title: Ki-67 staining for determination of rhesus macaque T cell proliferative responses ex vivo.
abstract: The capacity for robust proliferation upon re-infection is a hallmark of adaptive immunity and the basis of vaccination. A widely used animal model for the study of human disease is the rhesus macaque (RM), where capacity for proliferation can be assessed ex vivo using carboxyfluorescein succinimidyl ester (CFSE)-based dilution assays. However, we show over the course of the standard ex vivo proliferation assay that CFSE-labeling at commonly used dye concentrations induces significant cell death, but that this phenomenon is dose-dependent. Here, we describe an alternative semiquantitative method for estimating T cell proliferative responses that avoids the putative biases associated with chemical modification. RM peripheral blood mononuclear cells were stimulated ex vivo with cognate peptides for 5 days, immunostained for intracellular Ki-67, and then analyzed by flow cytometry. We describe a gating strategy using Ki-67 and side light scatter, also a marker of blastogenesis, which correlates strongly with data from CFSE dilution. We show that this method is a valid tool for measuring RM antigen-specific cellular proliferation ex vivo and can be used as an alternative to CFSE dilution assays.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Devon J
authors.initials: DJ
authors.lastname: Shedlock
authors.forename: Kendra T
authors.initials: KT
authors.lastname: Talbott
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Morrow
authors.forename: Bernadette
authors.initials: B
authors.lastname: Ferraro
authors.forename: David A
authors.initials: DA
authors.lastname: Hokey
authors.forename: Karuppiah
authors.initials: K
authors.lastname: Muthumani
authors.forename: David B
authors.initials: DB
authors.lastname: Weiner
mesh_terms: D000818:Animals; D000941:Antigens / Q000737:chemistry; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D049109:Cell Proliferation; D004396:Coloring Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry; D017353:Gene Deletion; D019394:Ki-67 Antigen / Q000096:biosynthesis*; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008253:Macaca mulatta; D013388:Succinimides / Q000737:chemistry; D013601:T-Lymphocytes / Q000166:cytology* / Q000382:microbiology
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D000941:Antigens; D004396:Coloring Agents; D005452:Fluoresceins; D019394:Ki-67 Antigen; D013388:Succinimides