Multiparametric Flow Cytometric Analysis of Inter-Patient Variation in STAT1 Phosphorylation Following Interferon Alfa Immunotherapy (original) (raw)

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Correspondence to: William E. Carson III, MD, Division of Surgical Oncology, The Ohio State University, N924 Doan Hall, 410 W. 10th Ave., Columbus, OH 43210 (e-mail: carson-1@medctr.osu.edu )

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Received:

03 November 2003

Revision received:

28 June 2004

Published:

01 September 2004

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Gregory B. Lesinski, Sri Vidya Kondadasula, Tim Crespin, Lei Shen, Kari Kendra, Michael Walker, William E. Carson, Multiparametric Flow Cytometric Analysis of Inter-Patient Variation in STAT1 Phosphorylation Following Interferon Alfa Immunotherapy, JNCI: Journal of the National Cancer Institute, Volume 96, Issue 17, 1 September 2004, Pages 1331–1342, https://doi.org/10.1093/jnci/djh252
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Abstract

Background: Regulation of gene expression by signal transducer and activator of transcription 1 (STAT1) within host tissues mediates the antitumor effects of interferon alfa (IFN α). We used a novel flow cytometric assay to examine phosphorylation-mediated activation of STAT1 within immune effector cell subsets following in vitro or in vivo IFN α treatments. Methods: Peripheral blood mononuclear cells (PBMCs) isolated from healthy donors (n = 17) or melanoma patients (n = 19) were treated in vitro with interferon alfa-2b (IFN α-2b) or phosphate-buffered saline (PBS) and subjected to multiparametric flow cytometry to measure the levels of phosphorylated STAT1 (P-STAT1) within immune cell subsets. We similarly analyzed PBMCs isolated from melanoma patients before and 1 hour after immunotherapy with IFN α-2b. All statistical tests were two-sided. Results: P-STAT1 levels in all major immune cell subsets increased within 15 minutes of in vitro IFN α-2b treatment of PBMCs; the increase was most pronounced in T lymphocytes and monocytes. Relatively low doses of IFN α-2b (i.e., 10 2 –10 3 IU/mL) induced maximal STAT1 activation in vitro . Compared with melanoma patients, healthy donors had higher basal levels of P-STAT1 (specific fluorescence [Fsp]; i.e., Fsp PBS , the level of P-STAT1 in PBS-treated cells) in total PBMCs, natural killer (NK) cells, and T cells (mean Fsp PBS in total PBMCs: 5.5 in healthy donors versus 1.6 in patients, difference = 3.9, 95% confidence interval [CI] = 1.4 to 6.5, P = .004; mean Fsp PBS in NK cells: 4.6 in healthy donors versus 0.9 in patients, difference = 3.7, 95% CI = 1.7 to 5.7, P = .001; mean Fsp PBS in T cells: 6.8 in healthy donors versus 0.9 in patients, difference = 5.9, 95% CI = 2.5 to 9.3, P = .002). P-STAT1 was detected in the NK and T cells of two patients who received IFN α-2b immunotherapy (20 MU/m 2 [MU = million units], administered by intravenous injection). P-STAT1 levels in the PBMCs of a patient treated sequentially with 5 MU/m 2 and 10 MU/m 2 IFN α-2b (administered by subcutaneous injection) also increased in response to treatments with IFN α-2b but did not increase further with the increased dosage of IFN α-2b. Conclusion: This flow cytometry method can be used to monitor STAT1 activation within subsets of immune cells from patients undergoing IFN α immunotherapy.

© Oxford University Press

Topic:

• flow cytometry
• immunotherapy
• melanoma
• phosphorylation
• interferon alfa-2a
• interferon-alpha
• stat1 gene
• fluid flow
• donors

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