title: 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)-gamma 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-gamma, time to reach peak degranulation, and kinetics of IFN-gamma secretion relative to degranulation. Responder CD8+ T lymphocytes that underwent degranulation without IFN-gamma production (CD107a+IFN-gamma-) 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-gamma-secreting cells that did or did not undergo degranulation (CD107a+IFN-gamma+ or CD107a-IFN-gamma+). 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-gamma- responders may be an important determinant of their ability to control CMV replication.
authors.affiliation: Department of Immunology, New England Primate Research Center, Harvard Medical School, Southborough, MA 01772, USA.
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: Chan
authors.forename: Amitinder
authors.initials: A
authors.lastname: Kaur
mesh_terms: D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D018106:CD28 Antigens / Q000032:analysis; D015550:Cell Degranulation / Q000276:immunology*; D003587:Cytomegalovirus / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D017874:Immediate-Early Proteins / Q000276:immunology; D016130:Immunophenotyping; D007371:Interferon-gamma / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000032:analysis; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D052119:Lysosomal-Associated Membrane Protein 2 / Q000378:metabolism; D051907:Lysosomal Membrane Proteins / Q000032:analysis / Q000378:metabolism; D008253:Macaca mulatta; D008562:Membrane Glycoproteins / Q000378:metabolism; D008985:Monensin / Q000494:pharmacology; D010446:Peptide Fragments / Q000276:immunology; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins / Q000378:metabolism; D018089:Superantigens / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000276:immunology* / Q000502:physiology; D015534:Trans-Activators / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014764:Viral Proteins / Q000276:immunology; D019014:fas Receptor / Q000032:analysis
chemical_list: D018106:CD28 Antigens; D018984:Epitopes, T-Lymphocyte; C088645:IE1 protein, cytomegalovirus; C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D052118:Lysosomal-Associated Membrane Protein 1; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D052899:Pore Forming Cytotoxic Proteins; D018089:Superantigens; D015534:Trans-Activators; D014409:Tumor Necrosis Factor-alpha; D014764:Viral Proteins; D019014:fas Receptor; D054353:Perforin; D020126:Brefeldin A; D007371:Interferon-gamma; D008985:Monensin; D017493:Leukocyte Common Antigens