Differential use of autophagy by primary dendritic cells specialized in cross-presentation

Antigen-presenting cells survey their environment and present captured antigens bound to major histocompatibility complex (MHC) molecules. Formation of MHC-antigen complexes occurs in specialized compartments where multiple protein trafficking routes, still incompletely understood, converge. Autophagy is a route that enables the presentation of cytosolic antigen by MHC class II molecules. Some reports also implicate autophagy in the presentation of extracellular, endocytosed antigen by MHC class I molecules, a pathway termed “cross-presentation.” The role of autophagy in cross-presentation is controversial. This may be due to studies using different types of antigen presenting cells for which the use of autophagy is not well defined. Here we report that active use of autophagy is evident only in DC subtypes specialized in cross-presentation. However, the contribution of autophagy to cross-presentation varied depending on the form of antigen: it was negligible in the case of cell-associated antigen or antigen delivered via receptor-mediated endocytosis, but more prominent when the antigen was a soluble protein. These findings highlight the differential use of autophagy and its machinery by primary cells equipped with specific immune function, and prompt careful reassessment of the participation of this endocytic pathway in antigen cross-presentation.     

Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells

Immature dendritic cells (DCs) sample their environment for antigens and after stimulation present peptide associated with major histocompatibility complex class II (MHC II) to naive T cells. We have studied the intracellular trafficking of MHC II in cultured DCs. In immature cells, the majority of MHC II was stored intracellularly at the internal vesicles of multivesicular bodies (MVBs). In contrast, DM, an accessory molecule required for peptide loading, was located predominantly at the limiting membrane of MVBs. After stimulation, the internal vesicles carrying MHC II were transferred to the limiting membrane of the MVB, bringing MHC II and DM to the same membrane domain. Concomitantly, the MVBs transformed into long tubular organelles that extended into the periphery of the cells. Vesicles that were formed at the tips of these tubules nonselectively incorporated MHC II and DM and presumably mediated transport to the plasma membrane. We propose that in maturing DCs, the reorganization of MVBs is fundamental for the timing of MHC II antigen loading and transport to the plasma membrane.     

Identification of a novel antigen cross‐presenting cell type in spleen

Antigen-presenting cells (APC), like dendritic cells (DC), are essential for T-cell activation, leading to immunity or tolerance. Multiple DC subsets each play a unique role in the immune response. Here, a novel splenic dendritic-like APC has been characterized in mice that has immune function and cell surface phenotype distinct from other, described DC subsets. These were identified as a cell type continuously produced in spleen long-term cultures (LTC) and have an in vivo equivalent cell type in mice, namely ‘L-DC’. This study characterizes LTC-DC in terms of marker phenotype and function, and compares them with L-DC and other known splenic DC and myeloid subsets. L-DC display a myeloid dendritic-like phenotype equivalent to LTC-DC as CD11c^loCD11b^hiMHC-II⁻CD8α⁻ cells, distinct by high accessibility and endocytic capacity for blood-borne antigen. Both LTC-DC and L-DC have strong antigen cross-presentation ability leading to strong activation of CD8⁺ T cells, particularly after exposure to lipopolysaccharide. However, they have weak ability to stimulate CD4⁺ T cells in antigen-specific responses. Evidence is presented here for a novel DC type produced by in vitro haematopoiesis which has distinct antigen-presenting potential and reflects a DC subset present also in vivo in spleen.     

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.     

Proteasome and class I antigen processing and presentation

The recent discovery of two proteasome homologous genes,LMP2 andLMP7, in the class II region of the human MHC, has implicated this multi-subunit protease in an early step of the immune response; the degradation of intracellular and viral proteins. Short peptides produced by the proteasome are transported into the ER by the product of another set of MHC class II genes,TAP1 andTAP2, where they bind and stabilise HLA class I molecules. Antigenic peptides displayed at the cell surface by HLA class I molecules mark cells for destruction by cytotoxic T lymphocytes. The role of the proteasome in antigen processing was questioned when mutant cells, which lack theLMP genes, were able to process and present antigens normally. The discovery that two proteasome -subunits, delta andMB1, highly homologous toLMP2 andLMP7 and expressed in reciprocal manner, is now consistent with a role for the proteasome in antigen processing. The incorporation of different -subunits into the proteasome may be a mechanism to modulate catalytic activity of the proteasome complex, allowing production of peptides that are more suitable to enter into the ER by the TAP transporters and to bind HLA class I molecules. But, in the absence of the LMPs, the other subunits permit processing of most antigens reasonably efficiently.Abbreviations ABC ATP-binding cassete - 2m 2-microglobulin - ER endoplasmic reticulum - IFN interferon - LMP low molecular weight peptide - MHC major histocompatibility complex - TAP transporter associated with antigen processing     

Interaction between antigen presenting cells and autoreactive T cells derived from BXSB mice with murine lupus

Systemic lupus erythematosus （SLE） is a typical autoimmune disease involving multiple systems and organs. Ample evidence suggests that autoreactive T cells play a pivotal role in the development of this autoimmune disorder. This study was undertaken to investigate the mechanisms of interaction between antigen presenting cells （APCs） and an autoreactive T cell （ATLI） clone obtained from lupus-prone BXSB mice. ATLI cells, either before or after 7-ray irradiation, were able to activate naive B cells, as determined by B cell proliferation assays. Macrophages from BXSB mice were able to stimulate the proliferation of resting ATL 1 cells at a responder/stimulator （R/S） ratio of 1/2.5. Dendritic cells （DCs） were much more powerful stimulators for ATLI cells on a per cell basis. The T cell stimulating ability ofmacrophages and B cells, but not DCs, was sensitive to T-ray irradiation. Monoclonal antibodies against mouse MHC-Ⅱ and CD4 were able to block DC-mediated stimulation of ATL 1 proliferation, indicating cognate recognition between ATL 1 and APCs. Our data suggest that positive feedback loops involving macrophages, B cells and autoreactive T cells may play a pivotal role in keeping the momentum of autoimmune responses leading to autoimmune diseases.     

Employing proteomics in the study of antigen presentation: an update

Introduction: Our immune system discriminates self from non-self by examining the peptide cargo of human leukocyte antigen (HLA) molecules displayed on the cell surface. Successful recognition of HLA-bound non-self peptides can induce T cell responses leading to, for example, the destruction of infected cells. Today, largely due to advances in technology, we have an unprecedented capability to identify the nature of these presented peptides and unravel the true complexity of antigen presentation.

Areas covered: In addition to conventional linear peptides, HLA molecules also present post-translationally modified sequences comprising a wealth of chemical and structural modifications, including a novel class of noncontiguous spliced peptides. This review focuses on these emerging themes in antigen presentation and how mass spectrometry in particular has contributed to a new view of the antigenic landscape that is presented to the immune system.

Expert Commentary: Advances in the sensitivity of mass spectrometers and use of hybrid fragmentation technologies will provide more information-rich spectra of HLA bound peptides leading to more definitive identification of T cell epitopes. Coupled with improvements in sample preparation and new informatics workflows, studies will access novel classes of peptide antigen and allow interrogation of rare and clinically relevant samples.     

The autophagy machinery restrains iNKT cell activation through CD1D1 internalization

Invariant natural killer T (iNKT) cells are innate T cells with powerful immune regulatory functions that recognize glycolipid antigens presented by the CD1D protein. While iNKT cell-activating glycolipids are currently being explored for their efficacy to improve immunotherapy against infectious diseases and cancer, little is known about the mechanisms that control CD1D antigen presentation and iNKT cell activation in vivo. CD1D molecules survey endocytic pathways to bind lipid antigens in MHC class II-containing compartments (MIICs) before recycling to the plasma membrane. Autophagosomes intersect with MIICs and autophagy-related proteins are known to support antigen loading for increased CD4⁺ T cell immunity. Here, we report that mice with dendritic cell (DC)-specific deletion of the essential autophagy gene Atg5 showed better CD1D1-restricted glycolipid presentation in vivo. These effects led to enhanced iNKT cell cytokine production upon antigen recognition and lower bacterial loads during Sphingomonas paucimobilis infection. Enhanced iNKT cell activation was independent of receptor-mediated glycolipid uptake or costimulatory signals. Instead, loss of Atg5 in DCs impaired clathrin-dependent internalization of CD1D1 molecules via the adaptor protein complex 2 (AP2) and, thus, increased surface expression of stimulatory CD1D1-glycolipid complexes. These findings indicate that the autophagic machinery assists in the recruitment of AP2 to CD1D1 molecules resulting in attenuated iNKT cell activation, in contrast to the supporting role of macroautophagy in CD4⁺ T cell stimulation.     

Murine spleen contains a diversity of myeloid and dendritic cells distinct in antigen presenting function

The spleen contains multiple subsets of myeloid and dendritic cells (DC). DC are important antigen presenting cells (APC) which induce and control the adaptive immune response. They are cells specialized for antigen capture, processing and presentation to naïve T cells. However, DC are a heterogeneous population and each subset differs subtly in phenotype, function and location. Similarly, myeloid cell subsets can be distinguished which can also play an important role in the regulation of immunity. This review aims to characterize splenic subsets of DC and myeloid cells to better understand their individual roles in the immune response.     

Antigen traffic pathways in dendritic cells

Dendritic cells (DC) are now believed to be the principal initiators of T cell-mediated immune responses. Their location in body tissues, migratory behaviour in response to inflammatory stimuli, endocytic properties, expression of MHC molecules and key T cell stimulatory molecules and many other attributes place these remarkable cells in a unique and influential position in the immune system. Progress in DC culture methods has recently allowed in-depth studies on the cell biological features that enable them to fulfil their crucial role in the immune response.     

The dendritic cell receptor for endocytosis, DEC-205, can recycle and enhance antigen presentation via major histocompatibility complex class II-positive lysosomal compartments

Many receptors for endocytosis recycle into and out of cells through early endosomes. We now find in dendritic cells that the DEC-205 multilectin receptor targets late endosomes or lysosomes rich in major histocompatibility complex class II (MHC II) products, whereas the homologous macrophage mannose receptor (MMR), as expected, is found in more peripheral endosomes. To analyze this finding, the cytosolic tails of DEC-205 and MMR were fused to the external domain of the CD16 Fcgamma receptor and studied in stable L cell transfectants. The two cytosolic domains each mediated rapid uptake of human immunoglobulin (Ig)G followed by recycling of intact CD16 to the cell surface. However, the DEC-205 tail recycled the CD16 through MHC II-positive late endosomal/lysosomal vacuoles and also mediated a 100-fold increase in antigen presentation. The mechanism of late endosomal targeting, which occurred in the absence of human IgG, involved two functional regions: a membrane-proximal region with a coated pit sequence for uptake, and a distal region with an EDE triad for the unusual deeper targeting. Therefore, the DEC-205 cytosolic domain mediates a new pathway of receptor-mediated endocytosis that entails efficient recycling through late endosomes and a greatly enhanced efficiency of antigen presentation to CD4(+) T cells.     

Processing of proteins in autophagy vesicles of antigen-presenting cells generates citrullinated peptides recognized by the immune system

Our laboratory has been investigating for some time the nature of the response of T lymphocytes in autoimmunity in the reactions against self-proteins that result in a number of diseases, such as type 1 diabetes, multiple sclerosis, rheumatoid arthritis (RA) and others. T cells recognize peptides generated from proteins that are processed by antigen-presenting cells (APC). The peptides may derive from exogenous proteins or from the normal catabolism of self-proteins. The peptides complexed to major histocompatibility complex (MHC) molecules constitute the chemical entity that is engaged by the antigen-receptor of T cells. An important hypothesis postulates that self-peptides that suffer post-translational modifications in the APC may form neo-antigens that are recognized by the immune system and form the target of autoimmunity. Our interest in citrullination in the context of antigen processing and presentation stemmed from studies suggesting that an immune response to citrullinated self-peptides may be involved in autoimmunity. In a first publication, we found T cells that specifically recognized citrullinated peptides after immunization of inbred mice with standard foreign proteins. We used the small protein hen-egg white lysozyme. These T cells only recognized the citrullinated peptide and not the unmodified one, thus proving that a neo-epitope had been created by this modification. But how this modification took place was not known. Our recent report describes a central role for autophagy in citrullination of peptides by APC.     

Processing of proteins in autophagy vesicles of antigen-presenting cells generates citrullinated peptides recognized by the immune system

Our laboratory has been investigating for some time the nature of the response of T lymphocytes in autoimmunity in the reactions against self-proteins that result in a number of diseases, such as type 1 diabetes, multiple sclerosis, rheumatoid arthritis (RA) and others. T cells recognize peptides generated from proteins that are processed by antigen-presenting cells (APC). The peptides may derive from exogenous proteins or from the normal catabolism of self-proteins. The peptides complexed to major histocompatibility complex (MHC) molecules constitute the chemical entity that is engaged by the antigen-receptor of T cells. An important hypothesis postulates that self-peptides that suffer post-translational modifications in the APC may form neo-antigens that are recognized by the immune system and form the target of autoimmunity. Our interest in citrullination in the context of antigen processing and presentation stemmed from studies suggesting that an immune response to citrullinated self-peptides may be involved in autoimmunity. In a first publication, we found T cells that specifically recognized citrullinated peptides after immunization of inbred mice with standard foreign proteins. We used the small protein hen-egg white lysozyme. These T cells only recognized the citrullinated peptide and not the unmodified one, thus proving that a neo-epitope had been created by this modification. But how this modification took place was not known. Our recent report describes a central role for autophagy in citrullination of peptides by APC.     

Antigen presentation between T cells drives Th17 polarization under conditions of limiting antigen

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Designing Peptide Vaccines for Cellular Cross-Presentation

Synthetic peptides are safe and relatively cheap vaccine components. However, the efficiency of peptide vaccines is limited by peptide interaction with non-professional antigen-presenting cells, which may hamper induction of productive T-cell responses. This paper argues that peptide vaccines should be modified for exclusive uptake by cells with the capacity to prime T-cell responses. Moreover, design of peptide vaccines should take intracellular antigen processing into account and exploit cellular mechanisms of proteolysis, transport and HLA class I assembly of antigenic peptides to enhance efficiency of T-cell priming and stimulation.     

溶酶体途径在细胞自噬过程中的功能意义

溶酶体具有高度保守的异质性,是细胞自噬的关键细胞器。细胞质中的蛋白质和细胞器最终在溶酶体降解,故溶酶体在维持细胞结构和功能的平衡方面起着重要生理作用。通过自噬溶酶体途径,细胞可清除某些病原体并参与抗原呈递。细胞自噬与异噬经溶酶体密切联系。自噬过程中溶酶体功能障碍与某些疾病和衰老等相关。对细胞自噬的溶酶体途径及其功能意义作了概述。     

Tumor antigen presentation: changing the rules

 Cell-based tumor vaccines have been developed on the basis of the hypothesis that tumor cells can be genetically modified to present antigen to T lymphocytes directly. Contrary to expectations, cross-priming is the predominant pathway for activation of tumor-specific CD8⁺ T cells, while direct presentation of antigen dominates activation of tumor-specific CD4⁺ T cells. These results pose interesting paradoxes for the generation of immune responses, and have definite implications for the development of anti-cancer vaccines. Received: 10 October 1997 / Accepted 10 January 1998     

树突状细胞和癌症的免疫学治疗

树突状细胞(dendritc cells,DC)是一种抗原提呈细胞,能特异地引发和调控机体免疫。它具有抗原呈现功能而不损害免疫系统,不仅能够激活CD4^ 辅助T细胞和CD8^ 细胞毒性T细胞,还能活化B细胞和自然杀伤细胞。已有的研究让人们看到了癌症疫苗的希望,但还处于早期阶段,有许多尚未确定的因素。因此有关DC疫苗用于对肿瘤的保护性和治疗性免疫还有待于进一步的研究。     

抗原处理相关转运体蛋白的研究进展

抗原处理相关转运体(transporter associated with antigen processing,TAP)蛋白在抗原提呈途径中发挥重要作用,它负责将内源性抗原从胞浆运送到内质网(endoplasmic reticulum,ER),以便主要组织相容性复合体(major histocompatibility complex,MHC)I结合多肽。TAP属于ATP结合盒(ATP-binding cassette,ABC)转运蛋白超家族B族,是由TAP1和TAP2两个亚基构成的异二聚体蛋白,其每个亚基各含有一个亲水的核酸结合区和一个疏水的跨膜结构域,并具有促进肽段转运的结构域。TAP参与MHC I类分子的组装,并在人获得性免疫系统中起着至关重要的作用。TAP基因具有多态性,因而增加了个体对疾病的易感性。TAP基因的突变及其调节机制的缺陷都可以导致其活性和表达下调,从而影响病毒性感染和肿瘤等疾病的发生。