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CD8+ T cytotoxic cells are the immune system's main effector cells in eliminating intracellular pathogens, such as viruses and some species of bacteria. CD8+ T cytotoxic cells are activated via Major Histocompatability Complex (MHC) Class I and co-receptors on dendritic cells.
MHC Class I processes intracellular antigen found in the cytosol (4). Antigen follows the endogenous processing pathway by being degraded in the protesome of the host cell. Surviving peptides of between 8-10 amino acids (1) are transported to the endoplasmic reticulum (ER) by TAP1 (transporter associated with antigen processing 1) and TAP2 (2,3), where they are loaded onto a MHC Class I molecule. These molecules are transported to the surface of the cell via the Golgi apparatus. Cross-presentation is the process where extracellular peptides are loaded onto MHC Class I molecules when usually they are loaded onto MHC Class II molecules (5).
MHC Class I is constitutively expressed on all tissues in the body (7), and therefore is presented bound to foreign peptides if a cell is infected. T cells require co-stimulation in order to perform their effector functions, and this cannot be achieved with regular tissue cells. Activation requires dendritic cell help (6). Therefore their must be a mechanism by which antigen from infected tissue cells is transported to a dendritic cell to initiate an adaptive immune response. This process is termed cross-presentation. Despite cross-presentation playing a vital role in protection against viruses and tumours (8), the exact mechanism by which it occurs is not fully known. Several possible models have been suggested, but none of these have been definitively proved nor accepted.
Firstly I intend to describe the potential mechanisms by which peptides are presented to MHC Class I molecules for cross-presentation. I then intend to evaluate sources of this extracellular antigen and the proposed mechanisms by which it enters the MHC Class I processing pathway.
The ER functions include the production of proteins from mRNA and transportation of these proteins to the Golgi apparatus. An important aspect of these functions is the correct folding of these proteins, and also the identification of misfolded proteins or proteins generated from viral RNA (9). If these are identified, the proteins are transported to the cytosol through a membrane pore (10). They then enter Endoplasmic Reticulum Associated Protein Degradation (ERAD) system, where they are degraded in the phagosome (11). The ERAD system targets unfolded proteins for ubiquitination by labelling them for degradation in the phagosome, before transporting them to the cytosol (5, 11).
The ER has been implicated in MHC Class I cross-presentation, with the ERAD protein Sec61 being an ER-associated protein (10, 12). Transportation between phagosomes, the cytosol and the ER is an unclear and complex system, but it seems to be vital in allowing peptides to undergo cross-presentation. It has been shown that several Toll-like receptors (TLRs) produced in the ER, such as TLR3, can access endosomes and lysosomes after TLR activation (13,14). On this basis it may be possible that other ER-derived proteins, such as parts of the MHC Class I processing machinery, may also be transported to these intracellular compartments after TLR activation of dendritic cells (10). Cross-presentation through this mechanism requires the recruitment of TAP1 and TAP2 to the phagosome. These proteins may then be used as a source of peptide for MHC Class I cross-presentation, and antigen binding to more than one TLR has been shown to increase CD8+ T cyctotoxic cell responses (13).
Sec61 may play an important role in MHC Class I cross-presentation. The phagosome containing material from another cell fuses with the endoplasmic reticulum, allowing recruitment of Sec61 (15,16,17). Sec61 forms a complex which acts as a translocon, forming a translocation channel from endosomes and phagosomes to the cytosol (18) and possibly from the ER to the cytoplasm (19). When the antigen is present in the cytosol, it is taken up and degraded by the proteosome before being transported to the ER. Antigen then follows the traditional MHC Class I processing pathway and is transported to the Golgi apparatus to be expressed on the surface of the cell (20). An example of this is shown in Leishmania infections. Sec61 may be responsible for transportation of Leishmania antigen across parasitophorous vacuoles into the cytosol of dendritic cells (21).
Alkalization of the Phagosome
Cross-presentation requires alkalization of the phagosome by NOX2, the NADPH oxidase (22). Alkalization prevents proteases in the lysosome from becoming activated (23). This allows for peptide survival in the lysosme, and these peptides can be processed and presented by MHC Class I molecules (17,21,24). Production of reactive oxygen species stopped after peptides were ready for cross-presentation, and this allowed phagosomal acidification by proteases from the lysosome (25). This switches the route of particulate antigen presentation from loading onto MHC Class I to MHC Class II (26). Therefore a lower acidity in the autophagosome results in MHC Class I cross-presentation, while a higher acidity results in MHC Class II presentation (27).
Phosphatidylinositol 3-phosphate (PI(3)P) is required to recruit the NADPH-oxidase subunits p40phox and p47phox to the phagosome (28). These increase the degradation of the contents of the phagosome via the phagosomeal proteases (29). Levels of PI(3)P in individual phagosomes appears to differ significantly even when at similar stages of maturation (29). This affects NADPH-oxidase activity and therefore alters the rate of peptide degradation (29). This would produce phagosomes with differing pH levels, with some being suitable for MHC Class I and others Class II. The cause of this is currently unknown, but in conjunction with the previously mentioned data this strengthens the possibility of alkalization of the phagosome being important in cross-presentation.
Whether soluble antigen is processed for MHC Class I or II appears to depend on the intracellular compartment it is present in (30). Therefore the mechanism by which the antigen enters the may be a determining factor for MHC presentation (27). It seems that antigen which is presented by MHC Class I is directed into early endosomes (a TAP-independent process), while peptide displayed on MHC Class II is transported in lysosomes (a TAP-dependant process) (31). As previously mentioned this may be partially time-dependant, with late endosomes believed to fuse with lysosomes, from which peptides are presented on MHC Class II (26).
Two receptors have been shown to support this mechanism, the mannose receptor and a scavenger receptor. The mannose receptor bound soluble antigen and this formed an early endosomal compartment, leading to MHC Class I presentation (27,31). Antigen binding to the scavenger receptors are pinocytosed and cause the formation of a lysosomal compartment, leading to MHC Class II presentation (27). This suggests that the receptor which bind antigen and whether the antigen is routed to an early endosome of a lysosome are factors which determine antigen presentation (31,33,34)
Like many haemopoietic cells, dendritic cells appear to have subsets, with different subsets performing different functions. In murine models, the CD8α+ dendritic cells appear to be the only dendritic cell subset which efficiently cross-present antigen bound to MHC Class I molecules (35,36). Another potentially important characteristic of CD8α+ dendritic cells is that they do not migrate from the lymphoid tissues, unlike conventional dendritic cells (37). CD8α+ dendritic cells appear to be commonly found in lymphoid tissues (38). This has two important implications. Firstly, they are the cells most likely to present antigen loaded onto MHC Class I to naïve CD8+ T cells. Secondly, as they have reduced migratory function, it is likely that they require other dendrite cells or macrophages as a source of antigen to be presented (39). This emphasises the importance of efficient transfer of antigen to the MHC Class I processing pathway, as improper transfer of antigen could severely limit immune responses to intracellular pathogens.
Monocytes/Macrophages in Cross-presentation
While dendritic cells are the only known cell that can cross-present MHC Class I molecules, it may require another haemaotpoeitic cell in order for this to occur (39,42). Monocytes from the blood migrate into tissues where they differentiate into macrophages (40). Macrophages are important in phagocytosing pathogens and cell debris (40). They also present antigen to T helper cells, but they seem to be unable to cross-present antigen to CD8+ cytotoxic T cells (35,36).
It has been suggested that some monocytes develop into dendritic cells after exposure to antigen (39,41). These cells then migrate from the site of infection to the lymphatic vessels, and then to the lymph nodes (39). Here, they present antigen to activated T cells and, most importantly, transfer peptide and MHC complexes to other dendritic cells capable of generating CD8+ T cytotoxic cell responses (43). This argument is strengthened as MHC-deficient monocytes are unable to assist in cross-presentation (39). The transfer is likely to be through endosomes rather than gap junctions, but this is still contentious (44). This system would concur with the previously mentioned CD8α+ dendritic cell proposal, whereby monocyte derived dendritic cells migrate to the lymph nodes where the present antigen to CD8α+ cells.
Antigen Acquisition for Cross-presentation
Autophagy is a mechanism by which components a cell are packaged into lysosomes in order to be broken down (45). This is often used when a cell is damaged or under stress (46). It has been suggested that during this process foreign peptides may be contained in this lysosome (47), which may be transferred into a dendritic cell and processed and presented on MHC Class I (48).
During a period of cellular stress, the ER may initiate the unfolded protein response (UPR) (50). This is due to an unusually high demand for protein production in the ER, often cause by viral manipulation of the host cell. In response to UPR, it is proposed that autophagy is induced and proceeds to envelope sections of the ER (49). These sections may contain parts of the endogenous processing pathway or MHC Class I molecules themselves (51). This would allow direct presentation of antigen in the autophagosome to MHC Class I molecules. Indeed, MHC Class I loading complexes have been found in dendritic cell phagosomes (17,52), and autophagy may have had a role in this process.
Chaperone-mediated autophagy (CMA) is an induced process as it requires certain genes to be upregulated, and this may be in response to infection (54). Atg4 and Atg7 are required for Atg8 to allow Atg3 to integrate into the autophagosomal membrane. This requires help from the Atg12-Atg5-Atg16 complex and Atg9 (2,53). This membrane engulfs some of the cytosol and organelles before joining with a lysosome membrane. This forms an autolysosome where esterases, lipases, and proteases degrade the contents (2).
CMA involves proteins from the cytosol binding to a chaperone complex consisting of several heat-shock proteins, including hsc70 (55,56). This complex binds to the lysosome-associated membrane protein 2A (LAMP) on the lysosomal membrane, and allows it to be degraded by the lysosomal enzymes (57). Chaperone proteins allow antigen loading in the ER for MHC Class I (58). Macroautophagy can be regulated by the PI3 inhibitors rapamycin, 3-MA and wortmannin (59). Used together, it was shown that increased macroautophagy increased MHC Class I cross-presentation, while inhibition of macroautophagy reduced cross-presentation (59).
Autophagy is required for clearance of Toxoplasma gondii from infected macrophages (60,61). CD8+ T cytotoxic cells are the main form of defence against T. gondii, and therefore MHC Class I cross-presentation is required. T. gondii or its proteins must enter an autophagosome, where they are degraded and transported to the MHC Class I processing pathway (10). Autophagosomes are known to destroy viruses and intracellular pathogens (51), and intact peptides from these may be used in cross-presentation. DC aggresome-like structures (DALIS) bind to proteins in autophagosomes and peptides from these can be presented to MHC Class I molecules (62)
While this suggests a role for CMA in cross-presentation, there are problems with this mechanism. NH4Cl prevents the acidification of lysosomes and their fusion with autophagosomes (2). When added to infected cells, cross-presentation was inhibited. This is an unexpected result and indicates further research is required.
Heat Shock Proteins
Heat shock proteins may play a role in MHC Class I cross-presentation and are associated with autophagy (63). The heat shock proteins are activated during times of cell stress, and therefore may be activated during infection (64). Dendritic cells express oxidized low-density lipoprotein receptor 1 (LOX-1) which is ligated by low-density lipoproteins (OxLDLs) on epithelial cells (65). This allows the recruitment of heat shock protein 70 (Hsp70). Hsp70 has two important roles in cross-presentation. Firstly, it binds to peptides to prevent aggregation and maintain their usefulness for presentation. (66) Secondly, it acts as a chaperone protein, transferring bound peptides into the dendritic cell (67). A similar function has been shown in Hsp90 (68). After ligation of appropriate receptors, Hsp90 delivers its bound peptides to the early endosome in the dendritic cell, rather than the endoplasmic reticulum (69). This may allow processing for MHC Class I presentation as previously described.
Gap junctions are intracellular channels by which molecules from one cell can be transported to another neighbouring cell (71). They are non specific channels, which allow peptides with a molecular weight of less than 1,000 Daltons to be transported between cells (70). This has been shown to allow communication between cells by the transfer of ions and molecules such as Ca2+ and IP3 (72). This has led to the theory that an infected cell may be able to transfer foreign peptide to the cytosol of a neighbouring dendrtic cell via a gap junction (70,73), before antigen is processed and expressed with MHC Class I on the dendritic cell. This theory is aided as connexin 43 gap junctions allow short peptides related to the immune response to be transported between cells (74). These are only present on haematopoietic cells, which include dendritic cells.
Pathogens have evolved to limit the function of gap junctions. Proteins produced by the herpes viruses HSV-2 (75) and HPV-16 (76) have been shown to close gap junctions between cells. Tumour cells have also been shown to have the same effect (77). If gap junctions are important in MHC Class I cross-presentation, then this would explain the poor CD8+ cytotoxic T cell responses to these diseases (5).
Although the transfer of ions through gap junctions is mainly unaffected, peptides are likely to be degraded by high cytosolic peptidase concentrations within the cell cytoplasm (78). Therefore the majority of foreign peptides will not be able to be processed for MHC Class I presentation by the DC. For gap junctions to have any impact on MHC Class I presentation, there would have to be a very high expression of these transferred peptides. Transfer of antigen via gap junctions is an inefficient process, and this suggests that while it may be a possible route for cross-presentation, it may not be the most important mechanism.
Phagocytosis of dead cells or cell debris may be an important source of antigen for MHC Class I cross-presentation (79). Cells which have undergone apoptosis compartmentalise their organelles and cytoplasm to prevent unwanted proteins being released into surrounding tissues (80). These compartments are referred to as apoptotic bodies, which are derived from the plasma membrane and contain cytoplasm from the apoptotic cell (80). Immature dendritic cells can phagosytose the apoptotic bodies, and these are transported to the endoplasmic reticulum or phagosomes to allow processing for MHC Class I presentation (18,81). This has been displayed with Mycobacterium tuberculosis infection (82).
Exit of Antigen From Intracellular Compartments
Lipid bilayers are not rigid or impermeable structures, allowing for changing their shape, function and enabling them to fuse with other membrane structures (10). Therefore although these membranes are designed to keep the contents of endosomes, lysosomes and phagosomes within the organelles, some molecules may escape. Transportation of peptides from these organelles to the cytosol may allow for cross-presentation of antigen, and therefore the mechanisms by which peptides escape may be vital to understand (10).
It has been suggested that some proteins may be able to pass through intact phagosomal membranes, or escape when the membrane is damaged (10). An isoform of the HIV-1 Tat protein can interact with the phagosomal membrane, forming a pore by which it can enter the cytosol (83). This pore is not specific for Tat, and therefore other proteins or partially degraded peptides may also escape the phagosome. A similar function has been found for other proteins which escape endosomes (84) and ER (85). The membrane of such organelles may become damaged as a direct result of containing a pathogen, or may become damaged due to homeostatic reasons, such as failing to control lysosomal pH (10). While uncontrolled release of the contents of phagosomes and lysosomes may cause the death of the cell (86) (both by pathogens and their products, but also host proteases and other enzymes), it is also a route by which antigen can access the MHC Class I processing pathway.
MHC Class I Recycling
MHC Class I recycling from the cell surface may be a mechanism by which MHC Class I molecules present on the surface of dendritic cells manage to enter the exogenous processing pathway (87). While immature dendtritic cells sample their surrounding area, molecules on their cell surface are internalised into endosomes of lysosomes (3,88). While there are many different molecules that will be present in these intracellular compartments, it is likely that cell surface receptors, including MHC Class I (89), will also be internalised. If the MHC Class I molecules are bound to antigen, this may be released and degraded in the lysosome (3). The surviving MHC Class I molecules are then transported to the cell surface with MHC Class II molecules bearing appropriate antigen. During this journey, the MHC Class I molecules may bind new peptides and display them on the cell surface (3). This has been shown to be TAP-independent, Brefeldin A (BFA) resistant and NH4Cl sensitive process, (90).
Evasion of the Immune System
Strains of the herpes simplex virus are known to undergo periods of latency, where they can survive in the human host with little or no immune response against the virus (91,92). As they are viruses, clearance of the infection relies upon increased CD8+ T cyctotoxic cell responses (93). Herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2) encode the production of ICP47 (94). ICP47 can enter the cytosol of dendritic cells after phagocytosis (10,94). This interferes with the TAP complex, inhibiting TAP1 and TAP 2-dependant peptide transfer (94 Although it is believed that cross-presentation may also occur in a TAP-independent fashion, blocking of the TAP-dependant pathway may contribute to the successful survival of these pathogens.