Figure 2
Figure 2. Antigen cross-presentation by neutrophils. (A) LB27.4 cells, BMNs, PENs, macrophages, and DCs pulsed for 2, 4, 6, or 8 hours with 0.4 mM Ova were incubated with B3Z cells. B3Z stimulation was measured by quantifying the release of β-galactosidase. Results are expressed in SI as mean (± SD) of 5 experiments. (B; Left panel) PENs, BMNs, and LB27–4 were incubated (white histogram) or not (gray histogram) for 20 minutes at 37°C with 0.4 mM Texas Red–Ova. (Middle panel) Cells were incubated with anti–H-2Kb mAb (white histogram) or isotype control mAb (gray histogram). (Right panel) Cells were pulsed with 0.4 mM Ova for 2, 4, and 8 hours, respectively, at 37°C before detection by FACS of SIINFEKL in MHC I molecules, using the 25-D1.16 mAb (white histogram) or isotype control mAb (gray histogram). Results are representative of 1 of 3 experiments. (C) DCs (□), macrophages (▵), PENs (■), BMNs (●), and LB27.4 (○) pulsed with the indicated concentrations of Ova for 2 hours (BMNs), 4 hours (PENs), or 8 hours (macrophages, DCs, and LB27.4) were incubated with B3Z cells. (D) Different numbers of DCs (□), macrophages (▵), PENs (■), and BMNs (●) pulsed with 0.4 mM Ova for 2 hours (BMNs), 4 hours (PENs), or 8 hours (macrophages and DCs) were incubated with B3Z cells. (E) BMNs (left panel), macrophages (top right panel), and DCs (bottom right panel) pulsed with the indicated concentrations of Ova, either soluble (□) or coated to beads (♦), were incubated with B3Z cells. (F) BMNs from wild-type mice either untreated (■) or treated with lactacystin (▩) and BMNs from TAP−/− mice (□) pulsed with 0.2 mM Ova for 2 hours were incubated with B3Z cells. (A,C-E) Results are expressed in SI as mean (± SD) of 5 experiments. (G) HLA-A2+ human neutrophils pulsed with or without 0.1 to 10 μg/mL HCV1a-NS3 protein (■) or with Ova (□), for 4 hours, were cultured with a human CD8+ T-cell clone specific for the HCV-NS3 peptide 1406-1415. IFN-γ production was quantified by ELISA in the 16-hour supernatants. Results are expressed in ng/mL as mean (± SD) of triplicate values and are representative of the data obtained with the neutrophils of 1 of 3 subjects.

Antigen cross-presentation by neutrophils. (A) LB27.4 cells, BMNs, PENs, macrophages, and DCs pulsed for 2, 4, 6, or 8 hours with 0.4 mM Ova were incubated with B3Z cells. B3Z stimulation was measured by quantifying the release of β-galactosidase. Results are expressed in SI as mean (± SD) of 5 experiments. (B; Left panel) PENs, BMNs, and LB27–4 were incubated (white histogram) or not (gray histogram) for 20 minutes at 37°C with 0.4 mM Texas Red–Ova. (Middle panel) Cells were incubated with anti–H-2Kb mAb (white histogram) or isotype control mAb (gray histogram). (Right panel) Cells were pulsed with 0.4 mM Ova for 2, 4, and 8 hours, respectively, at 37°C before detection by FACS of SIINFEKL in MHC I molecules, using the 25-D1.16 mAb (white histogram) or isotype control mAb (gray histogram). Results are representative of 1 of 3 experiments. (C) DCs (□), macrophages (▵), PENs (■), BMNs (●), and LB27.4 (○) pulsed with the indicated concentrations of Ova for 2 hours (BMNs), 4 hours (PENs), or 8 hours (macrophages, DCs, and LB27.4) were incubated with B3Z cells. (D) Different numbers of DCs (□), macrophages (▵), PENs (■), and BMNs (●) pulsed with 0.4 mM Ova for 2 hours (BMNs), 4 hours (PENs), or 8 hours (macrophages and DCs) were incubated with B3Z cells. (E) BMNs (left panel), macrophages (top right panel), and DCs (bottom right panel) pulsed with the indicated concentrations of Ova, either soluble (□) or coated to beads (♦), were incubated with B3Z cells. (F) BMNs from wild-type mice either untreated (■) or treated with lactacystin (▩) and BMNs from TAP−/− mice (□) pulsed with 0.2 mM Ova for 2 hours were incubated with B3Z cells. (A,C-E) Results are expressed in SI as mean (± SD) of 5 experiments. (G) HLA-A2+ human neutrophils pulsed with or without 0.1 to 10 μg/mL HCV1a-NS3 protein (■) or with Ova (□), for 4 hours, were cultured with a human CD8+ T-cell clone specific for the HCV-NS3 peptide 1406-1415. IFN-γ production was quantified by ELISA in the 16-hour supernatants. Results are expressed in ng/mL as mean (± SD) of triplicate values and are representative of the data obtained with the neutrophils of 1 of 3 subjects.

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