No abstract

Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A 2 inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A 2 (cPLA 2 ␣). Knocking down cPLA 2 ␣ expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA 2 ␣ fusion protein (EGFP-cPLA 2 ) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA 2 ␣ at Ser 505 . Transfection of a S505A mutant cPLA 2 ␣ showed that phosphorylation at Ser 505 is key for enzyme activity and LD formation. cPLA 2 ␣ contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA 2 ␣ inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA 2 ␣ in the formation of nascent LD from the endoplasmic reticulum.

Regulatory T cells (Treg cells), which maintain immune homeostasis and self-tolerance, form an immunological synapse (IS) with antigen-presenting cells (APCs). However, signaling events at the Treg IS remain unknown. Here we show that protein kinase C-η (PKC-η) associated with CTLA-4 and was recruited to the Treg IS. PKC-η-deficient Treg cells displayed defective suppressive activity, including suppression of tumor immunity but not autoimmune colitis. Phosphoproteomic analysis revealed an association between CTLA-4-PKC-η and the GIT-PIX-PAK complex, an IS-localized focal adhesion complex. Defective activation of this complex in PKC-η-deficient Treg cells was associated with reduced CD86 depletion from APCs by Treg cells. These results reveal a novel CTLA-4-PKC-η signaling axis required for contact-dependent suppression, implicating this pathway as a potential cancer immunotherapy target.