Journal Article

Establishment of the reversible peptide-major histocompatibility complex (pMHC) class I Histamer technology: tool for visualization and selection of functionally active antigen-specific CD8<sup>+</sup> T lymphocytes

Sabine Tischer, Till Kaireit, Constança Figueiredo, Oliver Hiller, Britta Maecker-Kolhoff, Renè Geyeregger, Stephan Immenschuh, Rainer Blasczyk and Britta Eiz-Vesper

in International Immunology Meeting Abstracts

Published on behalf of Japanese Society for Immunology

Volume 24, issue 9, pages 561-572
Published in print September 2012 |
Published online June 2012 | | DOI: http://dx.doi.org/10.1093/intimm/dxs059

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Multimers of soluble peptide-major histocompatibilty complex (pMHC) molecules are used in both basic and clinical immunology. They allow the specific visualization and isolation of antigen-specific T cells from ex vivo samples. Adoptive transfer of antigen-specific T cells sorted by pMHC multimers is an effective strategy for treatment of patients with malignancies or infectious diseases after transplantation. We developed a new reversible pMHC multimer called ‘Histamer’ to enable the specific detection and isolation of antiviral T cells from peripheral blood. HLA-A*02:01/CMVpp65 495–503 Histamer (A02/CMV Histamer) was generated by coupling 6xHis-tagged pMHC molecules onto cobalt-based magnetic beads. The specificity of the Histamer was evaluated by flow cytometry. Sorting of antiviral CD8+ cytotoxic T lymphocytes (CTLs) was performed by magnetic cell separation, followed by the monomerization of the Histamer after addition of the competitor l-histidine. Sorted T cells were analyzed for phenotype and function. The reversible pMHC Histamer proved to be highly specific and sensitive. CMV-specific T cells of up to 99.6% purity were isolated using the Histamer technology. Rapid and complete disassembly of the T-cell surface-bound A02/CMV Histamer followed by the subsequent dissociation of the pMHC monomers from CD8+ CTL receptors was achieved using 100 mM l-histidine. The function of CMV-specific T cells enriched by Histamer staining did not differ from CTLs induced by standard T-cell assays. This reversible T-cell staining procedure preserves the functionality of antigen-specific T cells and can be adapted to good manufacturing practice conditions. The pMHC Histamer technology offers full flexibility and fulfills all requirements to generate clinical-grade T lymphocytes.

Keywords: cytotoxic T lymphocytes; flow cytometry, human; immunomagnetic separation; pMHC Histamer; cytotoxic T lymphocytes

Journal Article.  8412 words.  Illustrated.

Subjects: Immunology ; Biochemical Immunology

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