Design of cassette vectors permitting cloning of all types of human TCR variable alpha and beta regions.

Journal article

T cell clones are an irreplaceable asset for the study of immune responses relevant to human pathologies. Such cells, however, cannot always be maintained in long-term culture. In order to reconstitute functional human T cell receptors (TCRs) into stable and fast growing hybridoma T cells, we developed a general approach based on a versatile cassette system, which allows cloning of all types of human T cell receptor variable alpha and beta region genes fused to murine constant regions. These chimeric constructs are easily excised and transferred into expression vectors that can be used to transfect a human CD4-expressing murine T cell hybridoma recipient. The resulting transfectants are highly stable both in terms of T cell receptor-CD3 expression and IL-2 response to the specific antigenic stimulus. Using these cassette vectors, we reconstituted the original HLA-restricted antigen specificity for two human T cell clones, one recognizing an immunodominant epitope of HIV-1 gp120, and the other recognizing an immunodominant epitope of HIV-1 reverse transcriptase. We found that the reconstituted hybridomas maintain the ability of the original T cell clones to recognize the appropriate epitope in the context of the relevant MHC either as a synthetic peptide or after processing. Their unlimited growth capacity makes them particularly suited for in vitro studies.

Authors: Caivano, A; D'Apice, L; Tiberio, C; Prisco, A; Acuto, O

• Publication status: Published
• Journal: Journal of immunological methods
• Volume: 255
• Issue: 1-2
• Pages: 125-134
• Publication date: 2001-09-01
• DOI: 10.1016/s0022-1759(01)00420-3
• EISSN: 1872-7905
• ISSN: 0022-1759
• Language: English
• Keywords: Peptide Fragments; Cloning, Molecular; Hybridomas; HIV Envelope Protein gp120; Immunodominant Epitopes; Mice; HIV Reverse Transcriptase; Receptors, Antigen, T-Cell, alpha-beta; Animals; Genetic Vectors; Recombinant Fusion Proteins; Humans; Mutagenesis, Insertional