Fig 2: Lysine 112 Is the Ubiquitination Site in T Cells Mediated by Nedd4. A Mass spectrometric analysis of RORγt ubiquitination site(s) using purified Myc-tagged proteins from HEK293T cells transfected with Flag-tagged Nedd4, Myc-tagged RORγt, and HA-tagged ubiquitin. B Anti-HA immunoblot analysis of Myc immunoprecipitates of HEK293T cells transfected with Flag-tagged Nedd4, Myc-tagged RORγt or RORγt KXR (K99R, K112R, K169R, K288R and K495R), and HA-tagged ubiquitin. C Anti-ubiquitin immunoblot analysis of RORγt immunoprecipitates of CD4+ T cells from Rorc–/– mice reconstituted with GFP-tagged RORγt or RORγt K112R, cultured under Th17-biased condition for two days, and restimulated with anti-CD3 and anti-CD28 for 15 min. D Th17 cell differentiation assay using naïve CD4+ T cells from Rorc–/– mice (n = 3 mice per group) reconstituted with GFP-tagged RORγt or RORγt K112R. **p < 0.01; student t test. Data are representative of three independent experiments for B, and two independent experiments for C and D

Fig 3: Nedd4 Targets RORγt for K27-Linked Polyubiquitination, thus Augmenting Its activity. A Anti-HA immunoblot analysis of Myc immunoprecipitates of lysates of HEK293T cells transfected with Flag-tagged Nedd4 or Nedd4 C867A mutant, together with Myc-tagged RORγt and HA-tagged ubiquitin. B Anti-ubiquitin immunoblot analysis of RORγt immunoprecipitates of lysates of CD4+ T cells from WT and Nedd4C854A mice stimulated under Th17-biased condition for three days, and restimulated with anti-CD3 and anti-CD28 for 15 min or left unstimulated. C EAE scores and Th1/Th17 responses of WT and Nedd4C854A mice (n = 3 mice per group) immunized with MOG35-55 in CFA. **p < 0.01; Mann–Whitney U test. D Anti-HA immunoblot analysis of Myc immunoprecipitates of lysates of HEK293T cells transfected with Flag-tagged Nedd4, Myc-tagged RORγt, and HA-tagged K48, K48R, K63, or K63R ubiquitin mutants. E Anti-HA immunoblot analysis of Myc immunoprecipitates of lysates of HEK293T cells transfected with Flag-tagged Nedd4, Myc-tagged RORγt, and HA-tagged K6, K11, K27, K29, or K33 ubiquitin mutants. F Anti-ubiquitin and anti-K27 ubiquitin immunoblot analyses of RORγt immunoprecipitates of Th17 cells from Cd4 Cre and Cd4 Cre-Nedd4f/f mice restimulated with anti-CD3 and anti-CD28 for 15 min or left unstimulated. G RORγt reporter assay using HEK293T cells transfected with RORγt reporter gene, Flag-tagged Nedd4 or Nedd4 C867A together with HA-tagged ubiquitin (left panel), or alternatively, transfected with RORγt reporter gene, Flag-tagged Nedd4, HA-tagged K27, or K27R ubiquitin mutant (right panel). H ROR binding to the RORE at CNS2 using nuclear lysates of CD4+ T cells stimulated under Th17-biased condition for three days, and restimulated with anti-CD3 and anti-CD28, assessed by EMSA. Data are representative of three independent experiments for A–F, and two independent experiments for G

Fig 4: Targeting lactate and BCL6 lactylation effectively alleviates LN disease.a–e PBMCs from SLE patients were adoptively transferred into NSG to develop humanized LN chimeras, followed by treatment with or without sodium oxamate (OXA, 500 mg/kg) every 3 days until 28 days (n = 6 (a, b) or 9 (e) independent biological samples). a The proportion of Tfh cells in the kidneys. b Serum human IgG anti-dsDNA. c Renal deposition of human IgG. White arrows (IgG). Scale bar: 100 μm. d HE staining. Black circle (Glomerulus). Scale bar: 100 μm. e Urine protein. f-j PBMCs from healthy donors were cultured with or without L-lac (10 mM) for 2 days. Stimulated PBMCs were injected into NSG mice to develop humanized chimeras (n = 6 (f, g) or 8 (j) independent biological samples). f The proportion of Tfh cells in the kidneys. g Serum human IgG anti-dsDNA. h Renal deposition of human IgG. White arrows (IgG). Scale bar: 100 μm. i HE staining. Black circle (Glomerulus). Scale bar: 100 μm. j Urine protein. k–o CD4+ T cells isolated from PBMCs of SLE patients were activated with anti-CD3/28 beads and transduced with shPCAF or shNC. After 24 h, PCAF-kd CD4+ T cells mixed with other PBMC components were adoptively transferred into NSG to develop humanized LN chimeras (n = 4 independent biological samples). k The proportion of Tfh cells in the kidneys. l Serum human IgG anti-dsDNA. m Renal deposition of human IgG. White arrows (IgG). Scale bar: 100 μm. n HE staining. Black circle (Glomerulus). Scale bar: 100 μm. o Urine protein. The data are represented as the mean ± SEM. Normality and lognormality are assessed using the Shapiro-Wilk test. Two-tailed paired t test (a, e, f, g, j, k, o), two-tailed Wilcoxon matched-pairs signed rank test (b, l). Source data are provided as a Source Data file.

Fig 5: Lactylation at Lys688 regulated by PCAF orchestrates the remodeling of BCL6 ubiquitination.a Healthy CD4+ T cells were co-cultured with MCs for 4 days and BCL6 lactylation was detected. b Healthy CD4⁺ T cells were co-cultured with GSK2837808A pretreated-MCs for 4 days. c Healthy CD4⁺ T cells were co-cultured with LN-associated MCs transfected with shLDHA or shNC for 4 days. d Healthy CD4⁺ T cells were co-cultured with UK5099 (20 μM) pretreated-MCs for 4 days. e MCs were transfected with siMPC1, siNC or MPC1-overexpression plasmids (after SLE plasma stimulation). Healthy CD4⁺ T cells were co-cultured with MCs for 4 days. f HEK293T cells were transfected with HA-BCL6, HA-BCL6K379R, HA-BCL6K688R and HA-BCL6K379K688R plasmids. After stimulation with LacNa, HA-BCL6 was immunoprecipitated for detecting lactylation. g HEK293T cells were transfected with HA-BCL6, HA-BCL6K688R, HA-BCL6K534K628R, HA-BCL6K534K628K688R and Myc-Ub plasmids. After stimulation with LacNa, HA-BCL6 was immunoprecipitated to detect ubiquitination and lactylation. h HEK293T cells were transfected with HA-BCL6, HA-BCL6K688R, and Myc-Ub plasmids. After stimulation with LacNa, HA-BCL6 was immunoprecipitated to detect ubiquitination. i HEK293T cells were transfected with HA-BCL6, Flag-GCN5, Flag-PCAF, Flag-CBP, Flag-P300 and Myc-TIP60 plasmids. HA-BCL6 was immunoprecipitated for detecting lactylation. j Anti-CD3/28-activitated healthy CD4+ T cells were stimulated with LacNa, PCAF expression and the interaction of PCAF with BCL6 was detected. k HEK293T cells were transfected with HA-BCL6, HA-BCL6K688R, Flag-PCAF, shPCAF and Myc-Ub plasmids. After stimulation with LacNa, HA-BCL6 was immunoprecipitated to detect ubiquitination and lactylation. l Anti-CD3/28-activated healthy CD4⁺ T cells were transduced with shPCAF or shNC, stimulated with LacNa. m Healthy CD4⁺ T cells transduced with shPCAF or shNC were co-cultured with LN-associated MCs (n = 6 independent biological samples). n Mice were immunized with immunogenic self-DNA (n = 7 independent biological samples). The data are represented as the mean ± SEM. Normality and lognormality are assessed using the Shapiro-Wilk test. Two-tailed unpaired t test (n), two-tailed paired t test (m). Source data are provided as a Source Data file.