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Dynamic interactions of macrophages with T cells during antigen presentation.

Underhill DM, Bassetti M, Rudensky A, Aderem A - J. Exp. Med. (1999)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle, Washington 98195, USA.

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.

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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.
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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.

Mentions: The rapid, antigen-specific expression of GFP in DO11-GFP cells allowed real-time visualization of productive APC–T cell interactions by video microscopy: we observed that macrophage–DO11-GFP cell interaction is dynamic. IFN-γ–stimulated RAW 264.7 cells were preloaded overnight with OVA, DO11-GFP cells were added, and the cells were mounted on a temperature-controlled microscope stage and observed for 3 h. At the beginning of the experiment, no cells were fluorescent, as shown in Fig. 4. After 3 h, many T cells in the field were expressing GFP.


Dynamic interactions of macrophages with T cells during antigen presentation.

Underhill DM, Bassetti M, Rudensky A, Aderem A - J. Exp. Med. (1999)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195713&req=5

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.
Mentions: The rapid, antigen-specific expression of GFP in DO11-GFP cells allowed real-time visualization of productive APC–T cell interactions by video microscopy: we observed that macrophage–DO11-GFP cell interaction is dynamic. IFN-γ–stimulated RAW 264.7 cells were preloaded overnight with OVA, DO11-GFP cells were added, and the cells were mounted on a temperature-controlled microscope stage and observed for 3 h. At the beginning of the experiment, no cells were fluorescent, as shown in Fig. 4. After 3 h, many T cells in the field were expressing GFP.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle, Washington 98195, USA.

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.

Show MeSH
Related in: MedlinePlus