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Multiple point mutations in an endogenous retroviral gene confer high immunogenicity on a drug-treated murine tumor
U Grohmann1, P Puccetti, M L Belladonna
1Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Italy.
Abstract:
Exposure in vivo of murine L5178Y lymphoma cells to cytoreductive triazene derivatives leads to the generation of immunogenic variant lines expressing new transplantation Ags recognized by CTL. In one such clonal variant (clone D), at least one subset of T cell neoepitopes are provided by proteins previously shown by serology to be products of endogenous retroviral env sequences. We report here on characterization of PCR-amplified gp70 env genes in clone D. Relative to known gp70 sequences in parental cells and in current databases, one gp70 sequence presented four distinct nucleotide changes, two of which were apparently unique to clone D DNA and cDNA upon differential hybridization analysis. Transfection experiments with the entire gp70 gene or subgenic fragments encompassing a single putative mutation showed that products of the mutated env gene or fragments may confer immunogenicity in vivo and susceptibility in vitro to lysis by clone D-primed, H-2Kd- or H-2Ld-restricted CTL. By skin test assay of mice primed with either clone D or three mutated synthetic peptides, evidence was obtained that amino acid substitutions at the relevant positions of the gp70 protein may produce immunogenic T cell epitopes and that these epitopes are expressed in vivo by clone D. These data point to the role of mutated retroviral tumor peptides as rejection Ags in histocompatible hosts.
Insights
Cytoreductive drugs create immunogenic lymphoma variants expressing new tumor antigens. Mutated retroviral env genes in these variants provide T cell neoepitopes, potentially acting as rejection antigens in cancer immunity.
Area of Science:
- Immunology
- Oncology
- Retroviral Research
Background:
- Cytoreductive triazene derivatives induce immunogenic variants in murine lymphoma cells.
- These variants express novel transplantation antigens recognized by cytotoxic T lymphocytes (CTL).
- Endogenous retroviral env sequences encode T cell neoepitopes in a specific variant, clone D.
Purpose of the Study:
- To characterize the mutated gp70 env genes in clone D.
- To investigate if mutated env gene products confer immunogenicity and CTL susceptibility.
- To determine if mutated retroviral peptides function as tumor rejection antigens.
Main Methods:
- Polymerase chain reaction (PCR) amplification and sequencing of gp70 env genes.
- Differential hybridization analysis to identify unique mutations.
- Transfection experiments with mutated gp70 gene fragments.
- Skin test assays in mice.
Main Results:
- One gp70 env sequence in clone D exhibited four distinct nucleotide changes, two unique to clone D DNA/cDNA.
- Transfection with mutated env gene fragments conferred in vivo immunogenicity and in vitro CTL lysis susceptibility.
- Mutated synthetic peptides elicited immune responses in mice, indicating expression of relevant T cell epitopes by clone D.
Conclusions:
- Mutated retroviral tumor peptides derived from env genes can act as rejection antigens.
- Amino acid substitutions in gp70 can generate immunogenic T cell epitopes.
- These findings highlight the role of mutated retroviral antigens in anti-tumor immunity.