SummaryThrough a network of progressively maturing vesicles, the endosomal system connects the cell’s interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle “cloud” and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell’s periphery. By drawing the endosomal system’s architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.

Resident memory T cells (TRM) inhabit peripheral tissues and are critical for protection against localized infections. Recently, it has become evident that CD103+ TRM are not only important in combating secondary infections, but also for the elimination of tumor cells. In several solid cancers, intratumoral CD103+CD8+ tumor infiltrating lymphocytes (TILs), with TRM properties, are a positive prognostic marker. To better understand the role of TRM in tumors, we performed a detailed characterization of CD8+ and CD4+ TIL phenotype and functional properties in non-small cell lung cancer (NSCLC). Frequencies of CD8+ and CD4+ T cell infiltrates in tumors were comparable, but we observed a sharp contrast in TRM ratios compared to surrounding lung tissue. The majority of both CD4+ and CD8+ TILs expressed CD69 and a subset also expressed CD103, both hallmarks of TRM. While CD103+CD8+ T cells were enriched in tumors, CD103+CD4+ T cell frequencies were decreased compared to surrounding lung tissue. Furthermore, CD103+CD4+ and CD103+CD8+ TILs showed multiple characteristics of TRM, such as elevated expression of CXCR6 and CD49a, and decreased expression of T-bet and Eomes. In line with the immunomodulatory role of the tumor microenvironment, CD8+ and CD4+ TILs expressed high levels of inhibitory receptors 2B4, CTLA-4, and PD-1, with the highest levels found on CD103+ TILs. Strikingly, CD103+CD4+ TILs were the most potent producers of TNF-α and IFN-γ, while other TIL subsets lacked such cytokine production. Whereas, CD103+CD4+PD-1low TILs produced the most effector cytokines, CD103+CD4+PD-1++ and CD69+CD4+PD-1++ TILs produced CXCL13. Furthermore, a large proportion of TILs expressed co-stimulatory receptors CD27 and CD28, unlike lung TRM, suggesting a less differentiated phenotype. Agonistic triggering of these receptors improved cytokine production of CD103+CD4+ and CD69+CD8+ TILs. Our findings thus provide a rationale to target CD103+CD4+ TILs and add co-stimulation to current therapies to improve the efficacy of immunotherapies and cancer vaccines.

Upon activation, conventional T (Tconv) cells undergo an mTOR-driven glycolytic switch. Regulatory T (Treg) cells reportedly repress the mTOR pathway and avoid glycolysis. However, here we demonstrate that human thymusderived (t)Treg cells can become glycolytic in response to tumor necrosis factor receptor 2 (TNFR2) costimulation. This costimulus increases proliferation and induces a glycolytic switch in CD3-activated tTreg cells, but not in Tconv cells. Glycolysis in CD3/TNFR2-activated tTreg cells is driven by PI3-kinase/mTOR signaling and supports tTreg cell identity and suppressive function. Contrary to glycolytic Tconv cells, glycolytic tTreg cells do not show net lactate secretion and shuttle glucose-derived carbon into the tricarboxylic acid cycle. Ex vivo characterization of blood-derived TNFR2 high CD4 + CD25 high CD127 low effector T cells, which were FOXP3 + IKZF2 + , revealed an increase in glucose consumption and intracellular lactate levels, identifying them as glycolytic tTreg cells. Our study links TNFR2 costimulation in human tTreg cells to metabolic remodeling, providing an additional avenue for drug targeting.receptor 2 (TNFR2, TNFRSF1B, CD120b) costimulation in tTreg and Tconv cells. TNFR2 was previously shown to be important for Treg cell responses and protection against autoimmunity in human and mouse 30,31 and is considered a clinical target for selective Treg expansion or inhibition in transplant rejection, autoimmunity, or cancer 9, 10 . We here report that CD3-activated tTreg cells selectively respond to TNFR2 costimulation by proliferation and a PI3K/mTOR-driven glycolytic switch that is important for tTreg cell identity and function. We also identify unique elements of the glycolytic program in tTreg cells and validate our findings in tTreg cells directly isolated from human blood. RESULTS A novel strategy allows for stable human Treg cell expansion in the absence of rapamycinHuman Treg cells occur in low frequency in the blood and therefore, expansion protocols are used for clinical application 32 . In such protocols, Treg cells are flow cytometrically sorted and expanded in presence of the mTOR inhibitor rapamycin that selectively inhibits proliferation of contaminating Tconv cells 12 . However, since rapamycin affects many aspects of metabolism, these expansion protocols are not suitable to generate Treg cells for metabolic studies. Also, such cultures may still be contaminated with pTreg cells that can convert back to Tconv cells and confound data interpretation. We therefore employed a novel method to purify stable human tTreg cells, based on the marker glycoprotein (GP)A33 33 . Among CD4 + T cells, naïve Tconv cells were purified by flow cytometry on the basis of a CD25 low CD127 high CD45RA + GPA33 int phenotype and naïve tTreg cells on the basis of a CD25 high CD127 low CD45RA + GPA33 high phenotype (Extended Data Figure 1a). Phenotypic analysis of these populations indicated that the naïve tTreg cells could be discriminated from Tconv cells as previously defined 34 by expressi...