Epigenetic regulation of Kcna3-encoding Kv1.3 potassium channel by cereblon contributes to regulation of CD4+ T-cell activation.

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.

Transition from heterotypic to homotypic PDK1 homodimerization is essential for TCR-mediated NF-κB activation.

Strong NF-κB activation requires ligation of both the CD28 coreceptor and TCR. Phosphoinositide-dependent kinase 1 (PDK1) acts as a scaffold by binding both protein kinase Cθ (PKCθ) and CARMA1, and is therefore essential for signaling to NF-κB. In this article, we demonstrate the importance of PDK1 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 (protein transduction domain [PTD]-PDK1-Thr(513)-Ile) bound the kinase domain, whereas 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.

Intestinal intraepithelial TCRγδ⁺ T cells are activated by normal commensal bacteria.

TCRγδ(+) T cells play a critical role in protecting the intestinal mucosa against pathogenic infection. In the absence of infection, TCRγδ(+) T cell activation must be continuously regulated by T regulatory cells (Treg) to prevent the development of colitis. However, the activation of intestinal TCRγδ(+) T cells under normal conditions has not been clearly resolved. In order to determine TCRγδ(+) T cell activation in vivo, we designed an NF-κB based reporter system. Using the recombinant lentiviral method, we delivered the NF-κB reporter to isolated TCRγδ(+) T cells, which were then adoptively transferred into normal mice. Our data indicate that the NF-κB activation level in TCRγδ(+) T cells is higher in the intestinal intraepithelial layer than in the lamina propria region. In addition, the surface expression level of lymphocyte activation marker CD69 in TCRγδ(+) T cells is also higher in the intestinal intraepithelial layer and this activation was reduced by Sulfatrim treatment which removes of commensal bacteria. Collectively, our data indicate that the TCRγδ(+) T cell population attached to the intestinal lumen is constitutively activated even by normal commensal bacteria.