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The ubiquitin-ligating enzyme (E3) Itch plays a crucial role in the regulation of inflammation, and Itch deficiency leads to severe airway inflammation. However, the molecular mechanisms by which Itch function is regulated remain elusive. In this study, we found that nontypeable Haemophilus influenzae induces the association of Itch with Ndfip1. Both Itch(-/-) and Ndfip1(-/-) mice exhibited severe airway inflammation in response to nontypeable Haemophilus influenza, which was associated with elevated expression of proinflammatory cytokines. Ndfip1 enhanced Itch ligase activity and facilitated Itch-mediated Tak1 ubiquitination. Mechanistically, Ndfip1 facilitated recruitment of ubiquitin-conjugating enzyme (E2) UbcH7 to Itch. The N-terminal region of Ndfip1 binds to UbcH7, whereas the PY motif binds to Itch. Hence, Ndfip1 acts as an adaptor for UbcH7 and Itch. Reconstitution of full-length Ndfip1 but not the mutants that fail to interact with either UbcH7 or Itch, restored the defect in Tak1 ubiquitination and inhibited elevated proinflammatory cytokine expression by Ndfip1(-/-) cells. These results provide new mechanistic insights into how Itch function is regulated during inflammatory signaling, which could be exploited therapeutically in inflammatory diseases., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The AP-1 family member JunB is a critical regulator of T cell function. JunB is a transcriptional activator of various cytokine genes, such as IL-2, IL-4, and IL-10; however, the post-translational modifications that regulate JunB activity in T cells are poorly characterized. We show here that JunB is conjugated with small ubiquitin-like modifier (SUMO) on lysine 237 in resting and activated primary T cells and T cell lines. Sumoylated JunB associated with the chromatin-containing insoluble fraction of cells, whereas nonsumoylated JunB was also in the soluble fraction. Blocking JunB sumoylation by mutation or use of a dominant-negative form of the SUMO-E2 Ubc-9 diminished its ability to transactivate IL-2 and IL-4 reporter genes. In contrast, nonsumoylable JunB mutants showed unimpaired activity with reporter genes controlled by either synthetic 12-O-tetradecanoylphorbol-13-acetate response elements or NF-AT/AP-1 and CD28RE sites derived from the IL-2 promoter. Ectopic expression of JunB in activated human primary CD4(+) T cells induced activation of the endogenous IL-2 promoter, whereas the nonsumoylable JunB mutant did not. Thus, our work demonstrates that sumoylation of JunB regulates its ability to induce cytokine gene transcription and likely plays a critical role in T cell activation.

The Ubc13 E2 ubiquitin-conjugating enzyme is essential for BCR-, TLR-, and IL-1 receptor (IL-1R)-mediated immune responses. Although Ubc13-deficient mice show defects in BCR-, TLR/IL-1R-, or CD40-mediated activation of mitogen-activated protein kinases, the function of Ubc13 in TCR-mediated signaling and responses remains uncertain. To address this, we here generated T cell-specific conditional Ubc13-deficient mice. The frequency of T lymphocytes was severely reduced in spleens from Ubc13-deficient mice. Moreover, Ubc13-deficient thymocytes displayed defective proliferation in response to anti-CD3/CD28 or PMA/ionophore stimulation. Regarding the signal transduction, although NF-kappaB activation was modestly affected, PMA/ionophore-induced activation of Jnk and p38 was profoundly impaired in Ubc13-deficient thymocytes. In addition, PMA/ionophore-mediated ubiquitination of NF-kappaB essential modulator (NEMO)/IkappaB kinase gamma (IKKgamma) and phosphorylation of TGF-beta-activated kinase 1 (TAK1) were nearly abolished in Ubc13-deficient thymocytes. Thus, Ubc13 plays an important role in thymocyte TCR-mediated signaling and immune responses.

Vaccination with class I tumor peptides has been performed to induce tumor-reactive CD8(+) T cells in vivo. However, the kinds of immune responses that vaccination might elicit in patients are not fully understood. In this study we tried to elucidate the mechanisms by which vaccination of class I binding tumor peptides into an HLA-A2(+) lung cancer patient elicited dramatic amounts of IgG1 and IgG2 specific to a nonamer peptide, ubiquitin-conjugated enzyme variant Kua (UBE2V)(43-51). The UBE2V(43-51) peptide contains cysteine at the sixth position. HLA-DR-restricted and UBE2V(43-51) peptide-recognizing CD4(+) T cells were induced from postvaccination, but not from prevaccination, PBMCs of the cancer patient. In addition, a CD4(+) T cell line (UB-2) and its clone (UB-2.3), both of which recognize the UBE2V(43-51) peptide in the context of HLA-DRB1*0403 molecules, were successfully established from postvaccination PBMCs. The peptide vaccination increased the frequency of peptide-specific T cells, especially CD4(+) T cells. In contrast, mass spectrometric analysis revealed that the vaccinated UBE2V(43-51) peptide contained both monomeric and dimeric forms. Both forms, fractionated by reverse phase HPLC, were recognized by UB-2 and UB-2.3 cells. Recognition by these CD4(+) T cells was observed despite the addition of a reduction reagent or the fixation of APC. Overall, these results indicate that vaccination with class I tumor peptides can induce HLA-DR-restricted CD4(+) T cells in vivo and elicit humoral immune responses, and that a cysteine-containing peptide can be recognized by CD4(+) T cells not only as a monomer, but also as a dimer.