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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.

Type

Journal article

Journal

J Immunol Methods

Publication Date

01/09/2001

Volume

255

Pages

125 - 134

Keywords

Animals, Cloning, Molecular, Genetic Vectors, HIV Envelope Protein gp120, HIV Reverse Transcriptase, Humans, Hybridomas, Immunodominant Epitopes, Mice, Mutagenesis, Insertional, Peptide Fragments, Receptors, Antigen, T-Cell, alpha-beta, Recombinant Fusion Proteins