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Flow cytometric measurement of STAT1 and STAT3 phosphorylation in CD4+ and CD8+ T cells—clinical applications in primary immunodeficiency diagnostics 

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Presentation on theme: "Flow cytometric measurement of STAT1 and STAT3 phosphorylation in CD4+ and CD8+ T cells—clinical applications in primary immunodeficiency diagnostics "— Presentation transcript:

1 Flow cytometric measurement of STAT1 and STAT3 phosphorylation in CD4+ and CD8+ T cells—clinical applications in primary immunodeficiency diagnostics  Michael Bitar, MSc, Andreas Boldt, PhD, Stefanie Binder, PhD, Michael Borte, MD, Karim Kentouche, MD, Stephan Borte, MD, PhD, Ulrich Sack, MD  Journal of Allergy and Clinical Immunology  Volume 140, Issue 5, Pages e9 (November 2017) DOI: /j.jaci Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Summary of pSTAT1 and pSTAT3 expression analysis by flow cytometry (n = 21). A, Percentage of cells positive for pSTAT1 expression. B, MFI of cells positive for pSTAT1 expression. C, Percentage of cells positive for pSTAT3 expression. D, MFI of cells positive for pSTAT3 expression. The bold line inside each box plot shows the median level, and upper and lower lines indicate the maximum and minimum values, respectively. (***P value = .001). stim, Stimulated; w/o, without stimulation. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Detection of pSTAT1 and pSTAT3 by flow cytometry in 4 patients with PID in comparison with healthy controls. Human peripheral blood was either left unstimulated (gray histogram) or stimulated for 15 minutes as indicated (black histogram). Percentages and MFI of cells positive for pSTAT1 and pSTAT3 are detailed within the histogram boxes. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 Flow cytometric gating strategy to exclude dead cells and doublets. PBMCs were stimulated with IFN-α for STAT1 phosphorylation, with IL-6 for STAT3 phosphorylation, or left unstimulated. Following staining with FVS450 according to manufacturer's specifications (BD Biosciences), live and dead cells can be discriminated by measurement of the fluorescence intensity. Dead cells have 10- to 20-fold higher fluorescence intensity than do live/intact cells (live gate—first left panel). Doublets were excluded on the basis of FSC area (FSC-A) versus FSC height (FSC-H) (singlets gate—second left picture). Additional staining for CD3+, CD4+, and CD8+ cells helps to identify the T-cell subsets (third left picture and right picture). FSC, Forward scatter. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 Detection of pSTAT3 in CD4+, CD8+ subsets by FCM and WB. A, Phosphorylation analysis of STAT3 by FCM; human peripheral blood was either left without stimulation (gray histogram) or stimulated with IL-6 (black histogram). STAT3 phosphorylation is presented as both percentage and the MFI of cells positive for pSTAT3. B, WB analysis, pSTAT3 bands (upper line at 92 KDa), β-actin blotting (bottom). stim, Stimulated; w/o, without stimulation. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 Evaluation of pSTAT3 in CD8+ CD45RA+ as naive cells and CD8+ CD45RA− as memory cells. After IL-6 stimulation, an efficient expression of pSTAT3 within CD8+ CD45RA+ naive cells was increased distinctly rather than memory cells CD45RA−. Percentages of naive and memory cells that were involved in phosphorylation of STAT3 are given within the histogram boxes. stim, Stimulated; w/o, without stimulation. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E4 Detection of pSTAT1 in CD4+, CD8+ subsets by FCM and WB. A, Phosphorylation analysis of STAT1 by FCM; human peripheral blood was either left without stimulation (gray histogram) or stimulated with IFN-α (black histogram). STAT1 phosphorylation is presented as both percentage and the MFI of cells positive for pSTAT1. B, WB analysis, pSTAT1 bands (upper line at 92 KDa), β-actin blotting (bottom). stim, Stimulated; w/o, without stimulation. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E5 Time-dependent STAT1 phosphorylation and its inhibition in a patient with GOF-STAT1 mutation compared with healthy control. PBMCs were isolated and stimulated with IFN-α for 15, 30, 60, and 90 minutes. After reaching the maximum of pSTAT1-MFI at 15 minutes, the phosphorylation decreased to a normal level in healthy control and in the patient, in both CD4+ (A) and CD8+ (C) T cells. Interestingly, the maximum pSTAT1-MFI was multiple higher in the GOF-STAT1 patient compared with healthy control (Fig E5, A and C). Furthermore, PBMCs were stimulated with IFN-α and after 15 minutes treated with staurosporine (B and D). Staurosporine treatment inhibited strongly the STAT1 phosphorylation in healthy control in contrast to the patient with GOF-STAT1 mutation (Fig E5, B and D). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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