Dynamic interactions of macrophages with T cells during antigen presentation

Abstract

We have established a method for real-time video analysis of the interaction of antigen-presenting cells (APCs) with T cells. Green fluorescent protein expression controlled by a nuclear factor of activated T cells (NFAT)-responsive promoter permits the visualization of productive antigen presentation in single T cells. The readout is rapid (within 2 h) and semiquantitative and allows analysis by video microscopy and flow cytometry. Using this approach, we demonstrate that macrophages have the capacity to simultaneously activate multiple T cells. In addition, the interaction of T cells with macrophages is extraordinarily dynamic: after initial stable contact, the T cells migrate continuously on the surface of the macrophage and from APC to APC during productive antigen presentation. Thus, T cells sum up signals from multiple interactions with macrophages during stimulation.

Figure 1

Productive antigen-specific interaction induces GFP expression in a T cell hybridoma. IFN-γ–stimulated RAW 264.7 cells were incubated for 2 h with (A and B) or without (C and D) 100 μg/ml of the OVA 323–339 peptide, and allowed to interact with a fivefold excess of DO11-GFP cells for 6 h. In the presence of peptide, T cells in contact with macrophages express GFP (B, arrows), whereas neighboring T cells not in direct contact with macrophages do not (B, arrowhead). In the absence of peptide, the DO11-GFP cells are not stimulated to express GFP (D, arrows) despite their adhesion to macrophages.

Figure 2

Analysis of antigen-induced GFP expression by flow cytometry. (A) RAW 264.7 cells were labeled with the fluorescent lipid DiI, stimulated for 48 h with IFN-γ, and loaded for 2 h with the indicated concentration of OVA 323–339 peptide. DO11-GFP cells were added at a 1:1 ratio, and the cells were allowed to interact for 6 h. GFP expression in the DO11-GFP cells was analyzed by flow cytometry. (B) IFN-γ–stimulated RAW 264.7 cells were preloaded for 2 h with the indicated dose of antigenic peptide, mixed 1:1 with CTO-labeled DO11-GFP cells, and allowed to interact for 6 h. The cells were harvested, and GFP expression in the T cell hybridoma was analyzed by flow cytometry. The percentage of T cells expressing GFP increased as a function of peptide concentration (○), as did the average fluorescence of the GFP-positive cells (□).

Figure 2

Analysis of antigen-induced GFP expression by flow cytometry. (A) RAW 264.7 cells were labeled with the fluorescent lipid DiI, stimulated for 48 h with IFN-γ, and loaded for 2 h with the indicated concentration of OVA 323–339 peptide. DO11-GFP cells were added at a 1:1 ratio, and the cells were allowed to interact for 6 h. GFP expression in the DO11-GFP cells was analyzed by flow cytometry. (B) IFN-γ–stimulated RAW 264.7 cells were preloaded for 2 h with the indicated dose of antigenic peptide, mixed 1:1 with CTO-labeled DO11-GFP cells, and allowed to interact for 6 h. The cells were harvested, and GFP expression in the T cell hybridoma was analyzed by flow cytometry. The percentage of T cells expressing GFP increased as a function of peptide concentration (○), as did the average fluorescence of the GFP-positive cells (□).

Figure 3

Kinetics of antigen-dependent stimulation of GFP expression in DO11-GFP cells. IFN-γ–stimulated RAW 264.7 cells were loaded overnight with OVA at a final concentration of 25 mg/ml (○), 2 mg/ml (□), or 0.5 mg/ml (▵). Control cells were also IFN-γ stimulated but were not incubated with antigen (▪). CTO-labeled DO11-GFP cells were added at a 1:1 ratio and allowed to interact with the RAW 264.7 cells for the indicated periods. The cells were harvested, and GFP expression in the T cell hybridoma was analyzed by flow cytometry.

Figure 4

Time-lapse imaging of macrophage–T cell interactions. IFN-γ–stimulated RAW 264.7 cells were loaded overnight with 10 mg/ml OVA. DO11-GFP cells were added at a 5:1 ratio to macrophages, the cells were mounted on a temperature-controlled microscope stage, and cell–cell interactions were continuously observed by video microscopy for 3 h. The panels show selected video images collected at the indicated time intervals after addition of DO11-GFP cells. At the beginning of the experiment (0 Hr.), no cells expressed GFP (top left). After 3 h, many T cells in the field were expressing GFP (bottom right). Individual T cells are labeled alphabetically.