Myeloid-derived suppressor cells (MDSCs) are well known regulators of regulatory T cells (Treg cells); however, the direct regulation of MDSCs by Treg cells has not been well characterized. We find that colitis caused by functional deficiency of Treg cells leads to altered expansion and reduced function of MDSCs. During differentiation of MDSCs in vitro from bone marrow cells, Treg cells enhanced MDSC function and controlled their differentiation through a mechanism involving transforming growth factor-β (TGF-β). TGF-β-deficient Treg cells were not able to regulate MDSC function in an experimentally induced model of colitis. Finally, we evaluated the therapeutic effect of TGF-β-mediated in-vitro-differentiated MDSCs on colitis. Adoptive transfer of MDSCs that differentiated with TGF-β led to better colitis prevention than the transfer of MDSCs that differentiated without TGF-β. Our results demonstrate an interaction between Treg cells and MDSCs that contributes to the regulation of MDSC proliferation and the acquisition of immunosuppressive functions.

Cereblon (CRBN) is a substrate receptor protein for the CRL4A E3 ubiquitin ligase complex. In this study, we report on a new regulatory role of CRBN in TLR4 signaling. CRBN overexpression leads to suppression of NF-κB activation and production of pro-inflammatory cytokines including IL-6 and IL-1β in response to TLR4 stimulation. Biochemical studies revealed interactions between CRBN and TAK1, and TRAF6 proteins. The interaction between CRBN and TAK1 did not affect the association of the TAB1 and TAB2 proteins, which have pivotal roles in the activation of TAK1, whereas the CRBN-TRAF6 interaction critically affected ubiquitination of TRAF6 and TAB2. Binding mapping results revealed that CRBN interacts with the Zinc finger domain of TRAF6, which contains the ubiquitination site of TRAF6, leading to attenuation of ubiquitination of TRAF6 and TAB2. Functional studies revealed that CRBN-knockdown THP-1 cells show enhanced NF-κB activation and p65- or p50-DNA binding activities, leading to up-regulation of NF-κB-dependent gene expression and increased pro-inflammatory cytokine levels in response to TLR4 stimulation. Furthermore, Crbn−/− mice exhibit decreased survival in response to LPS challenge, accompanied with marked enhancement of pro-inflammatory cytokines, such as TNF-α and IL-6. Taken together, our data demonstrate that CRBN negatively regulates TLR4 signaling via attenuation of TRAF6 and TAB2 ubiquitination.

The role of cereblon (CRBN) in T cells is not well understood. We generated mice with a deletion in Crbn and found cereblon to be an important antagonist of T-cell activation. In mice lacking CRBN, CD4+ T cells show increased activation and IL-2 production on T-cell receptor stimulation, ultimately resulting in increased potassium flux and calcium-mediated signaling. CRBN restricts T-cell activation via epigenetic modification of Kcna3, which encodes the Kv1.3 potassium channel required for robust calcium influx in T cells. CRBN binds directly to conserved DNA elements adjacent to Kcna3 via a previously uncharacterized DNA-binding motif. Consequently, in the absence of CRBN, the expression of Kv1.3 is derepressed, resulting in increased Kv1.3 expression, potassium flux, and CD4+ T-cell hyperactivation. In addition, experimental autoimmune encephalomyelitis in T-cell–specific Crbn-deficient mice was exacerbated by increased T-cell activation via Kv1.3. Thus, CRBN limits CD4+ T-cell activation via epigenetic regulation of Kv1.3 expression.

Summary The roles of Notch1 and Notch2 in T‐cell function have been well studied, but the functional roles of Notch in B cells have not been extensively investigated, except for Notch2 involvement in peripheral marginal zone B‐cell differentiation. This study examined the roles of Notch1 in murine primary B cells. During B‐cell activation by B‐cell receptor ligation, Notch1 was up‐regulated while Notch2 was not. In addition, Notch1 up‐regulation itself did not contribute to the further activation of B cells, but the Notch ligand was important for Notch1‐mediated further B‐cell activation. Moreover, Notch1 deficiency significantly decreased B‐cell activation and antibody secretion under the presence of Notch ligand. These data suggest that Notch1 is an important mediator for enhancing B‐cell activation and antibody secretion by Notch ligand.

Dyskerin pseudouridine synthase 1 (DKC1) is a conserved gene encoding the RNA-binding protein dyskerin, which is an essential component of the telomerase holoenzyme. DKC1 up-regulation is frequently observed in many different human cancers including hepatocellular carcinoma (HCC); however, its regulatory mechanisms remain unclear. Thus, we investigated the regulatory mechanism of DKC1 in HCC progression. We found that protein-disulfide isomerase-associated 3 (PDIA3) interacted with the DKC1 regulatory DNA in HCC cells but not in HCC cells with elevated reactive oxygen species (ROS) levels, using liquid chromatographic-tandem mass spectrometric analysis after isolating the DKC1 regulatory region binding proteins. PDIA3 repressed DKC1 expression in HCC cells by recognizing the G-quadruplex DNA at the DKC1 location. However, oxidative modification of PDIA3 induced by ROS redistributed this protein into the cytosolic regions, which stimulated DKC1 expression. We also identified Met338 in PDIA3 as the oxidatively modified residue and validated the effect of oxidative modification using an ectopic expression system, a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 knock-in system, and a xenograft mouse model. We observed that oxidatively modified PDIA3 promoted DKC1-mediated malignancy and survival of HCC cells in vitro and in vivo. HCC tissues showed a positive association with ROS, cytoplasmic PDIA3, and nuclear DKC1 levels. HCC patients with high PDIA3 protein and DKC1 mRNA levels also displayed reduced recurrence-free survival rates. Cumulatively, the results showed that cytoplasmic PDIA3 activity could be essential in raising DKC1 expression in HCC progression and predicting poor prognoses in HCC patients. Conclusion: Our study indicates that the elevated ROS levels in HCC modulate cytoplasmic PDIA3 levels, resulting in HCC cell survival through DKC1 up-regulation.

Strong NF-κB activation requires ligation of both the CD28 co-receptor and TCR. PDK1 acts as a scaffold by binding both PKCθ and CARMA1 and is therefore essential for signaling to NF-κB. Here, we demonstrate the importance of PDK1 threonine (Thr)-513 phosphorylation in regulating the intermolecular organization of PDK1 homodimers. Thr-513 is directly involved in heterotypic PDK1 homodimer formation, in which binding is mediated through the pleckstrin homology (PH) and kinase domains. Upon activation, phosphorylated Thr-513 instead mediates homotypic intermolecular binding through the PH domains. Consequently, cell permeable peptides with a Thr-513 to Ile derivative (PTD-PDK1-Thr-513-Ile) bound the kinase domain and while a Thr-513 to Asp peptide (PTD-PDK1-Thr-513-Asp) bound the PH domain. PTD-PDK1-Thr-513-Ile blocked binding between PDK1 and PKCθ, phosphorylation of PKCθ Thr-538, and activation of both NF-κB and AKT. In contrast, PTD-PDK1-Thr-513-Asp selectively inhibited binding between PDK1 and CARMA1 and blocked TCR/CD28 induced NF-κB activation. Therefore, Thr-513 phosphorylation regulates a critical intermolecular switch governing PDK1 homodimer structure and the capacity to interact with downstream signaling pathway components. Given the pleiotropic functions of PDK1, these data may open the door to the development of immunosuppressive therapies that selectively target the PDK1 to NF-κB pathway in T cell activation.

Phosphoinositide-dependent kinase 1 (PDK1) plays an important role in integrating the T cell antigen receptor (TCR) and CD28 signals to achieve efficient NF-κB activation. PDK1 is also an important regulator of T cell development, mediating pre-TCR induced proliferation signals. However, the role of PDK1 in B cell antigen receptor (BCR) signaling and B cell development remains largely unknown. In this study we provide genetic evidence supporting the role of PDK1 in B cell survival. We found PDK1 is required for BCR mediated survival in resting B cells, likely through regulation of Foxo activation. PDK1-dependent signaling to NF-κB is not crucial to resting B cell viability. However, PDK1 is necessary for triggering NF-κB during B cell activation and is required for activated B cell survival. Together these studies demonstrate that PDK1 is essential for BCR-induced signal transduction to Foxo and NF-κB and is indispensable for both resting and activated B cell survival.

HBx acts as a multifunctional regulator that modulates various cellular responses, which can lead to development and progression of hepatocellular carcinoma (HCC). Here, we show that the HBx protein is also localized to peroxisomes, and this increases cellular reactive oxygen species (ROS) to levels that are higher than when HBx is localized to other organelles. The elevated ROS strongly activated nuclear factor (NF)-κB. In addition, the peroxisome-localized HBx increased the expressions of matrix metalloproteinases and decreased the expression of E-cadherin, which increased the invasive ability of HCC cells. Thus, a specific distribution of HBx to peroxisomes may contribute to HCC progression by increasing the invasive ability of HCC cells through elevation of the cellular ROS level.