Article Figures & Data
Figures
- Figure 1.
iNKT-cell development in FTOC is blocked by IAP antagonists. A–D, Thymii from E16.5 embryos were cultured in the presence of IAP antagonists (LBW-242, LCL-161, LCJ-917), vehicle (−), or control compound (LCV-843) for 14 days. Control and LBW-242 were used at 500 nmol/L or as indicated; LCL-161 and LCJ-617 were used at 200 nmol/L. A, α-GalCer-loaded CD1d tetramer+ iNKT cells were identified in FTOC using flow cytometry. B, Quantification of results from three independent experiments performed and analyzed as in A. C, Dose–response curve for LBW-242 in FTOC. D, Cells recovered from FTOC were stimulated with anti-CD3 without further exposure to IAP antagonists; labels indicate the compounds added during thymic culture. IL2 was measured in the culture supernatant by ELISA. E, Foxp3-positive thymocytes were identified in LBW-242–treated cultures using thymi harvested from Foxp3-GFP knock-in embryos. F, γδ T cells were identified in LCL-161– and LCJ-917–treated cultures by δ chain expression. D–F, Error bars represent SEM. Results are representative of at least two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant versus control.
- Figure 2.
Mature iNKT cells persist in IAP antagonist–treated cultures. A, FTOC were performed as in Fig. 1 in the presence of LBW-242 or control compound (continuous treatment) or were cultured in the absence of compound until day 14 when LBW-242 or control compound were added (late addition). All cultures were harvested at day 16 and analyzed by flow cytometry. B, Spleen cells (5 × 105) were cultured in the presence of LBW-242 or control compound; after 24 hours, total viable cells were quantified by Trypan blue exclusion, and T cells and iNKT cells were identified by flow cytometry. Error bars present SEM. A and B, Results are representative of two independent experiments.
- Figure 3.
IAP antagonists enhance cytokine production from mature iNKT cells. A, Spleen cells (5 × 105) were treated with (α-GalCer) or vehicle in the presence of LBW-242 or control compound (LCV-843) for 24 hours. B, The iNKT-cell hybridomas 24.7, 24.8, and 24.9 were stimulated with anti-CD3 (10 μg/mL) for 24 hours in the presence of LBW-242 or control compound. C, Whole spleens from transnuclear mice (Vβ7A, Vβ7C, and Vβ8.2 on a RAG2−/− background) were inflated with PBS before homogenization and plating at 2 × 105 cells per well in a 96-well plate, with 1 μg/mL, 500 ng/mL, 100 ng/mL, 50 ng/mL, 10 ng/mL, 5 ng/mL, or 0 ng/mL α-GalCer with or without 500 nmol/L LCL-161, as indicated. Supernatants were collected after 24 hours. A–C, Cytokines were measured by ELISA. Error bars represent SEM. Results represent two independent experiments with six replicas per experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 versus control.
- Figure 4.
IAP antagonism costimulates cytokine production from conventional CD8+ and CD4+ T cells, as well as invariant NKT cells. CD8+ and CD4+ T cells were isolated from C57BL/6 mice by magnetic bead separation and plated at 1.5 × 105 cells per well in a 96-well plate. Whole spleens from Vα14 transnuclear mice were inflated with PBS, homogenized, and plated at 1.5 × 105 cells per well. LCL-161 depicts cells treated with 500 nmol/L LCL-161 without stimulation. C57BL6 CD4 and CD8 T cells were stimulated with anti-CD3/anti-CD28 beads, whereas Vα14 spleen cells were stimulated with 100 ng/mL α-GalCer. Supernatants were collected after 48 hours of culture and analyzed by cytokine bead array. Results are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
- Figure 5.
In vivo effects of IAP antagonist plus α-GalCer on cytokine production and on B16F10 lung metastases. A–C, All C57BL/6 mice were treated with vehicle or LCL-161 (75 mg/kg) by oral gavage at days −2 and day 0 of analysis. A, On day 0, LCL-161 or vehicle-treated mice were injected intraperitoneally with 1 μg α-GalCer and bled 2 hours after injection. Serum was collected and analyzed by ELISA. B, On day 0, LCL-161 or vehicle-treated mice were treated with 1 μg α-GalCer intraperitoneally; 4 hours after stimulation, mice were sacrificed and their spleens were homogenized and stained with anti-CD3 and PBS57-CD1d tetramer before being fixed, permeabilized, and stained intracellularly with anti-IL2 and anti-IFNγ. Representative flow plots are gated on CD3+Tetramer+ iNKT cells. C, Mice were treated with vehicle or LCL-161 (75 mg/kg) by oral gavage 2 days prior to, and on the day of tumor inoculation. Mice were injected with B16F10 cells (3 × 105) intravenously. Four hours after inoculation, and again on days 4 and 8, mice were treated with vehicle or 1 μg α-GalCer intraperitoneally. On day 14, mice were sacrificed, and lung nodules in the right lobe were enumerated and quantified. A–C, Results are representative of three independent experiments. *, P < 0.05; ***, P < 0.001.
- Figure 6.
Effect of IAP antagonist treatment on cultured human invariant NKT-cell IFNγ production and overall yield. A, B, Human peripheral blood mononuclear cells were depleted of CD14+ cells and cultured with α-GalCer (1 μg/mL) and LBW-242 (500 nmol/L) as indicated. IFNγ (A) and IL-4 (B) production were determined by ELISA of 48-hour culture supernatants. C, Human peripheral blood mononuclear cells were cultured with α-GalCer with or without LBW-242 or were left untreated. After 2 weeks, invariant NKT cells were identified by flow cytometry using coexpression of Vα24 and Vβ11, and total cultured cells were quantified by Trypan blue exclusion. Results are representative of three independent experiments. **, P < 0.01; ****, P < 0.0001.
Additional Files
Supplementary Data
- Supplemental Figure Legends - Figure legends
- Figure S1 - S1: Structure of IAP antagonists
- Figure S2 - S2: IAP antagonist treatment skewsT-cell development toward the CD8 lineage in fetal thymic organ culture (FTOC).
- Figure S3 - S3: IAP antagonism alone does not induce apoptosis of iNKT cells or conventional T cells.
- Figure S4 - S4: CD1d knockout mice show a significant contribution of the costimulatory effects of LBW-242 on cytokine production by CD4+ T cells.
Volume 6, Issue 1
