Multiparametric flow cytometric analysis of inter-patient variation in STAT1 phosphorylation following interferon Alfa immunotherapy.
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.


CONCLUSIONS
This flow cytometry method can be used to monitor STAT1 activation within subsets of immune cells from patients undergoing IFN alpha immunotherapy.
Lesinski
Gregory B
GB
Department of Human Cancer Genetics, Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University, Columbus, OH 43210, USA.
Kondadasula
Sri Vidya
SV
Crespin
Tim
T
Shen
Lei
L
Kendra
Kari
K
Walker
Michael
M
Carson
William E
WE
D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D016022:Case-Control Studies; D004268:DNA-Binding Proteins / Q000097:blood* / Q000187:drug effects; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000008:administration & dosage / Q000494:pharmacology*; D007694:Killer Cells, Natural / Q000378:metabolism; D008545:Melanoma / Q000097:blood / Q000188:drug therapy / Q000276:immunology / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050794:STAT1 Transcription Factor; D050795:STAT2 Transcription Factor; D015398:Signal Transduction; D012878:Skin Neoplasms / Q000097:blood / Q000188:drug therapy / Q000276:immunology / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism; D015534:Trans-Activators / Q000097:blood* / Q000187:drug effects
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050795:STAT2 Transcription Factor; D015534:Trans-Activators