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S. M. Pantanelli, Z. Li, B. Liu, S. Yeh, J. Lew, R. Nussenblatt; Single Cell Cytokine Profiling of Non-Infectious Uveitis Patients and Normal Controls Using a Multi-Color Intracellular Staining Strategy. Invest. Ophthalmol. Vis. Sci. 2008;49(13):814.
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To investigate the potential applications of single-cell cytokine profiling for understanding molecular mechanism of autoimmune uveitis.
Blood from 32 patients with diagnoses of non-infectious uveitis and 24 healthy donors was used for cytokine profiling. Peripheral blood mononuclear cells were isolated from whole blood and stimulated by PMA and ionomycin. A 3-color staining scheme for surface expression of CD3, intracellular interferon gamma, and simultaneous staining for either intracellular IL-2, 4, 5, 10, 12, IL-17 or TNF-alpha was used for cytokine profiling. The staining of intracellular cytokines was enhanced by concomitant blocking of Golgi transporter. After acquiring the data with a flow cytometer, CD3+ T-cells were examined for expression of the cytokines described above. Th1, Th2, and Th17 cytokine expression profiles were plotted and analyzed to identify differential expressions between patients with non-infectious uveitis and normal controls.
Interferon gamma, TNF-alpha and IL-2 were among the cytokines expressed most strongly in both uveitis patients and normal controls, but IL-4, 5, 10, 12, and 17 were all detectable at the single-cell level. The 3-color scheme allowed separation of CD3+ T-cells and CD3- non-T cells. Addition of the 2 simultaneous cytokine stains (FITC for interferon gamma and PE for others) permitted identification of single positive and double positive cytokine-expressing cells. This strategy revealed a sub-population of CD3+IFNg+IL17+ cells in both normal donors and uveitis patients. There were also CD3+IFNg+IL-2+, CD3+IFNg+TNF-alpha+ and CD3+IFNg+IL-10+ sub-populations. Initial analysis revealed tremendous variation of cytokine expression within uveitis patients; variation within the normal donor population was less, suggesting a potential association between cytokine expression and uveitic disease.
Multi-color intracellular cytokine profiling provided a powerful way to analyze cytokine expression at the single-cell level for studying autoimmune uveitis. Initial results support the feasibility and high-throughput nature of this approach. Preliminary data revealed unique patterns of certain cytokine expressing sub-populations in human peripheral leukocytes. Broader application of cytokine profiling and more robust database mining strategies may be needed to further our understanding of this analytical strategy.
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