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

Karen Anderson, 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)
JournalBlood
Volume96
Issue number11 PART I
StatePublished - 2000
Externally publishedYes

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Adoptive Immunotherapy
T-cells
Lymphocytes
Cytotoxic T-Lymphocytes
Microspheres
Purification
Blood
Antigens
Peptides
T-Lymphocytes
Neoplasm Antigens
Human Herpesvirus 4
Flow Cytometry
T-Lymphoid Precursor Cells
Technology
Enzyme-Linked Immunospot Assay
Flow cytometry
Homologous Transplantation
Sorting
Bone Marrow Transplantation

ASJC Scopus subject areas

  • Hematology

Cite this

Anderson, K., Wucherpfennig, K. A., Nicholas Haining, W., Schultze, J. L., Maecker, B., Xia, Z., ... 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).

Potential of antigen-specific adoptive immunotherapy using peptide/mhc tetramers and microbeads for highyield purification of cytotoxic t lymphocytes from peripheral blood. / Anderson, Karen; Wucherpfennig, Kai A.; Nicholas Haining, W.; Schultze, Joachim L.; Maecker, Britta; Xia, Zhinan; Butler, Marcus O.; Vonderheide, Robert H.; Nadler, Lee M.

In: Blood, Vol. 96, No. 11 PART I, 2000.

Research output: Contribution to journalArticle

Anderson, K, Wucherpfennig, KA, Nicholas Haining, W, Schultze, JL, Maecker, B, Xia, Z, Butler, MO, Vonderheide, RH & Nadler, LM 2000, 'Potential of antigen-specific adoptive immunotherapy using peptide/mhc tetramers and microbeads for highyield purification of cytotoxic t lymphocytes from peripheral blood', Blood, vol. 96, no. 11 PART I.
Anderson, Karen ; Wucherpfennig, Kai A. ; Nicholas Haining, W. ; Schultze, Joachim L. ; Maecker, Britta ; Xia, Zhinan ; Butler, Marcus O. ; Vonderheide, Robert H. ; Nadler, Lee M. / Potential of antigen-specific adoptive immunotherapy using peptide/mhc tetramers and microbeads for highyield purification of cytotoxic t lymphocytes from peripheral blood. In: Blood. 2000 ; Vol. 96, No. 11 PART I.
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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.",
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T1 - Potential of antigen-specific adoptive immunotherapy using peptide/mhc tetramers and microbeads for highyield purification of cytotoxic t lymphocytes from peripheral blood

AU - Anderson, Karen

AU - Wucherpfennig, Kai A.

AU - Nicholas Haining, W.

AU - Schultze, Joachim L.

AU - Maecker, Britta

AU - Xia, Zhinan

AU - Butler, Marcus O.

AU - Vonderheide, Robert H.

AU - Nadler, Lee M.

PY - 2000

Y1 - 2000

N2 - 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.

AB - 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.

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