Otud7b facilitates T cell activation and inflammatory responses by regulating Zap70 ubiquitination

Abstract

Signal transduction from the T cell receptor (TCR) is crucial for T cell-mediated immune responses and, when deregulated, also contributes to the development of autoimmunity. How TCR signaling is regulated is incompletely understood. In this study, we demonstrate a ubiquitin-dependent mechanism in which the deubiquitinase Otud7b has a crucial role in facilitating TCR signaling. Upon TCR ligation, Otud7b is rapidly recruited to the tyrosine kinase Zap70, a central mediator of TCR-proximal signaling. Otud7b deficiency attenuates the activation of Zap70 and its downstream pathways and impairs T cell activation and differentiation, rendering mice refractory to T cell-mediated autoimmune and inflammatory responses. Otud7b facilitated Zap70 activation by deubiquitinating Zap70, thus preventing the association of Zap70 with the negative-regulatory phosphatases Sts1 and Sts2. These findings establish Otud7b as a positive regulator of TCR-proximal signaling and T cell activation, highlighting the importance of deubiquitination in regulating Zap70 function.

Figure 1.

Otud7b deficiency perturbs T cell homeostasis in older mice. (A–D) Flow cytometry analyses of the percentage of naive (CD44^loCD62L^hi) and memory (CD44^hiCD62L^lo) CD4⁺ T cells (A and C) or the percentage of naive (CD44^lo) and memory (CD44^hi) CD8⁺ T cells (B and D) in the spleen of young (A and B; 6–8-wk-old) or older (C and D; 12-mo-old) Otud7b^+/+ (+/+) and Otud7b^−/− (−/−) mice. Data are presented as representative plots (left) and summary graphs of the mean ± SD values of three independent experiments (n = 4 for each experiment; right). (E–H) ICS and flow cytometry analyses of Th1 (IFN-γ⁺) and Th17 (IL-17A⁺) effector cells (E and G) or T reg cells (Foxp3⁺; F and H) in the spleen (E and F) or lamina propria of small intestine (F and H) of Otud7b^+/+ and Otud7b^−/− mice (12 mo old). Data are percentage of CD4⁺CD44⁺ cells (E and F) or total CD4⁺ T cells (G and H) and presented as representative plots (left) and summary graphs of three independent experiments (n = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.

Otud7b is required for T cell responses to bacterial infection and lymphopenic conditions. (A and B) ICS and flow cytometry analyses of IFN-γ–producing CD4⁺ and CD8⁺ T cells in the spleen of WT and Otud7b^−/− mice infected with LM-OVA for 7 d. Splenocytes were restimulated for 5 h with LLO190-201 (A) or OVA257-264 (B) peptide in the presence of monesin before the analyses, and data are presented as a representative plot (left) or summary graph (right). (C) L. monocytogenes titer in the liver, expressed as CFUs. (D and E) Bodyweight loss (D; percent of starting bodyweight) and colon histology (E; H&E staining) of Rag1^−/− mice adoptively transferred with WT or Otud7b^−/− CD4⁺CD45RB^hi T cells for the indicated time periods (D) or 8 wk (E). Data in all panels are representative of three independent experiments (n = 5). *, P < 0.05; **, P < 0.01.

Figure 3.

Otud7b deficiency attenuates T cell activation and ameliorates EAE induction. (A) Mean clinical scores of WT and Otud7b^−/− mice after EAE induction by MOG_35-55 immunization. The data represent two independent experiments (n = 8 for each experiment). (B and C) Flow cytometry analysis of infiltrating CD4⁺ and CD8⁺ T cells, infiltrating monocytes (CD11b⁺CD45^hi), and resident microglia (CD11b⁺CD45^lo) in the CNS of day 14 EAE-induced Otud7b^+/+ and Otud7b^−/− mice, showing a representative plot (B) and a summary graph (C). (D–F) ICS and flow cytometry analysis of IFN-γ⁺ Th1 and IL-17A⁺ Th17 cells in the spleen, draining LN, and CNS of EAE-induced (day 14) WT and Otud7b^−/− mice. Data are presented as a representative plot (D) and summary graphs of the percentage (E) or absolute numbers (F) of Th1 and Th17 cells. (G and H) Recall responses of antigen-specific T cells in the splenocytes and draining LN (dLN) cells of EAE-induced (day 14) Otud7b^+/+ and Otud7b^−/− mice. Splenocytes and draining LN cells were stimulated with the indicated concentration (G) or 10 µg/ml (H) of MOG peptide for 2 d and subjected to cell proliferation assay (G; based on [³H]-thymidine incorporation) and ELISA analyses (H). Data in B–H are representative of two independent experiments (n = 3 for each experiment). (I) Mean clinical scores of WT and Otud7b^−/− chimeric mice (TCRβ/δ^−/− mice adoptively transferred with WT or Otud7b^−/− bone marrows mixed with TCRβ/δ^−/− bone marrows) induced for EAE by MOG_35-55 immunization (n = 6). Data are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.

Otud7b regulates T cell activation and Th1 cell differentiation in vitro. (A and B) Cell proliferation based on [³H]-thymidine incorporation (A) and ELISA analyses of the indicated cytokines in the supernatants (B) of WT or Otud7b^−/− naive CD4⁺ splenic T cells stimulated for 40 h with the indicated doses of agonistic anti-CD3 and anti-CD28 antibodies. (C) qRT-PCR analyses of WT or Otud7b^−/− naive T cells stimulated with anti-CD3 and anti-CD28 antibodies for the indicated time periods. (D) ICS and flow cytometry analyses of Th1 cells, Th17 cells differentiated under TGF-β– and IL-1β–stimulating conditions, and T reg cells generated by in vitro differentiation of naive CD4⁺ T cells, presented as representative plots (left) and summary graphs of mean ± SD values of multiple mice (right; n = 4). Data in all panels are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5.

Otud7b^−/− T cells have impaired TCR signaling. (A–E) IB analyses of the indicated phosphorylated (p-) and total proteins (A and C–E) and EMSA analysis of the indicated transcription factors in the nuclear extracts (B) of total T cells (A–C) or naive CD4⁺ (D) and naive CD8⁺ (E) T cells acutely stimulated with anti-CD3 plus anti-CD28 using an antibody cross-linking method. (F) IB analysis using whole-cell extracts of EL4 cells transduced with a nonsilencing shRNA control (EL4-shCtrl) or an Otud7b-specific shRNA (EL4-shOtud7b), acutely stimulated with anti-CD3 plus anti-CD28 using an antibody-cross-linking method. Data in all panels are representative of three independent experiments.

Figure 6.

Otud7b interacts with Zap70 and regulates Zap70 ubiquitination upon TCR stimulation. (A) Co-IP analysis of Zap70–Otud7b interaction (top two) and direct IB analysis of Zap70 and Otud7b expression (bottom two) in WT or Otud7b^−/− T cells acutely stimulated with anti-CD3 plus anti-CD28 using an antibody cross-linking method. (B) Co-IP analysis of Zap70–Otud7b interaction (top) and direct IB assays (bottom two) using lysates of HEK293 cells transfected with Zap70, along with expression vectors for Otud7b or its mutants. (C) IB analyses of the indicated phosphorylated (p-) or total proteins in whole-cell lysates of Otud7b-knockdown EL4 cells (EL4-shOtud7b) reconstituted with WT Otud7b or its mutants, stimulated with anti-CD3 plus anti-CD28 antibodies. (D–F) Zap70 ubiquitination assays in anti-CD3/anti-CD28–stimulated primary T cells derived from Otud7b^+/+ or Otud7b^−/− mice (D), control, or Otud7b-knockdown EL4 cells (E), and Otud7b-knockdown EL4 cells reconstituted with WT Otud7b or its mutants (F). Data are representative of three (A–E) or two (F) independent experiments.

Figure 7.

Otud7b regulates the interaction of Sts1/2 with Zap70. (A–C) Co-IP assays to detect Zap70–Sts1/2 interaction in whole-cell lysates of anti-CD3/anti-CD28–stimulated Otud7b^+/+ and Otud7b^−/− primary T cells (A), control and Otud7b-knockdown EL4 cells (B), or Otud7b-knockdown EL4 cells reconstituted with WT Otud7b or its mutants (C). (D) IB analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of control EL4 cells (EL4-shCtrl) or Otud7b-knockdown EL4 cells (EL4-shOtud7b) that were further transduced with a nonsilencing control shRNA (Ctrl shRNA) or two different Sts1 shRNAs (#1 and #2). (E) IB analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of WT or Otud7b^−/− T cells transduced with a nonsilencing control shRNA or Sts1 shRNA that were stimulated as indicated. Data are representative of three (A–D) or two (E) independent experiments.

Figure 8.

Zap70 ubiquitination at K544 mediates recruitment of Sts1/2. (A) Results of mass spectrometry analyses of Zap70 ubiquitination in EL4 cells stimulated for 5 min with anti-CD3 plus anti-CD28 (A) or 293 cells transfected with Zap70 and ubiquitin (B–D), showing the identified peptides and the ubiquitination sites (K544, K304, and K538) highlighted in red. (B) Zap70 ubiquitination assay using lysates of 293 cells transfected with WT Zap70 or its indicated mutants along with HA-tagged ubiquitin, either in the presence (+) or absence (−) of Otud7b. (C) Zap70 ubiquitination assay using lysates of Zap70-deficient Jurkat cell line (p116) reconstituted with WT or mutant forms of Zap70 fused to GFP, which were either not treated (−) or stimulated (+) for 10 min with anti-CD3 plus anti-CD28 antibodies. The p116 cells reconstituted with untagged Zap70 (p116c) was included as a positive control. (D) Co-IP assays to detect the Zap70/Sts1 binding in the p116 cells described in C. (E) IB analysis of the phosphorylated (p-) and total proteins in the p116 cells described in C. Data are representative of three independent experiments (B–E).