In response to Toll-like receptor ligands, dendritic cells (DCs) dramatically enhance their antigen presentation capacity by stabilizing at the cell-surface MHC II molecules. We demonstrate here that, in human monocyte-derived DCs, the RING-CH ubiquitin E3 ligase, membrane-associated RING-CH I (MARCH I), promotes the ubiquitination of the HLA-DR ␤-chain. Thus, in nonactivated DCs, MARCH I induces the surface internalization of mature HLA-DR complexes, therefore reducing their stability and levels. We further demonstrate that the maturation-dependent down-regulation of MARCH I is a key event in MHC class II up-regulation at the surface of LPS-activated DCs. MARCH I is, therefore, a major regulator of HLA-DR traffic, and its loss contributes to the acquisition of the potent immunostimulatory properties of mature human DCs.antigen presentation ͉ ubiquitin ligase ͉ endocytosis ͉ LPS ͉ TLR A ctivation of dendritic cells (DCs) by invading pathogens involves a process of maturation that converts an immature DC (iDC) to a mature DC (mDC) and a subsequent Toll-like receptor (TLR)-mediated immune response. This process is accompanied by a marked cellular reorganization (1, 2). Redistribution of MHC class II molecules (MHC class II) from late endosomal compartments to the plasma membrane (3) represents one of the major functional events observed during the acquisition of the immunostimulatory function by mDCs. Recently in mouse DCs, ubiquitination of lysine-225 in the cytoplasmic tail of MHC II ␤-chain has been shown to be determinant for the targeting and accumulation of the mature form of MHC II in the lysosomes of iDCs (4, 5). Interestingly, ubiquitination of MHC II in murine DCs ceases on maturation, thus contributing to the increased level of MHC II at the cell surface (4, 5). Membrane-associated RING-CH (MARCH) proteins represent a family of E3 ubiquitin ligases, which all contain a variant catalytical RING-finger domain (RING-CH domain, C4HC3) located at the N terminus (6-10). It has been recently reported that transgenic overexpression of MARCH VIII/c-MIR in mouse splenic antigen-presenting cells (APCs) leads to internalization of surface CD86/B7-2 and MHC II, and subsequent lysosomal degradation (10). Furthermore, deletion of MARCH I, another E3 ubiquitin ligase, promoted an increase of surface MHC II levels in mouse B cells, although a direct implication of this ligase in the process of MHC II internalization was excluded (11). We identify here human MARCH I as a potent regulator of HLA-DR surface expression and explore its role during human monocyte-derived DC (MoDC) activation. We further show that MARCH I expression is lost at late stages of DC maturation and thereby participates in the stabilization of peptideloaded HLA-DR at the cell surface. Our observations suggest that MARCH I is essential to coordinate MHC II-restricted antigen presentation during human DC activation by TLR ligands. Results MHC Class II Ubiquitination Is Regulated in MoDCs.The subcellular localization of MHC class II (HLA-DR) in CD14 ϩ M...

Energetic metabolism reprogramming is critical for cancer and immune responses. Current methods to functionally profile the global metabolic capacities and dependencies of cells are performed in bulk. We designed a simple method for complex metabolic profiling called SCENITH, for Single Cell ENergetIc metabolism by profilIng Translation inHibition. SCENITH allows for the study of metabolic responses in multiple cell types in parallel by flow cytometry. SCENITH is designed to perform metabolic studies ex vivo, particularly for rare cells in whole blood samples, avoiding metabolic biases introduced by culture media. We analyzed myeloid cells in solid tumors from patients and identified variable metabolic profiles, in ways that are not linked to their lineage nor their activation phenotype. SCENITH ability to reveal global metabolic functions and determine complex and linked immune-phenotypes in rare cell subpopulations will contribute to the information needed for evaluating therapeutic responses or patient stratification.