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Methods Mol Biol ; 2521: 95-128, 2022.
Article in English | MEDLINE | ID: mdl-35732995

ABSTRACT

The protocol describes the procedure of antigen-specific T cell generation and TCR identification for the use in adoptive T cell therapy. We describe two paths of generating antigen-specific T cells, first, T cell stimulation with autologous dendritic cells pulsed with antigen peptide, second, in vivo T cell stimulation with peptide or DNA by gene gun application in a suitable mouse model followed by in vitro enrichment of peptide-reactive T cells. Peptide-stimulated T cells are sorted by fluorescence-activated cell sorting for CD107α or IFNγ expression and subsequently isolated RNA is used in a 5' rapid amplification of cDNA ends (RACE )-PCR specific for TCR for TCR chain identification. After retroviral cloning, it is re-expressed on human T cells to test its applicability in adoptive T cell therapy.


Subject(s)
Antigens, Neoplasm , Neoplasms , Animals , Genetic Therapy , Mice , Neoplasms/genetics , Neoplasms/therapy , Peptides/genetics , Receptors, Antigen, T-Cell/genetics
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