A fast and efficient HLA multimer-based sorting procedure that induces little apoptosis to isolate clinical grade human tumor specific T lymphocytes

  • Régis Bouquié
  • Annabelle Bonnin
  • Karine Bernardeau
  • Amir Khammari
  • Brigitte Dréno
  • Francine Jotereau
  • Nathalie Labarrière
  • François Lang
Original Article


HLA multimers are now widely used to stain and sort CD8 T lymphocytes specific for epitopes from viral or tumoral antigens presented in an HLA class I context. However, the transfer of this technology to a clinical setting to obtain clinical grade CD8 T lymphocytes that may be used in adoptive cell transfer (ACT) is hindered by two main obstacles: the first obstacle is the use of streptavidin or derived products that are not available in clinical grade to multimerize HLA/peptide monomers and the second is the reported high degree of apoptosis that eventually occurs when T cell receptors are crosslinked by HLA multimers. In the present report, we describe new HLA multimers composed of immunomagnetic beads covalently coupled to a mAb specific for the AviTag peptide and coated with HLA/peptide monomers bearing the non biotinylated AviTag at the COOH terminus of the HLA heavy chain. Thus, all the components of this new reagent can be obtained in clinical grade. We compared these new multimers with the previously described multimers made with streptavidin beads coated with biotinylated HLA/peptide monomers, in terms of sorting efficiency, recovery of functional T cells, apoptosis and activation. We provide evidence that the new multimers could very efficiently sort pure populations of T lymphocytes specific for three different melanoma antigens (Melan-A, gp100 and NA17-A) after a single peptide stimulation of melanoma patients’ PBMC. The recovered specific T cells were cytotoxic against the relevant melanoma cell-lines and, in most cases, produced cytokines. In addition, in marked contrast with streptavidin-based multimers, our new multimers induced very little apoptosis or activation after binding specific T lymphocytes. Altogether, these new multimers fulfill all the necessary requirements to select clinical grade T lymphocytes and should facilitate the development of ACT protocols in cancer patients.


Immunotherapy Cell-sorting HLA multimers Melanoma T lymphocytes 



Adoptive cell therapy


Activation induced cell death




Epstein–Barr virus


Good manufacturing practice


Cytotoxic T lymphocyte


Dendritic cell


Human leucotyte antigen


Peripheral blood mononuclear cells


T cell receptor


Tumor inflitrating lymphocyte



This work was supported by a grant from the «Ligue Nationale contre le Cancer» (labellisation 2003–2007) and by a grant from INCa “Thérapie adoptive cellulaire du cancer”.


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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Régis Bouquié
    • 1
  • Annabelle Bonnin
    • 1
  • Karine Bernardeau
    • 1
  • Amir Khammari
    • 2
  • Brigitte Dréno
    • 1
    • 2
  • Francine Jotereau
    • 1
    • 3
  • Nathalie Labarrière
    • 1
  • François Lang
    • 1
    • 4
  1. 1.INSERM U892Nantes Cedex 1France
  2. 2.CHU of Nantes, Unit of Skin CancerNantesFrance
  3. 3.Faculté des SciencesUniversité de NantesNantesFrance
  4. 4.UFR des Sciences PharmaceutiquesUniversité de NantesNantesFrance

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