Efficient communication and drug efficacy in the immune system relies heavily on transport of proteins, such as cytokines, through the lymph node tissue matrix to reach receptors on target cells. The lymph node undergoes massive structural reorganization during and after an inflammatory event, and how these changes effect transport is unknown. Without quantitative data on protein diffusion and binding in the naive and inflamed lymph node, it is difficult to predict the efficacy or action of a new immunotherapy. To date, measurement of transport of bioactive proteins in the lymph node has not been studied, mainly because of its small size and its rapid cellular reactivity (min to hr), which prevent standard measurement techniques. Here, we developed a microfluidic platform to deliver nanogram quantities of fluorescently labelled cytokine to microscale regions within slices of murine lymph node and diffusion was monitored by the integrated optical imaging technique with fluorescence microscopy. The method was first validated by delivering 3,10, 40, and 70 kDa FITC-dextran to slices of agarose gel and comparing the resulting diffusion coefficients to those obtained using standard methods (FRAP and microinjection). Next, we quantified the diffusion of three cytokines: tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and interleukin-2 (IL-2), which are representative of two major structural classes, vary in multimerization, molecular weight, and bind to various elements of the extracellular matrix. Diffusion was analyzed in both the T-cell and B-cell region of the lymph node for each cytokine, which significantly impacted their rate of transport. Lastly, we quantified the extent to which inflammation altered transport through the lymph node tissue. This is the first method to directly measure cytokine transport in live tissue slices, and in the future, it could be expanded to study the effects of disease and cancer progression on transport of proteins and immunotherapies in the immune system.

Transport analysis of cytokines in live lymph node tissue on-chip

254th National Meeting (2017)

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