Gasdermin D-mediated pyroptosis is regulated by AMPK-mediated phosphorylation in tumor cells.

Gasdermin D (GSDMD) is a critical mediator of pyroptosis, which consists of a N-terminal pore-forming domain and a C-terminal autoinhibitory domain. Its cytolytic activity is sequestered by the intramolecular autoinhibitory mechanism. Upon caspase-1/11 mediated cleavage of GSDMD, the N-terminal pore-forming domain (GD-NT) is released to mediate pyroptosis. However, it remains unclear how GD-NT is regulated once it is generated. In the current study, we developed a TetOn system in which GD-NT was selectively induced in tumor cells to explore how the cytolytic activity of GD-NT is regulated. We found that the cytolytic activity of GD-NT was negatively regulated by the AMP-activated protein kinase (AMPK) and AMPK activation rendered tumor cells resistant to GD-NT-mediated pyroptosis. Mechanistically, AMPK phosphorylated GD-NT at the serine 46 (pS46-GD), which altered GD-NT oligomerization and subsequently eliminated its pore-forming ability. In our in vivo tumor model, AMPK-mediated phosphorylation abolished GD-NT-induced anti-tumor activity and resulted in an aggressive tumor growth. Thus, our data demonstrate the critical role of AMPK in negatively regulating the cytolytic activity of GD-NT. Our data also highlight an unexpected link between GSDMD-mediated pyroptosis and the AMPK signaling pathway in certain tumor cells.

Ox40-Cre-mediated deletion of BRD4 reveals an unexpected phenotype of hair follicle stem cells in alopecia.

BRD4 is a bromodomain extraterminal domain family member and functions primarily as a chromatin reader regulating genes involved in cell-fate decisions. Here, we bred Brd4fl/fl Ox40-Cre mice in which Brd4 was conditionally deleted in OX40-expressing cells to examine the role of BRD4 in regulating immune responses. We found that the Brd4fl/fl Ox40-Cre mice developed profound alopecia and dermatitis, while other organs and tissues were not affected. Surprisingly, lineage-tracing experiments using the Rosa26fl/fl-Yfp mice identified a subset of hair follicle stem cells (HFSCs) that constitutively express OX40, and deletion of Brd4 specifically in such HFSCs resulted in cell death and a complete loss of skin hair growth. We also found that death of HFSCs triggered massive activation of the intradermal γδ T cells, which induced epidermal hyperplasia and dermatitis by producing the inflammatory cytokine IL-17. Interestingly, deletion of Brd4 in Foxp3+ Tregs, which also constitutively express OX40, compromised their suppressive functions, and this, in turn, contributed to the enhanced activation of γδ T cells, as well as the severity of dermatitis and hair follicle destruction. Thus, our data demonstrate an unexpected role of BRD4 in regulating skin follicle stem cells and skin inflammation.

Suppression of FOXP3 expression by the AP-1 family transcription factor BATF3 requires partnering with IRF4.

FOXP3 is the lineage-defining transcription factor for Tregs, a cell type critical to immune tolerance, but the mechanisms that control FOXP3 expression in Tregs remain incompletely defined, particularly as it relates to signals downstream of TCR and CD28 signaling. Herein, we studied the role of IRF4 and BATF3, two transcription factors upregulated upon T cell activation, to the conversion of conventional CD4+ T cells to FOXP3+ T cells (iTregs) in vitro. We found that IRF4 must partner with BATF3 to bind to a regulatory region in the Foxp3 locus where they cooperatively repress FOXP3 expression and iTreg induction. In addition, we found that interactions of these transcription factors are necessary for glycolytic reprogramming of activated T cells that is antagonistic to FOXP3 expression and stability. As a result, Irf4 KO iTregs show increased demethylation of the critical CNS2 region in the Foxp3 locus. Together, our findings provide important insights how BATF3 and IRF4 interactions integrate activating signals to control CD4+ cell fate decisions and govern Foxp3 expression.

Genetically targeting the BATF family transcription factors BATF and BATF3 in the mouse abrogates effector T cell activities and enables long-term heart allograft survival.

T cells must be activated and become effectors first before executing allograft rejection, a process that is regulated by diverse signals and transcription factors. In this study, we studied the basic leucine zipper ATF-like transcription factor (BATF) family members in regulating T cell activities in a heart transplant model and found that mice deficient for both BATF and BATF3 (Batf-/- Batf3-/- mice) spontaneously accept the heart allografts long-term without tolerizing therapies. Similarly, adoptive transfer of wild type T cells into Rag1-/- hosts induced prompt rejection of heart and skin allografts, whereas the Batf-/- Batf3-/- T cells failed to do so. Analyses of graft-infiltrating cells showed that Batf-/- Batf3-/- T cells infiltrate the graft but fail to acquire an effector phenotype (CD44high KLRG1+ ). Co-transfer experiments in a T cell receptor transgenic TEa model revealed that the Batf-/- Batf3-/- T cells fail to expand in vivo, retain a quiescent phenotype (CD62L+ CD127+ ), and unable to produce effector cytokines to alloantigen stimulation, which contrasted sharply to that of wild type T cells. Together, our data demonstrate that the BATF and BATF3 are critical regulators of T effector functions, thus making them attractive targets for therapeutic interventions in transplant settings.