Fig 2: Bcl-3 deficiency alleviates EAE progression through lactate. (A) Clinical score of Bcl-3-/- and WT mice after inducing EAE disease (n = 5). (B) Bcl-3-/- and WT mice were induced EAE, and one group of WT mice received 160 mg/kg lactate i.p. three days later; clinical score of EAE is evaluated every other day. (C) Spinal cords were isolated from mice on day 18 after immunization and fixed for haematoxylin & eosin staining, and infiltration of lymphocytes was highlighted with arrow. (D) Quantitative analysis of disease severity. (E) Spinal cords were isolated from mice on day 18 after immunization and fixed for LFB staining to analyze demyelination highlighted by arrow. (F) Representative staining of Th17 cells in CNS on day 18 post immunization. Represented dots were gated on TCRß+ CD4+ (G–H) statistical analysis of IL-17+ CD4+ cells and IFN-?+ CD4+ cells in the CNS. (I) Representative staining of Th17 cells in draining lymph nodes on day 14 post immunization. Represented dots were gated on CD4+ (J) statistical analysis of IL-17+ CD4+ cells in the draining lymph nodes. Data above are representative of 3 independent experiments. **P <0.01, ***P <0.001.

Fig 3: Bcl-3 depletion promotes glycolysis and inhibits mitochondrial respiration of Th17 cells. Naïve CD4+ T cells were isolated from the spleen of Bcl-3-/- and WT mice and induced Th17 differentiation by in vitro polarization assay and detected the metabolic signals by seahorse XF24 extracellular flux analyzer. (A) Glycolytic pathway was detected by extracellular acidification rate (ECAR). (B) Statistical analysis of ECAR (C) mitochondrial respiration was detected by oxygen consumption rate (OCR). (D) Statistical analysis of OCR. N = 3 for each group. Data above are representative of 3 independent experiments. *P <0.05, **P <0.01.

Fig 4: Bcl-3 regulates the differentiation of Th17 cells by lactate. (A) Th17 cells from the spleen lymphocytes of WT mice and Bcl-3-/- mice induced using in vitro polarization assay, 10 µM GNE-140 was added to Bcl-3-/- group for three days, and Th17 cells were analyzed by FACS. (B) Statistical analysis of Th17 cells. (C) Th17 cells from the spleen lymphocytes of WT mice and Bcl-3-/- mice induced using in vitro polarization assay, 10 µM GNE-140 was added to Bcl-3-/- group, 25 mM lactate were added to WT group for three days, and Th17 cells were analyzed by FACS. (D) Statistical analysis of Th17 cells. (E) IL-17 secretion in supernatant of Th 17 cells were detected by ELISA. (F) Lactate secretion in supernatant of Th 17 cells were detected by lactate detection kit. n = 3 for each group. Data above are representative of 3 independent experiments. *P <0.05, **P <0.01, ***P <0.001, “ns”, no significance.

Fig 5: Bcl-3 interact with Raptor. (A) Flag-Bcl-3 interacted with HA-Raptor in 293T cells. The cells were co-transfected with Flag-Bcl-3 and HA-Raptor plasmids for co-IP assays using the anti-Flag and anti-HA antibodies, respectively. (B) Endogenous Bcl-3 interacted with endogenous Raptor in 293T cells detected by co-IP assays. (C) RNC domain of Raptor is needed for Raptor to interact with Bcl-3. 293T cells were transduced with plasmids expressing WT or different deletion mutants of HA-Raptor together with Flag-Bcl-3 vector for co-IP assays. (D) The ID domain of ANK is required for Bcl-3 to interact with Raptor. 293T cells were transduced with vectors expressing WT or different deletion mutants of Flag-Bcl-3 vectors together with the HA-Raptor vector for co-IP assays. Data above are representative of 2 independent experiments.