Potential of antigen-specific adoptive immunotherapy using peptide/mhc tetramers and microbeads for highyield purification of cytotoxic t lymphocytes from peripheral blood

Karen S. Andersen, Kai A. Wucherpfennig, W. Nicholas Haining, Joachim L. Schultze, Britta Maecker, Zhinan Xia, Marcus O. Butler, Robert H. Vonderheide, Lee M. Nadler

Research output: Contribution to journalArticle

Abstract

Adoptive immunotherapy, such as donor lymphocyte infusions (DLI), is a potent approach for the treatment of cancer. Clinically, however, DLI is hampered by toxicity from GVHD. An alternative strategy may be the infusion of antigen-specific donor T lymphocytes, as exemplified by the use of anti-CMV or anti-EB V T cells for complications following allogeneic bone marrow transplantation. Tumor antigen-specific cytotoxic T lymphocyte (CTL) infusions have been more difficult, limited by the lack of widely applicable tumor antigens and the technical demands of isolating and expanding sufficient numbers of antigen-specific T cells. Here, we demonstrate the isolation of high numbers of purified antigen-specific T cells suitable for transfer using recombinant MHC/peptide tetramers and magnetic microbead cell sorting. CTL specific for one of two model antigenic peptides, influenza matrix MI-58 (flu-Mi) or EBV-BMLF1-280, were generated from PBMC of normal donors. CTL were expanded for up to two weeks ex vivo by stimulation with peptide-pulsed PBMC and cytokines. This increased the frequency of flu-M 1 tetramer+ CD3+ CD8+ T cells from <0.01% of PBMC to 6-10%. EBV-BMLF1 tetramer+ CD3+ CD8+ T cells increased from precursor frequencies of 0.02-0.05% of total PBMC to 22-27% (overall mean 814-fold increase). Peptide-specific CTL were sorted using MHC/peptide tetramers conjugated to PE, and anti-PE Miltenyi magnetic MACS microbeads. As determined by flow cytometry, purity ranged from 83-98% tetramer+ cetls, and recovery of antigen-specific CTL ranged between 36-93% (mean 59%). In comparison to different magnetic beads and flow cytometry based cell sorting, this tetramer/ microbead technology was significantly more rapid and efficient. Importantly, magnetic bead sorted CTL were functional as demonstrated by cytotoxicity of peptide-loaded target cells (40-96% lysis at 10:1 E:T ratio) and strong IFNy secretion in response to antigenic stimulation in ELISPOT analysis. Overall, for these two model antigens, up to 25 million antigen-specific CTL could be generated from 50 cc of peripheral blood. Combining non-specific techniques (such as mitogenic or CD3/CD28 stimulation) with this approach may further increase the yield of specific cells. With the ongoing discovery of tumor antigens suitable for clinical therapeutics, the evaluation of antigen-specific lymphocyte infusions, facilitated by tetramer/microbead technology, becomes an important next step in developing anti-tumor immunotherapy.

Original languageEnglish (US)
Pages (from-to)177a
JournalBlood
Volume96
Issue number11 PART I
StatePublished - Dec 1 2000
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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    Andersen, K. S., Wucherpfennig, K. A., Nicholas Haining, W., Schultze, J. L., Maecker, B., Xia, Z., Butler, M. O., Vonderheide, R. H., & Nadler, L. M. (2000). Potential of antigen-specific adoptive immunotherapy using peptide/mhc tetramers and microbeads for highyield purification of cytotoxic t lymphocytes from peripheral blood. Blood, 96(11 PART I), 177a.