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Cytokine Flow Cytometry: Understanding Cytokine Biology at the Single-Cell Level

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Abstract

In the past 4 years, cytokine flow cytometry has emerged as the premier technique for enumeration of cytokine producing T cells. The multiparameter capability of flow cytometry permits the simultaneous detection of two or more cytokines within a single cell, allowing true Th1 vs. Th2 determination. The high throughput inherent to flow cytometry has enormous advantages when applied to clinical research questions previously not amenable for study using labor intensive techniques such as ELISPOT, limiting dilution and T cell cloning. Furthermore, cytokine flow cytometry allows the study of individual T cells directly ex vivo, minimizing artifacts due to long term culture. As such, cytokine flow yields unique insights into cytokine biology heretofore not possible. We have used cytokine flow cytometry to examine coexpression of cytokines within the memory/effector CD4+, CD27− subset. Doing so, we have found distinct cytokine producing subsets that correlate with the previously described Th1, Th2 and Th0 subsets. The majority of cytokine producing cells were of these first two subsets with fewer cells coexpressing IFN-γ and IL-4. These results validate the Th1/Th2 hypothesis and demonstrate specific subsets of cytokine producing T cells in fresh ex vivo human T cells.

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REFERENCES

  1. Paul WE, Seder RA: Lymphocyte responses and cytokines. Cell 76(2):241-251, 1994

    Google Scholar 

  2. Mosmann TR, Coffman RL: TH1 and TH2 cells: Different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7:145-173, 1989

    Google Scholar 

  3. Muller W, Sinigaglia F: Single cell analysis: Sharpening the view through the magnifying glass. Curr Opin Immunol 7:255-257, 1995

    Google Scholar 

  4. Jung T, Schauer U, Heusser C, Neumann C, Rieger C: Detection of intracellular cytokines by flow cytometry. J Immunol Methods 159(1–2):197-207, 1993

    Google Scholar 

  5. Picker LJ, Singh MK, Zdraveski Z, Treer JR, Waldrop SL, Bergstresser PR, Maino VC: Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry. Blood 86(4):1408-1419, 1995

    Google Scholar 

  6. Prussin C, Metcalfe DD: Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J Immunol Methods 188(1):117-128, 1995

    Google Scholar 

  7. Assenmacher M, Schmitz J, Radbruch A: Flow cytometric determination of cytokines in activated murine T helper lymphocytes: Expression of interleukin-10 in interferon-gamma and in interleukin-4-expressing cells. Eur J Immunol 24(5):1097-1101, 1994

    Google Scholar 

  8. Openshaw P, Murphy EE, Hosken NA, Maino V, Davis K, Murphy K, O'Garra A: Heterogeneity of intracellular cytokine synthesis at the single-cell level in polarized T helper I and T helper 2 populations. J Exp Med 182(5):1357-1367, 1995

    Google Scholar 

  9. Sander B, Andersson J, Andersson U: Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol Rev 119:65-93, 1991

    Google Scholar 

  10. Tartakoff AM: Perturbation of vesicular traffic with the carboxylic ionophore monensin. Cell 32(4):1026-1028, 1983

    Google Scholar 

  11. Manz R, Assenmacher M, Pfluger E, Miltenyi S, Radbruch A: Analysis and sorting of live cells according to secreted molecules, relocated to a cell-surface affinity matrix. Proc Natl Acad Sci USA 92(6):1921-1925, 1995

    Google Scholar 

  12. Gray F, Kenney JS, Dunne JF: Secretion capture and report web: Use of affinity derivatized agarose microdroplets for the selection of hybridoma cells. J Immunol Methods 182(2):155-163, 1995

    Google Scholar 

  13. Assenmacher M, Scheffold A, Schmitz J, Segura Checa JA, Miltenyi S, Radbruch A: Specific expression of surface interferon-gamma on interferon-gamma producing T cells from mouse and man. Eur J Immunol 26(1):263-267, 1996

    Google Scholar 

  14. Schauer U, Jung T, Krug N, Frew A: Measurement of intracellular cytokines. Immunol Today 17:305-306, 1996

    Google Scholar 

  15. Bauer KD, Jacobberger JW: Analysis of intracellular proteins. Methods Cell Biol 41:351-376, 1994

    Google Scholar 

  16. Larsson L: Antibody specificity in immunochemistry. In Immunochemistry II, A Cuello (ed). Chichester, John Wiley, 1993, p 79

    Google Scholar 

  17. Elson LH, Nutman TB, Metcalfe DD, Prussin C: Flow cytometric analysis for cytokine production identifies T helper 1, T helper 2, and T helper 0 cells within the human CD4+CD27− lymphocyte subpopulation. J Immunol 154(9):4294-4301, 1995

    Google Scholar 

  18. Seder R, Prussin C: Are differential human T helper cells reversible? Int Archives Aller Immunol, 1997 (in press)

  19. Jung T, Schauer U, Rieger C, Wagner K, Einsle K, Neumann C, Heusser C: Interleukin-4 and interleukin-5 are rarely co-expressed by human T cells. Eur J Immunol 25(8):2413-2416, 1995

    Google Scholar 

  20. Sornasse T, Larenas PV, Davis KA, de Vries JE, Yssel H: Differentiation and stability of T helper 1 and 2 cells derived from naive human neonatal CD4+ T cells, analyzed at the single-cell level. J Exp Med 184(2):473-483, 1996

    Google Scholar 

  21. Murphy E, Shibuya K, Hosken N, Openshaw P, Maino V, Davis K, Murphy K, O'Garra A: Reversibility of T helper 1 and 2 populations is lost after long-term stimulation. J Exp Med 183(3):901-913, 1996

    Google Scholar 

  22. Krug N, Madden J, Redington AE, Lackie P, Djukanovic R, Schauer U, Holgate ST, Frew AJ, Howarth PH: T-cell cytokine profile evaluated at the single cell level in BAL and blood in allergic asthma [see comments]. Am J Respir Cell Mol Biol 14(4):319-326, 1996

    Google Scholar 

  23. Bendelac A: Mouse CD1-specific NK1 T cells. Development, specificity and function. Annu Rev Immunol 15 (in press), 1997

  24. Prussin C, Foster B: Vα244, Vβ111 natural T cells are the human analog of murine NK1.1 T cells and demonstrate a predominant Th1 phenotype. J Aller Clin Immunol 99(1):1049, 1997

    Google Scholar 

  25. Jung T, Lack G, Schauer U, Uberuck W, Renz H, Gelfand EW, Rieger CH: Decreased frequency of interferon-gamma-and interleukin-2-producing cells in patients with atopic diseases measured at the single cell level. J Allergy Clin Immunol 96(4):515-527, 1995

    Google Scholar 

  26. Nasert S, Burtchen N, Kussebi F, Millner M, Kroczek R, Jung T, Schwinzer R, Wahn U, Renz H: Stimulation of IgE and IgA production by CD45RA T helper cells in atopic patients. J Immunol 157(1):441-448, 1996

    Google Scholar 

  27. Elson LH, Shaw S, Van Lier RA, Nutman TB: T cell subpopulation phenotypes in filarial infections: CD27 negativity defines a population greatly enriched for Th2 cells. Int Immunol 6(7):1003-1009, 1994

    Google Scholar 

  28. De Jong R, Brouwer M, Hooibrink B, Van der Pouw-Kraan T, Miedema F, Van Lier RA: The CD27− subset of peripheral blood memory CD4+ lymphocytes contains functionally differentiated T lymphocytes that develop by persistent antigenic stimulation in vivo. Eur J Immunol 22(4):993-999, 1992

    Google Scholar 

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Prussin, C. Cytokine Flow Cytometry: Understanding Cytokine Biology at the Single-Cell Level. J Clin Immunol 17, 195–204 (1997). https://doi.org/10.1023/A:1027350226435

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