Characterization of T-cell subsets and T-cell receptor subgroups in pigtailed macaques using two- and three-color flow cytometry

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Abstract
In order to characterize macaque T-lymphocyte subsets, we used a chromophore from a dinoflagellate, peridinin chlorophyll A protein (PerCP), which, like fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE), can be excited by a 488-nm laser and emits light at 670 nm without spectral overlap with FITC and PE. Mouse monoclonal antibodies were conjugated with FITC, PE, and PerCP to detect CD4+ and CD8+ cells in macaque peripheral blood lymphocytes (PBL) subsets before and after activation and in nonactivated thymocytes. Resting and activated macaque blood CD4+ T-cells could be clearly delineated into discrete subsets with either CD28, CD45RA, or CD45RO as a second marker and CD26, CD29, CD44, or CD69 as a third marker. CD8+ cells were further subdivided by expression of similar combinations of markers. A subset of CD8+ CD28− T-cells in blood expressed the activation marker CD69, suggesting that they were already activated. Virtually all CD4+CD8+, CD4+CD8−, and CD4−CD8+ macaque thymocytes expressed CD2, CD3, and CD18 and not CD25, CD44, or CD45O, but macaque thymocyte subpopulations did differ in their expression of CD28 and CD29. The expression of T-cell receptor (TCR) subgroups on macaque PBL and thymocytes was analyzed before and after activation with staphylococcal enterotoxins (superantigens). The pattern of T-cell variable-region expression in macaques was similar to that seen in humans, with a high frequency of T cells expressing Vβ8. After superantigen stimulation, only minor changes in TCR Vβ expression were detectable in PBL. A dramatic increase in Vβ8 expression was seen after stimulation of macaque thymus with staphylococcal enterotoxin D (SE-D), a minor increase after toxic shock syndrome toxin 1 (TSST-1) stimulation, and a simultaneous decrease in Vβ6 levels.