Cell surface characterization of T lymphocytes and allergen-specific T cell clones: Correlation of CD26 expression with T H1 subsets Martin Willheim,

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Cell surface characterization of T lymphocytes and allergen-specific T cell clones: Correlation of CD26 expression with T H1 subsets Martin Willheim, MDa, Christof Ebner, MDa, Karin Baier a, Wolfgang Kern, MSca, Karl Schrattbauer, MDa, Ralf Theina, Dietrich Kraft, MDa, Heimo Breiteneder, PhDa, Walter Reinisch, MDb, Otto Scheiner, PhDa Journal of Allergy and Clinical Immunology Volume 100, Issue 3, Pages 348-355 (September 1997) DOI: 10.1016/S0091-6749(97)70248-3 Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 1 Established Bet v 1-specific TCCs (n = 16) were classified as T H1-, T H0-, or T H2-like as described in the Methods section. The clones were stimulated with rBet v 1 for 24 hours and subsequently stained for CD26. Data are presented as mean channel fluorescence. Expression of CD26 was significantly higher in T H1- than in T H2-like clones (p = 0.009), whereas no significant difference was found between T H1:T H0 ( p = 0.414) and T H0:T H2 ( p = 0.070). Journal of Allergy and Clinical Immunology 1997 100, 348-355DOI: (10.1016/S0091-6749(97)70248-3) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 2 Correlation between cytokine production and CD26 expression in Bet v 1-specific TCCs ( n = 36). TCCs were stimulated for 24 hours with PHA and subsequently analyzed for CD26 expression. IL-4 and IFN-γ in the culture supernatants were determined by ELISA. Ratio of IL-4/IFN-γ production and CD26 expression (left) correlated significantly ( p = 0.0122), as did as IL-4 production and CD26 expression (p = 0.0002). No correlation exists between IFN-γ production and CD26 expression. Journal of Allergy and Clinical Immunology 1997 100, 348-355DOI: (10.1016/S0091-6749(97)70248-3) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 2 Correlation between cytokine production and CD26 expression in Bet v 1-specific TCCs ( n = 36). TCCs were stimulated for 24 hours with PHA and subsequently analyzed for CD26 expression. IL-4 and IFN-γ in the culture supernatants were determined by ELISA. Ratio of IL-4/IFN-γ production and CD26 expression (left) correlated significantly ( p = 0.0122), as did as IL-4 production and CD26 expression (p = 0.0002). No correlation exists between IFN-γ production and CD26 expression. Journal of Allergy and Clinical Immunology 1997 100, 348-355DOI: (10.1016/S0091-6749(97)70248-3) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 2 Correlation between cytokine production and CD26 expression in Bet v 1-specific TCCs ( n = 36). TCCs were stimulated for 24 hours with PHA and subsequently analyzed for CD26 expression. IL-4 and IFN-γ in the culture supernatants were determined by ELISA. Ratio of IL-4/IFN-γ production and CD26 expression (left) correlated significantly ( p = 0.0122), as did as IL-4 production and CD26 expression (p = 0.0002). No correlation exists between IFN-γ production and CD26 expression. Journal of Allergy and Clinical Immunology 1997 100, 348-355DOI: (10.1016/S0091-6749(97)70248-3) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 3 CD26 expression in CD4 + lymphocytes producing IFN-γ or IL-4. PBMCs were stimulated for 4 hours with PMA and ionomycin in the presence of monensin, and subsequently stained for CD4, CD26, and the respective cytokine. Cells were gated as CD4 + lymphocytes. Upper panels show dot plot representations of cells stained for IFN-γ and CD26 (left), or IL-4 and CD26 (right). Cells were then gated as positive or negative for the respective cytokine. Overlay histograms for CD26 expression of those populations are shown in the lower panels. Journal of Allergy and Clinical Immunology 1997 100, 348-355DOI: (10.1016/S0091-6749(97)70248-3) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 4 Influence of cytokines on CD25 and CD26 expression on CD4+ T cells at different times. PHA-stimulated PBMCs from healthy and atopic donors were cultured for up to 14 days with or without the indicated cytokines. Data for IL-12 (100 U/ml, n = 8) are presented in the upper graphs, for IFN-γ (1000 U/ml, n = 6) in the middle graphs, and for IL-4 (0.5 ng/ml, n = 6) in the lower graphs. The statistical significance of the differences between control and cytokine-stimulated cultures is presented at the different time points. The asterisk indicates that the significant difference in CD25 expression was inverse to the difference in CD26 expression. Journal of Allergy and Clinical Immunology 1997 100, 348-355DOI: (10.1016/S0091-6749(97)70248-3) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 4 Influence of cytokines on CD25 and CD26 expression on CD4+ T cells at different times. PHA-stimulated PBMCs from healthy and atopic donors were cultured for up to 14 days with or without the indicated cytokines. Data for IL-12 (100 U/ml, n = 8) are presented in the upper graphs, for IFN-γ (1000 U/ml, n = 6) in the middle graphs, and for IL-4 (0.5 ng/ml, n = 6) in the lower graphs. The statistical significance of the differences between control and cytokine-stimulated cultures is presented at the different time points. The asterisk indicates that the significant difference in CD25 expression was inverse to the difference in CD26 expression. Journal of Allergy and Clinical Immunology 1997 100, 348-355DOI: (10.1016/S0091-6749(97)70248-3) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 4 Influence of cytokines on CD25 and CD26 expression on CD4+ T cells at different times. PHA-stimulated PBMCs from healthy and atopic donors were cultured for up to 14 days with or without the indicated cytokines. Data for IL-12 (100 U/ml, n = 8) are presented in the upper graphs, for IFN-γ (1000 U/ml, n = 6) in the middle graphs, and for IL-4 (0.5 ng/ml, n = 6) in the lower graphs. The statistical significance of the differences between control and cytokine-stimulated cultures is presented at the different time points. The asterisk indicates that the significant difference in CD25 expression was inverse to the difference in CD26 expression. Journal of Allergy and Clinical Immunology 1997 100, 348-355DOI: (10.1016/S0091-6749(97)70248-3) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 5 Cytokine production of CD4 + T cells on day 7. PHA-stimulated PBMCs from healthy ( n = 3) and atopic (n = 3) donors were cultured for 7 days with or without the indicated cytokines. After restimulation with PMA and ionomycin in the presence of monensin, percentages of cytokine cells were evaluated by intracellular cytokine detection. Dot plots from one representative experiment are shown. Mean (±SD) values from six experiments are indicated in the respective quadrants. Cells were gated as CD4 +lymphocytes. In each dot plot populations of cells only producing IL-4 (and not IFN-γ) are presented in the upper left quadrant, and cells producing only IFN-γ (and not IL-4) are shown in the lower right quadrant. Populations producing both cytokines are presented in the upper right quadrants. Journal of Allergy and Clinical Immunology 1997 100, 348-355DOI: (10.1016/S0091-6749(97)70248-3) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 5 Cytokine production of CD4 + T cells on day 7. PHA-stimulated PBMCs from healthy ( n = 3) and atopic (n = 3) donors were cultured for 7 days with or without the indicated cytokines. After restimulation with PMA and ionomycin in the presence of monensin, percentages of cytokine cells were evaluated by intracellular cytokine detection. Dot plots from one representative experiment are shown. Mean (±SD) values from six experiments are indicated in the respective quadrants. Cells were gated as CD4 +lymphocytes. In each dot plot populations of cells only producing IL-4 (and not IFN-γ) are presented in the upper left quadrant, and cells producing only IFN-γ (and not IL-4) are shown in the lower right quadrant. Populations producing both cytokines are presented in the upper right quadrants. Journal of Allergy and Clinical Immunology 1997 100, 348-355DOI: (10.1016/S0091-6749(97)70248-3) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 5 Cytokine production of CD4 + T cells on day 7. PHA-stimulated PBMCs from healthy ( n = 3) and atopic (n = 3) donors were cultured for 7 days with or without the indicated cytokines. After restimulation with PMA and ionomycin in the presence of monensin, percentages of cytokine cells were evaluated by intracellular cytokine detection. Dot plots from one representative experiment are shown. Mean (±SD) values from six experiments are indicated in the respective quadrants. Cells were gated as CD4 +lymphocytes. In each dot plot populations of cells only producing IL-4 (and not IFN-γ) are presented in the upper left quadrant, and cells producing only IFN-γ (and not IL-4) are shown in the lower right quadrant. Populations producing both cytokines are presented in the upper right quadrants. Journal of Allergy and Clinical Immunology 1997 100, 348-355DOI: (10.1016/S0091-6749(97)70248-3) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 5 Cytokine production of CD4 + T cells on day 7. PHA-stimulated PBMCs from healthy ( n = 3) and atopic (n = 3) donors were cultured for 7 days with or without the indicated cytokines. After restimulation with PMA and ionomycin in the presence of monensin, percentages of cytokine cells were evaluated by intracellular cytokine detection. Dot plots from one representative experiment are shown. Mean (±SD) values from six experiments are indicated in the respective quadrants. Cells were gated as CD4 +lymphocytes. In each dot plot populations of cells only producing IL-4 (and not IFN-γ) are presented in the upper left quadrant, and cells producing only IFN-γ (and not IL-4) are shown in the lower right quadrant. Populations producing both cytokines are presented in the upper right quadrants. Journal of Allergy and Clinical Immunology 1997 100, 348-355DOI: (10.1016/S0091-6749(97)70248-3) Copyright © 1997 Mosby, Inc. Terms and Conditions