We report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A*0201–presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved “reverse immunology” strategy. Next to motif-based HLA-A*0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A*0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA100–108; SLYSFPEPEA, PRA142–151; ALYVDSLFFL, PRA300–309; and SLLQHLIGL, PRA425–433) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A*0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer.
Proteasomes are the major source for the generation of peptides bound by MHC class I molecules. To study the functional relevance of the IFN-γ-inducible proteasome subunits low molecular mass protein 2 (LMP2), LMP7, and mouse embryonal cell (MEC) ligand 1 in Ag processing and concomitantly that of immunoproteasomes, we established the tetracycline-regulated mouse cell line MEC217, allowing the titrable formation of immunoproteasomes. Infection of MEC217 cells with Adenovirus type 5 (Ad5) and analysis of Ag presentation with Ad5-specific CTL showed that cells containing immunoproteasomes processed the viral early 1B protein (E1B)-derived epitope E1B192–200 with increased efficiency, thus allowing a faster detection of viral entry in induced cells. Importantly, optimal CTL activation was already achieved at submaximal immunosubunit expression. In contrast, digestion of E1B-polypeptide with purified proteasomes in vitro yielded E1B192–200 at quantities that were proportional to the relative contents of immunosubunits. Our data provide evidence that the IFN-γ-inducible proteasome subunits, when present at relatively low levels as at initial stages of infection, already increase the efficiency of antigenic peptide generation and thereby enhance MHC class I Ag processing in infected cells.
CTL directed against the Moloney murine leukemia virus (MuLV) epitope SSWDFITV recognize Moloney MuLV-induced tumor cells, but do not recognize cells transformed by the closely related Friend MuLV. The potential Friend MuLV epitope has strong sequence homology with Moloney MuLV and only differs in one amino acid within the CTL epitope and one amino acid just outside the epitope. We now show that failure to recognize Friend MuLV-transformed tumor cells is based on a defect in proteasome-mediated processing of the Friend epitope which is due to a single amino acid substitution (N→D) immediately flanking the C-terminal anchor residue of the epitope. Proteasome-mediated digestion analysis of a synthetic 26-mer peptide derived from the Friend sequence shows that cleavage takes place predominantly C-terminal of D, instead of V as is the case for the Moloney MuLV sequence. Therefore, the C terminus of the epitope is not properly generated. Epitope-containing peptide fragments extended with an additional C-terminal D are not efficiently translocated by TAP and do not show significant binding affinity to MHC class I-Kb molecules. Thus, a potential CTL epitope present in the Friend virus sequence is not properly processed and presented because of a natural flanking aspartic acid that obliterates the correct C-terminal cleavage site. This constitutes a novel way to subvert proteasome-mediated generation of proper antigenic peptide fragments.
The polymorphic minor histocompatibility Ag HA-1 locus encodes two peptides, HA-1H and HA-1R, with a single amino acid difference. Whereas the immunogenicity of the HA-1R allele has not yet been shown, the nonameric HA-1H peptide induces HLA-A2-restricted cytotoxic T cells in vivo and in vitro. It is not known whether the mHag HA-1H or HA-1R associates with other HLA class I molecules. Therefore, the polymorphic regions of both HA-1 alleles were analyzed to identify HLA class I binding peptides that are properly processed by proteasomal degradation. Peptide binding analyses were performed for all nonameric HA-1H/R peptides for binding to nine HLA class I molecules with >10% prevalence in the Caucasian population and for seven nonameric/decameric HA-1H/R peptides predicted to bind to HLA-A3, -B14, and -B60. Only the nonameric KECVLH/RDDL and decameric KECVLH/RDDLL peptides showed strong and stable binding to HLA-B60. In vitro digestion of 29-aa-long HA-1 peptides by purified 20S proteasomes revealed proper cleavage at the COOH termini of both HLA-B60 binding HA-1H and HA-1R peptides. In subsequent analyses, dendritic cells pulsed with the nonameric HA-1R peptide did not induce CTLs that recognize the natural HLA-B60/HA-1R ligand. In contrast, dendritic cells pulsed with the nonameric HA-1H peptide induced IFN-γ-secreting T cells specific for the natural HLA-B60/HA-1H ligand in three HLA-B60+ HA-1RR individuals, demonstrating the immunogenicity of the HLA-B60/HA-1H ligand. In conclusion, this study shows a novel HLA-B60-restricted T cell epitope of the minor histocompatibility Ag HA-1 locus.
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