Toine ten Broeke

Identification of a novel ubiquitin conjugation motif, required for ligand-induced internalization of the growth hormone receptor.

In addition to its role in selective protein degradation, the conjugation of ubiquitin to proteins has also been implicated in the internalization of plasma membrane proteins, including the α‐factor receptor Ste2p, uracil permease Fur4p, epithelial sodium channel ENaC and the growth hormone receptor (GHR). Binding of GH to its receptor induces receptor dimerization, resulting in the activation of signal transduction pathways and an increase of GHR ubiquitination. Previously, we have shown that the ubiquitin conjugation system mediates GH‐induced GHR internalization. Here, we present evidence that a specific domain of the GHR regulates receptor endocytosis via the ubiquitin conjugation system. This ubiquitin‐dependent endocytosis (UbE) motif consists of the amino acid sequence DSWVEFIELD and is homologous to sequences in other proteins, several of which are known to be ubiquitinated. In addition, we show that GH internalization by a truncated GHR is independent of the presence of lysine residues in the cytosolic domain of this receptor, while internalization still depends on an intact ubiquitin conjugation system. Thus, GHR internalization requires the recruitment of the ubiquitin conjugation system to the GHR UbE motif rather than the conjugation of ubiquitin to the GHR itself.

Differential MHC class II synthesis and ubiquitination confers distinct antigen-presenting properties on conventional and plasmacytoid dendritic cells

The importance of conventional dendritic cells (cDCs) in the processing and presentation of antigen is well established, but the contribution of plasmacytoid dendritic cells (pDCs) to these processes, and hence to T cell immunity, remains unclear. Here we showed that unlike cDCs, pDCs continued to synthesize major histocompatibility complex (MHC) class II molecules and the MHC class II ubiquitin ligase MARCH1 long after activation. Sustained MHC class II–peptide complex formation, ubiquitination and turnover rendered pDCs inefficient in the presentation of exogenous antigens but enabled pDCs to continuously present endogenous viral antigens in their activated state. As the antigen-presenting abilities of cDCs and pDCs are fundamentally distinct, these two cell types may activate largely nonoverlapping repertoires of CD4+ T cells.

MHC II in dendritic cells is targeted to lysosomes or T cell-induced exosomes via distinct multivesicular body pathways.

Dendritic cells (DCs) express major histocompatibility complex class II (MHC II) to present peptide antigens to T cells. In immature DCs, which bear low cell surface levels of MHC II, peptide‐loaded MHC II is ubiquitinated. Ubiquitination drives the endocytosis and sorting of MHC II to the luminal vesicles of multivesicular bodies (MVBs) for lysosomal degradation. Ubiquitination of MHC II is abrogated in activated DCs, resulting in an increased cell surface expression. We here provide evidence for an alternative MVB sorting mechanism for MHC II in antigen‐loaded DCs, which is triggered by cognately interacting antigen‐specific CD4+ T cells. At these conditions, DCs generate MVBs with MHC II and CD9 carrying luminal vesicles that are secreted as exosomes and transferred to the interacting T cells. Sorting of MHC II into exosomes was, in contrast to lysosomal targeting, independent of MHC II ubiquitination but rather correlated with its incorporation into CD9 containing detergent‐resistant membranes. Together, these data indicate two distinct MVB pathways: one for lysosomal targeting and the other for exosome secretion.

Endocytosed Transferrin Receptors Recycle via Distinct Dynamin and Phosphatidylinositol 3-Kinase-dependent Pathways

Recycling of endocytosed membrane proteins involves passage through early endosomes and recycling endosomes. Previously, we demonstrated a role for clathrin-coated vesicles in transferrin receptor recycling. These clathrin-coated vesicles are formed from recycling endosomes in a process that was inhibited in dynamin-1G273D-overexpressing cells. Here we show a second transferrin recycling pathway, which requires phosphatidylinositol 3-kinase activity. Two unrelated phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, retained endocytosed transferrin in early endosomes but did not affect transfer through recycling endosomes. The inhibitory effects of LY294002 and dynamin-1G273D on transferrin recycling were additive. In combination with brefeldin A, a drug that prevents the formation of clathrin-coated buds at recycling endosomes, LY294002 inhibited transferrin recycling synergistically. Collectively, these data indicate two distinct recycling pathways. One pathway involves transfer from early endosomes to recycling endosomes, from where clathrin/dynamin-coated vesicles provide for further transport, whereas the other route bypasses recycling endosomes and requires phosphatidylinositol 3-kinase activity.

MHC Class II Antigen Presentation by Dendritic Cells Regulated through Endosomal Sorting

For the initiation of adaptive immune responses, dendritic cells present antigenic peptides in association with major histocompatibility complex class II (MHCII) to naïve CD4(+) T lymphocytes. In this review, we discuss how antigen presentation is regulated through intracellular processing and trafficking of MHCII. Newly synthesized MHCII is chaperoned by the invariant chain to endosomes, where peptides from endocytosed pathogens can bind. In nonactivated dendritic cells, peptide-loaded MHCII is ubiquitinated and consequently sorted by the ESCRT machinery to intraluminal vesicles of multivesicular bodies, ultimately leading to lysosomal degradation. Ubiquitination of newly synthesized MHCII is blocked when dendritic cells are activated, now allowing its transfer to the cell surface. This mode of regulation for MHCII is a prime example of how molecular processing and sorting at multivesicular bodies can determine the expression of signaling receptors at the plasma membrane.

Endosome-mediated autophagy: An unconventional MIIC-driven autophagic pathway operational in dendritic cells

Activation of TLR signaling has been shown to induce autophagy in antigen-presenting cells (APCs). Using high-resolution microscopy approaches, we show that in LPS-stimulated dendritic cells (DCs), autophagosomes emerge from MHC class II compartments (MIICs) and harbor both the molecular machinery for antigen processing and the autophagosome markers LC3 and ATG16L1. This ENdosome-Mediated Autophagy (ENMA) appears to be the major type of autophagy in DCs, as similar structures were observed upon established autophagy-inducing conditions (nutrient deprivation, rapamycin) and under basal conditions in the presence of bafilomycin A1. Autophagosome formation was not significantly affected in DCs expressing ATG4BC74A mutant and atg4b−/− bone marrow DCs, but the degradation of the autophagy substrate SQSTM1/p62 was largely impaired. Furthermore, we demonstrate that the previously described DC aggresome-like LPS-induced structures (DALIS) contain vesicular membranes, and in addition to SQSTM1 and ubiquitin, they are positive for LC3. LC3 localization on DALIS is independent of its lipidation. MIIC-driven autophagosomes preferentially engulf the LPS-induced SQSTM1-positive DALIS, which become later degraded in autolysosomes. DALIS-associated membranes also contain ATG16L1, ATG9 and the Q-SNARE VTI1B, suggesting that they may represent (at least in part) a membrane reservoir for autophagosome expansion. We propose that ENMA constitutes an unconventional, APC-specific type of autophagy, which mediates the processing and presentation of cytosolic antigens by MHC class II machinery, and/or the selective clearance of toxic by-products of elevated ROS/RNS production in activated DCs, thereby promoting their survival.

Fc receptor inside-out signaling and possible impact on antibody therapy

Fc receptors (FcR) are expressed on immune cells and bind to the Fc tail of antibodies. This interaction is essential for FcR‐mediated signaling and triggering of cellular effector functions. FcR activation is tightly regulated to prevent immune responses by non‐antigen bound antibodies or in the absence of ‘danger signals’. FcR activity may be modulated at the plasma membrane via cross‐talk with integrins. In addition, cytokines at the site of infection/inflammation can increase FcR avidity, a process referred to as inside‐out signaling. This regulatory mechanism has been described for FcγRI (CD64), FcγRIIa (CD32a), and FcαRI (CD89) and is also well‐known for integrins. Key cellular events during inside‐out signaling are (de)phosphorylation, clustering, cytoskeleton rearrangements, and conformational changes. The latter can be studied with antibodies that specifically recognize epitopes exposed by the active (high affinity) or inactive (low affinity) state of the FcR. These antibodies are important tools to investigate the role of FcR activation in disease settings. Research on FcR has gained momentum with the rise of monoclonal antibodies (mAb) entering the clinic for the treatment of cancer and other diseases. The clinical outcome of mAb therapy may be improved by increasing FcR avidity by cytokine stimulation.

Endosomally Stored MHC Class II Does Not Contribute to Antigen Presentation by Dendritic Cells at Inflammatory Conditions

Major histocompatibility complex (MHC) class II (MHCII) is constitutively expressed by immature dendritic cells (DC), but has a short half‐life as a consequence of its transport to and degradation in lysosomes. For its transfer to lysosomes, MHCII is actively sorted to the intraluminal vesicles (ILV) of multivesicular bodies (MVB), a process driven by its ubiquitination. ILV have, besides their role as an intermediate compartment in lysosomal transfer, also been proposed to function as a site for MHCII antigen loading and temporal storage. In that scenario, DC would recruit antigen‐loaded MHCII to the cell surface in response to a maturation stimulus by allowing ILV to fuse back with the MVB delimiting membrane. Other studies, however, explained the increase in cell surface expression during DC maturation by transient upregulation of MHCII synthesis and reduced sorting of newly synthesized MHCII to lysosomes. Here, we have characterized the relative contributions from the biosynthetic and endocytic pathways and found that the vast majority of antigen‐loaded MHCII that is stably expressed at the plasma membrane by mature DC is synthesized after exposure to inflammatory stimuli. Pre‐existing endosomal MHCII contributed only when it was not yet sorted to ILV at the moment of DC activation. Together with previous records, our current data are consistent with a model in which passage of MHCII through ILV is not required for antigen loading in maturing DC and in which sorting to ILV in immature DC provides a one‐way ticket for lysosomal degradation.

Trafficking of MHC Class II in Dendritic Cells is Dependent on but Not Regulated by Degradation of Its Associated Invariant Chain

In dendritic cells (DC), newly synthesized MHCII is directed to endosomes by its associated invariant chain (Ii). Here, Ii is degraded after which MHCII is loaded with peptides. In immature DC, ubiquitination of peptide‐loaded MHCII drives its sorting to lysosomes for degradation. Ubiquitination of MHCII is strongly reduced in response to inflammatory stimuli, resulting in increased expression of MHCII at the plasma membrane. Whether surface exposure of MHCII is also regulated during DC maturation by changing the rate of Ii degradation remained unresolved by conflicting results in the literature. We here pinpoint experimental problems that have contributed to these controversies and demonstrate that immature and mature DC degrade Ii equally efficient at proper culture conditions. Only when DC were cultured in glutamine containing media, endosome acidification and Ii degradation were restricted in immature DC and enhanced in response to lipopolysaccharide (LPS). These effects are caused by ammonia, a glutamine decomposition product. This artificial behavior could be prevented by culturing DC in media containing a stable dipeptide as glutamine source. We conclude that Ii degradation is a prerequisite for but not a rate limiting step in MHCII processing.

Simultaneous Targeting of FcγRs and FcαRI Enhances Tumor Cell Killing

The efficacy of anticancer monoclonal antibodies (mAbs) is limited by the exhaustion of cellular effector mechanisms. The combination of IgG and IgA to two different tumor targets leads to enhanced cytotoxicity, providing a basis for therapeutic mAb improvements. Efficacy of anticancer monoclonal antibodies (mAb) is limited by the exhaustion of effector mechanisms. IgG mAbs mediate cellular effector functions through FcγRs expressed on effector cells. IgA mAbs can also induce efficient tumor killing both in vitro and in vivo. IgA mAbs recruit FcαRI-expressing effector cells and therefore initiate different effector mechanisms in vivo compared with IgG. Here, we studied killing of tumor cells coexpressing EGFR and HER2 by the IgG mAbs cetuximab and trastuzumab and their IgA variants. In the presence of a heterogeneous population of effector cells (leukocytes), the combination of IgG and IgA mAbs to two different tumor targets (EGFR and HER2) led to enhanced cytotoxicity compared with each isotype alone. Combination of two IgGs or two IgAs or IgG and IgA against the same target did not enhance cytotoxicity. Increased cytotoxicity relied on the presence of both the peripheral blood mononuclear cell and the polymorphonuclear (PMN) fraction. Purified natural killer cells were only cytotoxic with IgG, whereas cytotoxicity induced by PMNs was strong with IgA and poor with IgG. Monocytes, which coexpress FcγRs and FcαRI, also displayed increased cytotoxicity by the combination of IgG and IgA in an overnight killing assay. Coinjection of cetuximab and IgA2-HER2 resulted in increased antitumor effects compared with either mAb alone in a xenograft model with A431-luc2-HER2 cells. Thus, the combination of IgG and IgA isotypes optimally mobilizes cellular effectors for cytotoxicity, representing a promising novel strategy to improve mAb therapy. Cancer Immunol Res; 3(12); 1316–24. ©2015 AACR.