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

• Published in: International immunology 2012-09, Vol.24 (9), p.561-572
• Main Authors: Tischer, Sabine, Kaireit, Till, Figueiredo, Constança, Hiller, Oliver, Maecker-Kolhoff, Britta, Geyeregger, Renè, Immenschuh, Stephan, Blasczyk, Rainer, Eiz-Vesper, Britta
• Format: Article
• Language: English
• Subjects: Adoptive Transfer; CD8-Positive T-Lymphocytes - immunology; Cobalt - metabolism; Cytotoxicity, Immunologic; Flow Cytometry; Histidine - metabolism; HLA-A2 Antigen - analysis; HLA-A2 Antigen - immunology; Humans; Immunomagnetic Separation - methods; Immunophenotyping; Peptide Fragments - analysis; Peptide Fragments - immunology; Phosphoproteins - immunology; Protein Binding; Protein Multimerization; Sensitivity and Specificity; Viral Matrix Proteins - immunology
• ISSN: 0953-8178; 1460-2377
• DOI: 10.1093/intimm/dxs059

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.