Immunophenotyping of Rhesus CMV-Specific CD8 T-Cell Populations

A vaccine to ameliorate cytomegalovirus (CMV)-related pathogenicity in transplantation patients is considered a top priority. A therapeutic vaccine must include components that elicit both neutralizing antibodies, and highly effective CD8 T-cell responses. The most important translational model of vaccine development is the captive-bred rhesus macaque (Macaca mulatta) of Indian origin. There is a dearth of information on rhesus cytomegalovirus (rhCMV)-specific CD8 T cells due to the absence of well-defined CD8 T-cell epitopes presented by classical MHC-I molecules. In the current study, we defined two CD8 T-cell epitopes restricted by high-frequency Mamu alleles: the Mamu-A1*002:01 restricted VY9 (VTTLGMALY aa291-299) epitope of protein IE-1, and the Mamu-A1*008:01 restricted NP8 (NPTDRPIP aa96-103) epitope of protein phosphoprotein 65-2. We developed tetramers and determined the level, phenotype, and functional capability of the two epitope-specific T-cell populations in circulation and various tissues. We demonstrated the value of these tetramers for in situ tetramer staining. Here, we first provided critical reagents and established a flow cytometric staining strategy to study rhCMV-specific T-cell responses in up to 40% of captive-bred rhesus macaques.