Differences in CD80 and CD86 transendocytosis reveal CD86 as a key target for CTLA-4 immune regulation.

CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity.

Availability of 25-hydroxyvitamin D(3) to APCs controls the balance between regulatory and inflammatory T cell responses.

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], the active form of vitamin D, exerts potent effects on several tissues including cells of the immune system, where it affects T cell activation, differentiation and migration. The circulating, inactive form of vitamin D, 25(OH)D(3), is generally used as an indication of vitamin D status. However, use of this precursor depends on its uptake by cells and subsequent conversion by the enzyme 25(OH)D(3)-1α-hydroxylase (CYP27B1) into active 1,25(OH)(2)D(3). Using human T cells, we show in this study that addition of inactive 25(OH)D(3) is sufficient to alter T cell responses only when dendritic cells (DCs) are present. Mechanistically, CYP27B1 is induced in DCs upon maturation with LPS or upon T cell contact, resulting in the generation and release of 1,25(OH)(2)D(3), which subsequently affects T cell responses. In most tissues, vitamin D binding protein acts as a carrier to enhance the use of vitamin D. However, we show that vitamin D binding protein modulates T cell responses by restricting the availability of inactive 25(OH)D(3) to DC. These data indicate that the level of free 25(OH)D(3) available to DCs determines the inflammatory/regulatory balance of ensuing T cell responses.

Constitutive clathrin-mediated endocytosis of CTLA-4 persists during T cell activation.

CTLA-4 is one of the most important negative regulators of the T cell immune response. However, the subcellular distribution of CTLA-4 is unusual for a receptor that interacts with cell surface transmembrane ligands in that CTLA-4 is rapidly internalized from the plasma membrane. It has been proposed that T cell activation can lead to stabilization of CTLA-4 expression at the cell surface. Here we have analyzed in detail the internalization, recycling, and degradation of CTLA-4. We demonstrate that CTLA-4 is rapidly internalized from the plasma membrane in a clathrin- and dynamin-dependent manner driven by the well characterized YVKM trafficking motif. Furthermore, we show that once internalized, CTLA-4 co-localizes with markers of recycling endosomes and is recycled to the plasma membrane. Although we observed limited co-localization of CTLA-4 with lysosomal markers, CTLA-4 was nonetheless degraded in a manner inhibited by lysosomal blockade. T cell activation stimulated mobilization of CTLA-4, as judged by an increase in cell surface expression; however, this pool of CTLA-4 continued to endocytose and was not stably retained at the cell surface. These data support a model of trafficking whereby CTLA-4 is constitutively internalized in a ligand-independent manner undergoing both recycling and degradation. Stimulation of T cells increases CTLA-4 turnover at the plasma membrane; however, CTLA-4 endocytosis continues and is not stabilized during activation of human T cells. These findings emphasize the importance of clathrin-mediated endocytosis in regulating CTLA-4 trafficking throughout T cell activation.

Interleukin-7 and interleukin-15 drive CD4+CD28null T lymphocyte expansion and function in patients with acute coronary syndrome.

AIMS: Inflammation has important roles in atherosclerosis. CD4+CD28null (CD28null) T cells are a specialized T lymphocyte subset that produce inflammatory cytokines and cytotoxic molecules. CD28null T cells expand preferentially in patients with acute coronary syndrome (ACS) rather than stable angina and are barely detectable in healthy subjects. Importantly, ACS patients with CD28null T-cell expansion have increased risk for recurrent acute coronary events and poor prognosis, compared to ACS patients in whom this cell subset does not expand. The mechanisms regulating CD28null T-cell expansion in ACS remain elusive. We therefore investigated the role of cytokines in CD28null T-cell expansion in ACS. METHODS AND RESULTS: High-purity sorted CD4+ T cells from ACS patients were treated with a panel of cytokines (TNF-α, IL-1ß, IL-6, IL-7, and IL-15), and effects on the number, phenotype, and function of CD28null T cells were analysed and compared to the control counterpart CD28+ T-cell subset. IL-7- and IL-15-induced expansion of CD28null T cells from ACS patients, while inflammatory cytokines TNF-α, IL-1ß, and IL-6 did not. The mechanisms underlying CD28null T-cell expansion by IL-7/IL-15 were preferential activation and proliferation of CD28null T cells compared to control CD28+ T cells. Additionally, IL-7/IL-15 markedly augmented CD28null T-cell cytotoxic function and interferon-γ production. Further mechanistic analyses revealed differences in baseline expression of component chains of IL-7/IL-15 receptors (CD127 and CD122) and increased baseline STAT5 phosphorylation in CD28null T cells from ACS patients compared to the control CD28+ T-cell subset. Notably, we demonstrate that CD28null T-cell expansion was significantly inhibited by Tofacitinib, a selective JAK1/JAK3 inhibitor that blocks IL-7/IL-15 signalling. CONCLUSION: Our novel data show that IL-7 and IL-15 drive the expansion and function of CD28null T cells from ACS patients suggesting that IL-7/IL-15 blockade may prevent expansion of these cells and improve patient outcomes.

An unmet actin requirement explains the mitotic inhibition of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is the major internalisation route for many different receptor types in mammalian cells. CME is shut down during early mitosis, but the mechanism of this inhibition is unclear. In this study, we show that the mitotic shutdown is due to an unmet requirement for actin in CME. In mitotic cells, membrane tension is increased and this invokes a requirement for the actin cytoskeleton to assist the CME machinery to overcome the increased load. However, the actin cytoskeleton is engaged in the formation of a rigid cortex in mitotic cells and is therefore unavailable for deployment. We demonstrate that CME can be 'restarted' in mitotic cells despite high membrane tension, by allowing actin to engage in endocytosis. Mitotic phosphorylation of endocytic proteins is maintained in mitotic cells with restored CME, indicating that direct phosphorylation of the CME machinery does not account for shutdown. DOI: http://dx.doi.org/10.7554/eLife.00829.001.

Comparison of the intracellular trafficking itinerary of ctla-4 orthologues.

CTLA-4 is an essential inhibitor of T cell immune responses. At steady state, most CTLA-4 resides in intracellular compartments due to constitutive internalisation mediated via a tyrosine based endocytic motif (YVKM) within the cytoplasmic domain. This domain is highly conserved in mammals suggesting strong selective pressure. In contrast, the C-terminal domain varies considerably in non-mammals such as fish, xenopus and birds. We compared the ability of the C-terminus of these species to direct the trafficking of CTLA-4 with human CTLA-4. Using a chimeric approach, endocytosis was found to be conserved between human, xenopus and chicken CTLA-4 but was reduced substantially in trout CTLA-4, which lacks the conserved YXXM motif. Nevertheless, we identified an alternative YXXF motif in trout CTLA-4 that permitted limited endocytosis. Post-internalisation, CTLA-4 was either recycled or targeted for degradation. Human and chicken CTLA-4, which contain a YVKM motif, showed efficient recycling compared to xenopus CTLA-4 which contains a less efficient YEKM motif. Specific mutation of this motif in human CTLA-4 reduced receptor recycling. These findings suggest evolutionary development in the endocytic and recycling potential of CTLA-4, which may facilitate more refined functions of CTLA-4 within the mammalian immune system.

Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsic function of CTLA-4.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an essential negative regulator of T cell immune responses whose mechanism of action is the subject of debate. CTLA-4 shares two ligands (CD80 and CD86) with a stimulatory receptor, CD28. Here, we show that CTLA-4 can capture its ligands from opposing cells by a process of trans-endocytosis. After removal, these costimulatory ligands are degraded inside CTLA-4-expressing cells, resulting in impaired costimulation via CD28. Acquisition of CD86 from antigen-presenting cells is stimulated by T cell receptor engagement and observed in vitro and in vivo. These data reveal a mechanism of immune regulation in which CTLA-4 acts as an effector molecule to inhibit CD28 costimulation by the cell-extrinsic depletion of ligands, accounting for many of the known features of the CD28-CTLA-4 system.