Highlights d CD8 + T cell exhaustion is correlated with a high cholesterol level d Tumor microenvironment is enriched with cholesterol d Cholesterol in the tumor microenvironment induces CD8 + T cell exhaustion d ER stress-XBP1 pathway is required for cholesterol-induced CD8 + T cell exhaustion

CD8+ T cells can be polarized into IL-9–secreting (Tc9) cells. Ma et al. show that Tc9 cell differentiation is associated with a low cholesterol reprogramming profile, and manipulating cholesterol content in polarizing Tc9 cells significantly affects Tc9 differentiation and antitumor response.

FOXP3 is a forkhead family transcriptional repressor important for the development and function of CD4+CD25+ regulatory T cells. In humans, FOXP3 is expressed as two isoforms, a full-length form and a smaller form lacking exon 2. These two isoforms are expressed in approximately equal amounts in circulating regulatory T cells, and are induced equally in freshly activated CD4+CD25− T cells. Herein, we show that FOXP3 interacts with retinoic acid receptor-related orphan receptor (ROR)α, and that this interaction inhibits transcriptional activation mediated by RORα. Full-length FOXP3, but not the isoform lacking exon 2, interacts with RORα, and the region of FOXP3 involved in the interaction is encoded by exon 2. Mutation of the LxxLL motif in FOXP3, located in exon 2, abolished interaction and repression by FOXP3. Additionally, the inhibition of RORα by FOXP3 does not require an intact forkhead domain, demonstrating a mode of FOXP3 function that is independent of DNA binding. Interestingly, expression of RORα in T cells leads to the expression of genes that define Th17 cells, and the expression of each of these gene was inhibited by coexpression of full-length, but not ΔEx2, FOXP3. These data expand the possible targets of FOXP3-mediated repression and demonstrate functional differences between FOXP3 isoforms.

Targeted knockout of genes in primary human cells using CRISPR-Cas9 mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a pro-inflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9 mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a pro-inflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli.

Granzyme B expression is essential for eliciting NK cell cytotoxicity and T cell function. However, its transcriptional regulatory mechanism is not well understood. In this report, we demonstrate in human NK cells and T cells that the NF-κB-signaling pathway is involved in such control. Furthermore, a novel downstream human granzyme B gene sequence (GGAGATTCCC) was identified for NF-κB binding. EMSA, luciferase, and chromatin immunoprecipitation assays in vitro and in vivo indicated that this NF-κB binding site is functional in an NK cell line and its primary counterpart. Our data also demonstrate that this binding site is functional in Jurkat T cells. Taken together, we identified a novel NF-κB binding site, which plays a pivotal role in controlling human granzyme B gene transcription.