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Keywords: MHC; HLA; antigen presentation; immunology; transplantation; autoimmunity EN MHC HLA antigen presentation immunology transplantation autoimmunity 301 311 11 09/19/22 20221001 NES 221001 This article has been translated into different languages. HLA-A, -B and -C are present on virtually all our cells (except red blood cells) while HLA-DR, HLA-DQ and HLA-DP are mainly on immune cells. This process is optimized by yet another type of MHC molecule (HLA-DM, which looks similar to MHC class II, and in some cells works in concert with HLA-DO, another class II-like molecule. GRAPH The most important HLA molecules for transplantation are termed HLA-A, HLA-B and HLA-C for MHC class I and HLA-DR, HLA-DQ and HLA-DP for MHC class II. [Extracted from the article]

Targeting CD8+ T cells to recurrent tumor-specific mutations can profoundly contribute to cancer treatment. Some of these mutations are potential tumor antigens although they can be displayed by non-spliced epitopes only in a few patients, because of the low affinity of the mutated non-spliced peptides for the predominant HLA class I alleles. Here, we describe a pipeline that uses the large sequence variety of proteasome-generated spliced peptides and identifies spliced epitope candidates, which carry the mutations and bind the predominant HLA-I alleles with high affinity. They could be used in adoptive T cell therapy and other anti-cancer immunotherapies for large cohorts of cancer patients. As a proof of principle, the application of this pipeline led to the identification of a KRAS G12V mutation-carrying spliced epitope candidate, which is produced by proteasomes, transported by TAPs and efficiently presented by the most prevalent HLA class I molecules, HLA-A*02:01 complexes. [ABSTRACT FROM AUTHOR]

C9orf82 protein, or conserved anti-apoptotic protein 1 or caspase activity and apoptosis inhibitor 1 (CAAP1) has been implicated as a negative regulator of the intrinsic apoptosis pathway by modulating caspase expression and activity. In contrast, an independent genome wide screen for factors capable of driving drug resistance to the topoisomerase II (Topo II) poisons doxorubicin and etoposide, implicated a role for the nuclear protein C9orf82 in delaying DSBs repair downstream of Topo II, hereby sensitizing cells to DSB induced apoptosis. To determine its function in a genetically defined setting in vivo and ex vivo, we here employed CRISPR/Cas9 technology in zygotes to generate a C9orf82 knockout mouse model. C9orf82ko/ko mice were born at a Mendelian ratio and did not display any overt macroscopic or histological abnormalities. DSBs repair dependent processes like lymphocyte development and class switch recombination (CSR) appeared normal, arguing against a link between the C9orf82 encoded protein and V(D)J recombination or CSR. Most relevant, primary pre-B cell cultures and Tp53 transformed mouse embryo fibroblasts (MEFs) derived from C9orf82ko/ko E14.5 and wild type embryos displayed comparable sensitivity to a number of DNA lesions, including DSBs breaks induced by the topoisomerase II inhibitors, etoposide and doxorubicin. Likewise, the kinetics of γH2AX formation and resolution in response to etoposide of C9orf82 protein proficient, deficient and overexpressing MEFs were indistinguishable. These data argue against a direct role of C9orf82 protein in delaying repair of Topo II generated DSBs and regulating apoptosis. The genetically defined systems generated in this study will be of value to determine the actual function of C9orf82 protein. [ABSTRACT FROM AUTHOR]

Abstract: Major histocompatibility complex (MHC) class II molecules (MHC II) present exogenous antigens to CD4+ T cells to modulate immune responses. To contact these antigens, MHC II is delivered to the late endosomal MHC class II compartment (MIIC). This compartment has a complex architecture and consists of internal membranes or vesicles surrounded by a limiting membrane. These subdomains have different protein and lipid content. Also MHC II peptide loading is spatially organized in MIIC as it interacts with DM on intralumenal vesicles (ILVs) to bind antigen. How this is controlled is only understood in a sketchy manner. This may involve ubiquitin modification of MHC II, possibly by E3 ligases of the March family. But other proteins are likely involved as well including E3 ligases, deubiquitylating enzymes (DUBs), adaptor, scaffold, motor and vesicular coat proteins. Our lab performed a genome-wide siRNA screen to define novel proteins and pathways involved in MHC II antigen presentation. The data set is used to select candidate proteins involved in targeting MHC II into MIIC. This process involves ubiquitin modifications and various new molecules not considered as yet in this complex pathway. These molecules may be targeted by drugs to manipulate MHC II responses in auto-immunity, transplantation and other disease states. [Copyright &y& Elsevier]

CD70 is a TNF-related transmembrane molecule expressed by mature dendritic cells (DCs), which present antigens to T cells via major histocompatibility complex (MHC) molecules. In DCs, CD70 localizes with MHC class II molecules in late endosomal vesicles, known as MHC class II compartments (MIICs). MIICs are transported to the immune synapse when a DC contacts an antigen-specific CD4+ T cell. Consequently, MHC class II and CD70 are simultaneously exposed to the T cell. Thereby, T-cell activation via the antigen receptor and CD70-mediated co-stimulation are synchronized, apparently to optimize the proliferative response. We report here that the invariant chain (Ii), a chaperone known to transport MHC class II to MIICs, performs a similar function for CD70. CD70 was found to travel by default to the plasma membrane, whereas Ii coexpression directed it to late endosomes and/or lysosomes. In cells containing the MHC class II presentation pathway, CD70 localized to MIICs. This localization relied on Ii, since transport of CD70 from the Golgi to MIICs was impeded in Ii-deficient DCs. Biophysical and biochemical studies revealed that CD70 and Ii participate in an MHC-class-II-independent complex. Thus, Ii supports transport of both MHC class II and CD70 to MIICs and thereby coordinates their delivery to CD4+ T cells. [ABSTRACT FROM AUTHOR]

Background: The eradication of facultative intracellular bacterial pathogens, like Salmonella typhi, requires the concerted action of both the humoral immune response and the cytotoxic CD8+ T cell response. Dendritic cells (DCs) are considered to orchestrate the cytotoxic CD8+ T cell response via cross-presentation of bacterial antigens onto MHC class I molecules. Cross-presentation of Salmonella by DCs however, is accompanied by the induction of apoptosis in the DCs. Besides antibody production, B cells are required to clear Salmonella infection for other unknown reasons. Methodology/Principal Findings: Here we show that Salmonella-specific B cells that phagocytose Salmonella upon BCR-ligation reactivate human memory CD8+ T cells via cross-presentation yielding a Salmonella-specific cytotoxic T cell response. The reactivation of CD8+ T cells is dependent on CD4+ T cell help. Unlike the DCs, B cell-mediated cross-presentation of Salmonella does not coincide with apoptosis. Conclusions/Significance: B cells form a new player in the activation of the cytotoxic effector arm of the immune response and the generation of effective adaptive immunity in Salmonella infection. [ABSTRACT FROM AUTHOR]

Cross-presentation of extracellular antigens by MHC class I molecules is required for priming cytotoxic T lymphocytes (CTLs) at locations remote from the site of infection. Various mechanisms have been proposed to explain cross-presentation. One such mechanism involves the fusion of the endoplasmic reticulum (ER) with the endosomal-phagosomal system, in which the machinery required for peptide loading of MHC class I molecules is introduced directly into the phagosome. Here, we discuss the evidence for and against the ER-phagosome concept as well as other possible mechanisms of cross-presentation. [ABSTRACT FROM AUTHOR]

Detection and elimination of virus-infected cells by cytotoxic T lymphocytes depends on recognition of virus-derived peptides presented by MHC class I molecules. A critical step in this process is the translocation of peptides from the cytoplasm into the endoplasmic reticulum by the transporter associated with antigen processing (TAP). Here, we identified the bovine herpesvirus 1-encoded UL49.5 protein as a potent inhibitor of TAP. The expression of UL49.5 results in down-regulation of MHC class I molecules at the cell surface and inhibits detection and lysis of the cells by cytotoxic T lymphocytes. UL49.5 homologs encoded by two other varicelloviruses, pseudorabies-virus and equine herpesvirus 1, also block TAP. Homologs of U 149.5 are widely present in herpesviruses, acting as interaction partners for glycoprotein M, but in several varicelloviruses U149.5 has uniquely evolved additional functions that mediate its participation in TAP inhibition. Inactivation of TAP by UL49.5 involves two events: inhibition of peptide transport through a conformational arrest of the transporter and degradation of TAP by proteasomes. UL49.5 is degraded along with TAP via a reaction that requires the cytoplasmic tail of UL49.5. Thus, UL49.5 represents a unique immune evasion protein that inactivates TAP through a unique two-tiered process. [ABSTRACT FROM AUTHOR]

Major histocompatibility complex (MHC) class I molecules present peptides that are derived from endogenous proteins. These antigens can also be transferred to professional antigen-presenting cells in a process called cross-presentation, which precedes initiation of a proper T-cell response; but exactly how they do this is unclear. We tested whether peptides can be transferred directly from the cytoplasm of one cell into the cytoplasm of its neighbour through gap junctions. Here we show that peptides with a relative molecular mass of up to~1,800 diffuse intercellularly through gap junctions unless a three-dimensional structure is imposed. This intercellular peptide transfer causes cytotoxic T-cell recognition of adjacent, innocent bystander cells as well as activated monocytes. Gap-junction-mediated peptide transfer is restricted to a few coupling cells owing to the high cytosolic peptidase activity. We present a mechanism of antigen acquisition for cross-presentation that couples the antigen presentation system of two adjacent cells and is lost in most tumours: gap-junction-mediated intercellular peptide coupling for presentation by bystander MHC class I molecules and transfer to professional antigen presenting cells for cross-priming. [ABSTRACT FROM AUTHOR]

MHC class I molecules bind short peptides and present them to CD8+ T cells. Contrary to textbook descriptions, the generation of MHC class-I-associated peptides from endogenous proteins is a highly dynamic and remarkably inefficient process. Here, we describe recent experiments that show how nascent and mature proteins are degraded into peptides that are trimmed, transported and trimmed again to enable presentation of a small portion of the generated peptides. By linking the failure rate of protein synthesis with antigen presentation, a rapid T-cell response is ensured, which is crucial in combating viral infections. Presentation on MHC class I molecules is achieved by less than 0.1% of the specific peptides that have survived intracellular destruction. The other peptides are converted into free amino acids that are used for recycling into new proteins. [ABSTRACT FROM AUTHOR]

Reports that the tripeptidyl peptidase II (TPPII) acts as an alternative to the proteosomes in the major histocompatibility complex (MHC) class I antigen presentation pathway and generates an HIV Nef epitope recognized by specific cytotoxic T lymphocytes. Events of the MHC class I antigen presentation pathway; Role of TPPII in the class I antigen presentation pathway; Function of cytosolic peptidase.

• Antigen processing for MHC-II relies on endosomal vesicle positioning and maturation. • Both the internal and global endosomal architecture can be reshaped upon immune cell activation. • Bidirectional endosomal movements drive MHC-II export to the cell surface. • Autophagy delivers self-antigens into the endosomal pathway and can lead to autoimmunity. Antigen-presenting cells (APCs) capture and present pathogens to T cells, thus arousing adaptive immune responses geared at the elimination of these invaders. In APCs, pathogens acquired from the extracellular space intersect with MHC class II (MHC-II) molecules in the endolysosomal system, where processing and loading of antigenic peptides occur. The resulting complexes can then be directed to the cell surface for recognition by T cells. To achieve this, the endosomal pathway of APCs must undergo dramatic rearrangements upon pathogen encounter. In this review we discuss recent strides in our understanding of how APCs modulate the organization and function of their endolysosomes to best suit different stages of antigen acquisition, processing and presentation cascade. [ABSTRACT FROM AUTHOR]

Since the discovery of MHC molecules, it has taken 40 years to arrive at a coherent picture of how MHC class I and MHC class II molecules really work. This is a story of the proteases and MHC-like chaperones that support the MHC class I and II molecules in presenting peptides to the immune system. We now understand that the MHC system shapes both the repertoire of presented peptides and the subsequent T cell response, with important implications ranging from transplant rejection to tumor immunotherapies. Here we present an illustrated review of the ins and outs of MHC class I and MHC class II antigen presentation. [ABSTRACT FROM AUTHOR]

The classical reagents to visualize, characterize and isolate specific CD8+ T cells are MHC I molecules multimerized on a streptavidin backbone. By labelling them with a fluorochrome these multimers can be used for analysis and isolation using flow cytometry. Folding of pMHC complexes is a laborious process that needs to be performed for every peptide separately, a hurdle that many attempted to be overcome by the development of a number of peptide exchange tricks. These methods allow us to fold one large batch of pMHCs and exchange the peptide by applying an appropriate trigger. We have recently published an exchange technology that does not rely on chemicals or UV that can damage the protein, but instead uses temperature to induce exchange. We have designed template peptides for HLA-A*02:01 and H-2Kb with an affinity that is high enough for efficient refolding at 4°C, but low enough for dissociation at elevated temperatures. Whereas preceding technologies required multimerization post exchange, this simple and flexible method can be applied on multimers, thus reducing the number of steps prior to T cell staining. Using this technology multiple MHC I multimers can be generated in parallel, to allow staining and isolation of large sets of CD8+ T cells. We are currently working on designing template peptides for other alleles and optimizing backbones and labelling strategies to further increase the multiplex performance of MHC I multimers. [ABSTRACT FROM AUTHOR]

HLA class I (HLA-I) molecules drive immune responses by presenting antigen-derived peptides to T-lymphocytes. Loading of these peptides into the HLA-I binding groove occurs via a multistep process which can be hijacked by viruses and tumors for immune escape. Using iterative genome-wide haploid genetic screening we identified the protease SPPL3 to enhance HLA-I antigen presentation by inhibiting B3GNT5 activity, an enzyme of the glycosphingolipid (GSL) synthesis pathway. GSL profiling by LCMS demonstrated SPPL3-deficient cells to express high levels of sialic acid containing B3GNT5-synthesized GSLs compared to wild type cells. These GSLs localize in close proximity to HLA-I molecules -as revealed by LCMS analysis of the local membrane surrounding HLA-I-thereby shielding membrane proximal HLA-I epitopes from epitope specific antibody recognition. On a functional level, HLA-I shielding also resulted in loss of ligand (LIR-1) binding and a defect in CD8+ T cells activation. HLA-I shielding GSLs are overexpressed on several subtypes of cancer, suggesting a novel form of immune escape. Moreover, pharmacological inhibition of these GSLs on AML cells abolished HLA-I shielding. Our findings demonstrate that the composition of the GSL repertoire determines the efficacy of antigen presentation and represents a potential treatment target in infection and cancer. [ABSTRACT FROM AUTHOR]

MHC class I and MHC class II molecules present peptides to the immune system to drive proper T cell responses. Pharmacological modulation of T-cell responses can offer treatment options for a range of immune-related diseases. Pharmacological downregulation of MHC molecules may find application in treatment of auto-immunity and transplantation rejection while pharmacological activation of antigen presentation would support immune responses to infection and cancer. Since the cell biology of MHC class I and MHC class II antigen presentation is understood in great detail, many potential targets for manipulation have been defined over the years. Here, we discuss how antigen presentation by MHC molecules can be modulated by pharmacological agents and how chemistry can further support the study of antigen presentation in general. The chemical biology of antigen presentation by MHC molecules shows surprising options for immune modulation and the development of future therapies. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Examines the role of P13-K- and Vav-dependent pathway in regulating PKC family translocation in T cells. Mechanism of mature T cells activation; Correlation between T cells and PKC enzymes; Importance of actin cytoskeleton organization in Vav/Rac pathway activity.

Abstract: MHC class II molecules (MHCII) are critical for presenting antigens to CD4+ T-cells. They control ignition of CD4+ T cells and are as such involved in most auto-immune diseases. To define proteins and pathways controlling MHCII antigen presentation and expression, we performed a genome-wide flow cytometry based RNAi screen. Hits were subsequently classified by two screens that monitored the intracellular distribution and transcription of MHCII. This multi-dimensional approach allowed subclassification of hits into functional groups as a first step to defining new pathways controlling MHCII antigen presentation. The datasets from this screen are used as a template for several follow-up studies. This overview focuses on how data from genome-wide screens can be used for target-lead finding, data mining, systems biology and systematic cell biology. [Copyright &y& Elsevier]