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

J Immunol Methods. 2001 Sep 1;255(1-2):125-34. doi: 10.1016/s0022-1759(01)00420-3.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • HIV Envelope Protein gp120
  • Immunodominant Epitopes
  • Peptide Fragments
  • Receptors, Antigen, T-Cell, alpha-beta
  • Recombinant Fusion Proteins
  • HIV Reverse Transcriptase