Generation and purification of CD8+ Melan-A-specific cytotoxic T lymphocytes for adoptive transfer in tumor immunotherapy

Mathias Oelke, Ursula Moehrle, Ji Li Chen, Dirk Behringer, Vincenzo Cerundolo, Albrecht Lindemann, Andreas Mackensen

Research output: Contribution to journalArticlepeer-review


Tumor antigens that might serve as potential targets for adoptive T-cell therapy have been defined in different tumor entities, especially in malignant melanoma. To generate conditions to induce primary T-cell responses against different HLA-A*0201-restricted melanoma peptides and to allow further expansion of peptide-specific T cells for adoptive transfer, CD8+- purified T cells from healthy donors were stimulated with Melan-A-pulsed autologous dendritic cells. Dendritic cells were generated in vitro from monocytes with granulocyte macrophage colony-stimulating factor, interleukin- 4, and transforming growth factor-β1. After 3-4 weekly stimulation cycles with Melan-A-pulsed DCs, we were able to induce a strong peptide-specific CTL response in vitro. MHC-peptide tetramer staining revealed a frequency of up to 3.5% CD8+/Melan-A+ T cells. Additional antigen-independent expansion with anti-CD3/anti-CD28 monoclonal antibodies together with interleukin-2 gave rise to 600-fold expansion of CD8+ CTLs that maintained Melan-A specificity and were able to efficiently lyse Melan-A-expressing melanoma cells. To enrich antigen-specific T cells in vitro, we used a recently established technology for analysis and sorting of live cells according to secreted cytokines. In the present study, we demonstrated that Melan-A- specific T cells can be purified by magnetic separation according to secreted IFN-γ. These cells revealed a very potent monospecific CTL response, even at low E:T ratios, against Melan-A-pulsed and Melan-A-expressing target cells. Altogether, our study demonstrated that we have developed an efficient method for generating large numbers of peptide-specific T cells in vitro that may be used for adoptive T-cell transfer in tumor immunotherapy.

Original languageEnglish (US)
Pages (from-to)1997-2005
Number of pages9
JournalClinical Cancer Research
Issue number5
StatePublished - May 2000
Externally publishedYes

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

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