Selection of CMV-specific CD8+ and CD4+ T cells by mini-EBV-transformed B cell lines |
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Authors: | Wiesner Martina Zentz Caroline Hammer Markus H Cobbold Mark Kern Florian Kolb Hans-Jochem Hammerschmidt Wolfgang Zeidler Reinhard Moosmann Andreas |
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Affiliation: | Department of Gene Vectors, GSF - National Research Center for Environment and Health, Munich, Germany. |
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Abstract: | Efficient protocols to generate cytomegalovirus (CMV)-specific T cells are required for adoptive immunotherapy. Recombinant Epstein-Barr virus (EBV) vectors called mini-EBV can be used to establish permanent B cell lines in a single step, which present the CMV antigen pp65 in a constitutive manner. These B cell lines, coined pp65 mini-LCL, were successfully used to reactivate and expand CMV-specific cytotoxic T cells. Here we evaluate this pp65 mini-EBV system in closer detail, focusing on (1) the quantification of T cells with specific effector function and (2) the identification of CMV-specific CD4(+) helper T cells. The co-expansion of various functional CMV epitope specificities was demonstrated by IFN-gamma enzyme-linked immunospot assay (ELISPOT) assays and HLA-peptide tetramer staining. Single-cell cloning resulted in both CD4(+) and CD8(+) T cell clones, the majority of which was CMV specific. Thus, mini-LCL present the pp65 antigen on HLA class I and II, mobilizing both arms of the T cell response. Using a peptide library covering the pp65 sequence for further analysis of T cell clones, we identified new pp65 CD8(+) and CD4(+) T cell epitopes. |
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Keywords: | Cytomegalovirus T cells Mini‐EBV Mini‐LCL Adoptive immunotherapy |
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