Design of cassette vectors permitting cloning of all types of human TCR variable alpha and beta regions.
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.
Caivano
A
A
Institute of Protein Biochemistry and Enzymology, CNR, Via Guglielmo Marconi 10, 80125, Naples, Italy.
D'Apice
L
L
Tiberio
C
C
Prisco
A
A
Acuto
O
O
Guardiola
J
J
De Berardinis
P
P
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D015699:HIV Envelope Protein gp120 / Q000276:immunology; D054303:HIV Reverse Transcriptase / Q000276:immunology; D006801:Humans; D006825:Hybridomas; D016056:Immunodominant Epitopes / Q000276:immunology; D051379:Mice; D016254:Mutagenesis, Insertional; D010446:Peptide Fragments / Q000235:genetics*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D011993:Recombinant Fusion Proteins
D015699:HIV Envelope Protein gp120; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011993:Recombinant Fusion Proteins; D054303:HIV Reverse Transcriptase