title: Total cellular protein presence of the transcription factor IRF8 does not necessarily correlate with its nuclear presence.
abstract: The transcription factor interferon regulatory factor-8 (IRF8) plays an essential role in myeloid differentiation and lineage commitment, based largely on molecular and genetic studies. The detection of IRF8 in specific cell populations by flow cytometry (FCM) has the potential to provide new insights into normal and pathologic myelopoiesis, but critical validation of this protein-based approach, particularly in human samples, is lacking. In this study, the assessment of total cellular IRF8 presence was compared to its specific nuclear presence as assessed by imaging flow cytometry (IFC) analysis. Peptide neutralization of the IRF8-specific antibody that has been predominantly used to date in the literature served as a negative control for the immunofluorescent labeling. Expression of total IRF8 was analyzed by total cellular fluorescence analogous to the mean fluorescence intensity readout of conventional FCM. Additionally, specific nuclear fluorescence and the similarity score between the nuclear image (DAPI) and the corresponding IRF8 image for each cell were analyzed as parameters for nuclear localization of IRF8. IFC showed that peptide blocking eliminated binding of the IRF8 antibody in the nucleus. It also reduced cytoplasmic binding of the antibody but not to the extent observed in the nucleus. In agreement with the similarity score data, the total cellular IRF8 as well as nuclear IRF8 intensities decreased with peptide blocking. In healthy donor peripheral blood subpopulations and a positive control cell line (THP-1), the assessment of IRF8 by total cellular presence correlated well with its specific nuclear presence and correlated with the known distribution of IRF8 in these cells. In clinical samples of myeloid-derived suppressors cells derived from patients with renal carcinoma, however, total cellular IRF8 did not necessarily correlate with its nuclear presence. Discordance was primarily associated with peptide blocking having a proportionally greater effect on the IRF8 nuclear localization versus total fluorescence assessment. The data thus indicate that IRF8 can have cytoplasmic presence and that during disease its nuclear-cytoplasmic distribution may be altered, which may provide a basis for potential myeloid defects during certain pathologies.
authors: Flow and Image Cytometry Shared Resource, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY, United States. Electronic address: hans.minderman@roswellpark.org.
authors: Hans
authors: H
authors: Minderman
authors: Flow and Image Cytometry Shared Resource, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY, United States.
authors: Orla
authors: O
authors: Maguire
authors: Flow and Image Cytometry Shared Resource, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY, United States.
authors: Kieran L
authors: KL
authors: O'Loughlin
authors: Dept of Urology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY, United States.
authors: Jason
authors: J
authors: Muhitch
authors: Flow and Image Cytometry Shared Resource, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY, United States.
authors: Paul K
authors: PK
authors: Wallace
authors: Dept of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY, United States.
authors: Scott I
authors: SI
authors: Abrams
mesh_terms: D000906:Antibodies / Q000494:pharmacology; D002277:Carcinoma / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D016022:Case-Control Studies; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000235:genetics* / Q000276:immunology / Q000648:ultrastructure; D003593:Cytoplasm / Q000235:genetics* / Q000276:immunology / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D006410:Hematopoiesis / Q000235:genetics* / Q000276:immunology; D006801:Humans; D019044:Image Cytometry / Q000379:methods; D050835:Interferon Regulatory Factors / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000276:immunology; D007680:Kidney Neoplasms / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D022423:Myeloid Cells; D010455:Peptides / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods; D000099185:Interferon Regulatory Factor-8
chemical_list: D000906:Antibodies; D050835:Interferon Regulatory Factors; D010455:Peptides; D000099185:Interferon Regulatory Factor-8
keywords: IRF8; Imaging flow cytometry intracellular localization