A non-AUG-defined alternative open reading frame of the intestinal carboxyl esterase mRNA generates an epitope recognized by renal cell carcinoma-reactive tumor-infiltrating lymphocytes in situ

A number of Ags recognized by tumor-reactive T cells have been characterize d, including nonmutated gene products and a variety of epitopes shown to ar ise from either mutated or alternatively processed transcripts, Here, we re port that the screening of a cDNA library with an HLA-B7-restricted renal c ell carcinoma-reactive T cell clone derived from tumor-infiltrating lymphoc ytes (TILs) that were clonally amplified in vivo (as assessed by TCRBV comp lementarity determining region-3 length distribution analysis) resulted in the isolation of a nonamer encoded by an alternative open reading frame (OR F) (a fl frameshift) of the intestinal carboxyl esterase gene. This peptide binds HLA-B*0702-presenting molecules as assessed in an immunofluorescence -based peptide binding assay using transfected T2 cells, Constitutive expre ssion of this alternative ORF protein was observed in all transformed HLA-B 7(+) renal cell lines that were recognized in cytotoxicity assays by the TI Ls, The intestinal carboxyl esterase gene is transcribed in renal cell carc inoma tumors as well as in normal liver, intestinal, or renal tissues, Muta tion of the natural ATG translation initiation site did not alter recogniti on, indicating that frameshifting (i.e., slippage of the ribosome forward) and recoding are not involved. In addition, a point mutation of the three A UG codons that may be used as alternative translation initiation sites in t he fl ORF did not abolish recognition, whereas mutation of an upstream ACG codon did, indicating that the latter codon initiates the translation of th e alternative ORF, These results further extend the types of Ags that can b e recognized by tumor-reactive TILs in situ (i.e., leading to clonal T cell expansion).