May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Single Cell Cytokine Profiling of Non-Infectious Uveitis Patients and Normal Controls Using a Multi-Color Intracellular Staining Strategy
Author Affiliations & Notes
  • S. M. Pantanelli
    Univ. of Rochester School of Medicine, Rochester, New York
    Laboratory of Immunology, National Eye Institute, Bethesda, Maryland
  • Z. Li
    Laboratory of Immunology, National Eye Institute, Bethesda, Maryland
  • B. Liu
    Laboratory of Immunology, National Eye Institute, Bethesda, Maryland
  • S. Yeh
    Laboratory of Immunology, National Eye Institute, Bethesda, Maryland
  • J. Lew
    Laboratory of Immunology, National Eye Institute, Bethesda, Maryland
  • R. Nussenblatt
    Laboratory of Immunology, National Eye Institute, Bethesda, Maryland
  • Footnotes
    Commercial Relationships  S.M. Pantanelli, None; Z. Li, None; B. Liu, None; S. Yeh, None; J. Lew, None; R. Nussenblatt, None.
  • Footnotes
    Support  NIH Intramural Research Program
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 814. doi:https://doi.org/
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    • Get Citation

      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. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : To investigate the potential applications of single-cell cytokine profiling for understanding molecular mechanism of autoimmune uveitis.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: inflammation • cytokines/chemokines • autoimmune disease 
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