Mutation in the 2m Gene is not a Frequent Event in Head and Neck Squamous Cell Carcinomas Manita Feenstra, Erik Rozemuller, Karen Duran, Ilonka Stuy, Jan van den Tweel, Pieter Slootweg, Roel de Weger, and Marcel Tilanus ABSTRACT: In cryostat sections of 84 head and neck squamous cell carcinomas (HNSCC) HLA class I and 2m expression was analysed using monomorphic and locus specific monoclonal antibodies. Loss of expression was heterogeneous and none of the tumours tested showed a total loss of HLA class I and/or 2m when analysed with W6/32, which recognises HLA class I determinants and anti-2m MoAbs. Weak HLA class I and 2m expression was found in 9 tumours (11%) and heterogeneous expres- sion was found in 2 tumours (2%). When analysed with locus-specific antibodies (HCA2 and HC10, anti-HLA-A and anti-HLA-B/C, respectively) 37 tumours (44%) showed a loss, weak or heterogeneous expression of one or both loci. Tumours showing a down-regulated HLA class I expression were analysed for mutations in either allele of the 2m gene by sequencing based mutation analysis (SBMA). Exon 1 and exons 2 and 3 were amplified sep- arately by PCR using M13-tailed intron-specific primers. PCR products were sequenced in two directions. In none of the tumours mutations in the 2m gene were detected. In 59% of the tumours with down-regulated HLA class I expression, lost or down-regulated TAP 1 expression was found when analysed with anti-TAP 1 antibodies. This indicates an important role for TAP in down-regulation of HLA class I expression in HNSCC. Human Immunology 60, 697–706 (1999). © American Society for Histocom- patibility and Immunogenetics, 1999. Published by Elsevier Science Inc. KEYWORDS: HLA class I; 2m; TAP 1; expression; tumors; SBMA ABBREVIATIONS HNSCC head and neck squamous cell carcinoma 2m beta2-microglobulin HLA human leukocyte antigens TAP transporter associated with antigen processing MoAb monoclonal antibody SBMA sequencing based mutation analysis INTRODUCTION HLA class I molecules are highly polymorphic trans- membrane glycoproteins composed of a heavy chain, encoded by the HLA-A, B and C genes and 2-micro- globulin (2m). HLA class I molecules present short peptides derived from cytosolic proteins to antigen spe- cific cytotoxic T cells. An 8 to10 residue peptide with an allele-specific consensus motif can be bound in the groove of the HLA class I-2m heterodimer and pre- sented to cytotoxic T cells. Recognition of a foreign peptide or a tumour antigen induces immune responses that eventually may result in the elimination of the cell [1, 2]. Cell surface expression of HLA class I molecules requires the rapid formation of HLA class I heavy chains and 2m heterodimers in the endoplasmatic reticulum (ER), a process chaperoned by calnexin and calreticulin [3]. The HLA class I-2m heterodimers bind to trans- porters associated with antigen processing (TAP), a het- erodimeric complex specialised in the translocation of peptides from the cytosol into the ER. Peptides bound by class I molecules are mainly generated from cytosolic proteins by a 20S catalytic proteinase complex, the pro- From the Department of Pathology, University Hospital, Utrecht, The Netherlands (all authors). Address reprint requests to: Dr. M. G. J. Tilanus, University Hospital Utrecht Department of Pathology, H04.312, P.O. Box 85500, 3508 GA Utrecht The Netherlands. Received July 14, 1998; revised December 9, 1998; accepted December 10, 1998. Human Immunology 60, 697–706 (1999) 0198-8859/99/$–see front matter © American Society for Histocompatibility and Immunogenetics, 1999 Published by Elsevier Science Inc. PII S0198-8859(99)00015-4