Fig 2: Hectd3 interacts and promotes non-degradative K27- and K29-linked polyubiquitination on Malt1 and NF-?B activation in CD4+ T cells. a Immunoblot of Hectd3, Malt1, and GAPDH following Malt1 or IgG immunoprecipitation of protein extracts from draining lymph node CD4+ T cells of WT mice 13 days following EAE induction. b Immunoblot of Malt1 following polyubiquitinated protein enrichment of extract from draining lymph node CD4+ T cells of wild-type Hectd3-/- or (WT) mice 13 days following EAE induction. About 1.5× the amount of total protein from Hectd3-/- CD4+ T cells, compared to WT CD4+ T cells, was used to normalize for the reduction in Malt1 protein level in Hectd3-/- CD4+ T cells. At least 700 µg of total protein from mouse primary CD4+ T cells were enriched for ubiquitinated protein with Ubiquitinated Protein Enrichment Kit or Anti-Ub TUBE2, Agarose following manufacturer’s protocol. c, d Percentage of p65 or RelB nuclear translocation using p65/DAPI similarity analysis from ImageStream, in CD4+ T cells isolated from draining lymph nodes of Hectd3-/- and WT mice, 13 days following EAE induction. e, f ImageStream fluorescence imaging of PMA/ionomycin-induced p65 nuclear translocation (e) or RelB (f) in CD4+ T cells isolated from draining lymph nodes of Hectd3-/- or WT mice, 13 days following EAE induction. g Immunoblot of HA, Flag, and Xpress following two-step Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected with the indicated HA-Ub K only mutants, Flag-Malt1A, and Xpress-Hectd3. HA-Ub K only mutant denote that the only lysine in the HA-tagged ubiquitin is at the indicated residue, and all other lysine residues are mutated to arginine. HA-Ub K0 mutant indicate that all seven lysine residues are mutated to arginine. a–g Immunoblots, ImageStream similarity analyses, and ImageStream fluorescence imaging are representative of at least three independent experiments. Source data are provided as a Source Data file

Fig 3: Th17 cell program and pStat3 Y705 are defective in Hectd3-deficient T helper cells during EAE. a Representative flow cytometry analysis of intracellular IL-17A and GM-CSF in CD4+ T cells from the CNS of Hectd3-/- and wild-type (WT) mice, 13 days following EAE induction. b Frequencies and absolute numbers of IL-17A+, GM-CSF+, IFN?+, and IL-17A+GM-CSF+ CD4+ T cells in the CNS of Hectd3-/- and WT mice, 13 days following EAE induction; n = 5 per group, from three independent experiments. c, d Representative flow cytometry histograms, frequencies, and MFIs of CCR6, CD29, and IL-23R in draining lymph node CD4+ T cells from the indicated groups of mice 10 days following EAE induction; data (n = 6) are representative of three independent experiments. e Representative flow cytometry analysis of ROR?t+ T-bet+ CD4+ T cells in the CNS of Hectd3-/- and WT mice, 13 days following EAE induction, n = 6 per group from three independent experiments. f Absolute numbers of ROR?t+ CD4+ T cells and ROR?t+ T-bet+ CD4+ T cells in the CNS of Hectd3-/- and WT mice, 13 days following EAE induction. g Representative immunoblot of pStat3 Y705 and Stat3 protein level in CD4+ T cells isolated from draining lymph nodes of Hectd3-/- and WT mice, 13 days following EAE induction, from five independent experiments. Data are mean ± SEM; p value was obtained from Student’s t test. Source data are provided as a Source Data file. Gating strategy is shown in Supplementary Fig. 9

Fig 4: Hectd3-deficient Th17 cells have reduced pathogenicity. EAE disease scores (mean ± SEM) of WT recipients transferred with in vitro IL-23 re-stimulated CD4+ T cells from either Hectd3-/- or WT mice, induced with EAE, as indicated in Materials and methods. Details on induction of passive EAE are provided in Materials and methods. Clinical scoring of recipient mice was performed using criteria listed in Material and methods, 5 days following adoptive/passive transfer of in vitro reactivated CD4+ T cells into recipient mice; n = 5 per group from two independent experiments. Mann–Whitney two-tailed test was used for statistical analysis of EAE clinical scores. Source data are provided as a Source Data file

Fig 5: Hectd3 interacts with and promotes Stat3 polyubiquitination. a Immunoblot of Stat3 and ubiquitin following polyubiquitinated protein enrichment of extracts from dLN CD4+ T cells of Hectd3-/- or wild-type (WT) mice, 13 days following EAE induction. b Immunoblot of Hectd3 and Stat3 following Stat3 or IgG immunoprecipitation of protein extracts from dLN CD4+ T cells of WT mice 13 days following EAE induction. a, b Immunoblots are representative of three independent experiments. Source data are provided as a Source Data file