The crosstalk between autophagic and endo-/exosomal pathways in antigen processing for MHC presentation in anticancer T cell immune responses

T cells recognize antigen fragments from proteolytic products that are presented to them in the form of peptides on major histocompatibility complex (MHC) molecules, which is crucial for the T cell to identify infected or transformed cells. Autophagy, a process that delivers cytoplasmic constituents for lysosomal degradation, has been observed to provide a substantial source of intra- and extracellular antigens for MHC presentation to T cells, which will impact the tumor-specific immune response. Meanwhile, extracellular components are transported to cytoplasm for the degradation/secretion process by the endo-/exosomal pathway and are thus involved in multiple physiological and pathological processes, including immune responses. Autophagy and endo-/exosomal pathways are intertwined in a highly intricate manner and both are closely involved in antigen processing for MHC presentation; thus, we propose that they may coordinate in antigen processing and presentation in anticancer T cell immune responses. In this article, we discuss the molecular and functional crosstalk between autophagy and endo-/exosomal pathways and their contributions to antigen processing for MHC presentation in anticancer T cell immune responses.     

Autophagy and antigen presentation

CD4(+) T cells co-ordinate adaptive immunity and are required for immunological memory establishment and maintenance. They are thought to primarily recognize extracellular antigens, which are endocytosed, processed by lysosomal proteases and then presented on major histocompatibility complex (MHC) class II. However, recent studies have demonstrated that viral, tumour and autoantigens can gain access to this antigen presentation pathway from within cells by autophagy. This review will discuss the autophagic pathways that contribute to endogenous MHC class II antigen processing. Furthermore, potential characteristics of autophagy substrates, qualifying them to access these pathways, and regulation of autophagy will be considered. Finally, I will suggest how antigen presentation after autophagy might contribute to immune surveillance of infected and transformed cells.     

Phosphoinositide 3-kinase activation by Igbeta controls de novo formation of an antigen-processing compartment

Antigens that bind B cell antigen receptor (BCR) are preferentially and rapidly processed for antigen presentation. The BCR is a multimeric complex containing a signaling module composed of Igalpha and Igbeta. Signaling pathways implicated in antigen presentation through the BCR are ill defined. Here we demonstrate that phosphoinositide 3-kinase (PI3K) inhibitors preclude antigen presentation induced by BCR or Igbeta but not Igalpha. Unraveling the mechanisms responsible for this inhibition, we show that PI3K inhibitors block neither antigen internalization nor degradation. Rather PI3K inhibitors block de novo formation of a multivesicular antigen processing compartment, which is induced by triggering of the BCR or Igbeta. Strikingly, we found using fluorescent probes binding specifically to PI3K products that BCR and Igbeta but not Igalpha induce PI3K activation in endocytic compartments wherein antigen is transported. Altogether, these results strongly suggest that Igbeta couples the BCR to PI3K activation that is instrumental for de novo formation of the antigen processing compartment and efficient antigen presentation.     

Sequential proteolytic processing of the capsular Caf1 antigen of Yersinia pestis for major histocompatibility complex class II-restricted presentation to T lymphocytes

We studied the mechanisms of antigen presentation of CD4 T cell epitopes of the capsular Caf1 antigen of Yersinia pestis using murine bone marrow macrophages as antigen presenting cells and T cell hybridomas specific for major histocompatibility complex (MHC) class II-restricted epitopes distributed throughout the Caf1 sequence. The data revealed diversity in the pathways used and the degrees of antigen processing required depending on the structural context of epitopes within the Caf1 molecule. Two epitopes in the carboxyl-terminal globular domain were presented by newly synthesized MHC class II after low pH-dependent lysosomal processing, whereas an epitope located in a flexible amino-terminal strand was presented by mature MHC class II independent of low pH and with no detectable requirement for proteolytic processing. A fourth epitope located between the two regions of Caf1 showed intermediate behavior. The data are consistent with progressive unfolding and cleavage of rCaf1 from the amino terminus as it traverses the endosomal pathway, the availability of epitopes determining which pool of MHC class II is preferentially loaded. The Caf1 capsular protein is a component of second generation plague vaccines and an understanding of the mechanisms and pathways of MHC class II-restricted presentation of multiple epitopes from this candidate vaccine antigen should inform the choice of delivery systems and adjuvants that target vaccines successfully to appropriate intracellular locations to induce protective immune responses against as wide a T cell repertoire as possible.     

Ionizing Radiation Drives Key Regulators of Antigen Presentation and a Global Expansion of the Immunopeptidome

Little is known about the pathways regulating MHC antigen presentation and the identity of treatment-specific T cell antigens induced by ionizing radiation. For this reason, we investigated the radiation-specific changes in the colorectal tumor cell proteome. We found an increase in DDX58 and ZBP1 protein expression, two nucleic acid sensing molecules likely involved in induction of the dominant interferon response signature observed after genotoxic insult. We further observed treatment-induced changes in key regulators and effector proteins of the antigen processing and presentation machinery. Differential regulation of MHC allele expression was further driving the presentation of a significantly broader MHC-associated peptidome postirradiation, defining a radiation-specific peptide repertoire. Interestingly, treatment-induced peptides originated predominantly from proteins involved in catecholamine synthesis and metabolic pathways. A nuanced relationship between protein expression and antigen presentation was observed where radiation-induced changes in proteins do not correlate with increased presentation of associated peptides. Finally, we detected an increase in the presentation of a tumor-specific neoantigen derived from Mtch1. This study provides new insights into how radiation enhances antigen processing and presentation that could be suitable for the development of combinatorial therapies. Data are available via ProteomeXchange with identifier PXD032003.     

Promiscuous binding of invariant chain-derived CLIP peptide to distinct HLA-I molecules revealed in leukemic cells

Antigen presentation by HLA class I (HLA-I) and HLA class II (HLA-II) complexes is achieved by proteins that are specific for their respective processing pathway. The invariant chain (Ii)-derived peptide CLIP is required for HLA-II-mediated antigen presentation by stabilizing HLA-II molecules before antigen loading through transient and promiscuous binding to different HLA-II peptide grooves. Here, we demonstrate alternative binding of CLIP to surface HLA-I molecules on leukemic cells. In HLA-II-negative AML cells, we found plasma membrane display of the CLIP peptide. Silencing Ii in AML cells resulted in reduced HLA-I cell surface display, which indicated a direct role of CLIP in the HLA-I antigen presentation pathway. In HLA-I-specific peptide eluates from B-LCLs, five Ii-derived peptides were identified, of which two were from the CLIP region. In vitro peptide binding assays strikingly revealed that the eluted CLIP peptide RMATPLLMQALPM efficiently bound to four distinct HLA-I supertypes (-A2, -B7, -A3, -B40). Furthermore, shorter length variants of this CLIP peptide also bound to these four supertypes, although in silico algorithms only predicted binding to HLA-A2 or -B7. Immunization of HLA-A2 transgenic mice with these peptides did not induce CTL responses. Together these data show a remarkable promiscuity of CLIP for binding to a wide variety of HLA-I molecules. The found participation of CLIP in the HLA-I antigen presentation pathway could reflect an aberrant mechanism in leukemic cells, but might also lead to elucidation of novel processing pathways or immune escape mechanisms.     

Nuclear membrane-derived autophagy,a novel process that participates in the presentation of endogenous viral antigens during HSV-1 infection

Complex membrane trafficking events are involved in the regulation of antigen processing and presentation of both endogenous and exogenous antigens. While these processes were believed to involve mainly organelles along the endo/phagocytic and the biosynthetic pathways, recent studies have shown that autophagy also participates actively in both innate and adaptive immunity. We have shown recently that, in macrophages infected with the Herpes simplex type 1 virus, autophagy plays a key role in the targeting of viral proteins to hydrolytic compartments, and their processing for presentation on MHC class I molecules. This pathway involves a novel type of autophagosomes formed by coiling of the nuclear membrane where viral proteins are highly enriched. The ability to enhance the contribution of autophagy to antigen presentation in various conditions suggests that this pathway could be used to boost the immune response against viral infection and develop new vaccines.     

The NeuARt II system: a viewing tool for neuroanatomical data based on published neuroanatomical atlases

Background

Activation of naïve B lymphocytes by extracellular ligands, e.g. antigen, lipopolysaccharide (LPS) and CD40 ligand, induces a combination of common and ligand-specific phenotypic changes through complex signal transduction pathways. For example, although all three of these ligands induce proliferation, only stimulation through the B cell antigen receptor (BCR) induces apoptosis in resting splenic B cells. In order to define the common and unique biological responses to ligand stimulation, we compared the gene expression changes induced in normal primary B cells by a panel of ligands using cDNA microarrays and a statistical approach, CLASSIFI (Cluster Assignment for Biological Inference), which identifies significant co-clustering of genes with similar Gene Ontology? annotation.

Results

CLASSIFI analysis revealed an overrepresentation of genes involved in ion and vesicle transport, including multiple components of the proton pump, in the BCR-specific gene cluster, suggesting that activation of antigen processing and presentation pathways is a major biological response to antigen receptor stimulation. Proton pump components that were not included in the initial microarray data set were also upregulated in response to BCR stimulation in follow up experiments. MHC Class II expression was found to be maintained specifically in response to BCR stimulation. Furthermore, ligand-specific internalization of the BCR, a first step in B cell antigen processing and presentation, was demonstrated.

Conclusion

These observations provide experimental validation of the computational approach implemented in CLASSIFI, demonstrating that CLASSIFI-based gene expression cluster analysis is an effective data mining tool to identify biological processes that correlate with the experimental conditional variables. Furthermore, this analysis has identified at least thirty-eight candidate components of the B cell antigen processing and presentation pathway and sets the stage for future studies focused on a better understanding of the components involved in and unique to B cell antigen processing and presentation.     

Purification and Properties of Alcohol Dehydrogenase from Tea Seeds

The process of antigen presentation is not well understood. We screened for drugs that distinguish presentation of allogeneic class 2 antigens and exogenous antigens. When spleen cells were used as antigen presenting cells (APC), leupeptin and antipain preferentially inhibited allogeneic class 2 presentation, while they did not affect presentation of exogenous antigen and T cell growth. In contrast, they inhibited both presentations when spleen adherent cells (SAC) were used as APC. Our results suggest that SAC (mainly macrophages) and splenic B cells use different pathways to present exogenous antigens and that pathways to present allogeneic class 2 molecules are similar.     

The MHC: relationship between linkage and function.

It is intriguing that several genes with associated functions, including all of class I and class II genes, as well as some genes affecting antigen presentation of both class I and class II pathways, are linked in the MHC. Recent observations have led to speculation that there may be a functional explanation for keeping these related genes together.     

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.     

Apoptotic cells deliver processed antigen to dendritic cells for cross-presentation

Antigen derived from engulfed apoptotic cells can be cross-presented by dendritic cells (DCs) for the generation of major histocompatibility class I/peptide complexes. While the early events of recognition and internalization of the dying cell have been characterized, the antigen-processing pathway or pathways remain unknown. We established a mouse model assaying for the activation of polyclonal T cells reactive to antigen derived from apoptotic cells, and demonstrated two distinct pathways for the trafficking of exogenous epitopes. In the first, exogenous antigen is dependent on the DC's expression of a functional transporter associated with antigen processing (TAP). Surprisingly, we found evidence that a second pathway exists in which transfer of processed antigen from the dying cell allows formation of major histocompatibility class I/peptide complexes in TAP-deficient DCs. In vivo data suggest that in situations of stress (e.g., viral infection), this latter pathway may be more efficient, illustrating that dying cells may preselect immunologically important antigenic determinants.     

Immunological responses and actin dynamics in macrophages are controlled by N-cofilin but are independent from ADF

Dynamic changes in the actin cytoskeleton are essential for immune cell function and a number of immune deficiencies have been linked to mutations, which disturb the actin cytoskeleton. In macrophages and dendritic cells, actin remodelling is critical for motility, phagocytosis and antigen presentation, however the actin binding proteins, which control antigen presentation have been poorly characterized. Here we dissect the specific roles of the family of ADF/cofilin F-actin depolymerizing factors in macrophages and in local immune responses. Macrophage migration, cell polarization and antigen presentation to T-cells require n-cofilin mediated F-actin remodelling. Using a conditional mouse model, we show that n-cofilin also controls MHC class II-dependent antigen presentation. Other cellular processes such as phagocytosis and antigen processing were found to be independent of n-cofilin. Our data identify n-cofilin as a novel regulator of antigen presentation, while ADF on the other hand is dispensable for macrophage motility and antigen presentation.     

Antigen presentation in Syrian hamster cells: substrate selectivity of TAP controlled by polymorphic residues in TAP1 and differential requirements for loading of H2 class I molecules

Expression of mouse major histocompatibility complex (MHC) class I molecules in different cell lines derived from Syrian hamsters has revealed antigen presentation deficiencies of some H2 allelic products in two cell lines (BHK and NIL-2) which were overcome by transient expression of the rat transporter associated with antigen processing (TAP; Lobigs et al. 1995). Here we show that in both cell lines the endogenous MHC class I cell surface expression was completely down-regulated. Lymphokine treatment induced endogenous and recombinant mouse MHC class I cell surface expression to levels similar to that in other Syrian hamster cell lines competent for antigen presentation through transduced H2 molecules. Accordingly, constitutive downregulation of expression of accessory molecules of the MHC class I pathway can reveal differences between H2 class I alleles in antigen presentation not encountered when the expression levels are augmented. In addition to the differential expression of MHC class I pathway genes, two cell lines representing competent (FF) and defective (BHK) antigen presentation phenotypes for mouse class I MHC restriction elements demonstrated substantial sequence polymorphism in Tap1 but not Tap2. Cytokine-treated FF or BHK cells and human TAP-deficient T2 cells transfected with FF or BHK TAP1 in combination with FF TAP2 differed in their preference for C-terminal peptide residues, as shown by an in vitro peptide transport assay. Thus, polymorphic residues in TAP1 can influence the substrate selectivity of the Syrian hamster peptide transporter.     

A deterministic model for the processing and presentation of bacteria-derived antigenic peptides

The amount and the dynamics of antigen supply to the cellular antigen processing and presentation machinery differ largely among diverse microbial antigens and various types of antigen presenting cells. The precise influence, however, of antigen supply on the antigen presentation pattern of cells is not known. Here, we provide a basic deterministic mathematical model of antigen processing and presentation of microbial antigens. The model predicts that different types of antigen presenting cells e.g. cells presenting or cross-presenting exogenous antigens, cells infected with replicating microbes, or cells in which microbial antigen synthesis is blocked after a certain period of time have inherently different antigen presentation patterns which are defined by the kinetics of antigen supply. The reevaluation of existing experimental data [Sijts, A.J., Pamer, E.G., 1997. Enhanced intracellular dissociation of major histocompatibility complex class I-associated peptides: a mechanism for optimizing the spectrum of cell surface-presented cytotoxic T lymphocyte epitopes. J. Exp. Med. 185, 1403-1411] describing the processing and presentation of two antigenic peptides derived from the p60 proteins of the facultatively intracellular bacterium Listeria monocytogenes shows that p60 proteins accumulating intracellularly during bacterial infection of cells play no measurable role as substrate for the cytosolic antigen presentation pathway.     

Specificity of human cathepsin S determined by processing of peptide substrates and MHC class II-associated invariant chain

Cathepsin S (CatS) is a lysosomal cysteine protease of the papain family, the members of which possess relatively broad substrate specificities. It has distinct roles in major histocompatibility complex (MHC) class II-associated peptide loading and in antigen processing in both the MHC class I and class II pathways. It may therefore represent a target for interference with antigen presentation, which could be of value in the therapy of (auto)immune diseases. To obtain more detailed information on the specificity of CatS, we mapped its cleavage site preferences at subsites S3-S1' by in vitro processing of a peptide library. Only five amino acid residues at the substrate's P2 position allowed for cleavage by CatS under time-limited conditions. Preferences for groups of amino acid residues were also observed at positions P3, P1 and P1'. Based on these results, we developed highly CatS-sensitive peptides. After processing of MHC class II-associated invariant chain (Ii), a natural protein substrate of CatS, we identified CatS cleavage sites in Ii of which a majority matched the amino acid residue preference data obtained with peptides. These observed cleavage sites in Ii might be of relevance for its in vivo processing by CatS.     

MHC class II presentation of endogenous tumor antigen by cellular vaccines depends on the endocytic pathway but not H2-M

We have developed cell-based cancer vaccines that activate anti-tumor immunity by directly presenting endogenously synthesized tumor antigens to CD4⁺ T helper lymphocytes via MHC class II molecules. The vaccines are non-conventional antigen-presenting cells because they express MHC class II, do not express invariant chain or H-2M, and preferentially present endogenous antigen. To further improve therapeutic efficacy we have studied the intracellular trafficking pathway of MHC class II molecules in the vaccines using endoplasmic reticulum-localized lysozyme as a model antigen. Experiments using endocytic and cytosolic pathway inhibitors (chloroquine, primaquine, and brefeldin A) and protease inhibitors (lactacystin, LLnL, E64, and leupeptin) indicate antigen presentation depends on the endocytic pathway, although antigen degradation is not mediated by endosomal or proteasomal proteases. Because H2-M facilitates presentation of exogenous antigen via the endocytic pathway, we investigated whether transfection of vaccine cells with H-2M could potentiate endogenous antigen presentation. In contrast to its role in conventional antigen presentation, H-2M had no effect on endogenous antigen presentation by vaccine cells or on vaccine efficacy. These results suggest that antigen/MHC class II complexes in the vaccines may follow a novel route for processing and presentation and may produce a repertoire of class II-restricted peptides different from those presented by professional APC. The therapeutic efficacy of the vaccines, therefore, may reside in their ability to present novel tumor peptides, consequently activating tumor-specific CD4⁺ T cells that would not otherwise be activated.