Translation of a Retained Intron in Tyrosinase-related Protein (TRP) 2 mRNA Generates a New Cytotoxic T Lymphocyte (CTL)-defined and Shared Human Melanoma Antigen Not Expressed in Normal Cells of the Melanocytic Lineage

Abstract

SummaryWe report here the identification of a new shared human melanoma antigen recognized by a human leukocyte antigen (HLA)-A*68011-restricted cytotoxic T lymphocyte clone (CTL 128). The cDNA encoding this antigen is composed of a partially spliced form of the melanocyte differentiation antigen tyrosinase-related protein ( TRP )-2 , containing exons 1-4 with retention of intron 2 and part of intron 4 ( TRP-2-INT2 ). The sequence coding for the antigenic epitope is located at the 5 Ј end of intron 2 and is available for translation in the same open reading frame of the fully spliced TRP-2 mRNA. This peptide is also recognized by CTL 128 when presented by the HLA-A*3301, a member of the HLA-A3-like supertype that includes the HLA-A*68011. Quantitative reverse transcription PCR analysis carried out on total and/or cytoplasmic mRNA demonstrated that, in contrast to the fully spliced TRP-2 mRNA expressed in melanomas, normal skin melanocytes, and retina, the TRP-2-INT2 mRNA could be detected at significant levels in melanomas but not in normal cells of the melanocytic lineage. Instead, in these normal samples, both the spliced and the unspliced transcript of gp100 were expressed at high levels. Absence of endogenous TRP-2-INT2 expression in melanocytes was also confirmed by lack of recognition of HLA-A*68011-transduced, TRP-2 ϩ melanocyte lines by CTL 128. These results indicate that a partially spliced form of a differentiation antigen mRNA, present in the cytoplasmic compartment of neoplastic but not normal cells of the melanocytic lineage, can be the source of a melanoma-restricted T cell epitope.

A novel DNA methyltransferase I–derived peptide eluted from soluble HLA‐A*0201 induces peptide‐specific, tumor‐directed cytotoxic T cells

“…8,9 Identification of peptide p1028 15 revealed a previously unknown immunogenic, HLA-A2-restricted peptide derived from DNMT-1, using a novel method of identifying peptides eluted from sMHC. It has been eluted from the sMHC of ovarian, prostate and breast cancer cell lines.…”

“…7 Many of the recently identified antigens are nonmutated differentiation antigens overexpressed in tumors, e.g., a peptide derived from protein tyrosinase that was shown to successfully induce the attack and lysis of melanoma cells from cancer patients. 8 Several strategies have evolved to identify novel TAAs, e.g., cloning of epitopes recognized by CTLs 9 and SEREX technology, 10 where a patient's antibodies are used to identify tumor antigens from a cDNA expression library made from mRNA of tumor specimens.…”

CD20 alternative splicing isoform generates immunogenic CD4 helper T epitopes

“…Briefly, these mice are H-2 Class I and IA Class II knockout, and their CD8 T and CD4 T cells are restricted by the sole HLA-A*0201 and HLA-DR1*0101 molecules, respectively. 11,15 For D393-CD20 [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] (20mer) and D393-CD20 33-41 (9mer) immunization, mice were injected twice with 100 lg of D393-CD20 20mer or 50 mg of D393-CD20 9mer were emulsified in incomplete Freund adjuvant (IFA, Sigma-Aldrich). All peptide vaccinations were done subcutaneously at the base of the tail at Days 1 and 14.…”

“…To evaluate the presence of D393-CD20 specific T cell in the human repertoire, the D393-CD20 [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] 20mer-peptide overlapping the splicing site was used to stimulate T cells. For this purpose, peripheral blood lymphocytes of healthy volunteers were in vitro stimulated twice using 10 mmol/L of D393-CD20 [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] in 96 well microplates during 14 days as described in the material and method section.…”

“…The three first amino-acids used to name these peptides are indicated in boldface. T cell lines were obtained from healthy donors' PBMCs after two rounds of in vitro stimulation in microplates with 10 lg/mL of D393-CD20 [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] peptide. Specific responses were assessed by CD4 and IFN-g staining.…”