title: 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.
abstract: 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.
authors: Institute for Transfusion Medicine, Hannover Medical School, Germany.
authors: Sabine
authors: S
authors: Tischer
authors: Till
authors: T
authors: Kaireit
authors: Constança
authors: C
authors: Figueiredo
authors: Oliver
authors: O
authors: Hiller
authors: Britta
authors: B
authors: Maecker-Kolhoff
authors: Renè
authors: R
authors: Geyeregger
authors: Stephan
authors: S
authors: Immenschuh
authors: Rainer
authors: R
authors: Blasczyk
authors: Britta
authors: B
authors: Eiz-Vesper
mesh_terms: D019264:Adoptive Transfer; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D003035:Cobalt / Q000378:metabolism; D003602:Cytotoxicity, Immunologic; D005434:Flow Cytometry; D015789:HLA-A2 Antigen / Q000032:analysis / Q000276:immunology*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D016130:Immunophenotyping; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology; D010750:Phosphoproteins / Q000276:immunology; D011485:Protein Binding; D055503:Protein Multimerization; D012680:Sensitivity and Specificity; D014763:Viral Matrix Proteins / Q000276:immunology
chemical_list: C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D010446:Peptide Fragments; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; D003035:Cobalt; D006639:Histidine