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Factors affecting the cytokine production of human T cells stimulated by different modes of activation  Yoshio Harada, MDa, Sumiko Watanabe, PhDa, Hans.

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Presentation on theme: "Factors affecting the cytokine production of human T cells stimulated by different modes of activation  Yoshio Harada, MDa, Sumiko Watanabe, PhDa, Hans."— Presentation transcript:

1 Factors affecting the cytokine production of human T cells stimulated by different modes of activation  Yoshio Harada, MDa, Sumiko Watanabe, PhDa, Hans Yssel, PhDb, Ken-ichi Arai, MD, PhDa  Journal of Allergy and Clinical Immunology  Volume 98, Issue 6, Pages S161-S173 (December 1996) DOI: /S (96) Copyright © 1996 Mosby, Inc. Terms and Conditions

2 FIG. 1 Profile of cytokine production of human T-cell clones SP-B21 (TH0-like) and TA20 and TA23 (TH1-like). Cells (2 × 105) were stimulated with (A) αCD3-mAb (10 μg/ml, coated on 96-well plate) or (B) PMA (1 ng/ml) and A23187 (500 ng/ml). After 48 hours of incubation at 37° C, supernatants were collected, and levels of cytokines were determined by cytokine-specific ELISA. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

3 FIG. 1 Profile of cytokine production of human T-cell clones SP-B21 (TH0-like) and TA20 and TA23 (TH1-like). Cells (2 × 105) were stimulated with (A) αCD3-mAb (10 μg/ml, coated on 96-well plate) or (B) PMA (1 ng/ml) and A23187 (500 ng/ml). After 48 hours of incubation at 37° C, supernatants were collected, and levels of cytokines were determined by cytokine-specific ELISA. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

4 FIG. 2 Effects of herbimycin on cytokine production of human T-cell clone SP-B21. Cells (2 × 105) were stimulated with soluble αCD3-mAb (1 μg/ml) or PMA (1 ng/ml)/A23187 (500 ng/ml) in presence of various concentrations of herbimycin. As described in legend to Fig. 1, levels of IFN-γ (A), IL-4 (B), and IL-5 (C) were measured by cytokine-specific ELISA. Results are expressed as percentage of control to facilitate comparison. Control levels of cytokine production in response to αCD3-mAb and PMA/A23187, respectively, were as follows: IFN-γ, 1.7 and 45.9 ng/ml; IL-4, 0.31 and 4.0 ng/ml; IL-5, 3.9 and 8.7 ng/ml. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

5 FIG. 2 Effects of herbimycin on cytokine production of human T-cell clone SP-B21. Cells (2 × 105) were stimulated with soluble αCD3-mAb (1 μg/ml) or PMA (1 ng/ml)/A23187 (500 ng/ml) in presence of various concentrations of herbimycin. As described in legend to Fig. 1, levels of IFN-γ (A), IL-4 (B), and IL-5 (C) were measured by cytokine-specific ELISA. Results are expressed as percentage of control to facilitate comparison. Control levels of cytokine production in response to αCD3-mAb and PMA/A23187, respectively, were as follows: IFN-γ, 1.7 and 45.9 ng/ml; IL-4, 0.31 and 4.0 ng/ml; IL-5, 3.9 and 8.7 ng/ml. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

6 FIG. 2 Effects of herbimycin on cytokine production of human T-cell clone SP-B21. Cells (2 × 105) were stimulated with soluble αCD3-mAb (1 μg/ml) or PMA (1 ng/ml)/A23187 (500 ng/ml) in presence of various concentrations of herbimycin. As described in legend to Fig. 1, levels of IFN-γ (A), IL-4 (B), and IL-5 (C) were measured by cytokine-specific ELISA. Results are expressed as percentage of control to facilitate comparison. Control levels of cytokine production in response to αCD3-mAb and PMA/A23187, respectively, were as follows: IFN-γ, 1.7 and 45.9 ng/ml; IL-4, 0.31 and 4.0 ng/ml; IL-5, 3.9 and 8.7 ng/ml. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

7 FIG. 3 Effects of CsA on cytokine production of SP-B21. Cells (2 × 105) were stimulated with αCD3-mAb (1 μg/ml) (A) or PMA (1 ng/ml)/αCD3-mAb (1 μg/ml) (B) in presence of various concentrations of CsA. As described in legend to Fig. 1, levels of IFN-γ, IL-4, and IL-5 were measured by cytokine-specific ELISA. Results are expressed as percentage of control to facilitate comparison. Control levels of cytokine production in response to αCD3-mAb and PMA/A23187, respectively, were as follows: IFN-γ, 1.7 and 19.7 ng/ml; IL-4, 0.31 and 5.2 ng/ml; IL-5, 3.9 and 9.3 ng/ml. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

8 FIG. 3 Effects of CsA on cytokine production of SP-B21. Cells (2 × 105) were stimulated with αCD3-mAb (1 μg/ml) (A) or PMA (1 ng/ml)/αCD3-mAb (1 μg/ml) (B) in presence of various concentrations of CsA. As described in legend to Fig. 1, levels of IFN-γ, IL-4, and IL-5 were measured by cytokine-specific ELISA. Results are expressed as percentage of control to facilitate comparison. Control levels of cytokine production in response to αCD3-mAb and PMA/A23187, respectively, were as follows: IFN-γ, 1.7 and 19.7 ng/ml; IL-4, 0.31 and 5.2 ng/ml; IL-5, 3.9 and 9.3 ng/ml. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

9 FIG. 4 Effects of A23187 on cytokine production of SP-B21. Cells (2 × 105) were stimulated with αCD3-mAb (1 μg/ml), PMA (1 ng/ml), or αCD3-mAb (1 μg/ml)/PMA (1 ng/ml) in presence of various concentrations of A As described in legend to Fig. 1, levels of IL-10 (A), IFN-γ (B), and IL-4 (C) were measured by cytokine-specific ELISA. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

10 FIG. 4 Effects of A23187 on cytokine production of SP-B21. Cells (2 × 105) were stimulated with αCD3-mAb (1 μg/ml), PMA (1 ng/ml), or αCD3-mAb (1 μg/ml)/PMA (1 ng/ml) in presence of various concentrations of A As described in legend to Fig. 1, levels of IL-10 (A), IFN-γ (B), and IL-4 (C) were measured by cytokine-specific ELISA. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

11 FIG. 4 Effects of A23187 on cytokine production of SP-B21. Cells (2 × 105) were stimulated with αCD3-mAb (1 μg/ml), PMA (1 ng/ml), or αCD3-mAb (1 μg/ml)/PMA (1 ng/ml) in presence of various concentrations of A As described in legend to Fig. 1, levels of IL-10 (A), IFN-γ (B), and IL-4 (C) were measured by cytokine-specific ELISA. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

12 FIG. 5 Effects of αIL-2Rα mAb (A) or TGF-β (B) on proliferation of SP-B21. Cells (2 × 104) were stimulated with soluble αCD3-mAb (1 μg/ml), PMA (1 ng/ml), or PMA (1 ng/ml)/A23187 (500 ng/ml) in presence or absence of αIL-2Rα mAb or TGF-β. [3H]Thymidine incorporation was measured from triplicate samples. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

13 FIG. 5 Effects of αIL-2Rα mAb (A) or TGF-β (B) on proliferation of SP-B21. Cells (2 × 104) were stimulated with soluble αCD3-mAb (1 μg/ml), PMA (1 ng/ml), or PMA (1 ng/ml)/A23187 (500 ng/ml) in presence or absence of αIL-2Rα mAb or TGF-β. [3H]Thymidine incorporation was measured from triplicate samples. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

14 FIG. 6 Effects of PGE2 on cytokine production and proliferation of SP-B21. Cells (2 × 105) were stimulated with αCD3-mAb (1 μg/ml) or PMA (1 ng/ml)/A23187 (500 ng/ml) and with or without PGE2 (10 μmol/L). Total RNA was isolated after 4 hours of incubation with or without PGE2. Cytokine mRNA was analyzed with reverse-transcription PCR. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

15 FIG. 7 Effects of dBcAMP on cytokine production of TH0-like (SP-B21) and TH1-like (TA20 and TA23) human T-cell clones. Cells (2 × 105) were stimulated with αCD3-mAb (1 μg/ml) (A and C) or PMA (1 ng/ml)/A23187 (500 ng/ml) (B and D) and with or without dBcAMP (1 mmol/L). As described in legend to Fig. 1, levels of IFN-γ (A and B) and IL-4 (C and D) were measured by cytokine-specific ELISA. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

16 FIG. 7 Effects of dBcAMP on cytokine production of TH0-like (SP-B21) and TH1-like (TA20 and TA23) human T-cell clones. Cells (2 × 105) were stimulated with αCD3-mAb (1 μg/ml) (A and C) or PMA (1 ng/ml)/A23187 (500 ng/ml) (B and D) and with or without dBcAMP (1 mmol/L). As described in legend to Fig. 1, levels of IFN-γ (A and B) and IL-4 (C and D) were measured by cytokine-specific ELISA. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

17 FIG. 7 Effects of dBcAMP on cytokine production of TH0-like (SP-B21) and TH1-like (TA20 and TA23) human T-cell clones. Cells (2 × 105) were stimulated with αCD3-mAb (1 μg/ml) (A and C) or PMA (1 ng/ml)/A23187 (500 ng/ml) (B and D) and with or without dBcAMP (1 mmol/L). As described in legend to Fig. 1, levels of IFN-γ (A and B) and IL-4 (C and D) were measured by cytokine-specific ELISA. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

18 FIG. 7 Effects of dBcAMP on cytokine production of TH0-like (SP-B21) and TH1-like (TA20 and TA23) human T-cell clones. Cells (2 × 105) were stimulated with αCD3-mAb (1 μg/ml) (A and C) or PMA (1 ng/ml)/A23187 (500 ng/ml) (B and D) and with or without dBcAMP (1 mmol/L). As described in legend to Fig. 1, levels of IFN-γ (A and B) and IL-4 (C and D) were measured by cytokine-specific ELISA. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

19 FIG. 8 Electrophoretic mobility shift assay of NF-κB complex. SP-B21 cells (5 × 106) were stimulated with αCD3-mAb (1 μg/ml) or PMA (1 ng/ml)/A23187 (500 ng/ml) and with or without (A) PGE2 (10 μmol/L) or (B) dBcAMP (1 mmol/L). At 4 hours after stimulation, nuclear extracts prepared from these cells were incubated with IgκB oligonucleotide labeled with [α-32P]dGTP at room temperature for 15 minutes, and DNA–protein complexes were electrophoresed at 100 V on 4% polyacrylamide gel. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

20 FIG. 8 Electrophoretic mobility shift assay of NF-κB complex. SP-B21 cells (5 × 106) were stimulated with αCD3-mAb (1 μg/ml) or PMA (1 ng/ml)/A23187 (500 ng/ml) and with or without (A) PGE2 (10 μmol/L) or (B) dBcAMP (1 mmol/L). At 4 hours after stimulation, nuclear extracts prepared from these cells were incubated with IgκB oligonucleotide labeled with [α-32P]dGTP at room temperature for 15 minutes, and DNA–protein complexes were electrophoresed at 100 V on 4% polyacrylamide gel. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

21 FIG. 9 Electrophoretic mobility shift assay of complex bound to NF-AT motif. SP-B21 cells (5 × 106) were stimulated with αCD3-mAb (1 μg/ml) and with or without PGE2 (10 μmol/L). Cells were harvested 4 hours after stimulation, and nuclear extracts were prepared and electrophoretic mobility shift assays performed as described in legend to Fig. 8. Journal of Allergy and Clinical Immunology  , S161-S173DOI: ( /S (96) ) Copyright © 1996 Mosby, Inc. Terms and Conditions

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