Erroneous expression of NKG2D on granulocytes detected by phycoerythrin-conjugated clone 149810 antibody.
cross-reactivity
false positive
flow cytometry
glioblastoma multiforme
granulocytes
natural killer group 2 member
Journal
Cytometry. Part B, Clinical cytometry
ISSN: 1552-4957
Titre abrégé: Cytometry B Clin Cytom
Pays: United States
ID NLM: 101235690
Informations de publication
Date de publication:
05 2022
05 2022
Historique:
revised:
12
02
2021
received:
29
05
2020
accepted:
08
03
2021
pubmed:
23
3
2021
medline:
18
5
2022
entrez:
22
3
2021
Statut:
ppublish
Résumé
The activating Natural killer group 2 member D (NKG2D) receptor is typically expressed on NK cells, CD8 T lymphocytes, γδ T cells and small subsets of CD4 T lymphocytes. During the course of an extensive flow cytometry phenotyping of immune cells in the peripheral blood of patients with glioblastoma multiforme (GBM) we noticed an unexpected expression of NKG2D receptor on granulocytes using the phycoerythrin (PE)-conjugated clone 149810 antibody. Peripheral blood samples from 35 patients with GBM and 22 age-matched healthy control (HC) donors were analyzed using flow cytometry, imaging cytometry and real-time quantitative reverse transcription PCR to validate the observed expression of NKG2D receptor on myeloid cells. Reactivity with PE-149810 was mostly observed on granulocytes from GBM patients on dexamethasone treatment where it correlated with inferior survival rates. Surprisingly, such NKG2D expression on granulocytes was not observed using the allophycocyanin (APC)-conjugate of the same clone 149810 antibody or an indirect staining procedure with unconjugated clone 149810 antibody. Moreover, the PE-conjugate of a different anti-NKG2D clone (1D11) also did not stain granulocytes. Imaging cytometry indicated cell surface and intracellular localization of PE-149810 but not of PE-1D11 in granulocytes. Our results uncover an erroneous and false positive reactivity of PE-labeled (but not of APC-labeled or unconjugated) anti-NKG2D antibody 149810 on granulocytes from dexamethasone-treated GBM patients and raise a note of caution for studies of NKG2D expression on non-lymphoid cells.
Sections du résumé
BACKGROUND
The activating Natural killer group 2 member D (NKG2D) receptor is typically expressed on NK cells, CD8 T lymphocytes, γδ T cells and small subsets of CD4 T lymphocytes. During the course of an extensive flow cytometry phenotyping of immune cells in the peripheral blood of patients with glioblastoma multiforme (GBM) we noticed an unexpected expression of NKG2D receptor on granulocytes using the phycoerythrin (PE)-conjugated clone 149810 antibody.
METHODS
Peripheral blood samples from 35 patients with GBM and 22 age-matched healthy control (HC) donors were analyzed using flow cytometry, imaging cytometry and real-time quantitative reverse transcription PCR to validate the observed expression of NKG2D receptor on myeloid cells.
RESULTS
Reactivity with PE-149810 was mostly observed on granulocytes from GBM patients on dexamethasone treatment where it correlated with inferior survival rates. Surprisingly, such NKG2D expression on granulocytes was not observed using the allophycocyanin (APC)-conjugate of the same clone 149810 antibody or an indirect staining procedure with unconjugated clone 149810 antibody. Moreover, the PE-conjugate of a different anti-NKG2D clone (1D11) also did not stain granulocytes. Imaging cytometry indicated cell surface and intracellular localization of PE-149810 but not of PE-1D11 in granulocytes.
CONCLUSION
Our results uncover an erroneous and false positive reactivity of PE-labeled (but not of APC-labeled or unconjugated) anti-NKG2D antibody 149810 on granulocytes from dexamethasone-treated GBM patients and raise a note of caution for studies of NKG2D expression on non-lymphoid cells.
Identifiants
pubmed: 33749106
doi: 10.1002/cyto.b.22001
doi:
Substances chimiques
NK Cell Lectin-Like Receptor Subfamily K
0
Phycoerythrin
11016-17-4
Dexamethasone
7S5I7G3JQL
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
228-238Informations de copyright
© 2021 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.
Références
Adamczyk, M., Mattingly, P. G., Shreder, K., & Yu, Z. (1999). Surface plasmon resonance (SPR) as a tool for antibody conjugate analysis. Bioconjugate Chemistry, 10, 1032-1037.
Andersen, M. N., Al-Karradi, S. N., Kragstrup, T. W., & Hokland, M. (2016). Elimination of erroneous results in flow cytometry caused by antibody binding to Fc receptors on human monocytes and macrophages. Cytometry. Part A, 89, 1001-1009.
Antal-Szalmas, P., Strijp, J. A., Weersink, A. J., Verhoef, J., & Van Kessel, K. P. (1997). Quantitation of surface CD14 on human monocytes and neutrophils. Journal of Leukocyte Biology, 61, 721-728.
Bruhns, P. (2012). Properties of mouse and human IgG receptors and their contribution to disease models. Blood, 119, 5640-5649.
Bukowska-Strakova, K., Baran, J., Gawlicka, M., & Kowalczyk, D. (2006). A false expression of CD8 antigens on CD4+ T cells in a routine flow cytometry analysis. Folia Histochemica et Cytobiologica, 44, 179-183.
Butler, W. T., & Rossen, R. D. (1973). Effects of corticosteroids on immunity in man. I. Decreased serum IgG concentration caused by 3 or 5 days of high doses of methylprednisolone. The Journal of Clinical Investigation, 52, 2629-2640.
Chitadze, G., Fluh, C., Quabius, E. S., Freitag-Wolf, S., Peters, C., Lettau, M., Bhat, J., Wesch, D., Oberg, H. H., Luecke, S., et al. (2017). In-depth immunophenotyping of patients with glioblastoma multiforme: Impact of steroid treatment. Oncoimmunology, 6, e1358839.
Gonzalez-Quintela, A., Alende, R., Gude, F., Campos, J., Rey, J., Meijide, L. M., Fernandez-Merino, C., & Vidal, C. (2008). Serum levels of immunoglobulins (IgG, IgA, IgM) in a general adult population and their relationship with alcohol consumption, smoking and common metabolic abnormalities. Clinical and Experimental Immunology, 151, 42-50.
Guerra, N., Tan, Y. X., Joncker, N. T., Choy, A., Gallardo, F., Xiong, N., Knoblaugh, S., Cado, D., Greenberg, N. M., & Raulet, D. H. (2008). NKG2D-deficient mice are defective in tumor surveillance in models of spontaneous malignancy. Immunity, 28, 571-580.
Hughes, M. J., McGettrick, H. M., & Sapey, E. (2020). Importance of validating antibody panels: Anti-PD-L1 clone binds AF700 fluorophore. Journal of Immunological Methods, 483, 112795.
Kalyan, S., Chandrasekaran, V., Quabius, E. S., Lindhorst, T. K., & Kabelitz, D. (2014). Neutrophil uptake of nitrogen-bisphosphonates leads to the suppression of human peripheral blood gammadelta T cells. Cellular and Molecular Life Sciences, 71, 2335-2346.
Karimi, M. A., Aguilar, O., Zou, B., Bachmann, M. H., Carlyle, J. R., Baldwin, C. L., & Kambayashi, T. (2014). A truncated human NKG2D splice isoform negatively regulates NKG2D-mediated function. Journal of Immunology, 193, 2764-2771.
Kim, M. S., & Kim, T. S. (2013). R-phycoerythrin-conjugated antibodies are inappropriate for intracellular staining of murine plasma cells. Cytometry. Part A, 83, 452-460.
Lee, J., Machin, M., Russell, K. E., Pavlidis, S., Zhu, J., Barnes, P. J., Chung, K. F., Adcock, I. M., & Durham, A. L. (2016). Corticosteroid modulation of immunoglobulin expression and B-cell function in COPD. The FASEB Journal, 30, 2014-2026.
Lettau, M., Armbrust, F., Dohmen, K., Drews, L., Poch, T., Dietz, M., Kabelitz, D., & Janssen, O. (2018). Mechanistic peculiarities of activation-induced mobilization of cytotoxic effector proteins in human T cells. International Immunology, 30, 215-228.
Lopez-Soto, A., Huergo-Zapico, L., Acebes-Huerta, A., Villa-Alvarez, M., & Gonzalez, S. (2015). NKG2D signaling in cancer immunosurveillance. International Journal of Cancer, 136, 1741-1750.
Lubeck, M. D., Steplewski, Z., Baglia, F., Klein, M. H., Dorrington, K. J., & Koprowski, H. (1985). The interaction of murine IgG subclass proteins with human monocyte Fc receptors. Journal of Immunology, 135, 1299-1304.
Nolan, T., Hands, R. E., & Bustin, S. A. (2006). Quantification of mRNA using real-time RT-PCR. Nature Protocols, 1, 1559-1582.
Nunley, W. C., Jr., Kitchin, J. D., 3rd, & Normansell, D. E. (1982). Effects of dexamethasone therapy on serum immunoglobulin levels and peripheral cell counts in women undergoing conservative benign gynecologic surgery. Fertility and Sterility, 38, 318-324.
Petrelli, F., De Stefani, A., Ghidini, A., Bruschieri, L., Riboldi, V., Dottorini, L., Iaculli, A., Zaniboni, A., & Trevisan, F. (2021). Steroids use and survival in patients with glioblastoma multiforme: A pooled analysis. Journal of Neurology, 268, 440-447.
Shrestha, D., Bagosi, A., Szollosi, J., & Jenei, A. (2012). Comparative study of the three different fluorophore antibody conjugation strategies. Analytical and Bioanalytical Chemistry, 404, 1449-1463.
Temming, A. R., Bentlage, A. E. H., de Taeye, S. W., Bosman, G. P., Lissenberg-Thunnissen, S. N., Derksen, N. I. L., Brasser, G., Mok, J. Y., van Esch, W. J. E., Howie, H. L., et al. (2020). Cross-reactivity of mouse IgG subclasses to human Fc gamma receptors: Antibody deglycosylation only eliminates IgG2b binding. Molecular Immunology, 127, 79-86.
van Schie, R. C., & Wilson, M. E. (2000). Evaluation of human FcgammaRIIA (CD32) and FcgammaRIIIB (CD16) polymorphisms in Caucasians and African-Americans using salivary DNA. Clinical and Diagnostic Laboratory Immunology, 7, 676-681.
Warren, H. S. (2005). The Eighth Human Leucocyte Differentiation Antigen (HLDA8) Workshop: Natural killer cell section report. Cellular Immunology, 236, 17-20.
Wensveen, F. M., Jelencic, V., & Polic, B. (2018). NKG2D: A master regulator of immune cell responsiveness. Frontiers in Immunology, 9, 441.
Zarco, M. A., Ribera, J. M., Villamor, N., Balmes, A., Urbano Ispizua, A., & Feliu, E. (1998). Phenotypic changes in neutrophil granulocytes after G-CSF administration in patients with acute lymphoblastic leukemia under chemotherapy. Haematologica, 83, 573-575.