Erroneous expression of NKG2D on granulocytes detected by phycoerythrin-conjugated clone 149810 antibody

Background The activating Natural killer group 2 member D (NKG2D) receptor is typically expressed on NK cells, CD8 T lymphocytes, gamma delta 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.

Automated summary

The study revealed false positive reactivity of PE-labeled anti-NKG2D antibody 149810 on granulocytes from dexamethasone-treated GBM patients, while no such reactivity was observed with the APC-labeled or unconjugated version of the same clone.