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Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery
Published on: October 4, 2018
Multiparametric flow cytometric analysis of inter-patient variation in STAT1 phosphorylation following interferon
Gregory B Lesinski1, Sri Vidya Kondadasula, Tim Crespin
1Department of Human Cancer Genetics, Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University, Columbus, OH 43210, USA.
Insights
Signal transducer and activator of transcription 1 (STAT1) activation in immune cells is crucial for interferon alfa (IFN alpha) antitumor effects. A novel flow cytometry assay effectively monitors STAT1 phosphorylation in immune cells during IFN alpha immunotherapy.
Area of Science:
- Immunology
- Molecular Biology
- Cancer Therapy
Background:
- Signal transducer and activator of transcription 1 (STAT1) regulates gene expression and mediates antitumor effects of interferon alfa (IFN alpha).
- IFN alpha immunotherapy is a key treatment for various cancers, including melanoma.
- Monitoring STAT1 activation is essential for understanding treatment efficacy.
Purpose of the Study:
- To develop and validate a novel flow cytometry assay for measuring STAT1 activation.
- To examine STAT1 phosphorylation in immune cell subsets following IFN alpha treatment.
- To compare STAT1 activation in healthy donors and melanoma patients.
Main Methods:
- Multiparametric flow cytometry was used to quantify phosphorylated STAT1 (P-STAT1) in immune cell subsets.
- Peripheral blood mononuclear cells (PBMCs) from healthy donors and melanoma patients were treated with IFN alpha-2b in vitro.
- PBMCs from melanoma patients were analyzed before and after IFN alpha-2b immunotherapy.
Main Results:
- IFN alpha-2b treatment rapidly increased P-STAT1 levels in all major immune cell subsets, particularly T lymphocytes and monocytes.
- Healthy donors exhibited higher basal P-STAT1 levels in PBMCs, NK cells, and T cells compared to melanoma patients.
- IFN alpha-2b immunotherapy induced STAT1 activation in melanoma patients' immune cells.
Conclusions:
- The developed flow cytometry method accurately monitors STAT1 activation in immune cell subsets.
- This assay can be utilized to assess the biological response to IFN alpha immunotherapy in cancer patients.
- Understanding STAT1 activation patterns can inform therapeutic strategies in cancer treatment.
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 alpha). 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 alpha 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 alpha-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 alpha-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 alpha-2b treatment of PBMCs; the increase was most pronounced in T lymphocytes and monocytes. Relatively low doses of IFN alpha-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 alpha-2b immunotherapy (20 MU/m2 [MU = million units], administered by intravenous injection). P-STAT1 levels in the PBMCs of a patient treated sequentially with 5 MU/m2 and 10 MU/m2 IFN alpha-2b (administered by subcutaneous injection) also increased in response to treatments with IFN alpha-2b but did not increase further with the increased dosage of IFN alpha-2b.
Conclusion:
This flow cytometry method can be used to monitor STAT1 activation within subsets of immune cells from patients undergoing IFN alpha immunotherapy.
