801:Humans; D007621:Karyotyping; D019656:Loss of Heterozygosity; D008545:Melanoma / Q000235:genetics* / Q000276:immunology; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D017422:Sequence Analysis, DNA; D012878:Skin Neoplasms / Q000235:genetics* / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D015789:HLA-A2 Antigen