Graduate Student University of Virginia Charlottesville, Virginia, United States
Introduction/Rationale: The lymph node (LN) is a spatially organized tissue where lymphocytes communicate by secreting cytokines in response to stimuli. Since most cytokines act locally, detection at the secretion site is needed to understand signaling within tissue. However, no current technologies detect cytokine spatial distribution in ex vivo stimulated tissue samples except for genetically modified mice; an expensive option with limited cytokines.
Methods: We show two methods to visualize cytokine distribution in vibratome-sectioned, 300µm thick, murine LN slices. In the first method, we developed a cell-surface based sandwich immunoassay using a bispecific antibody reagent to detect IFN-γ at its secretion site in slices using microscopy. In the second, we developed a procedure to block cytokine secretion in live slices using Brefeldin A (BFA). Through adding BFA to slices during stimulation, cytokines accumulated in cells for intracellular staining in the fixed tissue.
Results: With both methods, we successfully visualized IFN-γ distribution in LN slices after anti-CD3ε stimulation. We show that IFN-γ is located primarily in the interfollicular region. Using intracellular staining, we have multiplexed to include both IFN-γ and IL-17.
Conclusion: Next steps for both assays include applying this method to LN slices after in vivo vaccination and expanding it to human tonsil tissue. We envision that these assays will allow us to better understand cytokine signaling in complex disease states within live tissue samples.
