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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.The inhibitory receptor CTLA-4 recognizes two ligands on opposing antigen-presenting cells, CD80 and CD86. Sansom and colleagues show CTLA-4 captures ligands by transendocytosis, whereupon low-affinity CD86 releases CTLA-4 at low pH to promote CTLA-4 recycling; however, high-affinity CD80 remains bound and targets CTLA-4 for ubiquitination and destruction.

Manipulating CD28 co-stimulation is a key element of anti-tumour immune responses and treating autoimmune diseases. CD28 can reduce the T cell activation threshold but has a complex relationship with T cell receptor (TCR) signalling and an unclear role in specific T cell subsets. Using a series of in vitro stimulation assays, we have studied the relative contribution of CD28 and TCR signals in human CD4 + T cell responses. We show that not only the quantity of CD28 co-stimulation but also its intensity relative to TCR differentially impacts the division of naïve and memory T cells. We show that CD28 co-stimulation can have TCR-independent effects on memory T cell phenotype and cytokine production and in some settings can antagonize TCR-driven functions. These data highlight the complex relationship between CD28 co-stimulation and TCR signals and expose clear differences in their use by naïve and memory T cells.

In an adult human body, only a minority (∼1%) of cells are dividing; all others are either quiescent, senescent or terminally differentiated. Cellular quiescence, also called G0, is a reversible non-proliferative state in which cells, such as adult stem cells, exist until stimuli trigger their re-entry into the cell cycle. Quiescent cells are known to reside within microenvironment niches of specific extracellular matrix (ECM) composition, but the molecular mechanisms that control their entry and maintenance into G0 and their long-term survival are poorly understood. Here, using a reproducible and homogenous in vitro model of quiescence, ex vivo tissue histology, phosphoproteomics, and molecular cell biological assays, we revealed that Laminin 111 was sufficient to trigger i) reversible cell cycle exit into G0; ii) sustained and elevated MAPK/ERK signaling; and iii) long-term survival. We found that ERK was activated through the Rap1-BRAF-MEK arm underneath Laminin-binding Integrin α3β1. Activated pERK was scaffolded into the cytoplasm by IQGAP1, thereby blocking its translocation into the nucleus and the activation of proliferative transcription factors. Instead, cytoplasmic pERK inhibited pro-apoptotic protein BAD, which mediated the survival of quiescent cells even in absence of mitogen stimuli. Importantly, we confirmed that pERK was elevated and retained in the cytoplasm of Lgr5+ stem cells when they were located within Laminin α1-positive niches in porcine intestine. These findings uncovered a molecular mechanism that may explain how quiescent cell pools, such as dormant adult stem cells, can survive many years despite low mitogen stimuli and be resistant to apoptotic challenges, including chemotherapy. Laminin 111 is sufficient to induce cellular quiescence (G0) and long-term survival. Laminin 111 triggers the sustained and elevated activation of ERK during G0. ERK is activated not by growth factor receptors but through the Rap1-BRAF-MEK arm underneath Laminin-binding Integrin α3β1. Active, phosphorylated ERK (pERK) is scaffolded by IQGAP1, which prevents it from translocating into the nucleus and activating proliferative transcription factors. Instead, cytoplasmic pERK mediates the phosphorylation, and thus inhibition, of BAD, thereby raising the threshold at which G0 cells enter apoptosis.

The CTLA-4 and PD-1 checkpoints control immune responses to self-antigens and are key targets in cancer immunotherapy. Both pathways are connected via a cis interaction between CD80 and PD-L1, the ligands for CTLA-4 and PD-1 respectively. This cis interaction prevents PD-1 binding to PD-L1 but is reversed by CTLA-4 trans-endocytosis of CD80. However, the mechanism by which CTLA-4 selectively removes CD80 but not PD-L1 is unclear. Here we show that CTLA-4 – CD80 interactions are unimpeded by PD-L1 and that CTLA-4 binding with CD80 does not displace PD-L1 per se. Rather, both the rigidity and bivalency of the WT CTLA-4 molecule is required to orientate CD80 such that PD-L1 interactions are no longer permissible. Moreover, soluble CTLA-4 released PD-L1 only at specific expression levels of CD80 and PD-L1, whereas CTLA-4 trans-endocytosis released PD-L1 in all conditions. These data show that PD-L1 release from CD80 is driven by biophysical factors associated with orientation and bivalent cross-linking of proteins in the cell membrane and that trans-endocytosis of CD80 efficiently promotes PD-L1 availability.

CTLA-4 and PD-1 are key immune checkpoints that are targeted in the treatment of cancer. Recently it has emerged that there is a physical interaction between the ligands of these pathways (CD80 and PD-L1), which can prevent PD-L1 inhibitory functions. Since CTLA-4 captures and degrades its ligands via transendocytosis, we investigated how transendocytosis of CD80 is impacted by PD-L1 interaction. We find that transendocytosis of CD80 results in a time-dependent recovery of PD-L1 availability that correlates with CD80 removal. Moreover, CD80 transendocytosis is highly specific in that only CD80 is removed, and its heterodimeric PD-L1 partner remains on the APC. We found no evidence that CTLA-4 interactions with CD80 were inhibited by PD-L1, however efficient removal of CD80 required an intact CTLA-4 cytoplasmic domain, distinguishing this process from more general trogocytosis. We also show that simple binding of CTLA-4 to the CD80-PD-L1 heterodimer is insufficient to liberate PD-L1-PD-1 interactions, suggesting that transendocytosis of CD80 is required to effectively control PD-L1-PD-1 interactions.

Glaucoma drainage devices are used in surgical glaucoma therapy. Success of controlling the intraocular pressure is limited due to fibrous implant encapsulation and fibrin coating on the implant which lead to drainage obstructions. An innovative implant with a magnetically adjustable valve was developed. The valve opening of the implant should eliminate inflammatory products from the outflow area and affect fibrous tissue formation to achieve a sufficient long-term aqueous humour outflow. Lifting of this valve should disturb cell adhesion by exerting mechanical forces. Before testing this hypothesis, the flow characteristics of glaucoma drainage devices, especially the outflow resistance by regular IOP, should be considered in a pilot study, as they are important in preventing too low postoperative intraocular pressure known as ocular hypotony. Therefore, two prototypes of the innovative implant differing in their valve area design were examined regarding their flow characteristics in a limited animal experiment lasting two weeks. Ten healthy New Zealand White rabbits were divided into two groups (A & B) with different implanted prototypes. Daily, tonometry and direct ophthalmoscopy were performed to assess the intraocular pressure and the inflammatory reaction of the eye. After two weeks, the rabbits were euthanised to evaluate the initially histological inflammatory reaction to the implant. In group A, one case of hypotony emerged. When considering the entire observation period, a highly statistically significant difference between the intraocular pressure in the operated eye and that in the control eye was detected in group A (p < 0.0001) in contrast to group B (p = 0.0063). The postoperative inflammatory signs decreased within two weeks. Histologically, a typical but low level foreign body reaction with macrophages and lymphocytes as well as mild to moderate fibrosis was seen after the short experimental period. Based on our tonometric results, prototype B seems to be the system of choice for further research assessing its long-term function and biocompatibility.

Long-term treatment with 6-thioguanine (6-TG) for pediatric acute lymphoblastic leukemia (ALL) is associated with high rates of hepatic sinusoidal obstruction syndrome (SOS). Nevertheless, current treatment continues to use short-term applications of 6-TG with only sparse information on toxicity. 6-TG is metabolized by thiopurine methyltransferase (TPMT) which underlies clinically relevant genetic polymorphism. We analyzed the association between hepatic SOS reported as a serious adverse event (SAE) and short-term 6-TG application in 3983 pediatric ALL patients treated on trial AIEOP-BFM ALL 2000 (derivation cohort) and defined the role of TPMT genotype in this relationship. We identified 17 patients (0.43%) with hepatic SOS, 13 of which with short-term exposure to 6-TG ( P  < 0.0001). Eight of the 13 patients were heterozygous for low-activity TPMT variants, resulting in a 22.4-fold (95% confidence interval 7.1–70.7; P  ≤ 0.0001 ) increased risk of hepatic SOS for heterozygotes in comparison to TPMT wild-type patients. Results were supported by independent replication analysis. All patients with hepatic SOS after short-term 6-TG recovered and did not demonstrate residual symptoms. Thus, hepatic SOS is associated with short-term exposure to 6-TG during treatment of pediatric ALL and SOS risk is increased for patients with low-activity TPMT genotypes.

Neurodevelopmental abnormalities together with neurodegenerative processes contribute to schizophrenia, an etiologically heterogeneous, complex disease phenotype that has been difficult to model in animals. The neurodegenerative component of schizophrenia is best documented by magnetic resonance imaging (MRI), demonstrating progressive cortical gray matter loss over time. No treatment exists to counteract this slowly proceeding atrophy. The hematopoietic growth factor erythropoietin (EPO) is neuroprotective in animals. Here, we show by voxel-based morphometry in 32 human subjects in a placebo-controlled study that weekly high-dose EPO for as little as 3 months halts the progressive atrophy in brain areas typically affected in schizophrenia, including hippocampus, amygdala, nucleus accumbens, and several neocortical areas. Specifically, gray matter protection is highly associated with improvement in attention and memory functions. These findings suggest that a neuroprotective strategy is effective against common pathophysiological features of schizophrenic patients, and strongly encourage follow-up studies to optimize EPO treatment dose and duration.