Figure 1. Autoantigenic stimulation... | Rockefeller University Press

Figure 1.

$Figure 1. Autoantigenic stimulation primes iNKT cells for subsequent TCR-independent IFN-γ production. (A) Flow cytometric analysis of an experiment in which isolated human PBMCs were depleted of B cells, monocytes, and DCs and then incubated in medium alone (open histograms) or in medium containing a mixture of IL-12, IL-18, and IFN-α (closed histograms). The cells were stained with α-GalCer–loaded CD1d tetramer and anti-CD3 and then fixed, permeabilized, and stained for intracellular IFN-γ. Results shown are from one representative analysis out of eight. (B) Compiled results from analyses of eight different human peripheral blood samples, showing the percentage of the indicated cell populations in each sample that stained positively for IFN-γ after the treatment described in A. Horizontal bars indicate the mean. (C) Flow cytometric analysis showing the intracellular IFN-γ staining of the peripheral blood iNKT cell population of an unstimulated sample (light gray histograms) compared with a sample treated with IL-12, IL-18, and IFN-α (dark gray histograms) or PMA and ionomycin (dotted lines) at the indicated time points. Results are from one representative experiment out of three. (D) Compiled results from analyses of five different human peripheral blood samples for IFN-γ staining induced by IL-12, IL-18, and IFN-α. The first two plots show the fraction of cells positive for IFN-γ in the iNKT or NK-enriched subsets for freshly isolated samples (fresh) compared with that in samples that were rested for 5 d in the absence of APCs and then exposed to the cytokine mixture (after 5 d). The rightmost plot shows the fraction of iNKT cells compared with NK cells in each sample that retained the ability to produce IFN-γ after 5 d. (E) Cultured human iNKT cells are primed to produce innate IFN-γ by transient exposure to CD1d+ APCs. (top) The indicated iNKT clones were incubated with CD1d-transfected (CD1d+) or untransfected (CD1d−) APCs, and production of the cytokines shown on the x axes was quantitated by ELISA. The dashed lines show the limit of detection of the ELISAs (5 pg/ml). (bottom) iNKT clones were exposed to the indicated APCs for 4 h, and then the APCs were removed. The purified iNKT cells were then incubated with CD1d− APCs in medium containing IL-12 and IL-18, and cytokine secretion was quantitated by ELISA. Results are shown as the fold increase in cytokine production compared with iNKT cells that were not treated with IL-12 and IL-18 (IFN-γ concentrations were typically in the range of 0.2–2 ng/ml; for the purposes of these analyses, cytokine concentrations that were below the limit of detection of the ELISA were arbitrarily assigned a value of 1 pg/ml). The results shown are from a single experiment in which the indicated iNKT cell clones were tested in parallel; similar results were obtained in at least three additional experiments each for clones GG1.2, JC2.4, and PP1.10. Similar results were also obtained in multiple independent experiments using three additional human NKT cell clones (J3N.5, J24N.22, and GL1.4). (F) iNKT cells were exposed to CD1d+ APCs for 4 h, then the APCs were removed, and the iNKT cells were rested in medium for the indicated times and then exposed to IL-12 and IL-18. Results are expressed as the fold increase in cytokine secretion compared with control iNKT cells that were exposed to the APCs but not stimulated with IL-12 and IL-18. The results shown are representative of three independent analyses on two different iNKT cell clones.$

Autoantigenic stimulation primes iNKT cells for subsequent TCR-independent IFN-γ production. (A) Flow cytometric analysis of an experiment in which isolated human PBMCs were depleted of B cells, monocytes, and DCs and then incubated in medium alone (open histograms) or in medium containing a mixture of IL-12, IL-18, and IFN-α (closed histograms). The cells were stained with α-GalCer–loaded CD1d tetramer and anti-CD3 and then fixed, permeabilized, and stained for intracellular IFN-γ. Results shown are from one representative analysis out of eight. (B) Compiled results from analyses of eight different human peripheral blood samples, showing the percentage of the indicated cell populations in each sample that stained positively for IFN-γ after the treatment described in A. Horizontal bars indicate the mean. (C) Flow cytometric analysis showing the intracellular IFN-γ staining of the peripheral blood iNKT cell population of an unstimulated sample (light gray histograms) compared with a sample treated with IL-12, IL-18, and IFN-α (dark gray histograms) or PMA and ionomycin (dotted lines) at the indicated time points. Results are from one representative experiment out of three. (D) Compiled results from analyses of five different human peripheral blood samples for IFN-γ staining induced by IL-12, IL-18, and IFN-α. The first two plots show the fraction of cells positive for IFN-γ in the iNKT or NK-enriched subsets for freshly isolated samples (fresh) compared with that in samples that were rested for 5 d in the absence of APCs and then exposed to the cytokine mixture (after 5 d). The rightmost plot shows the fraction of iNKT cells compared with NK cells in each sample that retained the ability to produce IFN-γ after 5 d. (E) Cultured human iNKT cells are primed to produce innate IFN-γ by transient exposure to CD1d⁺ APCs. (top) The indicated iNKT clones were incubated with CD1d-transfected (CD1d⁺) or untransfected (CD1d⁻) APCs, and production of the cytokines shown on the x axes was quantitated by ELISA. The dashed lines show the limit of detection of the ELISAs (5 pg/ml). (bottom) iNKT clones were exposed to the indicated APCs for 4 h, and then the APCs were removed. The purified iNKT cells were then incubated with CD1d⁻ APCs in medium containing IL-12 and IL-18, and cytokine secretion was quantitated by ELISA. Results are shown as the fold increase in cytokine production compared with iNKT cells that were not treated with IL-12 and IL-18 (IFN-γ concentrations were typically in the range of 0.2–2 ng/ml; for the purposes of these analyses, cytokine concentrations that were below the limit of detection of the ELISA were arbitrarily assigned a value of 1 pg/ml). The results shown are from a single experiment in which the indicated iNKT cell clones were tested in parallel; similar results were obtained in at least three additional experiments each for clones GG1.2, JC2.4, and PP1.10. Similar results were also obtained in multiple independent experiments using three additional human NKT cell clones (J3N.5, J24N.22, and GL1.4). (F) iNKT cells were exposed to CD1d⁺ APCs for 4 h, then the APCs were removed, and the iNKT cells were rested in medium for the indicated times and then exposed to IL-12 and IL-18. Results are expressed as the fold increase in cytokine secretion compared with control iNKT cells that were exposed to the APCs but not stimulated with IL-12 and IL-18. The results shown are representative of three independent analyses on two different iNKT cell clones.