MOLECULAR APPROACHES FOR THE CHARACTERIZATION OF HLA CLASS I TUMOUR PHENOTYPES IN HEAD AND NECK SQUAMOUS CELL CARCINOMA CELL LINES.
Selma Wiertsema, Manita Feenstra, Erik Rozemuller, Jan van den Tweel, Pieter Slootweg, Roel de Weger, Marcel Tilanus. Department of Pathology, University Hospital, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. 

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Tumour cells frequently exhibit down-regulation or even a complete loss of expression of HLA class I antigens. Down regulation of HLA class I may provide tumour cells with an immune escape phenotype. In the process of carcinogenesis loss or reduction of HLA class I expression is postulated to be caused by multiple mechanisms i.e., mutations in the HLA class I genes and the b2m gene, loss of heterozygosity (LOH), and impaired transcription of HLA class I genes and factors involved in antigen presentation. We have analyzed HLA class I down-regulation in 4 head and neck squamous cell lines using monomorphic, locus- and allele- specific MoAbs. Molecular approaches have been set up to study the underlying mechanisms of down-regulated HLA class I expression. Mutations in the HLA class I genes and b2m genes have been studied by sequencing based mutation analyses. In addition, the tumour cell lines were studied for LOH using 5 microsatellite markers within the HLA coding sequence and 2 microsatellite markers flanking the b2m gene. To characterize interference with transcription, a semi-quantitative RT-PCR with HLA, b2M, TAP 1&2 and LMP 2&7 specific primers was developed and will be applied to mRNA derived from these cell lines.

Application of molecular approaches in combination with immunohistochemical techniques will enable us to define complex immunesurveillance escape phenotypes in tumours.