In vitro inflammasome induction in MDSCs leads to loss of suppressor function. Inflammasome induction in freshly cultured wild-type and ASC^−/− MDSC-IL13 was carried out by adding 0.2 µg/mL LPS for 3 hours, followed by addition of 2 mM ATP or 0.8 µg/mL poly(dT) transfection. (A) Culture supernatants were harvested after an additional 1 hour and assayed for IL-1β production by ELISA. Data are representative of 3 independent experiments. (B) Inflammasome-induced MDSC-IL13 were washed extensively and plated in a CFSE suppression assay at a 1:1 ratio; data are representative of gated CFSE-labeled CD8⁺ responder T cells (n = 6 samples/group from 2 independent experiments). NLRP3 indicates LPS + ATP treatment, and AIM2 indicates LPS + poly(dT) treatment; gray histogram represents the no-MDSC proliferation control. Gated CD4⁺ responder T cells shown in supplemental Figure 5. (C) Kaplan-Meier survival curve of the C57Bl/6 → BALB/c GVHD model using inflammasome-induced MDSC-IL13, treated as above. MDSC v no MDSC P < .0001, MDSC vs MDSC AIM2 P < .0001, MDSC v MDSC NLRP3 P = .0029. Data represent n = 18 animals per group, combined from 2 independent experiments. (D) Kaplan-Meier survival curve of GVHD using MDSC-IL13 generated from either wild-type or ASC^−/− mice as indicated. Data represent n = 30 animals per group in 3 independent experiments. MDSC vs no MDSC P = .0399, MDSC vs ASC^−/− MDSC P = .0006. (E) Histograms represent % divided CFSE-labeled responding T-cells when plated against recovered wild-type or ASC^−/− MDSC-IL13 from day 5 posttransplant at a ratio of 1:1 and collected on day 3. Significant P values (< .05) were found when comparing any single group to wild-type MDSC-IL13 recovered from GVHD mice. Data are representative of 2 independent experiments. wt, wild-type.