Identification of a 17β-hydroxysteroid dehydrogenase type 12 pseudogene as the source of a highly restricted BALB/c Meth A tumor rejection peptide

Abstract

Mass spectrometric analysis identified the pep-tide recognized by a cytotoxic T lymphocyte (CTL) specific for the chemically induced BALB/c Meth A sarcoma as derived from a 17β-hydroxysteroid dehydrogenase type 12 (Hsd17b12) pseudogene present in the BALB/c genome, but only expressed in Meth A sarcoma. The sequence of the peptide is TYDKIKTGL and corresponds to Hsd17b12114–122 with threonine instead of isoleucine at codon 114 and is designated Hsd17b12114T. Immunization of mice with an Hsd17b12114T peptide-pulsed dendritic cell-based vaccine or a non-viral plasmid construct expressing the Hsd17b12114T peptide protected the mice from lethal Meth A tumor challenge in tumor rejection assays. A Hsd17b12114–122 peptide-pulsed vaccine was ineffective in inducing resistance in mice to Meth A sarcoma. These results confirm the immunogenicity of the identified tumor peptide, as well as demonstrate the efficacies of these vaccine vehicles. These findings suggest that the role of the human homolog ofHsd17b12,HSD17B12, as a potential human tumor antigen be explored.

17β hydroxysteroid dehydrogenase type 12 (HSD17B12) is a marker of poor prognosis in ovarian carcinoma

“…To avoid nonspecific binding of the secondary antibody (Ab), tissue sections were incubated with a serum-free protein blocker for 45 min prior to the staining procedure. After washing with PBS, sections were incubated with either a polyclonal rabbit Ab specific for the HSD17B12 114-122 peptide at 5μg/mL or with Abs to estrogen receptor (ER; clone 1D5) or progesterone receptor (PR; clone PgR 626) both purchased from DakoCytomation, for 30 min at room temperature (RT) in a moisture chamber [22]. Next, slides were washed in 0.5% BSA and then incubated with secondary anti-rabbit Ab labeled with horseradish peroxidase (HRP) under the same conditions.…”

Identification of Hydroxysteroid (17β) dehydrogenase type 12 (HSD17B12) as a CD8+ T-cell-defined human tumor antigen of human carcinomas

“…The KS1 and an IgG1 isotype mAb were from Dr. Soldano Ferrone (University of Pittsburgh, Pittsburgh PA) [19]. The peptide-immunoaffinity–purified polyclonal rabbit anti-TY-DKIKTGL peptide antibody, which cross-reacts with the IYDKIKTGL (Hsd17b12/HSD17B12 114–122 ) peptide, has previously been described [16]. Peptides were synthesized using standard Fmoc chemistry and their sequences con-firmed by tandem MS.…”

“…The expression of HSD17B12 in human normal and tumor cell lines was analyzed by immunoblot using the purified rabbit antibody at a concentration of 1 μg/ml and developed using horseradish peroxidase-conjugated goat anti-rabbit IgG Fc fragment-specific antibody (Jackson Immuno-Research Laboratories, Inc. West Grove, PA) at 1:10,000 dilution and Western Lightning Plus-ECL (Perkin Elmer, Inc., Waltham MA) [16]. …”

“…Interest in this effort was further prompted by our previous identification of a highly restricted tumor rejection antigen (TRA) of the BALB/c mouse Meth A sarcoma as a CD8 + T-cell-defined peptide derived from a aberrantly expressed pseudogene related to Hsd17b12 , the mouse homolog of human HSD17B12 [16]. This study was initiated using the algorithm predicted, HLA-A2-binding HSD17B12 114–122 peptide (IYDKIKTGL), which is nearly identical to the Meth A TRA (TYDKIKTGL) peptide, designated Hsd17b12 114T , but differs from it in the amino acid exchange of threonine for isoleucine at position 1.…”

Pseudogene in cancer: real functions and promising signature

“…Examples of this kind have been reported in melanoma58 and in sarcoma 59. Though theoretically ‘self’, cancer cells can produce proteins that are spatiotemporally inappropriate, thus being recognised by the immune system as ‘non-self’.…”