Marlieke L.M. Jongsma

Towards a systems understanding of MHC class I and MHC class II antigen presentation

The molecular details of antigen processing and presentation by MHC class I and class II molecules have been studied extensively for almost three decades. Although the basic principles of these processes were laid out approximately 10 years ago, the recent years have revealed many details and provided new insights into their control and specificity. MHC molecules use various biochemical reactions to achieve successful presentation of antigenic fragments to the immune system. Here we present a timely evaluation of the biology of antigen presentation and a survey of issues that are considered unresolved. The continuing flow of new details into our understanding of the biology of MHC class I and class II antigen presentation builds a system involving several cell biological processes, which is discussed in this Review.

A Genome-wide Multidimensional RNAi Screen Reveals Pathways Controlling MHC Class II Antigen Presentation

MHC class II molecules (MHC-II) present peptides to T helper cells to facilitate immune responses and are strongly linked to autoimmune diseases. To unravel processes controlling MHC-II antigen presentation, we performed a genome-wide flow cytometry-based RNAi screen detecting MHC-II expression and peptide loading followed by additional high-throughput assays. All data sets were integrated to answer two fundamental questions: what regulates tissue-specific MHC-II transcription, and what controls MHC-II transport in dendritic cells? MHC-II transcription was controlled by nine regulators acting in feedback networks with higher-order control by signaling pathways, including TGFβ. MHC-II transport was controlled by the GTPase ARL14/ARF7, which recruits the motor myosin 1E via an effector protein ARF7EP. This complex controls movement of MHC-II vesicles along the actin cytoskeleton in human dendritic cells (DCs). These genome-wide systems analyses have thus identified factors and pathways controlling MHC-II transcription and transport, defining targets for manipulation of MHC-II antigen presentation in infection and autoimmunity.

Routes to manipulate MHC class II antigen presentation.

MHC class II molecules (MHC-II) present antigenic fragments acquired in the endocytic route to the immune system for recognition and activation of CD4+ T cells. This ignites a series of immune responses. MHC-II strongly correlates to most autoimmune diseases. Understanding the biology of MHC-II is therefore expected to translate into novel means of autoimmunity control or immune response improvement. Although the basic cell biology of MHC-II antigen presentation is well understood, many novel aspects have been uncovered in recent years including means of antigen delivery, preparation for MHC-II loading, transport processes and vaccination strategies. We will discuss past, present and future of these insights into the biology of MHC-II.

An ER-Associated Pathway Defines Endosomal Architecture for Controlled Cargo Transport

Summary Through a network of progressively maturing vesicles, the endosomal system connects the cell’s interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle “cloud” and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell’s periphery. By drawing the endosomal system’s architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.

On the move: organelle dynamics during mitosis

A cell constitutes the minimal self-replicating unit of all organisms, programmed to propagate its genome as it proceeds through mitotic cell division. The molecular processes entrusted with ensuring high fidelity of DNA replication and subsequent segregation of chromosomes between daughter cells have therefore been studied extensively. However, to process the information encoded in its genome a cell must also pass on its non-genomic identity to future generations. To achieve productive sharing of intracellular organelles, cells have evolved complex mechanisms of organelle inheritance. Many membranous compartments undergo vast spatiotemporal rearrangements throughout mitosis. These controlled organizational changes are crucial to enabling completion of the division cycle and ensuring successful progeny. Herein we review current understanding of intracellular organelle segregation during mitotic division in mammalian cells, with a focus on compartment organization and integrity throughout the inheritance process.

Multiple sclerosis-associated CLEC16A controls HLA class II expression via late endosome biogenesis

C-type lectins are key players in immune regulation by driving distinct functions of antigen-presenting cells. The C-type lectin CLEC16A gene is located at 16p13, a susceptibility locus for several autoimmune diseases, including multiple sclerosis. However, the function of this gene and its potential contribution to these diseases in humans are poorly understood. In this study, we found a strong upregulation of CLEC16A expression in the white matter of multiple sclerosis patients (n = 14) compared to non-demented controls (n = 11), mainly in perivascular leukocyte infiltrates. Moreover, CLEC16A levels were significantly enhanced in peripheral blood mononuclear cells of multiple sclerosis patients (n = 69) versus healthy controls (n = 46). In peripheral blood mononuclear cells, CLEC16A was most abundant in monocyte-derived dendritic cells, in which it strongly co-localized with human leukocyte antigen class II. Treatment of these professional antigen-presenting cells with vitamin D, a key protective environmental factor in multiple sclerosis, downmodulated CLEC16A in parallel with human leukocyte antigen class II. Knockdown of CLEC16A in distinct types of model and primary antigen-presenting cells resulted in severely impaired cytoplasmic distribution and formation of human leucocyte antigen class II-positive late endosomes, as determined by immunofluorescence and electron microscopy. Mechanistically, CLEC16A participated in the molecular machinery of human leukocyte antigen class II-positive late endosome formation and trafficking to perinuclear regions, involving the dynein motor complex. By performing co-immunoprecipitations, we found that CLEC16A directly binds to two critical members of this complex, RILP and the HOPS complex. CLEC16A silencing in antigen-presenting cells disturbed RILP-mediated recruitment of human leukocyte antigen class II-positive late endosomes to perinuclear regions. Together, we identify CLEC16A as a pivotal gene in multiple sclerosis that serves as a direct regulator of the human leukocyte antigen class II pathway in antigen-presenting cells. These findings are a first step in coupling multiple sclerosis-associated genes to the regulation of the strongest genetic factor in multiple sclerosis, human leukocyte antigen class II.

ER contact sites direct late endosome transport

Endosomes shuttle select cargoes between cellular compartments and, in doing so, maintain intracellular homeostasis and enable interactions with the extracellular space. Directionality of endosomal transport critically impinges on cargo fate, as retrograde (microtubule minus‐end directed) traffic delivers vesicle contents to the lysosome for proteolysis, while the opposing anterograde (plus‐end directed) movement promotes recycling and secretion. Intriguingly, the endoplasmic reticulum (ER) is emerging as a key player in spatiotemporal control of late endosome and lysosome transport, through the establishment of physical contacts with these organelles. Earlier studies have described how minus‐end‐directed motor proteins become discharged from vesicles engaged at such contact sites. Now, Raiborg et al. implicate ER‐mediated interactions, induced by protrudin, in loading plus‐end‐directed motor kinesin‐1 onto endosomes, thereby stimulating their transport toward the cells periphery. In this review, we recast the prevailing concepts on bidirectional late endosome transport and discuss the emerging paradigm of inter‐compartmental regulation from the ER‐endosome interface viewpoint.

The regulatory network behind MHC class I expression

The MHC class I pathway, presenting endogenously derived peptides to T lymphocytes, is hijacked in many pathological conditions. This affects MHC class I levels and peptide presentation at the cell surface leading to immune escape of cancer cells or microbes. It is therefore important to identify the molecular mechanisms behind MHC class I expression, processing and antigen presentation. The identification of NLRC5 as regulator of MHC class I transcription was a huge step forward in understanding the transcriptional mechanism involved. Nevertheless, many questions concerning MHC class I transcription are yet unsolved. Here we illuminate current knowledge on MHC class I and NLRC5 transcription, we highlight some remaining questions and discuss the use of quickly developing high-content screening tools to reveal unknowns in MHC class I transcription in the near future.

MS-associated gene CLEC16A uses the molecular machinery of late endosomal biogenesis to control HLA-II expression in APC

enzymatically active and inactive forms of PTX. Our results indicate that PTX, through its ADP-ribosyltransferase activity, induces two series of events upstream of IL-6: 1) the activation of TLR4 signaling in myeloid cells, leading to pro-IL-1β synthesis; and 2) the formation of a pyrindependent inflammasome that cleaves pro-IL-1β into its active form. In turn, IL-1β stimulates nearby stromal cells to secrete IL-6, which is known to induce vascular changes required for leukocyte adhesion. Without pyrin, PTX does not induce neutrophil adhesion to cerebral capillaries and is less effective at inducing EAE in transgenic mice with encephalitogenic T lymphocytes. This study identifies thefirstmicrobial molecule that activates pyrin, a mechanism by which infections may influence MS and a potential therapeutic target for immune disorders.

Identification of potential immunotherapeutic targets in antigen presentation and costimulation networks

Anti-tumor immune responses are often hampered by an excess of coinhibition of T cells, but also by a lack of MHC antigen presentation and costimulation. The latter are not yet successfully exploited as targets for immunotherapy, while augmenting MHC and costimulatory molecules may favorably direct adaptive immune responses against cancer. Thus we set off to identify novel molecular targets within the network of antigen presentation and costimulation. As our understanding of players of MHC class I (MHC-I) is far from complete, we initially performed an unbiased genome-wide screen to systematically decipher the rest of the MHC-I network. The screen is based on near-haploid human leukemic cells (KBM7), retrovirally modified to contain millions of different single-gene-knockout cells. As a result some of these cells became MHC-I high or low and were separately sorted out by FACS. Deep sequence analyses of the sorted cell populations versus unsorted control cells revealed that the MHC-I low population was enriched for knockouts of known key proteins in the MHC-I network, such as Tapasin, TAP1, TAP2 and β2M. Among the new hit proteins of both screens, we identified two enzymes: a novel peptidase and an E3 ligase, which were first validated using siRNA knockdown in different cell lines. Using cutting edge TALEN and CRISPR genome-editing technology, we generated different cell lines that are completely knockout for the peptidase and E3 ligase. As expected, MHC-I expression on the E3 ligase knockout cell line was increased. Expression on the peptidase knockout cell lines was drastically decreased and could be rescued by retroviral reconstitution of the peptidase. Importantly, reconstitution using a catalytic inactive peptidase did not rescue MHC-I expression, showing that the proteolytic activity is essential for its MHC-I related function. Treatment of peptidase knockout cells with IFN-γ revealed that MHC-I could still be up-regulated. Moreover, the peptidase itself was not IFN-γ inducible in contrast to known other MHC-I regulators, arguing for an unexpected immune-independent regulation of the antigen presentation pathway. We are currently in the process of performing similar screens for the cell surface expression of costimulatory molecules. In conclusion, using high-tech screening and the latest state-of-the-art genome-editing tools, we are building a near-complete map of the intracellular network behind MHC-I and costimulatory molecule cell surface expression. Thereby we have initiated new efforts to define novel potential targets for immunotherapy.