Identification of HLA a*0201 glioblastoma multiforme cell lines for immunotherapy by PCR-SSP and DNA sequencing.

Most tumor specific antigens characterized to date are restricted by HLA a*0201, which is the major HLA subtype in many ethnic groups. Cancer cells that express tumor antigens in association with the HLA a*0201 subtype have been shown to be responsive to various immunotherapies. We therefore sought to identify glioma cell lines that also express this HLA subtype and determine whether they had the molecular properties needed for tumor-peptide presentation. The HLA a*0201 allele was identified with PCR using sequence-specific primers followed by DNA sequencing. With this method, we screened 15 glioma cell lines to determine if they were of the HLA a*0201 genotype. Glioma cell lines that express the HLA a*0201 subtype were further studied for the expression of MHC class I and beta-2-microglobulin (beta2m) molecules by flow cytometry, and peptide presentation molecules TAP-1, TAP-2, and tapasin by RT-PCR. We identified six out of fifteen cell lines that were of the HLA a*0201 subtype. These cell lines are U87, T98, U373, U138, CRL2365 and UMN-4. All these six cell lines exhibited high levels of MHC class I and beta2m molecules. In addition, these cell lines all expressed molecules required for peptide presentation as shown by the presence of peptide presentation-related molecules TAP-1, TAP-2 and tapasin. The identification of glioma cell lines that express the HLA a*0201 subtype along with the necessary molecules for peptide-presentation will enable their use in developing new immunotherapeutic approaches for treating brain tumors. The method used to identify HLA a*0201 glioma cells is rapid and inexpensive, and suitable for screening tumor cells.