Baratin et al. 10.1073/pnas.0507355102. |
Fig. 6. Freshly isolated human peripheral blood mononuclear cells were cultured alone (0), or in the presence of the natural killer (NK) cell target K562, Plasmodium falciparum-infected red blood cells (3D7-RBC), or uninfected RBC (RBC). NK cell activation was analyzed by flow cytometry after gating on CD3-CD56+ NK cells. After 20-24 h of culture, the NK cell-surface expression of CD25 and CD69 was assayed. One representative experiment is shown as histogram plots for CD25 (Upper) or CD69 (Lower) staining.
Fig. 7. The NK92 human NK cell line directly interacts with Pf-RBC (FcR3CSA strain) as "rosettes," whereas normal erythrocytes do not.
Fig. 8. The cell-surface expression of HLA class I and class II molecules on dendritic cells (DC) and plasmacytoid BDCA2+ dendritic cells (PDC) is up-regulated upon coculture with 3D7-RBC. Purified DC (CD19-CD14-BDCA-1+ or BDCA-2+ or BDCA-3+ cells) or purified PDC (BDCA-2+) were cultured for 20 h in the presence of 3D7-RBC or RBC and stained for surface expression of HLA-A,B,C or HLA-DR.
Fig. 9. HEK293 reporter cells transfected with human Toll-like receptor (TLR) 2 were stimulated with 3D7-RBC for 18 h. Results are represented as optical density (OD) after subtraction of the background signal obtained with RBC or medium alone. Saturating concentrations of the anti-TLR2 mAb inhibit 3D7-RBC stimulation without any effect of the isotype control used at the same concentration.
Supporting Methods
Mice.
C57BL/6 (B6) mice were purchased from Charles River Breeding Laboratories. B6 Ly5.1+ mice were bred at the Centre d’Immunologie de Marseille-Luminy animal facility. ZGKR (CD3z/FcR g/RAGKO, DAP12KI) mice were generated by crossing DAP12 knock-in mice, CD3z/FcRgKO mice, and RAG-1KO mice (a kind gift of E. Tomasello). MyD88-deficient mice, all Toll-like receptor (TLR)-deficient mice, and IL-18R-deficient mice were backcrossed more than eight times on a B6 background and were genotyped in the laboratory of B.R. All experiments have been performed in accordance with protocols approved by national regulation and the European Directives.Antibodies and Reagents.
mAbs directed against human markers were as follow. PerCP-Cy5.5-CD3, FITC-CD69, PE-IFNg, FITC-CD107a and FITC-CD107b mAb, and PE-conjugated mouse IgG1 isotype controls were purchased from BD Biosciences. APC-CD56, PE-CD25, FITC-CD3, PE-Cy5- CD56, FITC-CD14, and PE-CD19 were purchased from Beckman Coulter. PE-BDCA-1, APC-BDCA-2, and PE-BDCA-3 were purchased from Miltenyi Biotec. mAbs directed against mouse molecules were purchased from BD Pharmingen: APC-CD3, FITC-DX5, FITC-NK1.1, and PE-IFNg. PE-conjugated rat IgG1 isotypic control, biotinylated Ly5.1, PerCP-streptavidin, and FITC-Ly5.2 mAbs were purchased from BD Biosciences. Neutralizing mouse anti-human IL-18 mAb (clone 125-2H, 2 mg/ml; MBL, Japan) or anti-TLR2 mAb (clone T2.5, 50 mg/ml) (1) were added 15 min before the initiation of the culture. Mouse IgG1 isotype controls were used in parallel at the same concentrations.Flow Cytometric Analysis.
For cell-surface staining, cells were washed with FACS buffer (1´ PBS/0.1% NaN3/2% FCS) and stained for 30 min at 4°C with appropriate combinations of labeled mAb. After two washes, stained cells were fixed in 2% paraformaldehyde in PBS for 10 min at room temperature in the dark. For intracytoplasmic staining, fixed cells were permeabilized and stained with anti-IFN-g mAbs, by using the cytofix/cytoperm kit according to the manufacturer’s instructions, and analyzed in a FACSCalibur flow cytometer by using CELLQUEST software (BD Biosciences).Cytokine Assay.
IL-18 was detected in supernatants by sandwich ELISA using the human IL-18 ELISA kit from MBL. CXCL8 was measured in cultures supernatants by using the multiplex protein array technology according to the manufacturer’s instructions (Bio-Rad).HEK293 Cell Transfectants and Stimulation.
Stable HEK293 transfectants expressing human (h) or mouse (m) TLR were generated by CAYLA (Toulouse, France), and were selected for the lowest background in the absence of stimulation together with a strong expression of the reporter gene (soluble alkaline phosphatase) upon stimulation with appropriate agonist. For TLR assay, HEK293 transfectants were cultured in HEK blue detection medium (InvivoGen) into 96-well flat-bottom tissue culture plates and incubated with 0.5, 1, or 2 × 106 Plasmodium falciparum red blood cells (Pf-RBC) or RBC at 37°C for 18-24 h or optimal concentration of specific TLR agonists (InvivoGen): Pam3CSK4 (palmitoyl-3-cysteine-serine-lysine-4) and FSL-1 {[S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam2CGDPKHPKSF]}, Poly:IC, lipopolysaccharide E. coli K12, flagellin, R848, ODN2006, ODN1828, Actinomycetes sp. extracts, uropathogenic E. coli extracts for h/mTLR2, hTLR3, hTLR4, hTLR5, hTLR7/8, hTLR9, mTLR9, hTLR10, and mTLR11, respectively. TLR engagement was monitored by using a blue colorimetric assay measured with a microplate reader at 650 nm.Cytoadhesion Assay.
Cells from the IL-2-dependent NK92 human cell line were washed once in RPMI medium 1640 and resuspended at a concentration of 1 × 106 per ml in RPMI medium 1640, 10% FCS, 100 units/ml penicillin, 100 mg/ml streptomycin, 1 mM sodium pyruvate, and 2 mM L-glutamine, in the absence of IL-2. Cells were distributed at 1 × 106 per tube and incubated with Pf-RBC (10 × 106) at 37°C under rolling conditions for 1 h. The adhesion of Pf-RBC on natural killer cells was observed between slide and cover glass with an inverted microscope (Eclipse TE 200, Nikon), connected through a Sony Exwave HDA color video camera to a monitor.1. Meng, G., Rutz, M., Schiemann, M., Metzger, J., Grabiec, A., Schwandner, R., Luppa, P. B., Ebel, F., Busch, D. H., Bauer, S., et al. (2004) J. Clin. Invest. 113, 1473-1481.