Figure 4. Nlrp3/Asc deficiency or IL-1β antagonism causes lower IL-17A production and alloreactive T cell expansion. (A) Splenocytes were isolated from untreated mice (n = 4) or Nlrp3+/+ or Nlrp3−/− mice (n = 6 per group) receiving TBI+BM and T cells (BALB/c → C57BL/6 model) and analyzed for CD4+ T cell cytokine production by intracellular FACS staining after PMA/Ionomycin/Brefeldin A restimulation (left). A representative histogram of IL-17A–stained cells is shown (right). The experiment was performed two times with similar results. (B) Intestinal LPL from C57BL/6 mice were sorted into CD11b+CD11c− and CD11b+CD11cmid populations. Sorting strategy and overlaid post-sorting FACS analysis of resulting CD11b+CD11c− (green) or CD11b+CD11cmid (orange) populations are shown on the left. Cells were stimulated with or without LPS (20 ng/ml) and Anakinra (10 µg/ml) and co-incubated with BALB/c splenic CD4+ T cells. After 120 h, supernatants were analyzed for IL-17A. One representative of two independent experiments is shown. (C) BALB/c splenic CD4+ T cells were co-incubated with C57/BL6 GMCSF-DCs that were preexposed to increasing LPS concentrations with or without Anakinra (10 µg/ml). After 120 h of co-culture, the supernatant was analyzed for IL-17A by ELISA. One representative of four independent experiments is shown. (D) BALB/c naive splenic CD4+ T cells were incubated for 120 h with Nlrp3+/+ or Nlrp3−/− GMCSF-DC that were preexposed to LPS at different concentrations and the supernatant was analyzed for IL-17A by ELISA. One representative of four independent experiments is shown. (E) CFSE-labeled BALB/c CD4+ T cells were co-cultured with LPS preexposed DC from Nlrp3+/+ or Nlrp3−/− mice at different ratios for 96 h. One representative of four independent experiments is shown. (F) BALB/c splenic CD4+ T cells were incubated for 120 h with Asc+/+ or Asc−/− GMCSF-DC that were preexposed to LPS at different concentrations and the supernatant was analyzed for IL-17A by ELISA. One representative of four independent experiments is shown. (G) T cells, as in D, were restimulated with PMA (1 µM), ionomycin (100 nM), and Brefeldin A after co-culture with unprimed or 20 ng/ml LPS-primed DCs (Nlrp3+/+, Nlrp3−/−, or Anakinra-treated Nlrp3+/+) and analyzed by FACS for intracellular IL-17A, IFN-γ, and FoxP3. One representative of three independent experiments is shown. ***, P < 0.0001; **, P < 0.001; *, P < 0.05.
Figure 4.

Nlrp3/Asc deficiency or IL-1β antagonism causes lower IL-17A production and alloreactive T cell expansion. (A) Splenocytes were isolated from untreated mice (n = 4) or Nlrp3+/+ or Nlrp3−/− mice (n = 6 per group) receiving TBI+BM and T cells (BALB/c → C57BL/6 model) and analyzed for CD4+ T cell cytokine production by intracellular FACS staining after PMA/Ionomycin/Brefeldin A restimulation (left). A representative histogram of IL-17A–stained cells is shown (right). The experiment was performed two times with similar results. (B) Intestinal LPL from C57BL/6 mice were sorted into CD11b+CD11c− and CD11b+CD11cmid populations. Sorting strategy and overlaid post-sorting FACS analysis of resulting CD11b+CD11c− (green) or CD11b+CD11cmid (orange) populations are shown on the left. Cells were stimulated with or without LPS (20 ng/ml) and Anakinra (10 µg/ml) and co-incubated with BALB/c splenic CD4+ T cells. After 120 h, supernatants were analyzed for IL-17A. One representative of two independent experiments is shown. (C) BALB/c splenic CD4+ T cells were co-incubated with C57/BL6 GMCSF-DCs that were preexposed to increasing LPS concentrations with or without Anakinra (10 µg/ml). After 120 h of co-culture, the supernatant was analyzed for IL-17A by ELISA. One representative of four independent experiments is shown. (D) BALB/c naive splenic CD4+ T cells were incubated for 120 h with Nlrp3+/+ or Nlrp3−/− GMCSF-DC that were preexposed to LPS at different concentrations and the supernatant was analyzed for IL-17A by ELISA. One representative of four independent experiments is shown. (E) CFSE-labeled BALB/c CD4+ T cells were co-cultured with LPS preexposed DC from Nlrp3+/+ or Nlrp3−/− mice at different ratios for 96 h. One representative of four independent experiments is shown. (F) BALB/c splenic CD4+ T cells were incubated for 120 h with Asc+/+ or Asc−/− GMCSF-DC that were preexposed to LPS at different concentrations and the supernatant was analyzed for IL-17A by ELISA. One representative of four independent experiments is shown. (G) T cells, as in D, were restimulated with PMA (1 µM), ionomycin (100 nM), and Brefeldin A after co-culture with unprimed or 20 ng/ml LPS-primed DCs (Nlrp3+/+, Nlrp3−/−, or Anakinra-treated Nlrp3+/+) and analyzed by FACS for intracellular IL-17A, IFN-γ, and FoxP3. One representative of three independent experiments is shown. ***, P < 0.0001; **, P < 0.001; *, P < 0.05.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal