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Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety
Published on: July 24, 2011
Involvement of somatically acquired ecotropic viruses in the immunogenicity of nude-transplanted NIH/3T3 transformed
M Sensi1, M Bergomi, P Panceri
1Division of Experimental Oncology D, Istituto Nazionale Tumori, Milan, Italy.
Abstract:
Immunogenic tumor variants were previously derived after transplantation in vivo into nude mice of NIH/3T3-transformed cell lines. Nude-passaged cell lines were rejected by immunocompetent H-2q NIH mice, were recognized by specific CTL clones, and expressed new retroviral Ag. The aim of the present work was to investigate whether somatically acquired proviral sequences were present in the genome of nude-passaged cells and to test directly for a causative relationship between murine leukemia virus (MuLV) expression and immunogenicity. Southern blot analysis of PstI-digested DNA indicated that in contrast to the parental NIH/3T3 transformed cell lines (pT, T12N/5a, NS-1) all the nude-passaged immunogenic variants (pT-nude, T12N/5a-nude, NS-1-nude) contained newly acquired ecotropic-related proviruses. Immediately after in vitro establishment, these tumors displayed multiple integration sites as assessed by analysis of 3' proviral-cellular junctions. Long term in vitro culture of one of the cell lines (pT-nude) resulted in a cell line (pT-nude/vitro) that was clonal or oligo-clonal with respect to viral integration. Northern blot analysis established that the new proviruses were actively transcribed in all the immunogenic variants. To assess whether the somatically acquired ecotropic proviral sequences encode for target structures recognized by specific CTL, obtained after immunization of NIH mice with pT-nude, the parental cell line pT was transfected with plasmids containing the entire AKV MuLV genome, the cloned AKV gag or env genes. Screening of transfectants for their ability to stimulate the production of TNF by anti-pT-nude effectors indicated that cells transfected with the entire ecotropic virus or with MuLV-env gene products could be recognized by an NIH anti-pT-nude CTL line and NIH anti-pT-nude Kq-restricted CTL clones as well as the immunizing target pT-nude.
Insights
Newly acquired retroviruses in tumor cells drive immunogenicity. These findings link murine leukemia virus (MuLV) expression to tumor rejection, offering insights into cancer immunology.
Area of Science:
- Immunology
- Virology
- Cancer Research
Background:
- Immunogenic tumor variants were previously generated from NIH/3T3-transformed cell lines transplanted into nude mice.
- These nude-passaged cell lines exhibited rejection by immunocompetent mice and recognition by cytotoxic T-lymphocyte (CTL) clones, alongside new retroviral antigen expression.
Purpose of the Study:
- To determine if nude-passaged cells harbor somatically acquired proviral sequences.
- To investigate the direct causal relationship between murine leukemia virus (MuLV) expression and tumor immunogenicity.
Main Methods:
- Southern blot analysis to detect newly acquired proviruses in nude-passaged cell genomes.
- Northern blot analysis to assess proviral transcription.
- Transfection of parental cell lines with MuLV constructs (whole genome, gag, env) to test for CTL recognition.
Main Results:
- All immunogenic nude-passaged variants contained newly acquired ecotropic-related proviruses, unlike parental cell lines.
- These new proviruses were actively transcribed in immunogenic variants.
- Transfection with the complete ecotropic MuLV or MuLV-env gene products rendered parental cells recognizable by specific CTLs.
Conclusions:
- Somatically acquired ecotropic proviruses are present in immunogenic tumor variants.
- MuLV expression, particularly env gene products, is directly responsible for the immunogenicity and CTL recognition of these tumor variants.
