Flow cytometric detection of degranulation reveals phenotypic heterogeneity of degranulating CMV-specific CD8+ T lymphocytes in rhesus macaques

Abstract

Flow-cytometric conditions for detection of lysosomal-associated membrane proteins (LAMPs) on the surface of recently degranulated cells were optimized for rhesus macaques and used to investigate the functional properties of rhesus cytomegalovirus (rhCMV)-specific CD8+ T lymphocytes with regards to cytotoxicity and interferon (IFN)-γ secretion in six asymptomatic CMV-seropositive rhesus macaques. Unlike humans, the rhesus macaque LAMP-1 protein CD107a underwent little or no endocytosis over a six to 18 h stimulation period. Following in vitro stimulation, rhCMV-specific CD8+ T lymphocytes were heterogeneous with regards to the composition of cells positive for CD107a and/or IFN-γ, time to reach peak degranulation, and kinetics of IFN-γ secretion relative to degranulation. Responder CD8+ T lymphocytes that underwent degranulation without IFN-γ production (CD107a + IFN-γ–) were predominantly composed of terminally differentiated effectors (CD28–CD45RA+). Moreover, they had significantly lower frequencies of effector memory (CD28–CD45RA–) cells compared to the IFN-γ-secreting cells that did or did not undergo degranulation (CD107a + IFN-γ + or CD107a–IFN-γ +). The perforin content of effector CD8+ T lymphocytes was significantly greater than that of effector memory CD8+ T lymphocytes in rhesus macaques, suggesting that they were more cytolytic. Our findings suggest that the composition of rhCMV-specific CD8+ T lymphocytes with regards to CD107a + IFN-γ– responders may be an important determinant of their ability to control CMV replication.