The importance of being “pure” neutrophils

The identification of polymorphonuclear neutrophils as cellular source of cytokines prompts researchers in the field to investigate whether the production of neutrophil-derived cytokines can be altered in human diseases.1Scapini P. Cassatella M.A. Social networking of human neutrophils within the immune system.Blood. 2014; 124: 710-719Crossref PubMed Scopus (266) Google Scholar For such a purpose, we developed a simple procedure that allows the isolation of more than 99.6% pure neutrophils (here defined as Neu) from either whole blood or buffy coats.2Zimmermann M. Aguilera F.B. Castellucci M. Rossato M. Costa S. Lunardi C. et al.Chromatin remodelling and autocrine TNFalpha are required for optimal interleukin-6 expression in activated human neutrophils.Nat Commun. 2015; 6: 6061Crossref PubMed Scopus (68) Google Scholar Our procedure includes a density gradient centrifugation step of whole blood/buffy coats over Ficoll-Paque PLUS (GE Healthcare, Little Chalfont, United Kingdom), dextran sedimentation, and red cell osmotic lysis of the granulocyte pellet, and a final immunomagnetic negative selection step using the “EasySep Human Neutrophil Enrichment Kit”2Zimmermann M. Aguilera F.B. Castellucci M. Rossato M. Costa S. Lunardi C. et al.Chromatin remodelling and autocrine TNFalpha are required for optimal interleukin-6 expression in activated human neutrophils.Nat Commun. 2015; 6: 6061Crossref PubMed Scopus (68) Google Scholar (StemCell Technologies, Vancouver, British Columbia, Canada). By doing so, we have recently reported that Neu incubated with 5 μM R848 (a TLR8 ligand) produce IL-6 and TNF-α starting from 6 hours, and at maximal levels after an overnight incubation (up to 200 pg/mL/5 × 106 cells).2Zimmermann M. Aguilera F.B. Castellucci M. Rossato M. Costa S. Lunardi C. et al.Chromatin remodelling and autocrine TNFalpha are required for optimal interleukin-6 expression in activated human neutrophils.Nat Commun. 2015; 6: 6061Crossref PubMed Scopus (68) Google Scholar Under the same experimental conditions, however, Neu neither express mRNA encoding IL-102 or interferon-stimulated genes (ISGs) such as interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) and ISG15,E1Zimmermann M. Arruda-Silva F. Bianchetto-Aguilera F. Finotti G. Calzetti F. Scapini P. et al.IFNalpha enhances the production of IL-6 by human neutrophils activated via TLR8.Sci Rep. 2016; 6: 19674Crossref PubMed Scopus (57) Google Scholar nor produce IL-10.3Tamassia N. Zimmermann M. Castellucci M. Ostuni R. Bruderek K. Schilling B. et al.Cutting edge: an inactive chromatin configuration at the IL-10 locus in human neutrophils.J Immunol. 2013; 190: 1921-1925Crossref PubMed Scopus (53) Google Scholar Recently, 1-step kits specifically designed to guarantee a rapid isolation of “unstressed,” highly pure, neutrophils from the blood, skipping any density gradient centrifugation step, have been released into the market. These kits include the “MACSxpress Neutrophil isolation kit” (Miltenyi Biotec, Bergisch Gladbach, Germany) and the “EasySep direct Human Neutrophil Isolation Kit,” the resulting neutrophils defined here as wbNeuM and wbNeuE, respectively. To our surprise, both wbNeuM and wbNeuE incubated with R848 for 20 hours were found to express remarkable levels of IL-10, IFIT1, and ISG15 mRNA (Fig 1, A), as well as to release detectable amounts of IL-10 (Fig 1, B). Furthermore, although both wbNeuM and wbNeuE accumulated IFIT1 and ISG15 transcripts also upon incubation with 50 μg/mL poly(I:C) (a TLR3 ligand) for 20 hours (Fig 1, A), Neu did not, consistent with the notion that human neutrophils lack TLR3.E2Hayashi F. Means T.K. Luster A.D. Toll-like receptors stimulate human neutrophil function.Blood. 2003; 102: 2660-2669Crossref PubMed Scopus (715) Google Scholar, 4Tamassia N. Le Moigne V. Rossato M. Donini M. McCartney S. Calzetti F. et al.Activation of an immunoregulatory and antiviral gene expression program in poly(I: C)-transfected human neutrophils.J Immunol. 2008; 181: 6563-6573Crossref PubMed Scopus (86) Google Scholar R848-treated wbNeuM and wbNeuE were also induced to accumulate and release elevated levels of IL-6 mRNA (Fig 1, A) and IL-6 protein (Fig 1, B), respectively, already after 4 hours of incubation, unlike Neu.2Zimmermann M. Aguilera F.B. Castellucci M. Rossato M. Costa S. Lunardi C. et al.Chromatin remodelling and autocrine TNFalpha are required for optimal interleukin-6 expression in activated human neutrophils.Nat Commun. 2015; 6: 6061Crossref PubMed Scopus (68) Google Scholar Similarly, TNF-α and CXCL8 mRNA expression (Fig 1, A) and production (Fig 1, B) were much higher in wbNeuM and wbNeuE than in Neu. In contrast, wbNeuM, wbNeuE, and Neu did not show remarkable differences upon incubation with 10 ng/mL TNF-α, in terms of either CXCL8 mRNA accumulation and production, or other cytokine expression (Fig 1). Given these unexpected results, we evaluated the precise purity of wbNeuM, wbNeuE, and Neu by flow cytometric analysis, using a panel of 13 markers allowing the identification of the most representative leukocyte types in the blood. Neu was confirmed to be 99.77% ± 0.15% pure,2Zimmermann M. Aguilera F.B. Castellucci M. Rossato M. Costa S. Lunardi C. et al.Chromatin remodelling and autocrine TNFalpha are required for optimal interleukin-6 expression in activated human neutrophils.Nat Commun. 2015; 6: 6061Crossref PubMed Scopus (68) Google Scholar whereas both wbNeuM and wbNeuE, although reaching a purity of 98.86% ± 0.25% and 97.46% ± 1.06%, respectively (confirming what is declared in their related kit datasheets), were found to be reproducibly contaminated by eosinophils and slan+CD16+-monocytes5Hofer T.P. Zawada A.M. Frankenberger M. Skokann K. Satzl A.A. Gesierich W. et al.slan-defined subsets of CD16-positive monocytes: impact of granulomatous inflammation and M-CSF receptor mutation.Blood. 2015; 126: 2601-2610Crossref PubMed Scopus (85) Google Scholar (also known as slanDCsE3Schakel K. Kannagi R. Kniep B. Goto Y. Mitsuoka C. Zwirner J. et al.6-Sulfo LacNAc, a novel carbohydrate modification of PSGL-1, defines an inflammatory type of human dendritic cells.Immunity. 2002; 17: 289-301Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar), the latter cells representing the majority of the wbNeuM- and wbNeuE-copurified CD16+-monocytes (see Table E1 in this article's Online Repository at www.jacionline.org). However, eosinophil contamination was found irrelevant because neutrophils isolated by Ficoll-Paque followed by dextran sedimentation and erythrocyte lysis (here defined as NeuF), although containing 1.52% ± 1.12% eosinophils, did not differ from Neu in terms of CXCL8 production, IL-6 and TNF-α mRNA expression in response to R848, or IFIT1 and ISG15 mRNA expression in response to either R848 or poly(:C) (see Fig E1 in this article's Online Repository at www.jacionline.org). slan+CD16+-monocytes were consequently hypothesized as most likely responsible for the abnormal cytokine-producing response by wbNeuM and wbNeuE, given their well-known capacity to produce remarkably elevated amounts of cytokines when stimulated by R848 or other TLR ligands.E4Hansel A. Gunther C. Ingwersen J. Starke J. Schmitz M. Bachmann M. et al.Human slan (6-sulfo LacNAc) dendritic cells are inflammatory dermal dendritic cells in psoriasis and drive strong TH17/TH1 T-cell responses.J Allergy Clin Immunol. 2011; 127 (e1-9): 787-794Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar Our prediction appeared to be correct as, after depletion of slan+CD16+-monocytes by saturating amounts of αM-DC8–linked microbeads (see Fig E2 in this article's Online Repository at www.jacionline.org), wbNeuM completely lost the capacity to produce IL-10 (Fig 2, A), as well to express IFIT1 and ISG15 mRNA in response to either R848 or poly(I:C) (Fig 2, B). The effect of slan+CD16+-monocyte-depletion was less effective in the case of wbNeuE, particularly for IFIT1 and ISG15 mRNA expression (Fig 2, B). Nonetheless, both wbNeuM and wbNeuE depleted of slan+CD16+-monocytes lost the capacity to express IL-6, and produced TNF-α at much lesser levels than did untouched wbNeuM and wbNeuE, after incubation with R848 for 4 hours (Fig 2, A and B). Notably, that slan+CD16+-monocytes, but not neutrophils, were the cells actually producing IL-10 in response to R848 was clearly evidenced by a specific “IL-10 secretion assay” showing that only slan+CD16+-monocytes stained positive for IL-10 within wbNeuE (see Fig E3 in this article's Online Repository at www.jacionline.org) or wbNeuM (data not shown). To identify which leukocyte type(s), in addition to slan+CD16+-monocytes, could be responsible for IFIT1 and ISG15 mRNA expression in wbNeuE treated with either R848 or poly(I:C), we performed additional depletion experiments also targeting B cells, or natural killer cells, or plasmacytoid dendritic cells (pDCs) (see Fig E4 in this article's Online Repository at www.jacionline.org). As shown in Fig 2, C, depletion of both slan+CD16+-monocytes and pDCs from wbNeuE treated with R848 almost completely abolished the expression of IFIT1 and ISG15 mRNAs, consistent with the capacity of pDCs to produce IFN-α in response to R848 stimulation and, in turn, express ISGs.E5Kadowaki N. Ho S. Antonenko S. Malefyt R.W. Kastelein R.A. Bazan F. et al.Subsets of human dendritic cell precursors express different Toll-like receptors and respond to different microbial antigens.J Exp Med. 2001; 194: 863-869Crossref PubMed Scopus (1677) Google Scholar In the case of poly(I:C) stimulation, removal of slan+CD16+-monocytes together with either natural killer cells or pDCs slightly, but not significantly, decreased the expression of IFIT1 and ISG15 mRNAs in wbNeuE (Fig 2, D), whereas removal of slan+CD16+-monocytes together with B cells was irrelevant. The latter findings indicate that other leukocyte subtypes, or combination of them, are responsible for IFIT1 and ISG15 mRNA induction in poly(I:C)-treated wbNeuE. Taken together, our data demonstrate that the profile of cytokines derived from the stimulation of wbNeuM and wbNeuE may appear profoundly altered as a consequence of their contamination with very small percentages of slan+CD16+-monocytes (0.1% to 0.6%), alone or in combination with pDCs or other leukocyte subtypes. Such an inconvenience becomes, in fact, evident if wbNeuM and wbNeuE are stimulated with TLR ligands, but not with TNF-α. These findings support what we have been always recommending,1Scapini P. Cassatella M.A. Social networking of human neutrophils within the immune system.Blood. 2014; 124: 710-719Crossref PubMed Scopus (266) Google Scholar, 6Scapini P. Calzetti F. Cassatella M.A. On the detection of neutrophil-derived vascular endothelial growth factor (VEGF).J Immunol Methods. 1999; 232: 121-129Crossref PubMed Scopus (84) Google Scholar namely, that it is absolutely mandatory to use highly purified populations of neutrophils when gene expression and/or neutrophil-derived cytokines are investigated. Hence, based on the data presented here, the use of commercial kits aimed at rapidly purifying neutrophils is certainly advisable, provided that these kits are used after a round of blood density gradient centrifugation. Without this step, which ensures the elimination of most PBMCs from granulocytes, in turn favoring an optimal negative selection of contaminating cells by these specific kits, researchers may inadvertently obtain false-positive results: these may include the detection of either cytokines/genes made by contaminating cells,7Lewkowicz N. Mycko M.P. Przygodzka P. Cwiklinska H. Cichalewska M. Matysiak M. et al.Induction of human IL-10-producing neutrophils by LPS-stimulated Treg cells and IL-10.Mucosal Immunol. 2016; 9: 364-378Crossref PubMed Scopus (56) Google Scholar or unusually high amounts of neutrophil-derived cytokines, as shown here or elsewhere.8Naegelen I. Plançon S. Nicot N. Kaoma T. Muller A. Vallar L. et al.An essential role of syntaxin 3 protein for granule exocytosis and secretion of IL-1α, IL-1β, IL-12b, and CCL4 from differentiated HL-60 cells.J Leukoc Biol. 2015; 97: 557-571Crossref PubMed Scopus (25) Google Scholar, 9Gaber T. Hahne M. Strehl C. Hoff P. Dorffel Y. Feist E. et al.Disentangling the effects of tocilizumab on neutrophil survival and function.Immunol Res. 2016; 64: 665-676Crossref PubMed Scopus (12) Google Scholar, E6Dalboni T.M. Abe A.E. de Oliveira C.E. Lara V.S. Campanelli A.P. Gasparoto C.T. et al.Activation profile of CXCL8-stimulated neutrophils and aging.Cytokine. 2013; 61: 716-719Crossref PubMed Scopus (17) Google Scholar Moreover, although it has been unequivocally demonstrated that, in human neutrophils, the IL-10 locus is in an inactive state and cannot be remodeled in response to TLR ligands,3Tamassia N. Zimmermann M. Castellucci M. Ostuni R. Bruderek K. Schilling B. et al.Cutting edge: an inactive chromatin configuration at the IL-10 locus in human neutrophils.J Immunol. 2013; 190: 1921-1925Crossref PubMed Scopus (53) Google Scholar articles reporting the detection of IL-10 in supernatants from TLR-activated neutrophils7Lewkowicz N. Mycko M.P. Przygodzka P. Cwiklinska H. Cichalewska M. Matysiak M. et al.Induction of human IL-10-producing neutrophils by LPS-stimulated Treg cells and IL-10.Mucosal Immunol. 2016; 9: 364-378Crossref PubMed Scopus (56) Google Scholar continue to be, since then, published. Given that wbNeuM or neutrophils with unascertained purity were used in these reports,7Lewkowicz N. Mycko M.P. Przygodzka P. Cwiklinska H. Cichalewska M. Matysiak M. et al.Induction of human IL-10-producing neutrophils by LPS-stimulated Treg cells and IL-10.Mucosal Immunol. 2016; 9: 364-378Crossref PubMed Scopus (56) Google Scholar caution should be undertaken on their reliability based on the data presented here. Neutrophils from the same donors were isolated by different methods, as follows: (1) by density gradient centrifugation of whole blood over Ficoll-Paque PLUS (GE Healthcare), dextran sedimentation, and erythrocyte osmotic lysis (NeuF); (2) similarly to NeuF, but with an additional immunomagnetic negative selection step, using the “EasySep Human Neutrophil Enrichment Kit” (StemCell Technologies) (Neu); (3) directly isolated from whole blood, by the “MACSxpress Neutrophil isolation kit” (Miltenyi Biotec) (wbNeuM); and (4) directly isolated from whole blood by the “EasySep direct Human Neutrophil Isolation Kit” (StemCell Technologies) (wbNeuE). In selected experiments, 8 × 106 wbNeuM or wbNeuE were incubated with or without 30 μL of αM-DC8 microbeads (Miltenyi Biotec) for 20 minutes on ice, and then slan+CD16+-monocytes were removed by separation via MACS LD column (Miltenyi Biotec). In other experiments, wbNeuE were stained by a combination of 10 μL of αM-DC8-fluorescein isothiocyanate (FITC) (Miltenyi Biotec) with or without 20 μL of αCD19-FITC (Miltenyi Biotec) or 20 μL of αCD56-FITC (Miltenyi Biotec) or 10 μL of αCD303-FITC (Miltenyi Biotec) for 20 minutes on ice. Removal of stained cells was then achieved by incubation with 20 μL of anti-FITC microbeads for 20 minutes on ice and subsequent passage through MACS LD column. Neutrophils were suspended at 5 × 106/mL in RPMI 1640 medium supplemented with 10% low-endotoxin FBS (<0.5 EU/mL, from Sigma, St Louis, Mo), treated with 5 μM R848 (Invivogen, San Diego, Calif), 50 μg/mL poly(I:C) (Invivogen), and 10 ng/mL TNF-α (Peprotech, Rocky Hill, NJ), and finally plated in 48-well tissue culture plates (Greiner Bio-One, Kremsmünster, Austria) for culture at 37°C, 5% CO2 atmosphere. After 4 or 20 hours, neutrophils were collected and spun at 300g for 5 minutes. Cell-free supernatants were immediately frozen in liquid nitrogen and stored at −80°C, while the corresponding pellets were extracted for total RNA. Cytokine concentrations in cell-free supernatants were measured by ELISA kits specific for IL-6 (eBioscience, San Diego, Calif), IL-10 (eBioscience), TNF-α (eBioscience), and CXCL8 (Mabtech, Nacka Strand, Sweden). Detection limits of these ELISAs were 8 pg/mL for IL-6, 4 pg/mL for IL-10 and TNF-α, and 8 pg/mL for CXCL8. Total RNA (0.1 μg) extracted by the RNeasy mini kit (Qiagen, Venlo, Limburg, The Netherlands) from 1 × 106 neutrophils was reverse transcribed for RT-quantitative PCR using gene-specific primer pairs (Life Technologies, Carlsbad, Calif) available in the public database RTPrimerDB (www.rtprimerdb.org) under the following entry codes: glyceraldehyde-3-phosphate dehydrogenase (3539), TNF-α (3551), CXCL8 (3553), IL-6 (3545), IL-10 (8230), IFIT1 (3540), and ISG15 (3547). Data were calculated by Q-Gene software (http://www.genequantification.de/download.html) and expressed as mean normalized expression units after glyceraldehyde-3-phosphate dehydrogenase normalization. A total of 105 freshly isolated neutrophil populations were suspended in 50 μL PBS containing 10% complement-inactivated human serum (for Fcγ receptor blocking). Neutrophils were then stained for 15 minutes at room temperature with different combinations of the following regents: αCD14-Vioblue (clone TÜK4), αCD3-PE-Vio770 (clone BW 264156), αCD19-APC (clone LT19), αCD56-PE (clone REA196), αM-DC8-FITC (clone DD1), αCD11c-Vioblue (clone MJ4-27G12), αCD303-FITC (clone AC144), αCD141-APC (clone AD5-14H12), αFCeR1a-PE (clone CRA1) antibodies from Miltenyi Biotec; αCD1c-Alexa488 (clone L161), αCD45 brilliant Violet 510 (clone H130), αCD16-PerCP-Cy5.5 (clone 3G8), and αHLA-DR-APC-CY7 (clone L243) antibodies from Biolegend (Sa Diego, Calif) and αCD123-PE (clone 9F5) antibodies from BD Bioscience (San Jose, Calif). After washings, sample fluorescence was measured by an 8-color MACSQuant Analyzer (Miltenyi Biotec), for which at least 40,000 cells were acquired. Data analysis was performed using FlowJo software Version 8.8.6 (Tree Star, Ashland, Ore). A total of 2 × 106 neutrophils were treated with or without 5 μM R848 or 10 ng/mL of TNF-α for 14 hours at 37°C, then washed, incubated for 5 minutes with IL-10–catching reagent (Miltenyi Biotec) on ice, immediately diluted with warm medium, and then left for 45 minutes at 37°C on constant rotation to let IL-10 secretion proceed. After extensive washings, cells were stained on ice with αIL-10PE, αM-DC8-FITC, and αHLA-DR APC-Cy7 Abs. Vybrant Violet (Life Technologies) was used as cell viability reagent. Neutrophils were gated as high SSChigh/Vybrant-negative cells, whereas slan+CD16+-monocytes were gated as M-DC8/HLA-DR+, Vybrant-negative cells. A total of 800,000 cells per sample were acquired, as average, by MACSQuant Analyzer.Fig E2Assessment of slan+CD16+-monocyte removal from wbNeuM and wbNeuE. After wbNeuM and wbNeuE neutrophils isolation, slan+CD16+-monocytes were removed by separation using αM-DC8-linked microbeads via MACS column. Flow cytometry analysis was then used to assess the levels of contaminant slan+CD16+-monocytes present in neutrophils preparations. Accordingly, contaminating PBMCs were first identified by SSClow/CD45 positivity (left panels, black gate), and then slan+CD16+-monocytes were selected among them, as M-DC8/CD16 double positive cells (right panels, red gate) and overlayed (red dots) on total CD45 cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3IL-10 secretion assay. 2 x 106 WbNeuE were treated with or without 5 μM R848 or 10 ng/mL TNFα for 14 h and then assayed for IL-10 secretion. Histograms, displaying 1 representative experiment out of 2 with similar results, show that only slan+CD16+-monocytes (right panel), but not neutrophils (left panel), secrete IL-10. Similar results were obtained using WbNeuM.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Assessment of slan+CD16+-monocyte, B cell, NK cell and pDC removal from wbNeuE. After isolation, wbNeuE were stained with FITC-conjugated antibodies towards M-DC8 (for slan+CD16+-monocytes) alone or in combination with CD19 (for B cells), CD56 (for NK cells) or CD303 (for pDCs). Panels show that removal of stained cells was efficiently achieved by anti-FITC microbeads via MACS column separation. In fact, contaminating PBMCs were first gated by SSClow/CD45 positivity (upper panels, black gate), and then slan+CD16+-monocytes (red dots), B cells (light blue dots), NK cells (green dots) and pDCs (purple dots) were identified, using specific CD markers overlaid on HLA-DR/CD123 dot plots.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Cellular composition of the neutrophil populations isolated from human blood by various proceduresCell typeNeu (Ficoll and Easysep kit) (n = 5)WbNeuM (MACSxpress kit) (n = 5)WbNeuE (Easysep direct kit) (n = 7)NeuF (Ficoll-Paque) (n = 3)Neutrophils*99.77 ± 0.1598.86 ± 0.2597.46 ± 1.0696.17 ± 2.66Eosinophils†0.07 ± 0.060.29 ± 0.370.53 ± 0.611.52 ± 1.12CD14+-monocytes‡0.00 ± 0.000.02 ± 0.020.04 ± 0.040.03 ± 0.03Total CD16+-monocytes§0.00 ± 0.000.38 ± 0.200.25 ± 0.180.00 ± 0.00slan+CD16+-monocytes¶0.00 ± 0.000.33 ± 0.160.22 ± 0.190.00 ± 0.00CD1c DCs‖0.00 ± 0.000.00 ± 0.000.00 ± 0.000.00 ± 0.00CD141 DCs#0.00 ± 0.000.01 ± 0.000.02 ± 0.020.00 ± 0.01pDCs**0.00 ± 0.000.00 ± 0.000.06 ± 0.030.00 ± 0.00T cells††0.05 ± 0.090.09 ± 0.050.13 ± 0.301.95 ± 1.81B cells‡‡0.00 ± 0.000.02 ± 0.020.33 ± 0.160.00 ± 0.01NK cells§§0.01 ± 0.010.11 ± 0.080.25 ± 0.180.27 ± 0.46Values are in %. Cell percentages were calculated on the basis of total CD45+ cells. The various leukocyte populations were identified by flow cytometry analysis using the following criteria/surface markers: *SSChi/CD16+ cells = neutrophils; †SSChi/CD16− cells = eosinophils; ‡CD14+/CD16− cells = CD14+-classical monocytes; §CD16+/HLA-DR+/CD56− cells = total CD16+-monocytes; ¶CD16+/HLA-DR+/M-DC8+/CD14dim/neg cells = slan+CD16+-monocytes; ‖CD1c+/FcεR1+/HLA-DR+ cells = CD1c DCs; #CD141+/HLA-DR+/CD11cdim cells = CD141 DCs; **CD303+/CD123+ cells = pDCs; ††CD3+ cells = T lymphocytes; ‡‡CD19+/HLA-DR+ cells = B lymphocytes; §§CD56+ cells = NK cells.DC, Dendritic cell; NK, natural killer. Open table in a new tab Values are in %. Cell percentages were calculated on the basis of total CD45+ cells. The various leukocyte populations were identified by flow cytometry analysis using the following criteria/surface markers: *SSChi/CD16+ cells = neutrophils; †SSChi/CD16− cells = eosinophils; ‡CD14+/CD16− cells = CD14+-classical monocytes; §CD16+/HLA-DR+/CD56− cells = total CD16+-monocytes; ¶CD16+/HLA-DR+/M-DC8+/CD14dim/neg cells = slan+CD16+-monocytes; ‖CD1c+/FcεR1+/HLA-DR+ cells = CD1c DCs; #CD141+/HLA-DR+/CD11cdim cells = CD141 DCs; **CD303+/CD123+ cells = pDCs; ††CD3+ cells = T lymphocytes; ‡‡CD19+/HLA-DR+ cells = B lymphocytes; §§CD56+ cells = NK cells. DC, Dendritic cell; NK, natural killer.