Ndfip-mediated degradation of Jak1 tunes cytokine signalling to limit expansion of CD4+ effector T cells

Abstract

Nedd4 family E3 ubiquitin ligases have been shown to restrict T-cell function and impact T-cell differentiation. We show here that Ndfip1 and Ndfip2, activators of Nedd4 family ligases, together limit accumulation and function of effector CD4+ T cells. Using a three-part proteomics approach in primary T cells, we identify stabilization of Jak1 in Ndfip1/2-deficient T cells stimulated through the TCR. Jak1 degradation is aborted in activated T cells that lack Ndfips. In wild-type cells, Jak1 degradation lessens CD4+ cell sensitivity to cytokines during TCR stimulation, while in Ndfip-deficient cells cytokine responsiveness persists, promoting increased expansion and survival of pathogenic effector T cells. Thus, Ndfip1/Ndfip2 regulate the cross talk between the T-cell receptor and cytokine signalling pathways to limit inappropriate T-cell responses.

Figure 1. Ndfip2−/− mice do not show signs of inflammation.

(a,b) Representative flow cytometry analysis of T-cell populations from thymus and spleen of 5–7-week-old Ndfip2−/− and age-matched control mice: (a) CD4+ and CD8+ cells, (b) CD44 and CD62L expression on these cells, as noted. (c) Intracellular cytokine staining for IL-4 and IFNγ in CD4+ T cells from Ndfip2−/− and WT spleens stimulated ex vivo with PMA and ionomycin in the presence of BFA. Representative of at least five mice per genotype, 5–7 weeks of age. (d) CD4+ T cells were stimulated in vitro for the indicated time periods with αCD3/CD28. Ndfip1 and Ndfip2 expression was analysed by qPCR. Ndfip1/Ndfip2 expression relative to Actb was normalized to expression in unstimulated CD4+ T cells. Representative of a minimum of three independent experiments.

Figure 2. Ndfip2 deficiency exacerbates inflammation in Ndfip1^fl/flCD4Cre+ mice.

(a) H&E-stained sections of oesophagus, lung and skin from representative 8-week-old control, Ndfip1^fl/flCD4 Cre+ (cKO) and Ndfip2−/− Ndfip1^fl/flCD4 Cre+ (cDKO) mice (bar represents 100 μm). (b,c) Body weight (b) and (c) spleen count of 5–7-week-old WT, cKO, Ndfip2−/− and cDKO mice. Mean±s.e.m. n=5–15 mice. (d,e) Representative flow cytometry analysis of splenic CD3+CD4+ T cells, showing (d) expression CD44 and CD62L and (e) intracellular levels of IL-4 after ex vivo stimulation with PMA/ionomycin in the presence of BFA. (f–i) Quantification of (f) the number of CD4+ T cells, (g) naive CD62L^high CD4+ T cells, (h) CD44+ CD4+ T cells and (i) IL-4+ CD4s from spleen analysed by flow cytometry. Mean±s.e.m., n=7–12 mice. (j,k) Quantification of percent IL-4+ or IFNγ+ CD4+ T cells among CD44 high T cells. Mean±s.e.m., n=4–7 mice. P values calculated by one-way ANOVA with Holm–Sidak test for multiple comparisons: *P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001.

Figure 3. Ndfip deficiency causes intrinsic CD4+ effector T-cell expansion.

(a–g) Mixed foetal liver chimeras using CD45.2 WT, Ndfip1−/−, Ndfip2−/− or Ndfip1/Ndfip2 DKO foetal liver were analysed 6 weeks following reconstitution. (a) Representative CD44 and CD62L staining of splenic CD4+ T cells from Ndfip1−/− mixed chimera (top) and DKO mixed chimera (bottom), previously gated on live, singlet CD3+CD4+CD45.1 or CD45.2+ cells. (b,c) Flow cytometry analysis of CD45.1+ and CD45.2+ splenic T cells from chimeras showing (b) percentages of CD44+ cells and (c) percentages of CD44+ that are Ki67+. (d–f) Ex vivo stimulated splenocytes were stained for IL-4. (d) Representative flow plots of IL-4+ CD4+ T cells, (e) combined data from d, (f) percentages of CD44+ CD4 T cells that are IL-4+. (g) Percentages of CD45.2 cells among various T-cell subsets, normalized for reconstitution, as determined by the ratio for CD45.2:CD45.1 IgM+B220+ B cells in the bone marrow. Compartments analysed are as follows: A=IgM+B cells in bone marrow, B=double positive thymocytes, C=single positive CD4+ thymocytes, D=CD44+ CD4+ T cells in spleen, E=CD44+ CD4+ T cells in lung. Data shown in b and e were pooled from two experiments, 7–8 chimeras per group; (c,f,g) have 4–5 chimeras per group. Quantifications are average ±s.e.m. P values calculated by two-way ANOVA (b,c,e,f) or repeated measures one-way ANOVA (g), with Holm–Sidak test for multiple comparisons: *P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001.

Figure 4. Ndfip1/Ndfip2-deficient CD4+ T cells cause increased colitis.

(a–e) 0.5 × 10⁶ sorted naive CD4+ T cells from WT, Ndfip2−/−, cKO and cDKO mice were transferred into 6-week-old Rag1−/− recipients. Mice were weighed twice weekly and killed 6 weeks after transfer when 20% weight loss was observed in multiple mice. Spleen weight and body weight were compared to generate an inflammation index (a) and colons were measured (b). H&E-stained sections of the distal colon were imaged on the × 20 objective, and crypt depth was quantified (c,d). Splenocytes were stained for intracellular IL-4 and IL-17 after ex vivo stimulation with PMA/ionomycin in the presence of BFA, and analysed by flow cytometry (e). Previously gated on live singlets, CD4+, dump gate-. Quantifications shown ±s.e.m. n=5–7 mice. Control (ctrl) mice did not receive T cells. P values calculated by ordinary one-way ANOVA with Holm–Sidak test for multiple comparisons: *P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001.

Figure 5. Identification of differential ubiquitylation by proteomics.

(a) Schematic of the three proteomics methods used. (b) Area proportional Venn diagram illustrating the reproducibility of proteins identified as having SILAC ratios after TUBE enrichment in three out of four biological replicates. (c) SILAC–TUBE ratios (WT/DKO) for each protein were corrected using ‘input' ratio (DKO/WT) as calculated by label-free quantification of whole proteome DKO and WT data sets. The average corrected ratio is plotted against the average number of unique peptides observed per protein across four whole proteome LC-MS/MS experiments. Plot limited to 40 average unique peptides for clarity. Itch and Nedd4-2 (indicated) have log2 transformed corrected ratio >1 at the protein level. Itch=1.57±0.17 and Nedd4-2=3.18±0.226. (d) Overlap of proteins with corrected SILAC–TUBE ratios (observed in at least three experiments) and proteins identified with at least one K-ɛ-GG peptide (in at least one of the three K-ɛ-GG immunoprecipitation experiments). (e) Heat map illustrating reproducibility of corrected SILAC–TUBE ratios (WT/DKO, log2 transformed, observed in at least three of four experiments) for proteins identified with K-ɛ-GG peptides. Reverse=SILAC labeling swapped.

Figure 6. Ndfip1 and Ndfip2 promote Itch and Nedd4-2 activity.

(a) Immunoblot of Itch and Nedd4-2 isolated from activated WT or Ndfip1/Ndfip2 DKO CD4+ T-cell lysates by GST pulldown of Ndfip1 and Ndfip2 cytosolic domain GST fusion proteins. Coomassie stain for the GST fusion proteins revealed full-length products for each construct as well as a GST cleavage product. (b) Itch activity, in the absence or presence of Ndfip1 or Ndfip2, was analysed via TR-FRET polyubiquitylation assay. (c) E2/E3 transthiolation assay was used to test whether Ndfip2 promotes ubiquitin ‘charging' of Itch. Biotinylated ubiquitin non-covalently bound to Itch was analysed by western blot using fluorescent streptavidin (top); total protein was visualized by Coomassie stain (bottom). (d,e) As in b, human Nedd4L activity was assessed alone or in the presence of Ndfip1 or Ndfip2 by TR-FRET. Data for each panel is representative of a minimum of three independent experiments.

Figure 7. Jak1 degradation is dependent on Ndfip1/Ndfip2.

(a–j) Immunoblotting of restimulated WT and cDKO CD4+ T cells. (a) Cycloheximide was added 2 h after CD3/CD28 stimulation; cells were then incubated for an additional 4 h. (b) Level of Jak1 was normalized to GAPDH. The stability of Jak1 was determined by normalizing the percent Jak1 remaining in cDKO or cKO cells to the percent remaining in experiment-matched control cells. Data shown are average ±s.e.m. from three to five biologic replicates in more than three experiments. (c,d) p-STAT5 was quantified relative to tubulin. Relative levels of p-STAT5 in cDKO and cKO CD4+ T cells were normalized to experiment-matched control cells at 2 and 6 h of restimulation. Data shown is average ±s.e.m. for two biologic replicates. (e,f) Immunoblotting of total STAT5 in restimulated WT and cDKO CD4+ T cells. Cells were stimulated for 2 h, and cycloheximide was added for an additional 2 h of stimulation. (f) Level of STAT5 was normalized to GAPDH. The stability of STAT5 was determined by normalizing the relative STAT5 remaining after stimulation to the amount of STAT5 at 2 h. Data shown are average ±s.e.m. from three biologic replicates. (g–j) Levels of Jak1, normalized to GAPDH, at various timepoints following restimulation of WT and cDKO CD4+ T cells relative to Jak1 in IL-2-rested cells at time 0. (h) Rate of Jak1 degradation in relative units per hour over 6 h of stimulation. (i,j) Levels of Jak1, normalized to GAPDH, at various timepoints following rest in the absence of IL-2/TCR, of WT and cDKO CD4+ T cells relative to Jak1 in IL-2-rested cells at time 0. (j) As in g and i, levels of Jak1, normalized to GAPDH, 24 h ±TCR stimulation of WT and cDKO CD4+ T cells relative to Jak1 in IL-2-rested cells at time 0. Data shown in g–j average ±s.e.m. from two to four biologic replicates in more than three experiments. P value calculated by two sample, unpaired t-test *P<0.05.

Figure 8. Ndfip-deficient T cells show persistent cytokine signalling.

(a–c) Sorted naive CD4+ T cells from WT and cDKO mice were stimulated with αCD3/CD28 in the presence of Jak inhibitor I (JAKi) and analysed on day 5 by flow cytometry. (a) Representative plots of CFSE dilution and viability staining in WT and cDKO CD4+ T cells ±Jak inhibitor (31.2 nM). (b,c) Quantification of (b) percent of cDKO and WT cells divided four or more times relative to DMSO-treated experiment-matched WT cells and (c) viability of cDKO and WT CD4+ T cells normalized to DMSO-treated experiment-matched WT cells. Data shown is average ±s.e.m. from six biologic replicates. P values calculated by multiple t-test with Holm–Sidak correction. (d,e) p-STAT5 staining in cDKO and WT CD4+ T cells rested in the absence of IL-2 overnight then treated ±αCD3/CD28 beads before addition of IL-2. (d) Representative flow cytometry histograms. (e) Quantification of p-STAT5 MFI for eight to nine biologic replicates and four independent experiments, showing average ±s.e.m. Patterned bars indicate addition of exogenous IL-2. P values calculated by two-way ANOVA with Holm–Sidak test for multiple comparisons. *P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001.