On the co-purification of 6-sulfo LacNAc(+) dendritic cells (slanDC) with NK cells enriched from human blood.
The ability of NK cells to directly recognize pathogens and be activated via Toll-like receptors (TLR) is increasingly recognized. Nevertheless, controversial results on the NK cell ability to be directly activated by lipopolysaccharide (LPS), the ligand of TLR4, have been recently reported. To start elucidating the reasons explaining the contrasting observations of the literature, we focused on the potential role of currently used NK cell purification procedures to condition putative NK cell responsiveness to LPS. To do so, human NK cells were isolated by negative selection, using three different commercial kits, to be comparatively evaluated for the production of IFNgamma in response to ultra-pure LPS and/or IL-2. Despite the lack of surface TLR4, we found that two out of the three NK cell-enriched populations released IFNgamma (and one of the two, IL-12p70 as well) in response to the LPS plus IL-2 combination, whereas the last one did not. However, the two LPS plus IL-2-responsive NK cell populations were found variably contaminated with 6-sulfo LacNAc(+) dendritic cells (slanDC), demonstrated responsible for triggering, via the production of IL-12p70 in response to LPS, the release of IFNgamma by IL-2-stimulated NK cells. Accordingly, slanDC depletion completely abrogated the capacity to produce both IL-12p70 and IFNgamma in response to LPS plus IL-2 by slanDC-containing NK cells. Taken together, our data uncover that two commercially available kits, specifically designed to isolate NK cells by negative selection, also co-purify variable amounts of slanDC. The latter cells may dramatically affect the outcome of experiments carried on to evaluate NK cell responsiveness to TLR agonists such as LPS.
Costantini
Claudio
C
Department of Pathology, University of Verona, Italy.
Calzetti
Federica
F
Perbellini
Omar
O
Cassatella
Marco A
MA
D000606:Amino Sugars / Q000032:analysis; D002469:Cell Separation / Q000379:methods; D003713:Dendritic Cells / Q000276:immunology*; D005434:Flow Cytometry; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D007376:Interleukin-2 / Q000276:immunology / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000276:immunology*; D008070:Lipopolysaccharides / Q000276:immunology; D011933:Reagent Kits, Diagnostic
C467438:6-sulfo-LacNac; D000606:Amino Sugars; D007376:Interleukin-2; D008070:Lipopolysaccharides; D011933:Reagent Kits, Diagnostic; D007371:Interferon-gamma