title: Influenza virus-specific TCR-transduced T cells as a model for adoptive immunotherapy.
abstract: Adoptive transfer of T lymphocytes equipped with tumor-antigen specific T-cell receptors (TCRs) represents a promising strategy in cancer immunotherapy, but the approach remains technically demanding. Using influenza virus (Flu)-specific T-cell responses as a model system we compared different methods for the generation of T-cell clones and isolation of antigen-specific TCRs. Altogether, we generated 12 CD8(+) T-cell clones reacting to the Flu matrix protein (Flu-M) and 6 CD4(+) T-cell clones reacting to the Flu nucleoprotein (Flu-NP) from 4 healthy donors. IFN-γ-secretion-based enrichment of antigen-specific cells, optionally combined with tetramer staining, was the most efficient way for generating T-cell clones. In contrast, the commonly used limiting dilution approach was least efficient. TCR genes were isolated from T-cell clones and cloned into both a previously used gammaretroviral LTR-vector, MP91 and the novel lentiviral self-inactivating vector LeGO-MP that contains MP91-derived promotor and regulatory elements. To directly compare their functional efficiencies, we in parallel transduced T-cell lines and primary T cells with the two vectors encoding identical TCRs. Transduction efficiencies were approximately twice higher with the gammaretroviral vector. Secretion of high amounts of IFN-γ, IL-2 and TNF-α by transduced cells after exposure to the respective influenza target epitope proved efficient specificity transfer of the isolated TCRs to primary T-cells for both vectors, at the same time indicating superior functionality of MP91-transduced cells. In conclusion, we have developed optimized strategies to obtain and transfer antigen-specific TCRs as well as designed a novel lentiviral vector for TCR-gene transfer. Our data may help to improve adoptive T-cell therapies.
authors: Research Department Cell and Gene Therapy; Department of Stem Cell Transplantation (SCT); University Medical Center (UMC) Hamburg-Eppendorf; Hamburg, Germany.
authors: Belinda
authors: B
authors: Berdien
authors: Henrike
authors: H
authors: Reinhard
authors: Sabrina
authors: S
authors: Meyer
authors: Stefanie
authors: S
authors: Spöck
authors: Nicolaus
authors: N
authors: Kröger
authors: Djordje
authors: D
authors: Atanackovic
authors: Boris
authors: B
authors: Fehse
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D015870:Gene Expression; D005822:Genetic Vectors; D064368:Healthy Volunteers; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D007371:Interferon-gamma / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins / Q000276:immunology*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000276:immunology*; D014161:Transduction, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014758:Viral Core Proteins / Q000276:immunology*; D014763:Viral Matrix Proteins / Q000276:immunology*
chemical_list: D007376:Interleukin-2; C488936:M1 protein, Influenza A virus; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins; D011948:Receptors, Antigen, T-Cell; D014409:Tumor Necrosis Factor-alpha; D014758:Viral Core Proteins; D014763:Viral Matrix Proteins; D007371:Interferon-gamma
keywords: T-cell receptor (TCR); TCR gene therapy; adoptive immunotherapy; gene transfer; influenza antigen; lentiviral vectors