Selection of CMV‐specific CD8+ and CD4+ T cells by mini‐EBV‐transformed B cell lines

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‐γ 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.