Background: CTLA-4 is an essential regulator of T cell immune responses with unusual intracellular trafficking.Results: Endocytosis of CTLA-4 is continuous with subsequent recycling and degradation.Conclusion: Clathrin-mediated endocytosis of CTLA-4 persists in activated T cells.Significance: This alters our understanding of CTLA-4 behavior and, therefore, how it might function.

In clathrin-mediated membrane traffic, clathrin does not bind directly to cargo and instead binds to adaptors that mediate this function. For endocytosis, the main adaptor is the adaptor protein (AP)-2 complex, but it is uncertain how clathrin contacts AP-2. Here we tested in human cells the importance of the three binding sites that have been identified so far on the N-terminal domain (NTD) of clathrin. We find that mutation of each of the three sites on the NTD, alone or in combination, does not block clathrin/AP-2-mediated endocytosis in the same way as deletion of the NTD. We report here the fourth and final site on the NTD that is required for clathrin/AP-2-mediated endocytic function. Each of the four interaction sites can operate alone to mediate endocytosis. The observed functional redundancy between interaction sites on the NTD explains how productivity of clathrin-coated vesicle formation is ensured.

A long-standing paradigm in cell biology is the shutdown of endocytosis during mitosis. There is consensus that transferrin uptake is inhibited after entry into prophase and that it resumes in telophase. A recent study proposed that endocytosis is continuous throughout the cell cycle and that the observed inhibition of transferrin uptake is due to a decrease in available transferrin receptor at the cell surface, and not to a shutdown of endocytosis. This challenge to the established view is gradually becoming accepted. Because of this controversy, we revisited the question of endocytic activity during mitosis. Using an antibody uptake assay and controlling for potential changes in surface receptor density, we demonstrate the strong inhibition of endocytosis in mitosis of CD8 chimeras containing any of the three major internalization motifs for clathrin-mediated endocytosis (YXXΦ, [DE]XXXL[LI], or FXNPXY) or a CD8 protein with the cytoplasmic tail of the cationindependent mannose 6-phosphate receptor. The shutdown is not gradual: We describe a binary switch from endocytosis being "on" in interphase to "off" in mitosis as cells traverse the G 2 /M checkpoint. In addition, we show that the inhibition of transferrin uptake in mitosis occurs despite abundant transferrin receptor at the surface of HeLa cells. Our study finds no support for the recent idea that endocytosis continues during mitosis, and we conclude that endocytosis is temporarily shutdown during early mitosis.

Small molecule inhibitors of clathrin-mediated endocytosis are highly desired for the dissection of membrane trafficking pathways in the lab and for potential use as anti-infectives in the clinic. One inhibition strategy is to prevent clathrin from contacting adaptor proteins so that clathrin-mediated endocytosis cannot occur. “Pitstop” compounds have been developed that block only one of the four functional interaction sites on the N-terminal domain of clathrin heavy chain. Despite this limitation, Pitstop 2 causes profound inhibition of clathrin-mediated endocytosis. In this study, we probed for non-specific activity of Pitstop 2 by examining its action in cells expressing clathrin heavy chain harbouring mutations in the N-terminal domain interaction sites. We conclude that the inhibition observed with this compound is due to non-specificity, i.e. it causes inhibition away from its proposed mode of action. We recommend that these compounds be used with caution in cells and that they should not be used to conclude anything of the function of clathrin's N-terminal domain.

SummaryAt small synapses in the brain, clathrin-mediated endocytosis (CME) is the dominant mode of synaptic vesicle retrieval following weak stimulation [1–4]. Clathrin cannot bind to membranes or cargo directly and instead uses adaptor proteins to do so [5]. Although the involvement of clathrin and dynamin in synaptic vesicle retrieval is clear, it is unknown which adaptor proteins are used to sort the essential components into the vesicle [1, 4, 6]. In nonneuronal cells, CME of the majority of transmembrane receptors is either directly or indirectly via the heterotetrameric AP-2 complex [5]. In neurons, RNAi of the μ2 subunit of AP-2 resulted in only minor inhibition of synaptic vesicle retrieval [7, 8], a result echoed in C. elegans [9]. These results suggest that alternative adaptors may be employed for vesicle retrieval. Here, we tested which adaptors are required for vesicle retrieval at hippocampal synapses using a targeted RNAi screen coupled with optical measurements. Stonin 2 emerged as a major adaptor, whereas AP-2 played only a minor role in endocytosis at the synapse. Moreover, using chemically induced rerouting of stonin 2 to mitochondria it was possible to switch endocytically competent synapses to an impaired state on a timescale of minutes.

Small molecule inhibitors of clathrin-mediated endocytosis are highly desired for the dissection of membrane trafficking pathways in the lab and for potential use as anti-infectives in the clinic. One inhibition strategy is to prevent clathrin from contacting adaptor proteins so that clathrin-mediated endocytosis cannot occur. ''Pitstop'' compounds have been developed that block only one of the four functional interaction sites on the N-terminal domain of clathrin heavy chain. Despite this limitation, Pitstop 2 causes profound inhibition of clathrin-mediated endocytosis. In this study, we probed for non-specific activity of Pitstop 2 by examining its action in cells expressing clathrin heavy chain harbouring mutations in the N-terminal domain interaction sites. We conclude that the inhibition observed with this compound is due to non-specificity, i.e. it causes inhibition away from its proposed mode of action. We recommend that these compounds be used with caution in cells and that they should not be used to conclude anything of the function of clathrin's N-terminal domain.

No abstract

The tumor microenvironment (TME) is a complex network, consisting of the tumor, blood vessels, stromal and immune cells, and soluble factors. The immune system plays an important role in combating tumor growth, and multiple studies associate raised immune infiltrate with beneficial outcome. Various tractable immuno-oncology targets have been identified, both in the TME and immune cells. It is critical to test novel immuno-modulators in assays involving multiple cell types, to understand the MOA and identify biomarkers before moving into the clinic. Charles River have developed a range of primary human assays to model the TME in vitro. This platform models multiple anti-tumor immune effector pathways and has been validated with standard of care therapeutics. The assays include: T cell or NK cell-mediated tumor killing, myeloid/macrophage assays, Th1/Th17/iTreg differentiation and regulatory T cell suppression assays. Tumor killing assays were performed using an IncuCyte ZOOM and PBMC were cultured with tumor monolayers with TCR ligation. Keytruda and Yervoy raised levels of tumor cell death, indicating that they enhanced T cell killing. To further model the TME, 3D ‘spheroids’ were used to screen for either T cell mediated or ADCC-mediated NK cell killing. For ADCC, PBMC were co-cultured with tumor spheroids and killing measured by monitoring spheroid diameter in the presence of Herceptin, which potentiated NK-driven tumour killing. Myeloid/macrophage cells were differentiated from monocytes in tumor-conditioned media (TCM). TCM drove the generation of immature cells which were CD25lo, CD127lo, CD184hi, CD80lo, CD163hi, CD68lo and MHCIIlo. These cells produced IL-10 and VEGF and were suppressive in a T cell assay. Phagocytosis assays were also performed using anti-CD47 as a control. The TME is associated with increased Treg numbers and low levels of Th1 or Th17 cells. Many therapeutics aim to shift the balance away from Treg, towards Th1 or Th17 cells. Assays were therefore performed by differentiating naïve CD4+ T cells into iTreg, in the presence or absence of a USP7 inhibitor. Reduced iTreg generation, without significant alteration in Th1/17 frequency was observed. The resulting iTreg were less able to suppress T cell proliferation when compared to non-treated iTreg. Suppression assays were also performed using nTreg. As before, the USP7 inhibitor partially reversed nTreg-mediated suppression. Charles River is pleased to present an immuno-oncology platform to model the TME in human cells in vitro, enabling partners to rapidly assess the immunomodulatory capacity of their therapeutics. The 3D assays represent an important complex cell model to support translational drug discovery, sitting alongside T cell-mediated tumor killing, myeloid/macrophage assays, Th1/Th17/ iTreg differentiation and nTreg assays and helps to define the diverse aspects of micro-environmental control of immune response. Citation Format: Louise S. Brackenbury, S. Rhiannon Jenkinson, Shilina Roman, Robert D. Nunan, Sylvie D. Hunt, Anna Willox, Neil A. Williams, Omar Aziz, Ian Waddell. A translational immuno-oncology platform to model the tumor microenvironment in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2811.