Cryptic CTL Epitope on a Murine Sarcoma Meth A Generated by Exon Extension as a Novel Mechanism

Abstract

Using the recently developed ELISPOT cloning methodology, we obtained cDNA clone S35 coding for the Ag epitope recognized by a murine sarcoma Meth A-specific CTL clone AT-1. Analysis of truncated S35 constructs and overlapping peptides revealed that the peptide epitope was LGAEAIFRL. AT-1 CTL lysed peptide-pulsed CMS8 cells at a nanomolar concentration, and the peptide strongly stimulated IFN-γ production in AT-1 CTL. Sequence homology indicated that the S35 was derived from a mouse homologue of human retinoic acid-regulated nuclear matrix-associated protein (ramp). The ramp gene consisted of 15 exons. The majority of the ramp mRNA was the transcript normally spliced between exons 14 and 15, but a minor population of mRNA with an extended exon 14 was also present in Meth A cells. The epitope was derived from the newly created open reading frame, which resulted from extension of exon 14 after splicing of the adjacent intronic sequence.

Murine leukemia RL1 and sarcoma Meth A antigens recognized by cytotoxic T lymphocytes (CTL)

“…21) By using this new approach, we succeeded in defining the CTL epitope on methylcholanthrane-induced Meth A sarcoma, as described below. 50)…”

“…21) Following three rounds of screening by large-scale ELISPOT assay and two rounds of screening by small-scale ELISPOT assay, the cDNA clone S35 of 937 bp was obtained. 50) AT-1 CTL were stimulated with S35-transfected CMS5a, CMS8, CMS13 and H-2D d , but not H-2L d transfected L cells. To identify the peptide epitope recognized by AT-1 CTL, various truncated S35 mutants were prepared and transfected into CMS8 cells (Fig.…”

Alternative tumour-specific antigens

“…Splice variant antigen frequency in cancer. Splice variant antigens are posttranscriptionally derived TSAs arising from alternative splicing events, including those from mRNA splice junction mutations [52][53][54][55][56][57] , intron retention [58][59][60][61][62][63] or dysregulation of the spliceosome machinery in the tumour cell 15,64,65 . Other types of post-transcriptionally derived TSAs include alternative ribosomal products (for example, ribosomal frameshifting 66,67 , non-canonical initiation [68][69][70][71] , termination codon read-through 69 , reverse-stand transcription 72 and doublet decoding 73 ) and post-translational splicing 74-76 -these two mechanisms are difficult to apply in anticancer therapies, given the lack of tools for predicting such products.…”

Alternative Translational Products and Cryptic T Cell Epitopes: Expecting the Unexpected

“…The pepton hypothesis suggesting that epitopes could result from transcription of short genetic regions by a novel polymerase, yielding antigenic pepton-RNA that associated directly with the class I molecule (12), was intriguing, but no/little evidence emerged to support this idea (13). Atypical epitopes may also arise from posttranscriptional regulatory events, e.g., human melanoma peptides generated by either incompletely spliced message (14), mutation of a normally noncoding intronic sequence (15), or exon extension as shown with a cryptic peptide derived from a murine sarcoma (16). At the translational level, mechanisms such as ribosomal frameshifting (17), internal initiation using internal ribosome entry sites (18,19), initiation codon scanthrough (20,21), doublet decoding (22), or initiation from non-AUG codons (23,24) may contribute to the biosynthesis of nontraditional peptides.…”