Tài liệu: Ai cần tớ giúp....

CD1 antigen-binding grooves
The antigen-binding groove of mouse CD1d contains two connecting pockets, A' and F', which together are large enough to accommodate lipids having an overall size of 32â?"40 methylene units, depending on the position of glycolipids in the groove8. Corresponding to this, the sphingolipid and diacylglycerol antigens that are presented by this isoform have an overall alkyl chain length in this range2, 39, 79. By contrast, the human CD1B antigen-binding groove is much larger and consists of four adjacent pockets â?" A', C', F' and the T' 'tunnel'9 (Fig. 1). For CD1B, there are two 'entrances' from the outer surface of the protein into the pocket â?" a large opening between the -helices, which is also present in CD1d and MHC molecules, and a small portal in the lateral wall of the C' pocket. Therefore, it is possible that the alkyl chains of larger lipids could protrude from the groove through this C' portal or other sites.
The crystal structure of CD1B provides important insights into how this non-polymorphic antigen-presenting molecule can bind such structurally diverse lipids, including those that range in length from C12 to C80 (Ref. 26) Crystallized, refolded CD1B proteins simultaneously bind three lipids â?" either GM2 or PI, together with two C16 lipids (Fig. 1). This modular architecture indicates that CD1B could bind diacylglycerol, sphingolipid or other small antigens with an overall alkyl chain length of up to C40 by inserting them directly into the A' and C' pockets, as seen in the crystal structure. In this case, the areas of the CD1B antigen-binding groove that are not involved in antigen binding could be occupied by one or more immunologically inert, groove-stabilizing lipids.
Antigens with alkyl chains in the range of C40â?"76 might occupy three or all four pockets. The loading of yet larger lipids, such as C80 GMM or free mycolic acid, is predicted to require insertion of the lipid moiety into all four pockets, and the lipid might also protrude slightly through the portal in the C' pocket. This process might require conformational changes to CD1B so that the bound lipid can make contact with all four pockets simultaneously. In ad***ion, insertion of long-chain lipids might involve the expulsion of more than one chaperone lipid (Fig. 1).
This, or other related aspects of antigen loading, could explain why only antigens with lipid-chain lengths in the range of C54â?"80 require endosomal presentation (Fig. 4). The low pH of late endosomes could promote relaxation of the -helices, which form the roof and sides of the groove, thereby facilitating access to the groove. Alternatively, endosomes could provide exchange proteins with functions that are analogous to those of HLA-DM in the peptide loading of MHC class II molecules. A third possibility is that endosomal lipases could cleave C80 mycolates so that shorter alkyl chains could fit more easily in four or fewer pockets. As endosomal pathways seem to be more efficient than non-endosomal pathways for the loading of antigens onto CD1B, further investigation of the molecular mechanisms that govern this process, including the role of lipid cleavage or effects of pH on CD1B folding, will be important.
These observations indicate that the trafficking of CD1B to late endosomes and lysosmes could be a specialized mechanism for selectively presenting lipids with long chain lengths, which naturally accumulate in these compartments26, 80. As mycobacterial mycolates have longer alkyl chains than self-sphingolipids or -diacylglycerols, the efficient presentation of long-chain antigens in endosomes might even be a mechanism to skew the T-cell response towards lipids that have a more intrinsically foreign structure26 (Fig. 4). This hypothesis can be tested more directly by analysing the chain lengths of lipids that are sorted to endosomes and complexed with CD1B. In ad***ion, it is now possible to begin to determine whether long- or short-chain antigens activate T cells more efficiently in vivo using lipid-loaded tetramers and related techniques to measure the frequencies of lipid-antigen-specific T-cell precursors in infected humans.
Importance of separate processing pathways
Viewed broadly, these studies of the cellular requirements for lipid-antigen presentation by CD1 molecules provide evidence of functionally separate endosomal and non-endosomal pathways for glycolipid-antigen presentation to T cells. For CD1D, the existence of endosomal and non-endosomal pathways could allow APCs to control separately the activation of NKT-cell populations with invariant (V14+) and diverse (V14-) TCRs23, 27. However, clearly defined differences between the normal functions of these two cell populations have not been determined yet, and the structures of the natural endogenous antigens that are presented to them are not known. It will be necessary to resolve these questions to propose an integrated model of how the apparently separate pathways of CD1D antigen processing could regulate immune responses in vivo.
By contrast, there is much information relating the biological origin of microbial antigens, their precise molecular structures and the role of these structures in antigen processing and T-cell activation (Fig. 4). The studies that are reviewed here point to an emerging picture of how the endosomal and non-endosomal CD1-mediated antigen-presentation pathways could control physiological immune responses to foreign and self-glycolipids. We speculate that CD1A, CD1B and CD1C present exogenously acquired foreign antigens to T cells that function in host defence against infection. Intracellular pathogens, including mycobacteria, can inhibit endosomal maturation81, 82. Certain antigens, such as mycobacterial mycolates, can accumulate preferentially in lysosomes, whereas others, such as polyisoprenyl phosphates, might be degraded in the low pH of this compartment26, 65. For these and other reasons, surveillance for pathogens might be optimized by the fact that CD1A, CD1B and CD1C are specialized to sample different parts of the endosomal network individually, but function together to sample the entire endosomal pathway for pathogens.
Even in the presence of an intracellular infection, microbial lipids form only a small proportion of the total lipids comprising the membranes of APCs. In ad***ion, it is probable that self-lipids are loaded onto CD1 proteins in the ER, before CD1 is exposed to foreign antigens in the endosomal network37, 38. Therefore, T-cell activation by foreign lipids during host defence probably requires that cells have mechanisms for removing self-lipids from the CD1 groove and selectively loading bacterial glycolipids onto CD1, analogous to known mechanisms for loading foreign peptides onto endosomal MHC class II proteins73, 83. In contrast to cytosolic, ER, secretory and cell-surface compartments, endosomes are likely to be enriched for foreign lipids, because this is the first compartment to acquire live intracellular bacteria or lipid components shed from extracellular pathogens84. In ad***ion, as certain PATTERN-RECOGNITION RECEPTORS can bind foreign lipids specifically, microbial lipids can be internalized selectively for delivery to endosomes78. Therefore, the ability of CD1A, CD1B and CD1C to load bacterial antigens selectively in endosomes could skew the repertoire of lipids that are presented by these molecules towards those of exogenous or foreign origin. This might be particularly true for CD1B, which most clearly requires a low pH for binding lipids and seems to have specialized mechanisms for preferentially presenting long-chain mycobacterial lipids4, 26, 29.
At the same time, CD1 molecules can also bind and present self-diacylglycerols and -sphingolipids, and many examples of CD1-restricted autoreactive T cells are known. This indicates the existence of cellular pathways for the loading and presentation of endogenous self-lipids. Most studies of the loading of self-lipids onto CD1 molecules show that this occurs readily at the cell surface or, using recombinant CD1 proteins, in neutral biological buffers11, 37, 39, 77 (Fig. 4). This indicates that cellular pathways for presenting self-antigens involve low-stringency loading mechanisms that occur in most subcellular compartments, rather than only the specialized low-pH environment of endosomes. Although the non-endosomal presentation mechanisms are more rapid and less stringent than endosomal mechanisms, in most studies so far, they have been shown to be less efficient. Exogenously administered gangliosides and sulphatides require micromolar concentrations to activate T cells, in contrast to endosomally presented microbial antigens, which can be recognized at low nanomolar concentrations10, 11, 13-15 So, the non-endosomal pathway might function to sample, on a more global scale, abundant self-glycolipids, a role that could be well adapted for T cells that function in immunosurveillance or immunoregulation. Thereby, endosomal and non-endosomal pathways could both carry out important, but separate, functions in an immune response.
Concluding remarks
A more complete understanding of these separate pathways of lipid-antigen processing will involve precisely defining the cellular subcompartments in which lipid antigens and CD1 proteins intersect and the detailed molecular basis for insertion of lipids into the CD1 groove. So far, most studies have focused on the dynamic trafficking patterns of CD1 proteins, rather than those of lipids. Nevertheless, it is clear that certain classes of CD1-presented lipid have a non-random distribution in cellular subcompartments. For example, certain polyisoprenoid lipids accumulate in the ER, whereas sphingolipid glycosylation can occur selectively in the Golgi apparatus. Diacylglycerols with long alkyl chains can be sorted selectively to lysosomes80, 85, 86. Cellular activation or apoptosis leads to the redistribution of anionic phospholipids to the outer leaflet of the cytoplasmic membrane, and microbial lipids are delivered selectively to endosomes by pattern-recognition receptors present on the surface of maturing dendritic cells78. A clearer understanding of how these intracellular patterns of trafficking and accumulation of lipids lead to loading onto CD1 proteins should provide new insights into the normal biological functions of lipid-specific T cells.
Concay

CD1 antigen-binding grooves
The antigen-binding groove of mouse CD1d contains two connecting pockets, A' and F', which together are large enough to accommodate lipids having an overall size of 32â?"40 methylene units, depending on the position of glycolipids in the groove8. Corresponding to this, the sphingolipid and diacylglycerol antigens that are presented by this isoform have an overall alkyl chain length in this range2, 39, 79. By contrast, the human CD1B antigen-binding groove is much larger and consists of four adjacent pockets â?" A', C', F' and the T' 'tunnel'9 (Fig. 1). For CD1B, there are two 'entrances' from the outer surface of the protein into the pocket â?" a large opening between the -helices, which is also present in CD1d and MHC molecules, and a small portal in the lateral wall of the C' pocket. Therefore, it is possible that the alkyl chains of larger lipids could protrude from the groove through this C' portal or other sites.
The crystal structure of CD1B provides important insights into how this non-polymorphic antigen-presenting molecule can bind such structurally diverse lipids, including those that range in length from C12 to C80 (Ref. 26) Crystallized, refolded CD1B proteins simultaneously bind three lipids â?" either GM2 or PI, together with two C16 lipids (Fig. 1). This modular architecture indicates that CD1B could bind diacylglycerol, sphingolipid or other small antigens with an overall alkyl chain length of up to C40 by inserting them directly into the A' and C' pockets, as seen in the crystal structure. In this case, the areas of the CD1B antigen-binding groove that are not involved in antigen binding could be occupied by one or more immunologically inert, groove-stabilizing lipids.
Antigens with alkyl chains in the range of C40â?"76 might occupy three or all four pockets. The loading of yet larger lipids, such as C80 GMM or free mycolic acid, is predicted to require insertion of the lipid moiety into all four pockets, and the lipid might also protrude slightly through the portal in the C' pocket. This process might require conformational changes to CD1B so that the bound lipid can make contact with all four pockets simultaneously. In ad***ion, insertion of long-chain lipids might involve the expulsion of more than one chaperone lipid (Fig. 1).
This, or other related aspects of antigen loading, could explain why only antigens with lipid-chain lengths in the range of C54â?"80 require endosomal presentation (Fig. 4). The low pH of late endosomes could promote relaxation of the -helices, which form the roof and sides of the groove, thereby facilitating access to the groove. Alternatively, endosomes could provide exchange proteins with functions that are analogous to those of HLA-DM in the peptide loading of MHC class II molecules. A third possibility is that endosomal lipases could cleave C80 mycolates so that shorter alkyl chains could fit more easily in four or fewer pockets. As endosomal pathways seem to be more efficient than non-endosomal pathways for the loading of antigens onto CD1B, further investigation of the molecular mechanisms that govern this process, including the role of lipid cleavage or effects of pH on CD1B folding, will be important.
These observations indicate that the trafficking of CD1B to late endosomes and lysosmes could be a specialized mechanism for selectively presenting lipids with long chain lengths, which naturally accumulate in these compartments26, 80. As mycobacterial mycolates have longer alkyl chains than self-sphingolipids or -diacylglycerols, the efficient presentation of long-chain antigens in endosomes might even be a mechanism to skew the T-cell response towards lipids that have a more intrinsically foreign structure26 (Fig. 4). This hypothesis can be tested more directly by analysing the chain lengths of lipids that are sorted to endosomes and complexed with CD1B. In ad***ion, it is now possible to begin to determine whether long- or short-chain antigens activate T cells more efficiently in vivo using lipid-loaded tetramers and related techniques to measure the frequencies of lipid-antigen-specific T-cell precursors in infected humans.
Importance of separate processing pathways
Viewed broadly, these studies of the cellular requirements for lipid-antigen presentation by CD1 molecules provide evidence of functionally separate endosomal and non-endosomal pathways for glycolipid-antigen presentation to T cells. For CD1D, the existence of endosomal and non-endosomal pathways could allow APCs to control separately the activation of NKT-cell populations with invariant (V14+) and diverse (V14-) TCRs23, 27. However, clearly defined differences between the normal functions of these two cell populations have not been determined yet, and the structures of the natural endogenous antigens that are presented to them are not known. It will be necessary to resolve these questions to propose an integrated model of how the apparently separate pathways of CD1D antigen processing could regulate immune responses in vivo.
By contrast, there is much information relating the biological origin of microbial antigens, their precise molecular structures and the role of these structures in antigen processing and T-cell activation (Fig. 4). The studies that are reviewed here point to an emerging picture of how the endosomal and non-endosomal CD1-mediated antigen-presentation pathways could control physiological immune responses to foreign and self-glycolipids. We speculate that CD1A, CD1B and CD1C present exogenously acquired foreign antigens to T cells that function in host defence against infection. Intracellular pathogens, including mycobacteria, can inhibit endosomal maturation81, 82. Certain antigens, such as mycobacterial mycolates, can accumulate preferentially in lysosomes, whereas others, such as polyisoprenyl phosphates, might be degraded in the low pH of this compartment26, 65. For these and other reasons, surveillance for pathogens might be optimized by the fact that CD1A, CD1B and CD1C are specialized to sample different parts of the endosomal network individually, but function together to sample the entire endosomal pathway for pathogens.
Even in the presence of an intracellular infection, microbial lipids form only a small proportion of the total lipids comprising the membranes of APCs. In ad***ion, it is probable that self-lipids are loaded onto CD1 proteins in the ER, before CD1 is exposed to foreign antigens in the endosomal network37, 38. Therefore, T-cell activation by foreign lipids during host defence probably requires that cells have mechanisms for removing self-lipids from the CD1 groove and selectively loading bacterial glycolipids onto CD1, analogous to known mechanisms for loading foreign peptides onto endosomal MHC class II proteins73, 83. In contrast to cytosolic, ER, secretory and cell-surface compartments, endosomes are likely to be enriched for foreign lipids, because this is the first compartment to acquire live intracellular bacteria or lipid components shed from extracellular pathogens84. In ad***ion, as certain PATTERN-RECOGNITION RECEPTORS can bind foreign lipids specifically, microbial lipids can be internalized selectively for delivery to endosomes78. Therefore, the ability of CD1A, CD1B and CD1C to load bacterial antigens selectively in endosomes could skew the repertoire of lipids that are presented by these molecules towards those of exogenous or foreign origin. This might be particularly true for CD1B, which most clearly requires a low pH for binding lipids and seems to have specialized mechanisms for preferentially presenting long-chain mycobacterial lipids4, 26, 29.
At the same time, CD1 molecules can also bind and present self-diacylglycerols and -sphingolipids, and many examples of CD1-restricted autoreactive T cells are known. This indicates the existence of cellular pathways for the loading and presentation of endogenous self-lipids. Most studies of the loading of self-lipids onto CD1 molecules show that this occurs readily at the cell surface or, using recombinant CD1 proteins, in neutral biological buffers11, 37, 39, 77 (Fig. 4). This indicates that cellular pathways for presenting self-antigens involve low-stringency loading mechanisms that occur in most subcellular compartments, rather than only the specialized low-pH environment of endosomes. Although the non-endosomal presentation mechanisms are more rapid and less stringent than endosomal mechanisms, in most studies so far, they have been shown to be less efficient. Exogenously administered gangliosides and sulphatides require micromolar concentrations to activate T cells, in contrast to endosomally presented microbial antigens, which can be recognized at low nanomolar concentrations10, 11, 13-15 So, the non-endosomal pathway might function to sample, on a more global scale, abundant self-glycolipids, a role that could be well adapted for T cells that function in immunosurveillance or immunoregulation. Thereby, endosomal and non-endosomal pathways could both carry out important, but separate, functions in an immune response.
Concluding remarks
A more complete understanding of these separate pathways of lipid-antigen processing will involve precisely defining the cellular subcompartments in which lipid antigens and CD1 proteins intersect and the detailed molecular basis for insertion of lipids into the CD1 groove. So far, most studies have focused on the dynamic trafficking patterns of CD1 proteins, rather than those of lipids. Nevertheless, it is clear that certain classes of CD1-presented lipid have a non-random distribution in cellular subcompartments. For example, certain polyisoprenoid lipids accumulate in the ER, whereas sphingolipid glycosylation can occur selectively in the Golgi apparatus. Diacylglycerols with long alkyl chains can be sorted selectively to lysosomes80, 85, 86. Cellular activation or apoptosis leads to the redistribution of anionic phospholipids to the outer leaflet of the cytoplasmic membrane, and microbial lipids are delivered selectively to endosomes by pattern-recognition receptors present on the surface of maturing dendritic cells78. A clearer understanding of how these intracellular patterns of trafficking and accumulation of lipids lead to loading onto CD1 proteins should provide new insights into the normal biological functions of lipid-specific T cells.
Concay

References
1.
Porcelli, S. A. The CD1 family: a third lineage of antigen-presenting molecules. Adv. Immunol. 59, 1-98 (1995). | PubMed | ISI | ChemPort |

2.
Kawano, T. et al. CD1d-restricted and TCR-mediated activation of V14 NKT cells by glycosylceramides. Science 278, 1626-1629 (1997).
Synthetic -galactosyl ceramides were discovered as the first known antigens for invariant NKT cells. Fine-specificity studies showed that the -anomeric linkage of the carbohydrate, a modification that is found rarely in mammalian ceramides, is crucial for T-cell activation. | Article | PubMed | ISI | ChemPort |

3.
Hiromatsu, K. et al. Induction of CD1-restricted immune responses in guinea pigs by immunization with mycobacterial lipid antigens. J. Immunol. 169, 330-339 (2002). | PubMed | ISI | ChemPort |

4.
Porcelli, S., Morita, C. T. & Brenner, M. B. CD1b restricts the response of human CD4-8- T lymphoyctes to a microbial antigen. Nature 360, 593-597 (1992). | PubMed | ISI | ChemPort |

5.
Beckman, E. M. et al. CD1c restricts responses of mycobacteria-specific T cells. Evidence for antigen presentation by a second member of the human CD1 family. J. Immunol. 157, 2795-2803 (1996). | PubMed | ISI | ChemPort |

6.
Rosat, J. P. et al. CD1-restricted microbial lipid antigen-specific recognition found in the CD8+ T-cell pool. J. Immunol. 162, 366-371 (1999). | PubMed | ISI | ChemPort |

7.
Spada, F. M., Koezuka, Y. & Porcelli, S. A. CD1d-restricted recognition of synthetic glycolipid antigens by human natural killer T cells. J. Exp. Med. 188, 1529-1534 (1998). | Article | PubMed | ISI | ChemPort |

8.
Zeng, Z. et al. Crystal structure of mouse CD1: an MHC-like fold with a large hydrophobic binding groove. Science 277, 339-345 (1997).
The first crystal structure of a CD1 protein shows that mouse CD1d has a large hydrophobic antigen-binding groove that is composed of two pockets, A' and F'. | Article | PubMed | ISI | ChemPort |

9.
Gadola, S. D. et al. Structure of human CD1b with bound ligands at 2.3 Ã., a maze for alkyl chains. Nature Immunol. 3, 721-726 (2002).
This study reports two crystal structures of refolded CD1B proteins bound to the ganglioside GM2 or phosphatidylinositol. These structures show that the aliphatic hydrocarbon chains of lipids are inserted into the CD1 groove. The human CD1B groove was shown to be larger than that of mouse CD1d, and it is composed of four pockets -- A', C', F' and T'. This modular structure shows how CD1B can bind lipids that vary in overall chain length by inserting the lipids into two or more pockets in the groove. | Article | PubMed | ISI | ChemPort |

10.
Beckman, E. M. et al. Recognition of a lipid antigen by CD1-restricted T cells. Nature 372, 691-694 (1994). | PubMed | ChemPort |

11.
Shamshiev, A. et al. Presentation of the same glycolipid by different CD1 molecules. J. Exp. Med. 195, 1013-1021 (2002). | Article | PubMed | ISI | ChemPort |

12.
Sieling, P. A. et al. CD1-restricted T-cell recognition of microbial lipoglycan antigens. Science 269, 227-230 (1995). | PubMed | ISI | ChemPort |

13.
Moody, D. B. et al. Structural requirements for glycolipid antigen recognition by CD1b-restricted T cells. Science 278, 283-286 (1997). | Article | PubMed | ISI | ChemPort |

14.
Moody, D. B. et al. CD1c-mediated T-cell recognition of isoprenoid glycolipids in Mycobacterium tuberculosis infection. Nature 404, 884-888 (2000). | Article | PubMed | ISI | ChemPort |

15.
Shamshiev, A. et al. Self glycolipids as T-cell autoantigens. Eur. J. Immunol. 29, 1667-1675 (1999).
This study provides the first clear functional evidence for the presentation of self-glycolipids by non-endosomal mechanisms. | Article | PubMed | ISI | ChemPort |

16.
Koseki, H. et al. Homogenous junctional sequence of the V14+ T-cell antigen receptor -chain expanded in unprimed mice. Proc. Natl Acad. Sci. USA 87, 5248-5252 (1990). | PubMed | ISI | ChemPort |

17.
Porcelli, S., Gerdes, D., Fertig, A. M. & Balk, S. P. Human T cells expressing an invariant V24-JQ TCR are CD4- and heterogeneous with respect to TCR expression. Hum. Immunol. 48, 63-67 (1996). | Article | PubMed | ISI | ChemPort |

18.
Grant, E. P. et al. Molecular recognition of lipid antigens by T-cell receptors. J. Exp. Med. 189, 195-205 (1999). | Article | PubMed | ISI | ChemPort |

19.
Behar, S. M., Podrebarac, T. A., Roy, C. J., Wang, C. R. & Brenner, M. B. Diverse TCRs recognize murine CD1. J. Immunol. 162, 161-167 (1999). | PubMed | ISI | ChemPort |

20.
Cardell, S. et al. CD1-restricted CD4+ T cells in major histocompatibility complex class II-deficient mice. J. Exp. Med. 182, 993-1004 (1995). | PubMed | ISI | ChemPort |

21.
Burdin, N. et al. Structural requirements for antigen presentation by mouse CD1. Proc. Natl Acad. Sci. USA 97, 10156-10161 (2000). | Article | PubMed | ISI | ChemPort |

22.
Jackman, R. M. et al. The tyrosine-containing cytoplasmic tail of CD1b is essential for its efficient presentation of bacterial lipid antigens. Immunity 8, 341-351 (1998). | PubMed | ISI | ChemPort |

23.
Chiu, Y. H. et al. Distinct subsets of CD1d-restricted T cells recognize self-antigens loaded in different cellular compartments. J. Exp. Med. 189, 103-110 (1999). | Article | PubMed | ISI | ChemPort |

24.
Sugita, M. et al. Separate pathways for antigen presentation by CD1 molecules. Immunity 11, 743-752 (1999). | PubMed | ISI | ChemPort |

25.
Sugita, M., van Der, W., Rogers, R. A., Peters, P. J. & Brenner, M. B. CD1c molecules broadly survey the endocytic system. Proc. Natl Acad. Sci. 97, 8445-8450 (2000). | Article | PubMed | ISI | ChemPort |

26.
Moody, D. B. et al. Lipid length controls antigen entry into endosomal and nonendosomal pathways for CD1b presentation. Nature Immunol. 3, 435-442 (2002).
This study provides direct evidence for the role of alkyl chain length in controlling whether lipids are presented by endosomal or non-endosomal pathways. | Article | PubMed | ISI | ChemPort |

27.
Chiu, Y. H. et al. Multiple defects in antigen presentation and T-cell development by mice expressing cytoplasmic-tail-truncated CD1d. Nature Immunol. 3, 55-60 (2002). | Article | PubMed | ISI | ChemPort |

28.
Roberts, T. J. et al. Recycling CD1d1 molecules present endogenous antigens processed in an endocytic compartment to NKT cells. J. Immunol. 168, 5409-5414 (2002). | PubMed | ISI | ChemPort |

29.
Ernst, W. A. et al. Molecular interaction of CD1b with lipoglycan antigens. Immunity 8, 331-340 (1998). | PubMed | ISI | ChemPort |

30.
Calabi, F. & Milstein, C. A novel family of human major histocompatibility complex-related genes not mapping to chromosome 6. Nature 323, 540-543 (1986). | PubMed | ISI | ChemPort |

31.
Woolfson, A. & Milstein, C. Alternative splicing generates secretory isoforms of human CD1. Proc. Natl Acad. Sci. USA 91, 6683-6687 (1994). | PubMed | ISI | ChemPort |

32.
Angenieux, C. et al. Characterization of CD1e, a third type of CD1 molecule expressed in dendritic cells. J. Biol. Chem. 275, 37757-37764 (2000). | Article | PubMed | ISI | ChemPort |

33.
Sugita, M., Porcelli, S. A. & Brenner, M. B. Assembly and retention of CD1b heavy chains in the endoplasmic reticulum. J. Immunol. 159, 2358-2365 (1997). | PubMed | ISI | ChemPort |

34.
Huttinger, R., Staffler, G., Majdic, O. & Stockinger, H. Analysis of the early biogenesis of CD1b: involvement of the chaperones calnexin and calreticulin, the proteasome and 2-microglobulin. Int. Immunol. 11, 1615-1623 (1999). | Article | PubMed | ISI | ChemPort |

35.
Karadimitris, A. et al. Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography. Proc. Natl Acad. Sci. USA 98, 3294-3298 (2001). | Article | PubMed | ISI | ChemPort |

36.
Altamirano, M. M., Blackburn, J. M., Aguayo, C. & Fersht, A. R. Ligand-independent assembly of recombinant human CD1 by using oxidative refolding chromatography. Proc. Natl Acad. Sci. USA 98, 3288-3293 (2001). | Article | PubMed | ISI | ChemPort |

37.
Joyce, S. et al. Natural ligand of mouse CD1d1: cellular glycosylphosphatidylinositol. Science 279, 1541-1544 (1998). | Article | PubMed | ISI | ChemPort |

38.
De Silva, A. D. et al. Lipid-protein interactions: the assembly of CD1d1 with cellular phospholipids occurs in the endoplasmic reticulum. J. Immunol. 168, 723-733 (2002). | PubMed | ISI |

39.
Gumperz, J. et al. Murine CD1d-restricted T-cell recognition of cellular lipids. Immunity 12, 211-221 (2000). | PubMed | ISI | ChemPort |

40.
Sugita, M. & Brenner, M. B. An unstable 2-microglobulin: major histocompatibility complex class I heavy chain intermediate dissociates from calnexin and then is stabilized by binding peptide. J. Exp. Med. 180, 2163-2171 (1994). | PubMed | ISI | ChemPort |

41.
Bauer, A. et al. Analysis of the requirement for 2-microglobulin for expression and formation of human CD1 antigens. Eur. J. Immunol. 27, 1366-1373 (1997). | PubMed | ISI | ChemPort |

42.
Bendelac, A. et al. CD1 recognition by mouse NK1+ T lymphocytes. Science 268, 863-865 (1995). | PubMed | ISI | ChemPort |

43.
Kang, S. J. & Cresswell, P. Regulation of intracellular trafficking of human CD1d by association with MHC class II molecules. EMBO J. 21, 1650-1660 (2002).
This paper provides evidence for the association of CD1D with MHC class II molecules and for a functional role for MHC class-II-invariant-chain complexes in control of the intracellular trafficking of CD1D. | Article | PubMed | ISI | ChemPort |

Concay

References
1.
Porcelli, S. A. The CD1 family: a third lineage of antigen-presenting molecules. Adv. Immunol. 59, 1-98 (1995). | PubMed | ISI | ChemPort |

2.
Kawano, T. et al. CD1d-restricted and TCR-mediated activation of V14 NKT cells by glycosylceramides. Science 278, 1626-1629 (1997).
Synthetic -galactosyl ceramides were discovered as the first known antigens for invariant NKT cells. Fine-specificity studies showed that the -anomeric linkage of the carbohydrate, a modification that is found rarely in mammalian ceramides, is crucial for T-cell activation. | Article | PubMed | ISI | ChemPort |

3.
Hiromatsu, K. et al. Induction of CD1-restricted immune responses in guinea pigs by immunization with mycobacterial lipid antigens. J. Immunol. 169, 330-339 (2002). | PubMed | ISI | ChemPort |

4.
Porcelli, S., Morita, C. T. & Brenner, M. B. CD1b restricts the response of human CD4-8- T lymphoyctes to a microbial antigen. Nature 360, 593-597 (1992). | PubMed | ISI | ChemPort |

5.
Beckman, E. M. et al. CD1c restricts responses of mycobacteria-specific T cells. Evidence for antigen presentation by a second member of the human CD1 family. J. Immunol. 157, 2795-2803 (1996). | PubMed | ISI | ChemPort |

6.
Rosat, J. P. et al. CD1-restricted microbial lipid antigen-specific recognition found in the CD8+ T-cell pool. J. Immunol. 162, 366-371 (1999). | PubMed | ISI | ChemPort |

7.
Spada, F. M., Koezuka, Y. & Porcelli, S. A. CD1d-restricted recognition of synthetic glycolipid antigens by human natural killer T cells. J. Exp. Med. 188, 1529-1534 (1998). | Article | PubMed | ISI | ChemPort |

8.
Zeng, Z. et al. Crystal structure of mouse CD1: an MHC-like fold with a large hydrophobic binding groove. Science 277, 339-345 (1997).
The first crystal structure of a CD1 protein shows that mouse CD1d has a large hydrophobic antigen-binding groove that is composed of two pockets, A' and F'. | Article | PubMed | ISI | ChemPort |

9.
Gadola, S. D. et al. Structure of human CD1b with bound ligands at 2.3 Ã., a maze for alkyl chains. Nature Immunol. 3, 721-726 (2002).
This study reports two crystal structures of refolded CD1B proteins bound to the ganglioside GM2 or phosphatidylinositol. These structures show that the aliphatic hydrocarbon chains of lipids are inserted into the CD1 groove. The human CD1B groove was shown to be larger than that of mouse CD1d, and it is composed of four pockets -- A', C', F' and T'. This modular structure shows how CD1B can bind lipids that vary in overall chain length by inserting the lipids into two or more pockets in the groove. | Article | PubMed | ISI | ChemPort |

10.
Beckman, E. M. et al. Recognition of a lipid antigen by CD1-restricted T cells. Nature 372, 691-694 (1994). | PubMed | ChemPort |

11.
Shamshiev, A. et al. Presentation of the same glycolipid by different CD1 molecules. J. Exp. Med. 195, 1013-1021 (2002). | Article | PubMed | ISI | ChemPort |

12.
Sieling, P. A. et al. CD1-restricted T-cell recognition of microbial lipoglycan antigens. Science 269, 227-230 (1995). | PubMed | ISI | ChemPort |

13.
Moody, D. B. et al. Structural requirements for glycolipid antigen recognition by CD1b-restricted T cells. Science 278, 283-286 (1997). | Article | PubMed | ISI | ChemPort |

14.
Moody, D. B. et al. CD1c-mediated T-cell recognition of isoprenoid glycolipids in Mycobacterium tuberculosis infection. Nature 404, 884-888 (2000). | Article | PubMed | ISI | ChemPort |

15.
Shamshiev, A. et al. Self glycolipids as T-cell autoantigens. Eur. J. Immunol. 29, 1667-1675 (1999).
This study provides the first clear functional evidence for the presentation of self-glycolipids by non-endosomal mechanisms. | Article | PubMed | ISI | ChemPort |

16.
Koseki, H. et al. Homogenous junctional sequence of the V14+ T-cell antigen receptor -chain expanded in unprimed mice. Proc. Natl Acad. Sci. USA 87, 5248-5252 (1990). | PubMed | ISI | ChemPort |

17.
Porcelli, S., Gerdes, D., Fertig, A. M. & Balk, S. P. Human T cells expressing an invariant V24-JQ TCR are CD4- and heterogeneous with respect to TCR expression. Hum. Immunol. 48, 63-67 (1996). | Article | PubMed | ISI | ChemPort |

18.
Grant, E. P. et al. Molecular recognition of lipid antigens by T-cell receptors. J. Exp. Med. 189, 195-205 (1999). | Article | PubMed | ISI | ChemPort |

19.
Behar, S. M., Podrebarac, T. A., Roy, C. J., Wang, C. R. & Brenner, M. B. Diverse TCRs recognize murine CD1. J. Immunol. 162, 161-167 (1999). | PubMed | ISI | ChemPort |

20.
Cardell, S. et al. CD1-restricted CD4+ T cells in major histocompatibility complex class II-deficient mice. J. Exp. Med. 182, 993-1004 (1995). | PubMed | ISI | ChemPort |

21.
Burdin, N. et al. Structural requirements for antigen presentation by mouse CD1. Proc. Natl Acad. Sci. USA 97, 10156-10161 (2000). | Article | PubMed | ISI | ChemPort |

22.
Jackman, R. M. et al. The tyrosine-containing cytoplasmic tail of CD1b is essential for its efficient presentation of bacterial lipid antigens. Immunity 8, 341-351 (1998). | PubMed | ISI | ChemPort |

23.
Chiu, Y. H. et al. Distinct subsets of CD1d-restricted T cells recognize self-antigens loaded in different cellular compartments. J. Exp. Med. 189, 103-110 (1999). | Article | PubMed | ISI | ChemPort |

24.
Sugita, M. et al. Separate pathways for antigen presentation by CD1 molecules. Immunity 11, 743-752 (1999). | PubMed | ISI | ChemPort |

25.
Sugita, M., van Der, W., Rogers, R. A., Peters, P. J. & Brenner, M. B. CD1c molecules broadly survey the endocytic system. Proc. Natl Acad. Sci. 97, 8445-8450 (2000). | Article | PubMed | ISI | ChemPort |

26.
Moody, D. B. et al. Lipid length controls antigen entry into endosomal and nonendosomal pathways for CD1b presentation. Nature Immunol. 3, 435-442 (2002).
This study provides direct evidence for the role of alkyl chain length in controlling whether lipids are presented by endosomal or non-endosomal pathways. | Article | PubMed | ISI | ChemPort |

27.
Chiu, Y. H. et al. Multiple defects in antigen presentation and T-cell development by mice expressing cytoplasmic-tail-truncated CD1d. Nature Immunol. 3, 55-60 (2002). | Article | PubMed | ISI | ChemPort |

28.
Roberts, T. J. et al. Recycling CD1d1 molecules present endogenous antigens processed in an endocytic compartment to NKT cells. J. Immunol. 168, 5409-5414 (2002). | PubMed | ISI | ChemPort |

29.
Ernst, W. A. et al. Molecular interaction of CD1b with lipoglycan antigens. Immunity 8, 331-340 (1998). | PubMed | ISI | ChemPort |

30.
Calabi, F. & Milstein, C. A novel family of human major histocompatibility complex-related genes not mapping to chromosome 6. Nature 323, 540-543 (1986). | PubMed | ISI | ChemPort |

31.
Woolfson, A. & Milstein, C. Alternative splicing generates secretory isoforms of human CD1. Proc. Natl Acad. Sci. USA 91, 6683-6687 (1994). | PubMed | ISI | ChemPort |

32.
Angenieux, C. et al. Characterization of CD1e, a third type of CD1 molecule expressed in dendritic cells. J. Biol. Chem. 275, 37757-37764 (2000). | Article | PubMed | ISI | ChemPort |

33.
Sugita, M., Porcelli, S. A. & Brenner, M. B. Assembly and retention of CD1b heavy chains in the endoplasmic reticulum. J. Immunol. 159, 2358-2365 (1997). | PubMed | ISI | ChemPort |

34.
Huttinger, R., Staffler, G., Majdic, O. & Stockinger, H. Analysis of the early biogenesis of CD1b: involvement of the chaperones calnexin and calreticulin, the proteasome and 2-microglobulin. Int. Immunol. 11, 1615-1623 (1999). | Article | PubMed | ISI | ChemPort |

35.
Karadimitris, A. et al. Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography. Proc. Natl Acad. Sci. USA 98, 3294-3298 (2001). | Article | PubMed | ISI | ChemPort |

36.
Altamirano, M. M., Blackburn, J. M., Aguayo, C. & Fersht, A. R. Ligand-independent assembly of recombinant human CD1 by using oxidative refolding chromatography. Proc. Natl Acad. Sci. USA 98, 3288-3293 (2001). | Article | PubMed | ISI | ChemPort |

37.
Joyce, S. et al. Natural ligand of mouse CD1d1: cellular glycosylphosphatidylinositol. Science 279, 1541-1544 (1998). | Article | PubMed | ISI | ChemPort |

38.
De Silva, A. D. et al. Lipid-protein interactions: the assembly of CD1d1 with cellular phospholipids occurs in the endoplasmic reticulum. J. Immunol. 168, 723-733 (2002). | PubMed | ISI |

39.
Gumperz, J. et al. Murine CD1d-restricted T-cell recognition of cellular lipids. Immunity 12, 211-221 (2000). | PubMed | ISI | ChemPort |

40.
Sugita, M. & Brenner, M. B. An unstable 2-microglobulin: major histocompatibility complex class I heavy chain intermediate dissociates from calnexin and then is stabilized by binding peptide. J. Exp. Med. 180, 2163-2171 (1994). | PubMed | ISI | ChemPort |

41.
Bauer, A. et al. Analysis of the requirement for 2-microglobulin for expression and formation of human CD1 antigens. Eur. J. Immunol. 27, 1366-1373 (1997). | PubMed | ISI | ChemPort |

42.
Bendelac, A. et al. CD1 recognition by mouse NK1+ T lymphocytes. Science 268, 863-865 (1995). | PubMed | ISI | ChemPort |

43.
Kang, S. J. & Cresswell, P. Regulation of intracellular trafficking of human CD1d by association with MHC class II molecules. EMBO J. 21, 1650-1660 (2002).
This paper provides evidence for the association of CD1D with MHC class II molecules and for a functional role for MHC class-II-invariant-chain complexes in control of the intracellular trafficking of CD1D. | Article | PubMed | ISI | ChemPort |

Concay

44.
Jayawardena-Wolf, J., Benlagha, K., Chiu, Y. H., Mehr, R. & Bendelac, A. CD1d endosomal trafficking is independently regulated by an intrinsic CD1d-encoded tyrosine motif and by the invariant chain. Immunity 15, 897-908 (2001).
This study provides evidence for two routes of trafficking of CD1D proteins to late endosomes. CD1D proteins can recycle rapidly from the cell surface to endosomes or they can be delivered to this compartment after association with MHC class-II-invariant-chain complexes. | PubMed | ISI | ChemPort |

45.
Briken, V., Jackman, R. M., Dasgupta, S., Hoening, S. & Porcelli, S. A. Intracellular trafficking pathway of newly synthesized CD1b molecules. EMBO J. 21, 825-834 (2002).
These experiments provide evidence for the physical association of the cytoplasmic tail of CD1B with AP2 and AP3 using surface plasmon-resonance assays. In ad***ion, this report provides evidence for the role of this interaction in the intracellular localization of and antigen-presenting function of CD1B. | Article | PubMed | ISI | ChemPort |

46.
Sugita, M. et al. Failure of trafficking and antigen presentation by CD1 in AP3-deficient cells. Immunity 16, 697-706 (2002).
Using a yeast two-hybrid system, this report provides evidence for the association of CD1B with AP2 and AP3. Human cells deficient in AP3 are shown to have a reduced efficiency of lipid-antigen presentation. | PubMed | ISI | ChemPort |

47.
Boehm, M. & Bonifacino, J. S. Genetic analyses of adaptin function from yeast to mammals. Gene 286, 175-186 (2002). | Article | PubMed | ISI | ChemPort |

48.
Kantheti, P. et al. Mutation in AP3 in the mocha mouse links endosomal transport to storage deficiency in platelets, melanosomes and synaptic vesicles. Neuron 21, 111-122 (1998). | PubMed | ISI | ChemPort |

49.
Dell'Angelica, E. C., Shotelersuk, V., Aguilar, R. C., Gahl, W. A. & Bonifacino, J. S. Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the 3A subunit of the AP3 adaptor. Mol. Cell 3, 11-21 (1999). | PubMed | ISI | ChemPort |

50.
Owen, D. J. & Evans, P. R. A structural explanation for the recognition of tyrosine-based endocytotic signals. Science 282, 1327-1332 (1998). | Article | PubMed | ISI | ChemPort |

51.
Ohno, H. et al. Interaction of tyrosine-based sorting signals with clathrin-associated proteins. Science 269, 1872-1875 (1995). | PubMed | ISI | ChemPort |

52.
Bonifacino, J. S. & Dell'Angelica, E. C. Molecular bases for the recognition of tyrosine-based sorting signals. J. Cell Biol. 145, 923-926 (1999). | Article | PubMed | ISI | ChemPort |

53.
Johnson, K. F. & Kornfeld, S. A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function. J. Biol. Chem. 267, 17110-17115 (1992). | PubMed | ISI | ChemPort |

54.
Letourneur, F. & Klausner, R. D. A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. Cell 69, 1143-1157 (1992). | PubMed | ISI | ChemPort |

55.
Rapoport, I., Chen, Y. C., Cupers, P., Shoelson, S. E. & Kirchhausen, T. Dileucine-based sorting signals bind to the -chain of AP-1 at a site distinct and regulated differently from the tyrosine-based motif-binding site. EMBO J. 17, 2148-2155 (1998). | Article | PubMed | ISI | ChemPort |

56.
Rodionov, D. G. & Bakke, O. Medium chains of adaptor complexes AP-1 and AP-2 recognize leucine-based sorting signals from the invariant chain. J. Biol. Chem. 273, 6005-6008 (1998). | Article | PubMed | ISI | ChemPort |

57.
Greenberg, M., DeTulleo, L., Rapoport, I., Skowronski, J. & Kirchhausen, T. A dileucine motif in HIV-1 Nef is essential for sorting into clathrin-coated pits and for downregulation of CD4. Curr. Biol. 8, 1239-1242 (1998). | PubMed | ISI | ChemPort |

58.
Salamero, J., Le Borgne, R., Saudrais, C., Goud, B. & Hoflack, B. Expression of major histocompatibility complex class II molecules in HeLa cells promotes the recruitment of AP-1 Golgi-specific assembly proteins on Golgi membranes. J. Biol. Chem. 271, 30318-30321 (1996). | Article | PubMed | ISI | ChemPort |

59.
Kang, S., Liang, L., Parker, C. D. & Collawn, J. F. Structural requirements for major histocompatibility complex class II invariant chain endocytosis and lysosomal targeting. J. Biol. Chem. 273, 20644-20652 (1998). | Article | PubMed | ISI | ChemPort |

60.
Zhong, G., Romagnoli, P. & Germain, R. N. Related leucine-based cytoplasmic targeting signals in invariant chain and major histocompatibility complex class II molecules control endocytic presentation of distinct determinants in a single protein. J. Exp. Med. 185, 429-438 (1997). | Article | PubMed | ISI | ChemPort |

61.
Kongsvik, T. L., Honing, S., Bakke, O. & Rodionov, D. G. Mechanism of interaction between leucine-based sorting signals from the invariant chain and clathrin-associated adaptor protein complexes AP1 and AP2. J. Biol. Chem. 277, 16484-16488 (2002). | Article | PubMed | ISI | ChemPort |

62.
Porcelli, S. et al. Recognition of cluster of differentiation 1 antigens by human CD4-CD8- cytolytic T lymphocytes. Nature 341, 447-450 (1989). | PubMed | ISI | ChemPort |

63.
Briken, V., Jackman, R. M., Watts, G. F., Rogers, R. A. & Porcelli, S. A. Human CD1b and CD1c isoforms survey different intracellular compartments for the presentation of microbial lipid antigens. J. Exp. Med. 192, 281-288 (2000). | Article | PubMed | ISI | ChemPort |

64.
Sieling, P. A. et al. Human double-negative T cells in systemic lupus erythematosus provide help for IgG and are restricted by CD1c. J. Immunol. 165, 5338-5344 (2000). | PubMed | ISI | ChemPort |

65.
Moody, D. B. Polyisoprenyl glycolipids as targets of CD1-mediated T-cell responses. Cell Mol. Life Sci. 58, 1461-1474 (2001). | PubMed | ISI | ChemPort |

66.
Rodionov, D. G., Nordeng, T. W., Pedersen, K., Balk, S. P. & Bakke, O. A critical tyrosine residue in the cytoplasmic tail is important for CD1d internalization but not for its basolateral sorting in MDCK cells. J. Immunol. 162, 1488-1495 (1999). | PubMed | ISI | ChemPort |

67.
Blumberg, R. S., Colgan, S. P. & Balk, S. P. CD1d: outside-in antigen presentation in the intestinal epithelium? Clin. Exp. Immunol. 109, 223-225 (1997). | PubMed | ISI | ChemPort |

68.
Rodionov, D. G., Nordeng, T. W., Kongsvik, T. L. & Bakke, O. The cytoplasmic tail of CD1d contains two overlapping basolateral sorting signals. J. Biol. Chem. 275, 8279-8282 (2000). | Article | PubMed | ISI | ChemPort |

69.
Mellman, I. & Steinman, R. M. Dendritic cells: specialized and regulated antigen-processing machines. Cell 106, 255-258 (2001). | PubMed | ISI | ChemPort |

70.
Boes, M. et al. T-cell engagement of dendritic cells rapidly rearranges MHC class II transport. Nature 418, 983-988 (2002). | Article | PubMed | ISI | ChemPort |

71.
Riese, R. J. et al. Regulation of CD1 function and NK1.1+ T-cell selection and maturation by cathepsin S. Immunity 15, 909-919 (2001). | PubMed | ISI | ChemPort |

72.
Honey, K. et al. Thymocyte expression of cathepsin L is essential for NKT-cell development. Nature Immunol. 3, 1069-1074 (2002). | Article | PubMed | ISI | ChemPort |

73.
Nakagawa, T. Y. & Rudensky, A. Y. The role of lysosomal proteinases in MHC class II-mediated antigen processing and presentation. Immunol. Rev. 172, 121-129 (1999). | PubMed | ISI | ChemPort |

74.
Sugita, M. et al. Cytoplasmic tail-dependent localization of CD1b antigen-presenting molecules to MIICs. Science 273, 349-352 (1996).
This report provides the first evidence that tyrosine-based sequences of CD1 proteins have a role in the delivery of CD1 proteins to late compartments in the endosomal network, including the MHC class II compartment. | PubMed | ISI | ChemPort |

75.
Moody, D. B., Reinhold, B. B., Reinhold, V. N., Besra, G. S. & Porcelli, S. A. Uptake and processing of glycosylated mycolates for presentation to CD1b-restricted T cells. Immunol. Lett. 65, 85-91 (1999). | Article | PubMed | ISI | ChemPort |

76.
Geho, D. H. et al. Glycosyl-phosphatidylinositol reanchoring unmasks distinct antigen-presenting pathways for CD1b and CD1c. J. Immunol. 165, 1272-1277 (2000). | PubMed | ISI | ChemPort |

77.
Shamshiev, A. et al. The T-cell response to self-glycolipids shows a novel mechanism of CD1b loading and a requirement for complex oligosaccharides. Immunity 13, 255-264 (2000). | PubMed | ISI | ChemPort |

78.
Prigozy, T. I. et al. The mannose receptor delivers lipoglycan antigens to endosomes for presentation to T cells by CD1b molecules. Immunity 6, 187-197 (1997). | PubMed | ISI | ChemPort |

79.
Brossay, L. et al. Structural requirements for galactosylceramide recognition by CD1-restricted NK T cells. J. Immunol. 161, 5124-5128 (1998). | PubMed | ISI | ChemPort |

80.
Mukherjee, S., Soe, T. T. & Maxfield, F. R. Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails. J. Cell Biol. 144, 1271-1284 (1999). | Article | PubMed | ISI | ChemPort |

81.
Ferrari, G., Langen, H., Naito, M. & Pieters, J. A coat protein on phagosomes involved in the intracellular survival of mycobacteria. Cell 97, 435-447 (1999). | PubMed | ISI | ChemPort |

82.
Pancholi, P., Mirza, A., Bhardwaj, N. & Steinman, R. M. Sequestration from immune CD4+ T cells of mycobacteria growing in human macrophages. Science 260, 984-986 (1993). | PubMed | ISI | ChemPort |

83.
Cresswell, P. Invariant chain structure and MHC class II function. Cell 84, 505-507 (1996). | PubMed | ISI | ChemPort |

84.
Schaible, U. E., Hagens, K., Fischer, K., Collins, H. L. & Kaufmann, S. H. Intersection of group I CD1 molecules and mycobacteria in different intracellular compartments of dendritic cells. J. Immunol. 164, 4843-4852 (2000). | PubMed | ISI | ChemPort |

85.
Rush, J. S., Sweitzer, T., Kent, C., Decker, G. L. & Waechter, C. J. Biogenesis of the endoplasmic reticulum in activated B lymphocytes: temporal relationships between the induction of protein N-glycosylation activity and the biosynthesis of membrane protein and phospholipid. Arch. Biochem. Biophys. 284, 63-70 (1991). | PubMed | ISI | ChemPort |

86.
Maccioni, H. J., Daniotti, J. L. & Martina, J. A. Organization of ganglioside synthesis in the Golgi apparatus. Biochim. Biophys. Acta 1437, 101-118 (1999). | Article | PubMed | ISI | ChemPort |

87.
Melian, A. et al. Molecular recognition of human CD1b antigen complexes: evidence for a common pattern of interaction with TCRs. J. Immunol. 165, 4494-4504 (2000). | PubMed | ISI | ChemPort |

Acknowledgements
We thank M. Sugita, M. Brenner, I. Wilson, T. Cheng, A. Rudensky, K. Honey, V. Cerundolo, Y. Jones and S. Gadola for providing data to illustrate figures and for communicating unpublished data.
Concay

44.
Jayawardena-Wolf, J., Benlagha, K., Chiu, Y. H., Mehr, R. & Bendelac, A. CD1d endosomal trafficking is independently regulated by an intrinsic CD1d-encoded tyrosine motif and by the invariant chain. Immunity 15, 897-908 (2001).
This study provides evidence for two routes of trafficking of CD1D proteins to late endosomes. CD1D proteins can recycle rapidly from the cell surface to endosomes or they can be delivered to this compartment after association with MHC class-II-invariant-chain complexes. | PubMed | ISI | ChemPort |

45.
Briken, V., Jackman, R. M., Dasgupta, S., Hoening, S. & Porcelli, S. A. Intracellular trafficking pathway of newly synthesized CD1b molecules. EMBO J. 21, 825-834 (2002).
These experiments provide evidence for the physical association of the cytoplasmic tail of CD1B with AP2 and AP3 using surface plasmon-resonance assays. In ad***ion, this report provides evidence for the role of this interaction in the intracellular localization of and antigen-presenting function of CD1B. | Article | PubMed | ISI | ChemPort |

46.
Sugita, M. et al. Failure of trafficking and antigen presentation by CD1 in AP3-deficient cells. Immunity 16, 697-706 (2002).
Using a yeast two-hybrid system, this report provides evidence for the association of CD1B with AP2 and AP3. Human cells deficient in AP3 are shown to have a reduced efficiency of lipid-antigen presentation. | PubMed | ISI | ChemPort |

47.
Boehm, M. & Bonifacino, J. S. Genetic analyses of adaptin function from yeast to mammals. Gene 286, 175-186 (2002). | Article | PubMed | ISI | ChemPort |

48.
Kantheti, P. et al. Mutation in AP3 in the mocha mouse links endosomal transport to storage deficiency in platelets, melanosomes and synaptic vesicles. Neuron 21, 111-122 (1998). | PubMed | ISI | ChemPort |

49.
Dell'Angelica, E. C., Shotelersuk, V., Aguilar, R. C., Gahl, W. A. & Bonifacino, J. S. Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the 3A subunit of the AP3 adaptor. Mol. Cell 3, 11-21 (1999). | PubMed | ISI | ChemPort |

50.
Owen, D. J. & Evans, P. R. A structural explanation for the recognition of tyrosine-based endocytotic signals. Science 282, 1327-1332 (1998). | Article | PubMed | ISI | ChemPort |

51.
Ohno, H. et al. Interaction of tyrosine-based sorting signals with clathrin-associated proteins. Science 269, 1872-1875 (1995). | PubMed | ISI | ChemPort |

52.
Bonifacino, J. S. & Dell'Angelica, E. C. Molecular bases for the recognition of tyrosine-based sorting signals. J. Cell Biol. 145, 923-926 (1999). | Article | PubMed | ISI | ChemPort |

53.
Johnson, K. F. & Kornfeld, S. A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function. J. Biol. Chem. 267, 17110-17115 (1992). | PubMed | ISI | ChemPort |

54.
Letourneur, F. & Klausner, R. D. A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. Cell 69, 1143-1157 (1992). | PubMed | ISI | ChemPort |

55.
Rapoport, I., Chen, Y. C., Cupers, P., Shoelson, S. E. & Kirchhausen, T. Dileucine-based sorting signals bind to the -chain of AP-1 at a site distinct and regulated differently from the tyrosine-based motif-binding site. EMBO J. 17, 2148-2155 (1998). | Article | PubMed | ISI | ChemPort |

56.
Rodionov, D. G. & Bakke, O. Medium chains of adaptor complexes AP-1 and AP-2 recognize leucine-based sorting signals from the invariant chain. J. Biol. Chem. 273, 6005-6008 (1998). | Article | PubMed | ISI | ChemPort |

57.
Greenberg, M., DeTulleo, L., Rapoport, I., Skowronski, J. & Kirchhausen, T. A dileucine motif in HIV-1 Nef is essential for sorting into clathrin-coated pits and for downregulation of CD4. Curr. Biol. 8, 1239-1242 (1998). | PubMed | ISI | ChemPort |

58.
Salamero, J., Le Borgne, R., Saudrais, C., Goud, B. & Hoflack, B. Expression of major histocompatibility complex class II molecules in HeLa cells promotes the recruitment of AP-1 Golgi-specific assembly proteins on Golgi membranes. J. Biol. Chem. 271, 30318-30321 (1996). | Article | PubMed | ISI | ChemPort |

59.
Kang, S., Liang, L., Parker, C. D. & Collawn, J. F. Structural requirements for major histocompatibility complex class II invariant chain endocytosis and lysosomal targeting. J. Biol. Chem. 273, 20644-20652 (1998). | Article | PubMed | ISI | ChemPort |

60.
Zhong, G., Romagnoli, P. & Germain, R. N. Related leucine-based cytoplasmic targeting signals in invariant chain and major histocompatibility complex class II molecules control endocytic presentation of distinct determinants in a single protein. J. Exp. Med. 185, 429-438 (1997). | Article | PubMed | ISI | ChemPort |

61.
Kongsvik, T. L., Honing, S., Bakke, O. & Rodionov, D. G. Mechanism of interaction between leucine-based sorting signals from the invariant chain and clathrin-associated adaptor protein complexes AP1 and AP2. J. Biol. Chem. 277, 16484-16488 (2002). | Article | PubMed | ISI | ChemPort |

62.
Porcelli, S. et al. Recognition of cluster of differentiation 1 antigens by human CD4-CD8- cytolytic T lymphocytes. Nature 341, 447-450 (1989). | PubMed | ISI | ChemPort |

63.
Briken, V., Jackman, R. M., Watts, G. F., Rogers, R. A. & Porcelli, S. A. Human CD1b and CD1c isoforms survey different intracellular compartments for the presentation of microbial lipid antigens. J. Exp. Med. 192, 281-288 (2000). | Article | PubMed | ISI | ChemPort |

64.
Sieling, P. A. et al. Human double-negative T cells in systemic lupus erythematosus provide help for IgG and are restricted by CD1c. J. Immunol. 165, 5338-5344 (2000). | PubMed | ISI | ChemPort |

65.
Moody, D. B. Polyisoprenyl glycolipids as targets of CD1-mediated T-cell responses. Cell Mol. Life Sci. 58, 1461-1474 (2001). | PubMed | ISI | ChemPort |

66.
Rodionov, D. G., Nordeng, T. W., Pedersen, K., Balk, S. P. & Bakke, O. A critical tyrosine residue in the cytoplasmic tail is important for CD1d internalization but not for its basolateral sorting in MDCK cells. J. Immunol. 162, 1488-1495 (1999). | PubMed | ISI | ChemPort |

67.
Blumberg, R. S., Colgan, S. P. & Balk, S. P. CD1d: outside-in antigen presentation in the intestinal epithelium? Clin. Exp. Immunol. 109, 223-225 (1997). | PubMed | ISI | ChemPort |

68.
Rodionov, D. G., Nordeng, T. W., Kongsvik, T. L. & Bakke, O. The cytoplasmic tail of CD1d contains two overlapping basolateral sorting signals. J. Biol. Chem. 275, 8279-8282 (2000). | Article | PubMed | ISI | ChemPort |

69.
Mellman, I. & Steinman, R. M. Dendritic cells: specialized and regulated antigen-processing machines. Cell 106, 255-258 (2001). | PubMed | ISI | ChemPort |

70.
Boes, M. et al. T-cell engagement of dendritic cells rapidly rearranges MHC class II transport. Nature 418, 983-988 (2002). | Article | PubMed | ISI | ChemPort |

71.
Riese, R. J. et al. Regulation of CD1 function and NK1.1+ T-cell selection and maturation by cathepsin S. Immunity 15, 909-919 (2001). | PubMed | ISI | ChemPort |

72.
Honey, K. et al. Thymocyte expression of cathepsin L is essential for NKT-cell development. Nature Immunol. 3, 1069-1074 (2002). | Article | PubMed | ISI | ChemPort |

73.
Nakagawa, T. Y. & Rudensky, A. Y. The role of lysosomal proteinases in MHC class II-mediated antigen processing and presentation. Immunol. Rev. 172, 121-129 (1999). | PubMed | ISI | ChemPort |

74.
Sugita, M. et al. Cytoplasmic tail-dependent localization of CD1b antigen-presenting molecules to MIICs. Science 273, 349-352 (1996).
This report provides the first evidence that tyrosine-based sequences of CD1 proteins have a role in the delivery of CD1 proteins to late compartments in the endosomal network, including the MHC class II compartment. | PubMed | ISI | ChemPort |

75.
Moody, D. B., Reinhold, B. B., Reinhold, V. N., Besra, G. S. & Porcelli, S. A. Uptake and processing of glycosylated mycolates for presentation to CD1b-restricted T cells. Immunol. Lett. 65, 85-91 (1999). | Article | PubMed | ISI | ChemPort |

76.
Geho, D. H. et al. Glycosyl-phosphatidylinositol reanchoring unmasks distinct antigen-presenting pathways for CD1b and CD1c. J. Immunol. 165, 1272-1277 (2000). | PubMed | ISI | ChemPort |

77.
Shamshiev, A. et al. The T-cell response to self-glycolipids shows a novel mechanism of CD1b loading and a requirement for complex oligosaccharides. Immunity 13, 255-264 (2000). | PubMed | ISI | ChemPort |

78.
Prigozy, T. I. et al. The mannose receptor delivers lipoglycan antigens to endosomes for presentation to T cells by CD1b molecules. Immunity 6, 187-197 (1997). | PubMed | ISI | ChemPort |

79.
Brossay, L. et al. Structural requirements for galactosylceramide recognition by CD1-restricted NK T cells. J. Immunol. 161, 5124-5128 (1998). | PubMed | ISI | ChemPort |

80.
Mukherjee, S., Soe, T. T. & Maxfield, F. R. Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails. J. Cell Biol. 144, 1271-1284 (1999). | Article | PubMed | ISI | ChemPort |

81.
Ferrari, G., Langen, H., Naito, M. & Pieters, J. A coat protein on phagosomes involved in the intracellular survival of mycobacteria. Cell 97, 435-447 (1999). | PubMed | ISI | ChemPort |

82.
Pancholi, P., Mirza, A., Bhardwaj, N. & Steinman, R. M. Sequestration from immune CD4+ T cells of mycobacteria growing in human macrophages. Science 260, 984-986 (1993). | PubMed | ISI | ChemPort |

83.
Cresswell, P. Invariant chain structure and MHC class II function. Cell 84, 505-507 (1996). | PubMed | ISI | ChemPort |

84.
Schaible, U. E., Hagens, K., Fischer, K., Collins, H. L. & Kaufmann, S. H. Intersection of group I CD1 molecules and mycobacteria in different intracellular compartments of dendritic cells. J. Immunol. 164, 4843-4852 (2000). | PubMed | ISI | ChemPort |

85.
Rush, J. S., Sweitzer, T., Kent, C., Decker, G. L. & Waechter, C. J. Biogenesis of the endoplasmic reticulum in activated B lymphocytes: temporal relationships between the induction of protein N-glycosylation activity and the biosynthesis of membrane protein and phospholipid. Arch. Biochem. Biophys. 284, 63-70 (1991). | PubMed | ISI | ChemPort |

86.
Maccioni, H. J., Daniotti, J. L. & Martina, J. A. Organization of ganglioside synthesis in the Golgi apparatus. Biochim. Biophys. Acta 1437, 101-118 (1999). | Article | PubMed | ISI | ChemPort |

87.
Melian, A. et al. Molecular recognition of human CD1b antigen complexes: evidence for a common pattern of interaction with TCRs. J. Immunol. 165, 4494-4504 (2000). | PubMed | ISI | ChemPort |

Acknowledgements
We thank M. Sugita, M. Brenner, I. Wilson, T. Cheng, A. Rudensky, K. Honey, V. Cerundolo, Y. Jones and S. Gadola for providing data to illustrate figures and for communicating unpublished data.
Concay

Review
1st Class Ticket to Class I: Protein Toxins as Pathfinders for Antigen Presentation
Daniel C. Smith 1 , J. Michael Lord 1 , Lynne M. Roberts 1 , Eric Tartour 2 and Ludger Johannes 3 , *

A number of bacterial toxins have evolved diverse strategies for crossing membrane barriers in order to reach their substrates in the mammalian cytosol. Recent studies show that this property can be exploited for the delivery of fused antigens into the major histocompatibility complex class I-restricted presentation pathway, with the goal of eliciting a specific immune response. Here we discuss the peculiarities of the trafficking pathways of a variety of toxins, and how these may allow the toxins to be used as delivery vehicles for therapeutic and diagnostic purposes.

The antibody response is the most familiar branch of the immune system and its therapeutic manipulation through the use of vaccines has been one of the major advances in modern medicine. However, for pathogens that replicate intracellularly and thereby evade this antibody response, classical vaccination has proven inefficient. In these cases, it is desirable to also stimulate the activity of CD8+ cytotoxic T lymphocytes (CTL), that are capable of directly destroying a target cell harbouring intracellular pathogens or presenting genetic or somatic abnormalities, as is the case for some tumour cells.
Most nucleated cells are able to load antigenic peptides derived from intracellular viral proteins or abnormal cellular proteins onto their major histocompatibility complex (MHC) class I proteins. These peptides are delivered to neosynthesized MHC class I molecules in the lumen of the endoplasmic reticulum (ER) via peptide transporters associated with antigen processing (TAP) in the ER membrane (1) (Figure 1). In this classical antigen presentation pathway, peptide-loaded MHC class I complexes are transported via the Golgi apparatus to the plasma membrane, where they are recognised by mature cytotoxic CD8+ T cells (Figure 1). In order to convert precursor CD8+ T cells in*****ch mature CTL capable of killing the virally infected cell, specialised cells of the immune system are required. Indeed, the induction of a primary CTL response against infectious agents or tumours relies on professional antigen-presenting cells (APC). These cells, among which dendritic cells (DC) play a prominent role (2,3), express on their surfaces not only MHC class I molecules but also MHC class II and costimulatory molecules. The MHC class II molecules of APC present peptides generated in a specialised endosomal compartment following endocytosis of any exogenous viral or tumour antigens. MHC class II presentation leads in turn to the activation of CD4+ helper T lymphocytes (TH1) and the secretion of the T-cell growth promoting cytokine interleukin 2 (IL-2). As a result, the antigen-activated precursor CD8+ T cells proliferate and differentiate into mature effector CTL that promote the killing of infected cells (Figure 2a). Interestingly, DC have the remarkable capacity to present exogenous material not only by MHC class II molecules but also by class I proteins in a process known as cross-priming (4). Cross-priming of DC, therefore, can in itself ensure a CTL response (Figure 2b).
Thus, the induction of protective cell-mediated immunity, in ad***ion to the humoral (antibody) response, is desirable to aid the clearance of certain viruses and certain tumours. This has led to a flurry of novel approaches designed to deliver material directly to the MHC class I processing and presentation machinery [for a review see (5); see also (6,7)]. Since cross-priming can be artificially provoked by antigen delivery vectors that target and enter DC, this too can be exploited for immunotherapy. One such approach exploits the intracellular trafficking and membrane translocation properties of particular protein toxins to which antigenic peptides have been fused.
This review focuses on several bacterial toxins that have been used in antigen presentation studies (see Table 1), including those that inhibit cell signalling (adenylate cyclase toxin (ACTx), anthrax toxin (ATx) and pertussis toxin (PTx)) and protein synthesis (diphtheria toxin (DTx), Pseudomonas exotoxin (PEx) and Shiga toxin (STx)). These holotoxins are all composed of an enzymatically active A- and one or more cell-binding B-subunit(s) (also denoted as fragments or chains). We have chosen to abbreviate the toxin subunits as A or B; e.g. STx is composed of STxA and STxB, etc. With the exception of ACTx, these toxins enter cells by endocytosis, although their precise routing and the mechanisms and sites of their membrane translocation to the cytosol are variable (Figure 3). Despite these differences, they can all transfer antigens or immunodominant peptides to MHC class I proteins within cells. In the following parts of the review, we will discuss how and to what extent the intracellular transport characteristics of these toxins can be exploited for antigen presentation (MHC class I and class II presentation, DC targeting, DC maturation, toxicity, immunogenicity) and for immunological and clinical use (induction of an efficient cellular immune response, tumour protection, etc.).
Getting into the Cytosol
Protein toxins must cross a membrane barrier in order to reach their cytosolic substrates in their respective target cells. This ability makes them ideal tools to tackle the major problem in deliberately targeting antigens into the MHC class I pathway: namely, delivering the antigen to the cytosol for proteasomal processing. As it turns out, protein toxins seem to have evolved a number of strategies to overcome this hurdle (Figure 3).

Translocation at the plasma membrane
ACTx is a major virulence factor of the whooping cough bacterium, Bordetella pertussis. After binding its cell-surface receptor, found on a subset of leucocytes (8), the toxin directly crosses the plasma membrane by an unknown mechanism and ACTxA subsequently deregulates cAMP levels (Figure 3, path 1). Recombinant ACTx toxoids bearing CD8+ T-cell epitopes within their mutated, and thereby detoxified, A-subunits were able to induce specific CTL responses in mice (9,10), and protection against experimental tumours has been demonstrated (11). Surface presentation of the ACTx-delivered epitopes occurred via the classical MHC class I pathway (Figure 3, path 1 broken line), as shown by the dependence on cytosolic proteasome activity and a functional TAP complex.

Endosomal membrane translocation
DTx, secreted by Corynebacterium diphtheriae, and ATx from Bacillus anthracis, enter cells by binding to specific surface receptors. Both proteins are subsequently endocytosed and translocate to their cytosolic targets from acidic endosomes (Figure 3, path 2). In the case of furin-cleaved DTx, the low pH of the endosome causes DTxB to undergo a well-characterised conformational change that facilitates DTxA translocation into the cytosol (12), carrying with it any fused viral peptides (13). Whereas DTx has not yet been actively pursued as an antigen carrier, ATx, has already been successfully used to induce T-cell immunity. This toxin consists of a monomeric cell binding protective antigen (PA) that can form a heptamer once activated by a cell surface protease. Heptameric PA can then bind the enzymatic toxin monomers, lethal factor (LF) and/or the edema factor (EF). In this form the toxin is internalized and at the low pH of endosomes the PA moiety creates a translocation pore in the membrane to mediate cytosolic delivery of LF/EF (14). If LF is truncated to disarm its destructive activity and genetically fused to T-cell epitopes, delivery into cells by PA induces CTL immunity (15-17) (Figure 3, path 2).

ER-associated membrane translocation
Several toxins that do not have any pore-forming ability, including the sialic acid-interacting PTx from Bordetella pertussis, and PEx from Pseudomonas aeruginosa, follow a retrograde route from the cell surface through endosomes and the Golgi apparatus, to the ER (18,19) (Figure 3, path 3). Interestingly, STx (from Shigella dysenteriae), by-passes the degrading environment of the late endocytic pathway (20) and traffics directly to the Golgi from the early endosome (Figure 3, path 3a). The presence of molecular chaperones and protein disulphide isomerase in the ER lumen to aid unfolding of the toxins and, in particular, the process of ER-associated degradation (ERAD) (21), probably all contribute to the favourability of this compartment for membrane translocation. ERAD eliminates misfolded proteins from the ER via the Sec61p translocon. This dislocation is coupled to their cytosolic proteasome-dependent degradation. While parasitising this pathway would obviously enable access to the cytosol, the toxin must, to some extent, escape the ensuing degradation during a natural infection. Strikingly, lysine residues, which direct a protein to the proteasome following ubiquitination, are under-represented in their catalytic A-subunits (22), a feature which may attenuate targeting to proteasomes.
However, the predominant coupling of the ERAD pathway to proteasomes is of potential interest in terms of antigen presentation since antigenic peptides fused to the catalytic A-subunits, or detoxified versions thereof, might be expected to be targeted to the endogenous MHC class I pathway via the proteasome and the TAP transporter. Indeed, it has been shown that antigenic peptides fused to the A-chain of STx can be presented in a class I-dependent manner at the cell surface (23). For PTxA-dependent antigen presentation it has been shown that it is blocked by brefeldin A (BFA), a reagent known to perturb the Golgi stack. This suggested the involvement of the Golgi apparatus in either a failure of anterograde peptide-loaded MHC class I transport to the plasma membrane or a requirement for the Golgi in retrograde transport of the toxin (25). Surprisingly however, peptide presentation in this instance was not prevented by proteasome inhibitors and also occurred in TAP-deficient cells, suggesting that processing did not occur in the cytosol and that ad***ional quality control or e***ing pathways may exist in post-Golgi compartments (26). It remains to be determined to what extent this observation holds true for the other ER-targeted toxins.
TAP- and proteasome-independent processing also seem to occur in the case of PEx, which is endocytosed following binding to the 2-macroglobulin receptor. However, in this case, antigen presentation via detoxified PExA was also BFA-insensitive (27,28), implying that transport through the Golgi was not required prior to peptide processing, even though the holotoxin normally reaches the ER via this compartment (29). This suggested that antigen processing and loading must occur earlier in the retrograde pathway, possibly in endosomes, involving MHC class I molecules that undergo recycling from the cell surface. Consistent with an endosomal site for PE-peptide processing, it has been shown that PEx can also internalise a MHC class II-specific antigen where processing occurs within specialised endosomes (30).

Unconventional transport pathways
Recent work with STx has suggested that the same toxin can have different transport itineraries in different cell types (e.g. HeLa cells and APC), thereby affecting the way antigen may reach the cytosol. However, subsequent proteasomal processing and presentation via the classical MHC class I pathway still occurred (31,32). In these experiments, the use of quantitative biochemical methods revealed that in human monocyte-derived DC, STxB does not appear to follow the retrograde pathway to the Golgi apparatus or the ER (33) as it does in other cell types (e.g. HeLa cells). Therefore, delivery to the cytosol in DC cannot involve the ERAD pathway. Whether the same is true for other types of DC remains to be studied. The differential transport of STxB in monocyte-derived human DC and HeLa cells could be correlated with a role of lipid microdomains ('rafts') in the endosome-to-Golgi trafficking of this lipid-binding toxin. STxB failed to associate with such microdomains in DC, potentially precluding transport to the Golgi and beyond, but it could associate with rafts in HeLa cells. Native STx is not toxic to human monocytes (34), from which DC are derived, although it is potently toxic towards HeLa cells. Together, these observations suggest that STx becomes proteolytically processed within the endosome/lysosome system of DC, and that STxB (plus any fused antigen) can then escape to the cytosol by an unknown mechanism. This would imply the existence of a membrane translocation mechanism for STxB (but not the holotoxin) at the level of endosomes or lysosomes in DC. At the moment, it is not clear whether this pathway is the same as the one operating on phagocytosed insoluble material (35) or endocytosed antigen-antibody complexes (36,37), which also enter the cytosol from endosomes.

Concay
Được Milou sửa vào 06:00 ngày 18/06/2003

Review
1st Class Ticket to Class I: Protein Toxins as Pathfinders for Antigen Presentation
Daniel C. Smith 1 , J. Michael Lord 1 , Lynne M. Roberts 1 , Eric Tartour 2 and Ludger Johannes 3 , *

A number of bacterial toxins have evolved diverse strategies for crossing membrane barriers in order to reach their substrates in the mammalian cytosol. Recent studies show that this property can be exploited for the delivery of fused antigens into the major histocompatibility complex class I-restricted presentation pathway, with the goal of eliciting a specific immune response. Here we discuss the peculiarities of the trafficking pathways of a variety of toxins, and how these may allow the toxins to be used as delivery vehicles for therapeutic and diagnostic purposes.

The antibody response is the most familiar branch of the immune system and its therapeutic manipulation through the use of vaccines has been one of the major advances in modern medicine. However, for pathogens that replicate intracellularly and thereby evade this antibody response, classical vaccination has proven inefficient. In these cases, it is desirable to also stimulate the activity of CD8+ cytotoxic T lymphocytes (CTL), that are capable of directly destroying a target cell harbouring intracellular pathogens or presenting genetic or somatic abnormalities, as is the case for some tumour cells.
Most nucleated cells are able to load antigenic peptides derived from intracellular viral proteins or abnormal cellular proteins onto their major histocompatibility complex (MHC) class I proteins. These peptides are delivered to neosynthesized MHC class I molecules in the lumen of the endoplasmic reticulum (ER) via peptide transporters associated with antigen processing (TAP) in the ER membrane (1) (Figure 1). In this classical antigen presentation pathway, peptide-loaded MHC class I complexes are transported via the Golgi apparatus to the plasma membrane, where they are recognised by mature cytotoxic CD8+ T cells (Figure 1). In order to convert precursor CD8+ T cells in*****ch mature CTL capable of killing the virally infected cell, specialised cells of the immune system are required. Indeed, the induction of a primary CTL response against infectious agents or tumours relies on professional antigen-presenting cells (APC). These cells, among which dendritic cells (DC) play a prominent role (2,3), express on their surfaces not only MHC class I molecules but also MHC class II and costimulatory molecules. The MHC class II molecules of APC present peptides generated in a specialised endosomal compartment following endocytosis of any exogenous viral or tumour antigens. MHC class II presentation leads in turn to the activation of CD4+ helper T lymphocytes (TH1) and the secretion of the T-cell growth promoting cytokine interleukin 2 (IL-2). As a result, the antigen-activated precursor CD8+ T cells proliferate and differentiate into mature effector CTL that promote the killing of infected cells (Figure 2a). Interestingly, DC have the remarkable capacity to present exogenous material not only by MHC class II molecules but also by class I proteins in a process known as cross-priming (4). Cross-priming of DC, therefore, can in itself ensure a CTL response (Figure 2b).
Thus, the induction of protective cell-mediated immunity, in ad***ion to the humoral (antibody) response, is desirable to aid the clearance of certain viruses and certain tumours. This has led to a flurry of novel approaches designed to deliver material directly to the MHC class I processing and presentation machinery [for a review see (5); see also (6,7)]. Since cross-priming can be artificially provoked by antigen delivery vectors that target and enter DC, this too can be exploited for immunotherapy. One such approach exploits the intracellular trafficking and membrane translocation properties of particular protein toxins to which antigenic peptides have been fused.
This review focuses on several bacterial toxins that have been used in antigen presentation studies (see Table 1), including those that inhibit cell signalling (adenylate cyclase toxin (ACTx), anthrax toxin (ATx) and pertussis toxin (PTx)) and protein synthesis (diphtheria toxin (DTx), Pseudomonas exotoxin (PEx) and Shiga toxin (STx)). These holotoxins are all composed of an enzymatically active A- and one or more cell-binding B-subunit(s) (also denoted as fragments or chains). We have chosen to abbreviate the toxin subunits as A or B; e.g. STx is composed of STxA and STxB, etc. With the exception of ACTx, these toxins enter cells by endocytosis, although their precise routing and the mechanisms and sites of their membrane translocation to the cytosol are variable (Figure 3). Despite these differences, they can all transfer antigens or immunodominant peptides to MHC class I proteins within cells. In the following parts of the review, we will discuss how and to what extent the intracellular transport characteristics of these toxins can be exploited for antigen presentation (MHC class I and class II presentation, DC targeting, DC maturation, toxicity, immunogenicity) and for immunological and clinical use (induction of an efficient cellular immune response, tumour protection, etc.).
Getting into the Cytosol
Protein toxins must cross a membrane barrier in order to reach their cytosolic substrates in their respective target cells. This ability makes them ideal tools to tackle the major problem in deliberately targeting antigens into the MHC class I pathway: namely, delivering the antigen to the cytosol for proteasomal processing. As it turns out, protein toxins seem to have evolved a number of strategies to overcome this hurdle (Figure 3).

Translocation at the plasma membrane
ACTx is a major virulence factor of the whooping cough bacterium, Bordetella pertussis. After binding its cell-surface receptor, found on a subset of leucocytes (8), the toxin directly crosses the plasma membrane by an unknown mechanism and ACTxA subsequently deregulates cAMP levels (Figure 3, path 1). Recombinant ACTx toxoids bearing CD8+ T-cell epitopes within their mutated, and thereby detoxified, A-subunits were able to induce specific CTL responses in mice (9,10), and protection against experimental tumours has been demonstrated (11). Surface presentation of the ACTx-delivered epitopes occurred via the classical MHC class I pathway (Figure 3, path 1 broken line), as shown by the dependence on cytosolic proteasome activity and a functional TAP complex.

Endosomal membrane translocation
DTx, secreted by Corynebacterium diphtheriae, and ATx from Bacillus anthracis, enter cells by binding to specific surface receptors. Both proteins are subsequently endocytosed and translocate to their cytosolic targets from acidic endosomes (Figure 3, path 2). In the case of furin-cleaved DTx, the low pH of the endosome causes DTxB to undergo a well-characterised conformational change that facilitates DTxA translocation into the cytosol (12), carrying with it any fused viral peptides (13). Whereas DTx has not yet been actively pursued as an antigen carrier, ATx, has already been successfully used to induce T-cell immunity. This toxin consists of a monomeric cell binding protective antigen (PA) that can form a heptamer once activated by a cell surface protease. Heptameric PA can then bind the enzymatic toxin monomers, lethal factor (LF) and/or the edema factor (EF). In this form the toxin is internalized and at the low pH of endosomes the PA moiety creates a translocation pore in the membrane to mediate cytosolic delivery of LF/EF (14). If LF is truncated to disarm its destructive activity and genetically fused to T-cell epitopes, delivery into cells by PA induces CTL immunity (15-17) (Figure 3, path 2).

ER-associated membrane translocation
Several toxins that do not have any pore-forming ability, including the sialic acid-interacting PTx from Bordetella pertussis, and PEx from Pseudomonas aeruginosa, follow a retrograde route from the cell surface through endosomes and the Golgi apparatus, to the ER (18,19) (Figure 3, path 3). Interestingly, STx (from Shigella dysenteriae), by-passes the degrading environment of the late endocytic pathway (20) and traffics directly to the Golgi from the early endosome (Figure 3, path 3a). The presence of molecular chaperones and protein disulphide isomerase in the ER lumen to aid unfolding of the toxins and, in particular, the process of ER-associated degradation (ERAD) (21), probably all contribute to the favourability of this compartment for membrane translocation. ERAD eliminates misfolded proteins from the ER via the Sec61p translocon. This dislocation is coupled to their cytosolic proteasome-dependent degradation. While parasitising this pathway would obviously enable access to the cytosol, the toxin must, to some extent, escape the ensuing degradation during a natural infection. Strikingly, lysine residues, which direct a protein to the proteasome following ubiquitination, are under-represented in their catalytic A-subunits (22), a feature which may attenuate targeting to proteasomes.
However, the predominant coupling of the ERAD pathway to proteasomes is of potential interest in terms of antigen presentation since antigenic peptides fused to the catalytic A-subunits, or detoxified versions thereof, might be expected to be targeted to the endogenous MHC class I pathway via the proteasome and the TAP transporter. Indeed, it has been shown that antigenic peptides fused to the A-chain of STx can be presented in a class I-dependent manner at the cell surface (23). For PTxA-dependent antigen presentation it has been shown that it is blocked by brefeldin A (BFA), a reagent known to perturb the Golgi stack. This suggested the involvement of the Golgi apparatus in either a failure of anterograde peptide-loaded MHC class I transport to the plasma membrane or a requirement for the Golgi in retrograde transport of the toxin (25). Surprisingly however, peptide presentation in this instance was not prevented by proteasome inhibitors and also occurred in TAP-deficient cells, suggesting that processing did not occur in the cytosol and that ad***ional quality control or e***ing pathways may exist in post-Golgi compartments (26). It remains to be determined to what extent this observation holds true for the other ER-targeted toxins.
TAP- and proteasome-independent processing also seem to occur in the case of PEx, which is endocytosed following binding to the 2-macroglobulin receptor. However, in this case, antigen presentation via detoxified PExA was also BFA-insensitive (27,28), implying that transport through the Golgi was not required prior to peptide processing, even though the holotoxin normally reaches the ER via this compartment (29). This suggested that antigen processing and loading must occur earlier in the retrograde pathway, possibly in endosomes, involving MHC class I molecules that undergo recycling from the cell surface. Consistent with an endosomal site for PE-peptide processing, it has been shown that PEx can also internalise a MHC class II-specific antigen where processing occurs within specialised endosomes (30).

Unconventional transport pathways
Recent work with STx has suggested that the same toxin can have different transport itineraries in different cell types (e.g. HeLa cells and APC), thereby affecting the way antigen may reach the cytosol. However, subsequent proteasomal processing and presentation via the classical MHC class I pathway still occurred (31,32). In these experiments, the use of quantitative biochemical methods revealed that in human monocyte-derived DC, STxB does not appear to follow the retrograde pathway to the Golgi apparatus or the ER (33) as it does in other cell types (e.g. HeLa cells). Therefore, delivery to the cytosol in DC cannot involve the ERAD pathway. Whether the same is true for other types of DC remains to be studied. The differential transport of STxB in monocyte-derived human DC and HeLa cells could be correlated with a role of lipid microdomains ('rafts') in the endosome-to-Golgi trafficking of this lipid-binding toxin. STxB failed to associate with such microdomains in DC, potentially precluding transport to the Golgi and beyond, but it could associate with rafts in HeLa cells. Native STx is not toxic to human monocytes (34), from which DC are derived, although it is potently toxic towards HeLa cells. Together, these observations suggest that STx becomes proteolytically processed within the endosome/lysosome system of DC, and that STxB (plus any fused antigen) can then escape to the cytosol by an unknown mechanism. This would imply the existence of a membrane translocation mechanism for STxB (but not the holotoxin) at the level of endosomes or lysosomes in DC. At the moment, it is not clear whether this pathway is the same as the one operating on phagocytosed insoluble material (35) or endocytosed antigen-antibody complexes (36,37), which also enter the cytosol from endosomes.

Concay
Được Milou sửa vào 06:00 ngày 18/06/2003

Dendritic Cell Targeting
Dendritic cells have a central role in the induction of a primary immune response because of their ability to present externally derived antigens in a MHC class I and class II-dependent manner and because they possess the necessary costimulatory molecules (2,3). Targeting to DC is therefore desirable for any vector developed as a reagent for immunotherapy. ACTx and STxB bind to receptors that are expressed on DC (8,33), which may explain the efficiency of these toxins to induce T-cell immunity (9,10,32). Indeed, immune system cells that express antigen but lack costimulatory molecules are most often incapable of priming T cells and may actually induce tolerance (4). Thus, toxins that associate with ubiquitously expressed receptors are likely to be inefficient in activating T cells, although they may be of interest for the development of treatments for auto-immune diseases. Mature DC are also considered to be more potent than their immature precursors in boosting a specific immune response (38). Vectors with the ability to not only deliver exogenous antigen into the cytosol of DC, but also to induce their maturation were shown to be particularly efficient in eliciting therapeutic immunity against tumours. It may therefore be of significance that some toxins with proven efficacy in vivo, i.e. ACTx and STx, may intervene in DC maturation: ACTx induces DC maturation in vitro (8), and it has been demonstrated that STxB stimulates the production of TNF (39), a cytokine which acts as a maturation factor for DCs.

Connections Between MHC Class I and MHC Class II Presentation
Due to transport into the endosomal pathway, some toxins might be capable of introducing a combination of antigenic peptides into both the MHC class I- and the MHC class II-restricted pathways (30). This may contribute to better in vivo performance, since CD4+ T cells are necessary to elicit a long-lasting memory T-cell immunity (40) and have been shown to contribute to an efficient anti-tumour response (41). For PEx, class II-restricted antigen presentation has been demonstrated (30), and the fact that STxB enters the lysosomal pathway in DC (33) suggests that this vector may also mediate targeting to the class II pathway.

Practical Issues
In spite of the promise of the targeting mechanisms discussed here, there are some practical issues that will have to be addressed in developing therapeutic strategies. For example, there is the question of toxicity. In most of the cases described above, antigenic peptides were coupled to catalytic A-subunits disarmed by point mutations or deletions. However, total catalytic inactivation is rarely achieved, and even partially active toxins may present health risks that need to be thoroughly addressed before entering clinical evaluation. On the other hand, a mild toxic effect resulting in a localised nonspecific immune response, particularly where it might aid the maturation of DC, could be beneficial. Indeed, native cholera toxin (CTx) from Vibrio cholerae, has been shown to promote the maturation of DC, leading to the priming of an antibody response (42). An alternative approach has been to exploit the non-toxic B subunits as vectors in isolation of their catalytic A chains. This is exemplified by the successful use of STxB as an antigen carrier, whose intracellular transport characteristics are indistinguishable from that of the parent holotoxin (31,32).
Another consideration is the potential production of antitoxin antibodies that might neutralise the targeting and transport of the vector. Indeed, in the case of immunotoxins (anti-tumour antibody-toxin conjugates) the production of neutralising antibodies has been one of the major problems in the clinic. This may be less of a problem with the protein toxins alone, however, as they have low immunogenicity. Although this is thought to be linked to low numbers of lysine residues within the toxin A-subunits sequences [allowing them to escape ubiquitin-modification and processing by the proteasome (22)], this is probably not the only reason, since even for STxB which has a normal lysine content, most MHC alleles are not very immunogenic (43). Even in human subjects who have been exposed to STx, only a fraction produce antibodies (44). These data support the idea that some toxins have evolved the ability to evade the antibody response.

Perspectives
The ability of different toxins to target peptides derived from exogenous antigens into the MHC class I-restricted antigen presentation pathway has been the rational basis for their use as vectors in presenting exogenous antigens for the induction of T-cell immunity (5). Indeed, ACTx, ATx, and STxB have been shown to elicit a CTL response in mice when coupled to antigenic peptides or proteins (Table 1) (17,32,45). Protective anti-viral and anti-tumour immunity has also been observed after immunisation with ATx and ACTx (10,46). However, in some cases, e.g. PEx, exogenous peptides were introduced into the MHC class I pathway but failed to induce CTL in vivo (27,47). This observation suggests that in ad***ion to the delivery of antigen into the MHC class I pathway, other factors will need to be taken into consideration for the future use of these toxins as vectors for vaccine development. Some toxins (CTx, PTx) are already in use in vaccine protocols because of their effectiveness as adjuvants, such that when mixed with antigen they enhance the immunogenicity of that antigen. Although the adjuvant capacity of these toxins is not linked to their ability to deliver antigens (48,49), it is expected that toxins that combine these two properties will be most suitable for immunotherapy.
Until now, immunotherapy protocols in humans have been mostly based on live vectors such as viruses and bacteria. However, the risk that these vectors pose to immunosuppressed patients and to the environment limits their broad use in vaccine protocols. In ad***ion, their high intrinsic immunogenicity strongly decreases their efficiency after the first immunisation, necessitating the development of prime-boost protocols that use more than one type of virus (50,51). For these reasons, synthetic vectors such as toxins that are tailored for immunotherapy are eagerly awaited in the field of vaccine development.



Acknowledgments Go to: Choose Top of page Getting into the Cytosol... Dendritic Cell Targeting... Connections Between MHC C... Practical Issues Perspectives Acknowledgments << References
This work was supported by a research grant from the 'Association pour la Recherche sur le Cancer' (no. 9028) to E.T. and L.J., and a Wellcome Trust Programme Grant to L.M.R. and J.M.L. We thank Christophe Lamaze for reading the manuscript.

Concay

Dendritic Cell Targeting
Dendritic cells have a central role in the induction of a primary immune response because of their ability to present externally derived antigens in a MHC class I and class II-dependent manner and because they possess the necessary costimulatory molecules (2,3). Targeting to DC is therefore desirable for any vector developed as a reagent for immunotherapy. ACTx and STxB bind to receptors that are expressed on DC (8,33), which may explain the efficiency of these toxins to induce T-cell immunity (9,10,32). Indeed, immune system cells that express antigen but lack costimulatory molecules are most often incapable of priming T cells and may actually induce tolerance (4). Thus, toxins that associate with ubiquitously expressed receptors are likely to be inefficient in activating T cells, although they may be of interest for the development of treatments for auto-immune diseases. Mature DC are also considered to be more potent than their immature precursors in boosting a specific immune response (38). Vectors with the ability to not only deliver exogenous antigen into the cytosol of DC, but also to induce their maturation were shown to be particularly efficient in eliciting therapeutic immunity against tumours. It may therefore be of significance that some toxins with proven efficacy in vivo, i.e. ACTx and STx, may intervene in DC maturation: ACTx induces DC maturation in vitro (8), and it has been demonstrated that STxB stimulates the production of TNF (39), a cytokine which acts as a maturation factor for DCs.

Connections Between MHC Class I and MHC Class II Presentation
Due to transport into the endosomal pathway, some toxins might be capable of introducing a combination of antigenic peptides into both the MHC class I- and the MHC class II-restricted pathways (30). This may contribute to better in vivo performance, since CD4+ T cells are necessary to elicit a long-lasting memory T-cell immunity (40) and have been shown to contribute to an efficient anti-tumour response (41). For PEx, class II-restricted antigen presentation has been demonstrated (30), and the fact that STxB enters the lysosomal pathway in DC (33) suggests that this vector may also mediate targeting to the class II pathway.

Practical Issues
In spite of the promise of the targeting mechanisms discussed here, there are some practical issues that will have to be addressed in developing therapeutic strategies. For example, there is the question of toxicity. In most of the cases described above, antigenic peptides were coupled to catalytic A-subunits disarmed by point mutations or deletions. However, total catalytic inactivation is rarely achieved, and even partially active toxins may present health risks that need to be thoroughly addressed before entering clinical evaluation. On the other hand, a mild toxic effect resulting in a localised nonspecific immune response, particularly where it might aid the maturation of DC, could be beneficial. Indeed, native cholera toxin (CTx) from Vibrio cholerae, has been shown to promote the maturation of DC, leading to the priming of an antibody response (42). An alternative approach has been to exploit the non-toxic B subunits as vectors in isolation of their catalytic A chains. This is exemplified by the successful use of STxB as an antigen carrier, whose intracellular transport characteristics are indistinguishable from that of the parent holotoxin (31,32).
Another consideration is the potential production of antitoxin antibodies that might neutralise the targeting and transport of the vector. Indeed, in the case of immunotoxins (anti-tumour antibody-toxin conjugates) the production of neutralising antibodies has been one of the major problems in the clinic. This may be less of a problem with the protein toxins alone, however, as they have low immunogenicity. Although this is thought to be linked to low numbers of lysine residues within the toxin A-subunits sequences [allowing them to escape ubiquitin-modification and processing by the proteasome (22)], this is probably not the only reason, since even for STxB which has a normal lysine content, most MHC alleles are not very immunogenic (43). Even in human subjects who have been exposed to STx, only a fraction produce antibodies (44). These data support the idea that some toxins have evolved the ability to evade the antibody response.

Perspectives
The ability of different toxins to target peptides derived from exogenous antigens into the MHC class I-restricted antigen presentation pathway has been the rational basis for their use as vectors in presenting exogenous antigens for the induction of T-cell immunity (5). Indeed, ACTx, ATx, and STxB have been shown to elicit a CTL response in mice when coupled to antigenic peptides or proteins (Table 1) (17,32,45). Protective anti-viral and anti-tumour immunity has also been observed after immunisation with ATx and ACTx (10,46). However, in some cases, e.g. PEx, exogenous peptides were introduced into the MHC class I pathway but failed to induce CTL in vivo (27,47). This observation suggests that in ad***ion to the delivery of antigen into the MHC class I pathway, other factors will need to be taken into consideration for the future use of these toxins as vectors for vaccine development. Some toxins (CTx, PTx) are already in use in vaccine protocols because of their effectiveness as adjuvants, such that when mixed with antigen they enhance the immunogenicity of that antigen. Although the adjuvant capacity of these toxins is not linked to their ability to deliver antigens (48,49), it is expected that toxins that combine these two properties will be most suitable for immunotherapy.
Until now, immunotherapy protocols in humans have been mostly based on live vectors such as viruses and bacteria. However, the risk that these vectors pose to immunosuppressed patients and to the environment limits their broad use in vaccine protocols. In ad***ion, their high intrinsic immunogenicity strongly decreases their efficiency after the first immunisation, necessitating the development of prime-boost protocols that use more than one type of virus (50,51). For these reasons, synthetic vectors such as toxins that are tailored for immunotherapy are eagerly awaited in the field of vaccine development.



Acknowledgments Go to: Choose Top of page Getting into the Cytosol... Dendritic Cell Targeting... Connections Between MHC C... Practical Issues Perspectives Acknowledgments << References
This work was supported by a research grant from the 'Association pour la Recherche sur le Cancer' (no. 9028) to E.T. and L.J., and a Wellcome Trust Programme Grant to L.M.R. and J.M.L. We thank Christophe Lamaze for reading the manuscript.

Concay