T helper type 1 (Th1) cells are a subset of CD4+ effector T cells that is required for host defense against intracellular viral and bacterial pathogens. Th1 cells develop from activated, naïve CD4+ T cells in the presence of IL-27, IL-12, and IFN-gamma. They can be distinguished from other CD3+CD4+CD8- T cells based on the cell surface expression of IL-12 R beta 2, IL-27 R alpha/WSX-1, IFN-gamma R2, IL-18 R, CCR5, and CXCR3, and the expression of the transcriptional regulators, STAT4 and T-bet, the latter of which is considered to be the master transcriptional regulator required for Th1 cell development. Th1-secreted cytokines induce classical macrophage activation, as well as the activation of natural killer cells and cytotoxic CD8+ T cells. If unregulated, Th1 cells can contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, psoriasis, and type 1 diabetes.
Detection of Cell Surface and Intracellular Markers on Human Th1 Cells by Flow Cytometry. CD4+ T cells were isolated from total human peripheral blood mononuclear cells using a cell selection protocol, such as the one found in the MagCellect™ Human CD4+ T Cell Isolation Kit (R&D Systems, Catalog # MAGH102). Isolated cells were incubated at 37 ˚C for five days in media containing Recombinant Human IL-12 (R&D Systems, Catalog # 219-IL) and Anti-Human IL-4 Antibody (R&D Systems, Catalog # AB-204-NA), followed by stimulation with 50 ng/mL PMA, 200 ng/mL calcium ionomycin, and 3 µM monensin for several hours. Live, single, CD4+ cells are shown in the dot plots, determined using a fixable viability dye, doublet exclusion, and staining with an Alexa Fluor® 700-conjugated Mouse Anti-Human CD4 Monoclonal Antibody (R&D Systems, Catalog # FAB3791N). The cells were additionally stained using an Alexa Fluor® 594-conjugated Rat Anti-Human TIM-3 Monoclonal Antibody (R&D Systems, Catalog # FAB2365T), a PE-conjugated Mouse Anti-Human/Mouse IL-12 R beta 2 Monoclonal Antibody (R&D Systems, Catalog # FAB1959P), a PerCP-conjugated Mouse Anti-Human IFN-gamma Monoclonal Antibody (R&D Systems, Catalog # IC285C) and an Alexa Fluor® 488-conjugated Mouse Anti-Human T-bet Monoclonal Antibody (R&D Systems, Catalog # IC53851G). To facilitate intracellular staining, cells were fixed and permeabilized with FlowX FoxP3 Fixation & Permeabilization Buffer Kit (R&D Systems, Catalog # FC012). Dot plots show the relative TIM-3+, IL-12 R beta 2+, T-bet+, and IFN-gamma+ cells in CD4+ resting (blue dots, lower left quadrant) and Th1-differentiated (orange dots, right quadrants) cell populations. Quadrant markers were set based on staining with the appropriate isotype controls (R&D Systems, Catalog # IC003N, # IC006T, # IC002P, # IC0041C, and # IC002G). Note: The five fluorochrome-conjugated antibodies and buffers used for cell surface and intracellular staining of human Th1 cells using this protocol are also provided together in the FlowX™ Human Th1 Cell Multi-Color Flow Cytometry Kit (R&D Systems, Catalog # FMC009B).