Understanding the lineage differentiation of memory T cells is a central question in immunology. We investigated this issue by analysing the expression of the chemokine receptor CCR7, which defines distinct subsets of naive and memory T lymphocytes with different homing and effector capacities and antiviral immune responses to HIV and cytomegalovirus. Ex vivo analysis of the expression of CD45RA and CCR7 antigens, together with in vitro analysis of the cell-division capacity of different memory CD8+ T-cell populations, identified four subsets of HIV- and CMV-specific CD8+ T lymphocytes, and indicated the following lineage differentiation pattern: CD45RA+ CCR7+ --> CD45RA- CCR7+ --> CD45RA- CCR7- --> CD45RA+ CCR7-. Here we demonstrate through analysis of cell division (predominantly restricted to the CCR7+ CD8+ T-cell subsets) that the differentiation of antigen-specific CD8+ T cells is a two-step process characterized initially by a phase of proliferation largely restricted to the CCR7+ CD8+ cell subsets, followed by a phase of functional maturation encompassing the CCR7- CD8+ cell subsets. The distribution of these populations in HIV- and CMV-specific CD8+ T cells showed that the HIV-specific cell pool was predominantly (70%) composed of pre-terminally differentiated CD45RA- CCR7- cells, whereas the CMV-specific cell pool consisted mainly (50%) of the terminally differentiated CD45RA+ CCR7- cells. These results demonstrate a skewed maturation of HIV-specific memory CD8+ T cells during HIV infection.

Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide. Since its expression is upregulated during inflammation, NAMPT represents a novel clinical biomarker in acute lung injury, rheumatoid arthritis, and Crohn's disease. However, its role in disease progression remains unknown. We report here that NAMPT is a key player in inflammatory arthritis. Increased expression of NAMPT was confirmed in mice with collagen-induced arthritis, both in serum and in the arthritic paw. Importantly, a specific competitive inhibitor of NAMPT effectively reduced arthritis severity with comparable activity to etanercept, and decreased pro-inflammatory cytokine secretion in affected joints. Moreover, NAMPT inhibition reduced intracellular NAD concentration in inflammatory cells and circulating TNFα levels during endotoxemia in mice. In vitro pharmacological inhibition of NAMPT reduced the intracellular concentration of NAD and pro-inflammatory cytokine secretion by inflammatory cells. Thus, NAMPT links NAD metabolism to inflammatory cytokine secretion by leukocytes, and its inhibition might therefore have therapeutic efficacy in immune-mediated inflammatory disorders.

Furin is a subtilisin-related endoprotease which processes a wide range of bioactive proteins. Furin is concentrated in the trans-Golgi network (TGN), where proteolytic activation of many precursor proteins takes place. A significant fraction of furin, however, cycles among the TGN, the plasma membrane, and endosomes, indicating that the accumulation in the TGN reflects a dynamic localization process. The cytosolic domain of furin is necessary and sufficient for TGN localization, and two signals are responsible for retrieval of furin to the TGN. A tyrosine-based (YKGL) motif mediates internalization of furin from the cell surface into endosomes. An acidic cluster that is part of two casein kinase II phosphorylation sites (SDSEEDE) is then responsible for retrieval of furin from endosomes to the TGN. In addition, the acidic EEDE sequence also mediates endocytic activity. Here, we analyzed the sorting of furin in polarized epithelial cells. We show that furin is delivered to the basolateral surface of MDCK cells, from where a significant fraction of the protein can return to the TGN. A phenylalanine-isoleucine motif together with the acidic EEDE cluster is required for basolateral sorting and constitutes a novel signal regulating intracellular traffic of furin.Furin, a member of a family of mammalian enzymes related to the yeast Kex2p and the bacterial subtilisins, is a calciumdependent serine endoprotease that cleaves proproteins at the C terminus of multibasic sites (reviewed in references 28 and 35).Although furin is concentrated in the trans-Golgi network (TGN) in the steady state, a significant fraction of the protease cycles among the plasma membrane, endosomes, and the TGN (2, 26). Rat furin is a type I integral membrane glycoprotein composed of a 714-residue luminal domain, a 21-residue transmembrane region, and a 58-amino-acid cytosolic tail (8, 23). The cytosolic tail of furin is necessary and sufficient for TGN localization (2,5,26,33,40). Several signals that control trafficking of furin have been identified in the cytosolic domain (see Fig. 2). Internalization from the cell surface involves a classical tyrosine-based signal (YKGL) and an acidic amino acid cluster (SDSEEDE) (33,39,40). The serine residues in the acidic cluster are subject to phosphorylation by casein kinase II (CKII) (12), and phosphorylation regulates the retrieval of the endoprotease from endosomes to the TGN (12,25,39). In PC12 cells, inactivation of the CKII site results in the transfer of furin into mature secretory granules from which the protease is normally excluded (6). The furin tail interacts with the TGN-enriched clathrin adapter AP-1, probably via the connector protein PACS-1 (41). Since the interaction with AP-1 is dependent on the phosphorylation state of the serines in the CKII site (6), removal of furin from mature secretory granules may be mediated by AP-1 and clathrin.Little is known about trafficking of furin in epithelial cells, where the protease may be delivered from the TGN to the apical or the basolateral plasm...

We assessed de novo T-cell generation by determining T-cell receptor-rearrangement excision circles (TRECs) based on patient age and on stage of HIV-1 infection. TRECs were measured in purified CD4 and CD8 T cells of a large cohort of HIV-1-infected subjects (n ‫؍‬ 297) with chronic infection but no previous antiretroviral treatment and of a control group of HIV-negative subjects (n ‫؍‬ 120). HIV-1-infected subjects were stratified on the basis of CD4 T-cell counts in 3 groups, early-stage disease (more than 500 CD4 T

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