title: Characterization of mitogen-stimulated porcine lymphocytes using a stable fluorescent dye (PKH2) and multicolor flow cytometry.
abstract: Stimulation of lymphocyte proliferation using mitogens or specific antigens is a method that is used frequently to assess immune responsiveness. While useful, lymphocyte blastogenesis, or [3H]-thymidine incorporation, provides little information regarding the response of specific subsets to the stimulant. Here, we report that the fluorescent cell membrane probe, PKH2, is a useful tool for measuring the proliferation of porcine lymphocyte subpopulations by utilizing multicolor flow cytometry. For this study, mitogen-induced proliferation of porcine peripheral blood mononuclear cells (PBMCs) was measured using [3H]-thymidine incorporation as well as a flow cytometric-based proliferation assay. From the [3H]-thymidine incorporation data alone, it was observed that PBMC stimulated with either concanavalin A (Con A), phytohemagglutinin (PHA) or pokeweed mitogen (PWM) demonstrated greater proliferation on day 3 than on day 5 of culture. Using the PKH dye and flow cytometric analysis, the responsiveness of specific lymphocyte subsets to mitogen stimulation was detected. The predominant subsets of porcine lymphocytes responding to Con A or PHA stimulation were CD4(+)CD8(+), CD4(-)CD8alpha(hi), CD4(-)CD8alpha(lo) and gammadelta TCR(+) cells. PWM stimulation induced responses by CD4(+)CD8(+), CD4CD8alpha(hi) but not by CD4(-)CD8alpha(lo) or gammadelta TCR(+) cells. Con A stimulation resulted in a sustained proliferation of CD8alpha(hi) cells over the 5-day period while PHA stimulation resulted in proliferation that peaked within the first 3 days. Little or no proliferative responses were detected within the IgM(+) population (e.g. B cells). This is the first study to define the contribution of individual lymphocyte subsets to mitogen-induced proliferation of porcine PBMCs.
authors.affiliation: Veterinary Medical Research Institute, Iowa State University, 1802 Elwood Drive, Ames, IA 50011, USA. adorn@iastate.edu
authors.forename: A D
authors.initials: AD
authors.lastname: Dorn
authors.forename: W R
authors.initials: WR
authors.lastname: Waters
authors.forename: V M
authors.initials: VM
authors.lastname: Byers
authors.forename: B A
authors.initials: BA
authors.lastname: Pesch
authors.forename: M J
authors.initials: MJ
authors.lastname: Wannemuehler
mesh_terms: D000818:Animals; D003208:Concanavalin A / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology*; D016131:Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism; D009930:Organic Chemicals; D010835:Phytohemagglutinins / Q000276:immunology; D011043:Pokeweed Mitogens / Q000276:immunology; D013552:Swine / Q000097:blood / Q000276:immunology*; D013936:Thymidine / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D009930:Organic Chemicals; C068241:PKH-2; D010835:Phytohemagglutinins; D011043:Pokeweed Mitogens; D003208:Concanavalin A; D013936:Thymidine