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Antigen presentation in virus infection
Antigen presentation in virus infection Nicholas Murray and Andrew McMichael Institute of M o l e c u l a r M e d i c i n e , John Radcliffe Hospital, O x f o r d , UK Important recent advances have been made in our understanding of antigen processing of cytoplasmic antigens and presentation by class I molecules of the MHC. Peptide transporter-like molecules encoded within the MHC have been characterized and have, by transfection, corrected some of the presentation-mutant cell lines. The nature of peptide-MHC class I interactions has been clarified by further resolution of the HLA A2 and B27 crystals and elution of peptides. The differences between antigenicity and immunogenicity for viral antigens have been highlighted by studies in transgenic animals. Current Opinion in Immunology 1992, 4:401-407
Introduction This review addresses two issues: antigen processing in non-specialist cells that are infected with a virus and the nature of viral antigen processing in vivo. Our comments are confined to the antigen processing system that involves the class I molecules of the MHC, not because class II MHC antigen processing is unimportant in virus infections, but because in the latter system virus proteins are probably processed in the same way as other externally derived antigens [1]. Class I restricted cytotoxic T lymphocytes (CTLs) are particularly important in virus infections, either eliminating virus or controlling persisting infection. A considerable amount of detail is n o w known about processing of cytoplasmic virus antigens to peptide epitopes that bind to class I MHC molecules. Rather less is known about h o w these virus-specific CTL responses are initiated in vivo. a critical question is whether specialized antigen-presenting cells (APCs) are required.
Presentation of virus antigen in infected cells Zinkernagel and Doherty [2] showed that virus-specific CTLs recognized virus antigens in association with class I MHC molecules. The p h e n o m e n o n was explained when it was shown that CTLs recognized peptides derived from virus proteins [3,4] that were usually internal and not displayed intact on the cell surface. A critical experiment was the demonstration that cells transfected with fragments of influenza nucleoprotein (NP) were recognized by NPspecific CTLs [5]; the exact epitopes were dependent on the MHC type involved and the fragments recognized shared no signal sequence that could be involved in translocation across a membrane. Further illumination
came when the mutant cell-line RMA-S was shown to be capable of presenting externally added peptides to CTLs but not internally derived antigen from infecting virus [6]. A similar defect was shown in the cell-line 721.174 and its derivative T2 [7]-; these cells had a deletion in their class II MHC region, implying that there are genes in this region involved in processing of virus antigens. Further support came from the finding of a similar defect in the mutant cell line 721.134 [8]. All o f these mutant cell-lines also failed to express most class I molecules in a stable form at the cell-surface, although abundant free heavy chain and ]3-2 microglobulin was present in the endoplasmic reticulum (ER). Because most class I MHC molecules depend on b o u n d peptides for their stability and associate in the ER, it was argued that these mutant cell-lines were defective in generation or transport o f peptides into the ER [9]. Mapping of the MHC class II regions in humans and rodents soon revealed that there were genes coding for proteins o f the ATP-binding cassette family, already known to be involved in intracellular transport processes [8,1(>12]. The two genes, known under various names (Table 1), were shown to code for two chains o f a heterodimer with sequence characteristics o f a transporter protein [13]. Further, the defect in the mutant cell-line 721.134 was reversed by transfection of one of these, the P~fl (TAP 1) gene [13,14~ Another human mutant cell-line 36.1, with a similar phenotype, was corrected by transection of the TAP-2 gene as was the mouse cell-line RMA-S. So far, the 721.174 cell-line has not been corrected, but this may lack other important genes in this region. Monaco et at [15] showed in 1984 that there were genes in the MHC class II region that coded for low molecular weight proteins (LMPs); these showed some limited polymorphisms [16o]. The
Abbreviations APC antigen-presenting cell; CIM--class I modifying; CT~cytotoxic T lymphocyte; DC~endritic cell; ER-~endoplasmic reticulum; GP--glycoprotein; ICAM--intercellular adhesion molecule; IFN interferon; IL--interleukin; LCMV--lymphocytic choriomeningitis virus; LFA--lymphocyte function associated antigen; L M ~ l o w molecular weight protein; MHC--major histocompatibility complex; N~nucleoprotein. (~ Current Biology Ltd ISSN 0952-7915
401
402
Immunity to infection LMPs have subsequently been shown to be components of multicatalytic protease complexes, proteasomes, and two components, from a total of around twenty are encoded in both the human and mouse MHC class II regions [17"-19"]. These might be the proteases that digest viral proteins to generate antigenic peptides; however, this has yet to be proven.
Table 1. Antigen-processing genes in the MHC. Revised name
TAP-'I
TAP-2
Original name
Species
Reference
Ring-4 Psf-1 Ham-1 Mtp-1
Human Human Mouse
[12] [8] [11]
Rat
[10]
Ring-'l 1 Psf-2 Ham-2 Mtp-2
Human Human Mouse Rat
[12] ]8] [11] [10]
These findings have led to the view that antigen processing for the class I MHC pathway is almost entirely controlled by MHC genes. However, it must be stressed that this pathway is still hypothetical. It is not certain that the 'transporters' transport; Levy et a t [20,.] have found, using microsome preparations in vitro, that peptide transport was not ATP-dependent, although class I assembly was. If this microsome system is representative of the ER, this simple view may not be quite correct. The transporters, and to a lesser extent the proteasomes, show genetic polymorphisms [15]. In the rat this has been associated with differences in antigen processing, such that identical class I molecules in cells from two congenic rat strains, which differ only in their class II MHC regions, present different peptide epitopes and behave as alloantigens [21,22-]. The class I molecules differ in their rates of assembly, glycosylation and transport to the cell surface. More revealing was the high performance liquid chromatography hydrophobicity profile of the peptides eluted from the class I molecules from the two rat strains; the molecules that assembled more rapidly contained more hydrophilic peptides. The MHC class II regions of these rat strains showed allelic differences in the TAP-2 (MtlY2) genes. Transfection of the M~t>2 transporter gene of the dominant (fast-assembling) phenotype into a cell line that showed the recessive phenotype (slow-assembling) changed the bound peptides from a hydrophobic to hydrophilic pattern [23..]. A somewhat similar antigen processing polymorphism has been described in a human family, but this has not yet been firmly assigned to a MHC-linked gene, raising the possibility that non-MHC genes could also be involved in these processes [24.]. Such polymorphism adds a further layer of functional polymorphism to the class I MHC antigen-presentation system. Class I molecules present antigenic peptides to the Tcell receptors. The peptides bind in a groove on the
membrane-distal surface of the class I molecule, as revealed by the crystal structure, now resolved at 2.6A_ [25"]. It is clear that the fine structure of this groove determines the nature of the peptide bound. The ends of the groove, which form two pockets, A and F, are conserved and bind the amino and carboxyl terminals of the peptides respectively. Evidence for this comes from both crystallographic [26 o'] and functional sources [27.]. Four additional pockets in the middle region of the cleft, B,C,D and E, are polymorphic and differ considerably between different class I MHC molecules [28]. For each class I molecule, these pockets determine the shared (anchor) residues [29 "~ of the epitope peptides, because they can accommodate their side chains. For instance, HLA B27 allows only an arginine side chain in its B pocket [26".], whereas the HLA A2 B pocket can accomodate leucine or isoleucine [29-o1. In the latter case, this requirement has been confirmed by analysis of a very large number of eluted peptides by mass spectroscopy [30"]. Close to the F pocket is a critical amino acid at position 116 that plays an important role in determining the nature of the side chain of the carboxyl amino acid [27"]; for instance, residue 116 is aspartate in B27 and the commonest carboxyl terminal amino acid is arginine or lysine [31"], residue 116 is tyrosine in HLA A2 and the last residue of the peptide is valine or leucine [29~176Thus, it becomes clear how the structure of the groove selects the peptide epitope and it should soon be possible to predict peptides that bind at relatively high affinity with some accuracy. The peptide is an integral part of the class I molecule. The mutant cell lines that lack functional transporters do not fill their class I molecules and they may be retained in the ER or appear on the cell surface in an unstable, and probably empty, form [32]. Townsend's group [9,33~ -] has shown that the association between a newly synthesized class I molecule and peptide occurs in the ER and is a critical step in the maturation of the class I molecule. The peptides that bind to class I molecules have been shown to be remarkably consistent in their length, nine +/-one, amino acids [36,37]. It has been found that the epitope peptides tend to bind to class I molecules with relatively high affinity [37] and nonamer peptides bind with greater avidity than longer or shorter peptides [34",35"]. However, not all peptides (made synthetically) that bind with high affinity under in vitro conditions are necessarily epitopes [35"]. Naturally processed high affinity binding peptides may not be epitopes because they are cross-reactive with self peptides (and therefore tolerize reactive T cells). Also, not all theoretical nonamers may be generated by the antigen-processing system. In support of the latter, various groups have demonstrated that flanking sequences in a protein can affect epitope generation [38%39",40]. Another likely factor is competition between self and virus peptides in the ER or for the transporters. Analysis of peptides, eluted from class I molecules, by mass spectroscopy suggests that over 200 different peptides may be present in HLA A2 purified from a single cell line [30"]. The threshold for antigen recognition by CTL has been estimated to be 200 molecules per cell [41]; therefore, to be antigenic peptides have to bind to > 0.2% of surface class I molecules; affinity and concentration are likely to be im-
Antigen presentation in virus infection Murray and McMichael portant in this competitive process. Finally, as discussed below there may be differences between antigenicity and immunogenicity dependent on cell type or state (e.g., activation) and this will also determine whether a particular peptide sequence stimulates a CTL response.
over several months. This difference could be accounted for by dosage effects if the transgene peptides mix with those derived from cellular proteins and compete for transport into the ER and subsequent binding. However, when Oldstone et al. injected CTL clones specific for the relevant class I molecules plus virus peptide into the transgenic animals they showed that these CTLs were localized to the pancreatic islets in the transgenics (and not in controls), indicating that in the absence of virus infection or other inflammatory stimulus at least some of the cells in the islets were expressing class I complexes containing transgene peptide. It is worth making the piont here that in the presence of inflammation cytokines are released, particularly IFN-7, that induce the upregulation of MHC class I expression by cells, thereby increasing the number and variety of peptide-class I complexes available to T cells; concievably this might occasionally lead to presentation of self peptides not normally seen, resulting in autoimmune reactions.
Immunogenicity in M H C class I restricted T-cell responses Assembly of a trimolecular complex of heavy chain, ~2-microglobulin and peptide and its delivery to the cell surface for interaction with the T-cell receptor creates a structure which is antigenic, but not necessarily immunogenic. That is, the presence of antigenic cellsurface class-I peptide complexes does not guarantee T-cell recognition and activation i n v i v a Recent experiments with transgenic mice have made this point clearly [42.-44..].
Experiments with mice transgenic for both a virus protein under a tissue specific promoter and for a T-cell receptor specific for host MHC class I together with a peptide from that protein, have demonstrated that there can be circulating T cells with high affinity for the anti genic class I complex without tissue damage [43"~ In the allo-reactive systems, where expression is relatively high, peripheral anergy seems to operate, implying some stimulus to the T cells, albeit negative. In the islet cell experiments, the T cells appear to be 'ignorant', demonstrating that the presence of an antigenic complex capable of a biologically relevant interaction with circulating T cells is on its own not sufl~cient for T-cell activation.
Ohashi e t aL [42 "~ created mice transgenic for lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) under the rat insulin promoter and showed that the protein was expressed only in pancreatic islet cells. These H-2b mice were phenotypicaUy normal and did not develop diabetes, even when crossed with mice expressing a transgenic T-cell receptor known to recognize a peptide from LCMV GP in the context of H-2D b. However, when the mice were infected with LCMV they developed fatal diabetes rapidly, over 9-11 days in the single transgenics, and 4-5 days in the double transgenics. Oldstone e t al. [43 ~ made very similar transgenic animals using LCMV GP or LCMV NP, but in their system expression may have been at a lower level, in that immunohistochemical staining of the pancreas for the virus proteins was negative in contrast to the transgenic mice generated by Ohahsi e t aL [42~ Nevertheless, northern blots revealed transgene mRNA which was confined to the pancreas. The majority of these mice (94%) did not progress to diabetes, unless infected with LCMV, when the time course of disease onset was very different from that seen by Ohashi e t al. - - the disease appeared only
Cytosol Virus protein
Endoplasmic reticulum
Peptides t
")~
Golgi
What are the requirements for presentation of class I MHC and virus antigen in immunogenic form? This issue has rarely been addressed but we can extrapolate from studies involving allogeneic responses. The most potent of the so called 'professional APCs' is the dendritic cell (DC); cell fractionation experiments have shown that for allo responses the ability to induce CTL responses lies almost exclusively in this population [45]. It has been possible to prime CTL responses to influenza virus i n
Infected cell membrane
CTL membrane
] ,
Proteolysis
Transport /
MHC I peptide
Fig. 1. The diagram shows the pathway within the cell by which virus antigens are processed and peptides presented at the cell surface by class I MHC molecules. MHC encoded genes may contribute to the proteolysis (proteasome complex) and peptide transport (TAP-1 and TAP-2). Viral peptides and unfolded HLA class I heavy and light ([32-microglobulin) chains associate in the endoplasmic reticulum. Accessory molecules and their ligands, including CD8, lymphocyte function associated antigen (LFA)-I and CD2, are necessary for the interaction with cytotoxic T lymphocytes (CTLs) leading to lysis. Additional accessory molecules, such as CD28 interacting with B7, are probably necessary to activate CTL precursors and to initiate primary responses.
I•A•ccessory
//
activators 1
403
404
Immunityto infection vitro by exposing T cells to infected DCs [46]; similar in vitro primary CTL responses have also been made to human immunodeficiency virus [47"]. In vitro priming
has been achieved by pulsing DCs with epitope peptides [46,47"]; however, similarly pulsed macrophages or spleen cells did not prime CTL responses to influenza virus [46]. Culture of mouse spleen cells at high density with peptide initiated anti-influenza responses [48], but the cell types involved were not studied. Fewer experiments have been carried out in vivo, Kast et al. [49] have used virus-infected DCs to prime CTLs in non-responsive mice. There have been many attempts to prime for CTLs with non-infectious vaccines with varying success, but the cells involved have not been analyzed and detailed discussion of vaccines is beyond the scope of this review. There could be several reasons for the special effectiveness of DCs in CTL priming. The high level of MHC class II expression may not be relevant because virus-specific CTLs can be induced in mice depleted of CD4 + T cells [50]. Boog et al. [51] showed that DCs express MHC class I with a much lower level of sialation (i.e. glycosylation with negatively charged sialic acid residues) than other cells, and that treatment of monocytes with neuraminidase conferred the capacity to induce class I specific allo responses in vitro. Decreased glycosylation reduces surface negative charge and this may facilitate the interaction between T cells and their targets. DCs also have very high levels of adhesion molecules [52], which again may increase the affinity of interactions between these APCs and T cells compared with normal cells. Notwithstanding this potentially increased affinity, evidence from MHC class II based systems strongly suggests that the interactions between APCs and responder T cells are in fact qualitatively different from those with other cells [53-56]. Engagement of the CD28 surface receptor (present on around 85% of CD4 + cells and 50% of CD8 + cells) results in specific intracellular events that are different from, but complementary to, those due to signalling from the T-cell receptor. Cytokine mRNA is specifically stabilized, and these effects are not blocked by cyclosporin (whereas intracellular events arising from CD3 complex activation are) [53-56]. The ligand for CD28 has been identified as B7 [57",58], a molecule found on activated B cells; its distribution among other APCs is not clear. Transfection of B7 into a B7-negative Burkitts lymphoma cell line, which is incapable of inducing class I specific allo responses, reconstituted the ability to stimulate alloantigen-induced proliferation [59"]. Furthermore, the presence of CD28 has been shown to be sufficient to prevent induction of anergy in T-cell clones [60"]. B ceils have not been investigated for their ability to prime virus-specific CTL responses, but might take up virus following specific recognition by surface antibody, even when they do not express the normal ligand for viral entry. It is interesting to note that the molecule CTLA-4, which was defined by subtraction cloning from cDNA from a mouse CTL line, has strong homology with CD28, and in fact appears to bind with a higher affinity to B7 than does CD28 [61.-]. Other T-cell molecules that may also be involved in cell--cell signalling in antigen presentation include CD5 [62,63] (the ligand for which, CD72,
seems to be confined to B cells [64..]); CD2 (the ligand for which is lymphocyte function associated antigen (LFA)-3 [65]); and the ligand for heat stable antigen (recently defined as a costimulatory molecule in CD4 + Tcell responses [66"]). Molecules such as CD8 (which binds the ~z 3 domain of class I [67]) and LFA-1 (interacts with intercellular adhesion molecule (ICAM)-I and -2 [65]), whose ligands are not confined to specialized APCs are also capable of delivering intracellular signals, but in view of the ubiquity of ICAM-1 these sig nals cannot be sufficient to confer immunogenicity upon peptide-class I complex. tafferty in 1983 [68] argued that transplantation rejection was initiated by passenger APCs of a haemopoietic lineage, and that without effective antigen presentation a mismatched transplant remained unseen by an immune system perfectly capable of rejecting it. Similarly, workers on a number of tumor models have shown that a tumor may be manipulated into acting as its own APC, in general by transfection of the gene for one of an increasing number of cytokines, including interleukin (IL)-2 [69"*,70], IL4 [71], IL-7 [72], IFN-y [73,74], tumor necrosis factor-a [75,76], and granulocyte-macrophage colony stimulating factor [77]; despite this, tumors are in general 'potentially antigenic' rather than immunogenic. As an example, one might argue that viral warts, where multiple warts will often disappear simultaneously, are antigenic but unseen by the immune system until some triggering event (viral uptake by a Langerhans cell?) occurs.
Conclusions There is clearly a processing pathway for presentation of peptide breakdown products of vires (and cellular) proteins that is common to all cells that express class I MHC; although the processing machinery is likely to be 'idling' at very low turnover levels in uninfected, non-immune system cells in the absence of IFNs or other cytokines. However, only certain cells, specialized APCs, are likely to be able to activate T cells to induce effector function, although once this activation step is taken the resulting CTL is capable of interacting with any cell bearing the appropriate class I molecule in combination with peptide. This provides a mechanism by which some self proteins, or rather their derivative peptides, can become antigenic by cross-reacting with an immunogenic antigen, such as a virus. It could explain why potentially antigenic tumour cells escape T-cell surveillance. On the other hand, it is less clear how viruses which do not infect specialist APCs initiate CTL responses; all virus infections so far examined stimulate CTL responses. Possibly low levels of infection are sufficient, particularly in the environment of a lymph node. Alternatively, perhaps DCs or B cells can process non-infectious antigen into the class I MHC antigen processing pathway.
Acknowledgements We are grateful to the Imperial Cancer Research Fund (N.M) and Medical Research Council (A.M.) for support.
A n t i g e n p r e s e n t a t i o n in v i r u s i n f e c t i o n M u r r a y a n d M c M i c h a e l
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: 9 of special interest 99 of outstanding interest 1.
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18.
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33. ..
ELLIOTrT, CERUNDOLOV, ELVINJ, TOWNSENDA: Peptide Induced Conformational Change of a Class I Heavy Chain. Nature 1991, 351:402-405. Demonstration that the peptide induces folding of the free heavy chain and stabilizes preformed heavy chain-~-2 microglobulin complexes; only short peptides alter conformation of the heavy chain. 34. ..
CERUNDOIDV, ELLIOTrT, BASTINJ, RAMMENSEEH-G, TOWNSEND Aa The Binding Affinity and Dissociation Rates of Peptides for Class I Major Histocompatibility Complex Molecules. Eur J Immunol 1991, 21:2069-2076. A comparison of binding of peptides of different lengths to the H-2Db molecule; shows that nonamer peptides bind with higher atl~nity than longer or shorter peptides. ELVlNJ, CERUNDOLO V, ELLIOT T, TOWNSEND A: A Quantitative Assay of Peptide-dependent Class I Assembly. Eur J Immunol 1991, 21:202~2032. A simple assay for screening peptides for binding to class I molecules.
OHASHI PS, OEHEN S, BUERKI K, PIRCHER H, OHASHI CT, ODERMATI ~ B, MA1JSSEN B, ZINKERNAGELRM, HENGARTNER H: Ablation of 'Tolerance' and Induction of Diabetes by Virus Infection in Viral Antigen Transgenic Mice. Cell 1991. 65:305-317. Describes the construction of mice that express LCMV GP, under the control of a rat insulin promoter, in the pancreas. When crossed with mice transgenic for a T-cell specific for LCMV GP the resultant mice show peripheral tolerance and do not develop diabetes; however, infection with LCMV results in tolerance being overcome and the development of diabetes. 42. oo
43. 9.
OLDSTONEMB, NERENBERGM, SOUTHERNP, PRICEJ, LEWICKIH: Virus Infection Triggers Insulin-dependent Diabetes Mellitus in a Transgenic Model: Role of Anti-self (Virus) Immune Response. Cell 1991, 65:319-331. Similar experiments to those of Ohashi et al. [42"o], but differences in the pattem of tolerance breakdown are described. Infection with LCMV results in the appearance of diabetes over several months, rather than days as in [42o"]. 44.
SCHONRICHG, KALINKE U, MOMBURG F, MALISSENM, SCHMI'IT VA, MALISSENB, HAMMERL1NGGJ, ARNOLDB: Down-regulation of T Cell Receptors on Self-reactive T Cells as a Novel Mechanism for Extrathymic Tolerance Induction. Cell 1991, 65:293-304. Examines the nature of tolerance in a mouse transgenic for both Kb (expressed in the CNS) and for a Kb-specific T-cell receptor and shows that T cell receptor down-regulation is one mechanism of tolerance. oo
45.
YOUNGJW, STEINMANRM: Dendritic Cells Stimulate Primary Human Cytolytic Lymphocyte Responses in the Absence of CD4 + Helper T Cells. J Exp Med 1990, 171:1315-1332.
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MACATONIASE, TAYLORPM, KNIGHT SC, ASKONASBA: Primary Stimulation by Dendritic Ceils Induces Anti-viral Proliferative and Cytotoxic T Ceil Responses in Vitro. J Exp Med 1989, 169:1255-1264.
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VAN BLEEKGM, NATHENSONSM: Isolation of an Endogenously Processed Immunodominant Viral Peptide from the Class I H2Kb Molecule. Nature 1990, 348:213 216.
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ROTZSCHKEO, FALK K, DERES K, SCHILD H, RAMMENSEE HG: Isolation and Analysis of Naturally Processed Viral Peptides as Recognized by Cytotoxic T Cells. Nature 1990, 348: 252-254.
38. .
HAHNYS, BRACtALEV, BRACIALEV: Presentation of Viral Antigen to Class I Major Histocompatibility Complex-restricted Cytotoxic T Lymphocyte. Recognition of an Immunodominant Influenza Haemagglutinin Site by Cytotoxic T Lymphocyte is Independent of the Position of the Site in the Haemagglutinin Translation Product. J Exp Med 1991, 174:733-736. Using reconbinant DNA technology, a segment of the gene encoding an H-2Kd-restricted epitope was inserted into the gene encoding influenza hemagglutinin in six different positions and expressed by recombinant vaccinia vires. All were presented to CTLs as well as the wild-type epi tope, implying that flanking sequences were not important. 39. o,
DELVAL M, SCHtaCHT H-J, RUPPERT T, REDDEHASE mJ, KOSZINOWSKIUH: Efficient Processing of an Antigenic Sequence for Presentation by MHC Class I Molecule D e -
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MACATONIASE, PATTERSONS, KNIGHTSC: Primary Proliferative and Cytotoxic T-ceil Responses to HIV Induced in Vitro by Human Dendritic Cells. Immunology 1991, 74:399-406. A demonstration of the ability of DCs to initiate vires-specific CTL responses in vitro. 48.
CARBONEF, MOORE M, SHEILJ, BEVAN M: Induction of Cytolytic T Lymphocytes by Primary In Vitro Stimulation with Peptides. J Exp Med 1988, 167:1767-1780.
49.
KAST WM, BOOG CJP, ROEP BO, VOORDOUWAC, MELIEF CJM: Failure or Success in the Restoration of Virus Specific Cytotoxic T Lymphocyte Response Defects by Dendritic Ceils. J lmmunol 1988, 140:3186-3193.
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LIGHTMAN S, COBBOLD S, WALl)MANN H, ASKONAS BA: Do L3T4 + T Cells Act as Effector Cells in Protection Against Influenza Virus Infection. Immunology 1987, 62:139-144.
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BOOGCJ, NEEFJESJJ, BOES J, PLOEGH HJ, MEtaEF CJ: Specific Immune Responses Restored by Alteration in Carbohydrate Chains of Surface Molecules on Antigen-presenting Cells. Eur J Immunol 1989, 19:53~542.
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FREUDENTHALPS, STEINMAN RM: The Distinct Surface of Human Blood Dendritic Cells, as Observed after an Improved Isolation Method. Proc Natl Acad Sci USA, 1990, 87:7698-7702.
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LINDSTEINT, JUNE CH, LEDBETI'ERJA, STELLA G, THOMPSON CB: Regulation of Lymphokine Messenger RNA Stability by a Surface-mediated T Cell Activation Pathway. Science 1989, 244:339-343.
54.
THOMPSONCB, LINDSTENT, LEDBETFERJA, KUNKELSL, YOUNG HA, EMERSONSG, LEIDENJM, JUNE CJ: CD28 Activation Pathway Regulates the Production of Multiple T-cell-derived Lymphokines/Cytokines. Proc Natl Acad Sci USA, 1989, 86:1333-1337.
55.
56.
JUNECH, LEDBETI'ERJA, LINDSTENT, THOMPSON CB: Evidence for the Involvement of Three Distinct Signals in the Induction of IL-2 Gene Expression in Human T LymphocTtes. J Immunol 1989, 143:153-161. JUNE CH, LEDBETTERJA+ LINSLEYPS, THOMPSON CB: Role of the CD28 Receptor in T-ceU Activation. Immunol Today, 1990, 11:211-216.
57.
LINSLEYPS, BRADY W, GROSMA1RE L, ARUEFO A, DAMLE NK, LEDBETI'ERJA: Binding of the B Cell Activation Antigen B7 to CD28 Costimulates T Cell Proliferation and Interleukin 2 mRNA Accumulation. J Exp Med 1991, 173:721-730. Shows that B7 delivers a signal to the T cells which has additional, complementary effects on signals transduced through the T-cell receptor complex. 9 o
58.
LINSLEYPS, CLARK EA, LEDBETFER JA: T-cell Antigen CD28 Mediates Adhesion with B Cells by Interacting with Activation Antigen B7/BB-1. Proc Natl Acad Sci U S a 1990, 87:5031-5035.
59. 9.
AZUMAM, CAYABYABM, BUCK D, PmLLIPSJH, LANIERLL: CD28 interaction with B7 Costimulates Primary Allogeneic Proliferative Responses and Cytotoxicity Mediated by Small, Resting T Lymphocytes. J Exp Med, 1992, 175:353-360. Demonstrates the importance of costimulatory function in T-cell activation by reconstitution of the ability of a B7- Burkitts lymphoma cell line to simulate alloantigen-induced proliferation after transfection of B7. HARDINGFA, MCARTHUR JG, GROSS JA, RAULET DH: CD28mediated Signalling Co-stimulates Murine T Cells and Prevents Induction of Energy in T-cell Clones. Nature 1992, 356:607-609. Demonstration that the presence or absence of CD28 may be significant in the interactions of T cells with cells bearing peptide-class I complexes. The presence of CD28 prevents the induction of anergy in T-cell clones
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FEARONER, PARDOLL DM, ITAYA T, GOLUMBEK P, LEVITSKY HI, S1MONS JW, KARAStJYAMA H, VOGELSTEIN B, FROST P: Interleukin-2 production by Tumor Cells Bypasses T Helper Function in the Generation of an Antitumor Response. Cell 1990, 60:397-403. Demonstrates that IL-2 gene transfection may confer the ability to present antigen on the surface of non-APes. 70.
GANSBACHERB, ZIER K, DANIELS B, CRONIN K, BANNERJI R, GILBOA E: Interleukin 2 Gene Transfer into Tumor Cells Abrogates Tumorigenicity and Induces Protective Immunity. J Exp Med 1990, 172:1217-1224.
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WATANABE Y, KURIBAYASHI K, MIYATAKE S, NISHIHARA N, NAKAYAMAE, TANIYAMAT, SAKATAT: Exogenous Expression of Mouse Interferon Gamma eDNA in Mouse Neuroblastoma C1300 Cells Results in Reduced Tumorigenicity by Augmented Anti-tumor Immunity. Proc Natl Acad Sci U S A 1989, 86:9456-9460.
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ASHERAL, MULE JJ, KASID A, RESTIFO NP, SALO NJ, REICHERT CM, JAFFE G, FENDLYB, KRIEGLERM, ROSENBERGSA: Murine Tumor cells Transduced with the Gene for Tumor Necrosis Factor-alpha. Evidence for Paracrine Immune Effects of Tumor Necrosis Factor against Tumors. J Immunol 1991, 146:3227-3234.
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BLANKENSTEINT, Q1N ZH, UBERLAK, MULLERW, ROSENH, VOLK HD, D1AMANTSTEINT: Tumor Suppression after T u m o r Celltargeted Tumor Necrosis Factor Alpha Gene Transfer. J Exp Med 1991, 173:1047-1052.
77.
COLOMBO MP, FERRARI G, STOPPACCIARO A, PARENZA M, RODOLEO M, MAVUaO F, PAP,MtANI G: Granulocyte Colonystimulating Factor Gene Transfer Suppresses Tumorigenicity of a Murine Adenocarcinoma In Vivo. J Exp Med 1991, 173:889-897.
78.
GLYNNER, POW1S SH, BECK S, KELLYA, KERR LA: A Proteasome Related Gene b e t w e e n the Two ABC Transporter loci in the Class II Region of the Human MHC. Nature 1991, 353:357--360.
60. 9
LINSLEYPS, BRADY W, URNES M, GROSMAIRE IS, DAMLE NK, LEDBETTERJA: CTLA-4 is a Second Receptor for the B Cell Activation Antigen B7. J Exp Med 1991, 174:561-569. Is CTLA-4 a CTL-specific ligand for the B7 activation molecule?
61. +o
62.
63.
64. 9.
VANDENBERGHEP, CEUPPENS JL: Immobilized anti-CD5 Together with Prolonged Activation of Protein Kinase C Induce Interleukin 2-dependent T Cell Growth: Evidence for Signal Transduction through CD5. E u r J Immunol, 1991 21:251-259. SPERTINIF, STOHLW, RAMESHN, MOODYC, GEHARS: Induction of Human T Cell Proliferation by a Monoclonal Antibody to CD5. J Immunol 1991, 146:4~52.
VANDEVELDEH, VANHOEGEN I, LUO W, PARNESJR, THIELEMANS K: The B-cell Surface Protein CD72/Lyb-2 is the Ligand for CD5 [see comments]. Nature 1991, 351:662q565. This paper presents evidence that the CD5 molecule is likely to be relevant to interactions between lymphocytes. Signalling through this receptor is complementary to signalling through the CD3-T-cell receptor complex. The ligand CD27 has not been identified on cells other than B lymphocytes.
SPRINGERTA: Adhesion Receptors of the Immune System. Nature 1990, 346:425-434.
N Murray and A McMichael, Molecular Immunology Group, Insitute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK.
407
Introduction This review addresses two issues: antigen processing in non-specialist cells that are infected with a virus and the nature of viral antigen processing in vivo. Our comments are confined to the antigen processing system that involves the class I molecules of the MHC, not because class II MHC antigen processing is unimportant in virus infections, but because in the latter system virus proteins are probably processed in the same way as other externally derived antigens [1]. Class I restricted cytotoxic T lymphocytes (CTLs) are particularly important in virus infections, either eliminating virus or controlling persisting infection. A considerable amount of detail is n o w known about processing of cytoplasmic virus antigens to peptide epitopes that bind to class I MHC molecules. Rather less is known about h o w these virus-specific CTL responses are initiated in vivo. a critical question is whether specialized antigen-presenting cells (APCs) are required.
Presentation of virus antigen in infected cells Zinkernagel and Doherty [2] showed that virus-specific CTLs recognized virus antigens in association with class I MHC molecules. The p h e n o m e n o n was explained when it was shown that CTLs recognized peptides derived from virus proteins [3,4] that were usually internal and not displayed intact on the cell surface. A critical experiment was the demonstration that cells transfected with fragments of influenza nucleoprotein (NP) were recognized by NPspecific CTLs [5]; the exact epitopes were dependent on the MHC type involved and the fragments recognized shared no signal sequence that could be involved in translocation across a membrane. Further illumination
came when the mutant cell-line RMA-S was shown to be capable of presenting externally added peptides to CTLs but not internally derived antigen from infecting virus [6]. A similar defect was shown in the cell-line 721.174 and its derivative T2 [7]-; these cells had a deletion in their class II MHC region, implying that there are genes in this region involved in processing of virus antigens. Further support came from the finding of a similar defect in the mutant cell line 721.134 [8]. All o f these mutant cell-lines also failed to express most class I molecules in a stable form at the cell-surface, although abundant free heavy chain and ]3-2 microglobulin was present in the endoplasmic reticulum (ER). Because most class I MHC molecules depend on b o u n d peptides for their stability and associate in the ER, it was argued that these mutant cell-lines were defective in generation or transport o f peptides into the ER [9]. Mapping of the MHC class II regions in humans and rodents soon revealed that there were genes coding for proteins o f the ATP-binding cassette family, already known to be involved in intracellular transport processes [8,1(>12]. The two genes, known under various names (Table 1), were shown to code for two chains o f a heterodimer with sequence characteristics o f a transporter protein [13]. Further, the defect in the mutant cell-line 721.134 was reversed by transfection of one of these, the P~fl (TAP 1) gene [13,14~ Another human mutant cell-line 36.1, with a similar phenotype, was corrected by transection of the TAP-2 gene as was the mouse cell-line RMA-S. So far, the 721.174 cell-line has not been corrected, but this may lack other important genes in this region. Monaco et at [15] showed in 1984 that there were genes in the MHC class II region that coded for low molecular weight proteins (LMPs); these showed some limited polymorphisms [16o]. The
Abbreviations APC antigen-presenting cell; CIM--class I modifying; CT~cytotoxic T lymphocyte; DC~endritic cell; ER-~endoplasmic reticulum; GP--glycoprotein; ICAM--intercellular adhesion molecule; IFN interferon; IL--interleukin; LCMV--lymphocytic choriomeningitis virus; LFA--lymphocyte function associated antigen; L M ~ l o w molecular weight protein; MHC--major histocompatibility complex; N~nucleoprotein. (~ Current Biology Ltd ISSN 0952-7915
401
402
Immunity to infection LMPs have subsequently been shown to be components of multicatalytic protease complexes, proteasomes, and two components, from a total of around twenty are encoded in both the human and mouse MHC class II regions [17"-19"]. These might be the proteases that digest viral proteins to generate antigenic peptides; however, this has yet to be proven.
Table 1. Antigen-processing genes in the MHC. Revised name
TAP-'I
TAP-2
Original name
Species
Reference
Ring-4 Psf-1 Ham-1 Mtp-1
Human Human Mouse
[12] [8] [11]
Rat
[10]
Ring-'l 1 Psf-2 Ham-2 Mtp-2
Human Human Mouse Rat
[12] ]8] [11] [10]
These findings have led to the view that antigen processing for the class I MHC pathway is almost entirely controlled by MHC genes. However, it must be stressed that this pathway is still hypothetical. It is not certain that the 'transporters' transport; Levy et a t [20,.] have found, using microsome preparations in vitro, that peptide transport was not ATP-dependent, although class I assembly was. If this microsome system is representative of the ER, this simple view may not be quite correct. The transporters, and to a lesser extent the proteasomes, show genetic polymorphisms [15]. In the rat this has been associated with differences in antigen processing, such that identical class I molecules in cells from two congenic rat strains, which differ only in their class II MHC regions, present different peptide epitopes and behave as alloantigens [21,22-]. The class I molecules differ in their rates of assembly, glycosylation and transport to the cell surface. More revealing was the high performance liquid chromatography hydrophobicity profile of the peptides eluted from the class I molecules from the two rat strains; the molecules that assembled more rapidly contained more hydrophilic peptides. The MHC class II regions of these rat strains showed allelic differences in the TAP-2 (MtlY2) genes. Transfection of the M~t>2 transporter gene of the dominant (fast-assembling) phenotype into a cell line that showed the recessive phenotype (slow-assembling) changed the bound peptides from a hydrophobic to hydrophilic pattern [23..]. A somewhat similar antigen processing polymorphism has been described in a human family, but this has not yet been firmly assigned to a MHC-linked gene, raising the possibility that non-MHC genes could also be involved in these processes [24.]. Such polymorphism adds a further layer of functional polymorphism to the class I MHC antigen-presentation system. Class I molecules present antigenic peptides to the Tcell receptors. The peptides bind in a groove on the
membrane-distal surface of the class I molecule, as revealed by the crystal structure, now resolved at 2.6A_ [25"]. It is clear that the fine structure of this groove determines the nature of the peptide bound. The ends of the groove, which form two pockets, A and F, are conserved and bind the amino and carboxyl terminals of the peptides respectively. Evidence for this comes from both crystallographic [26 o'] and functional sources [27.]. Four additional pockets in the middle region of the cleft, B,C,D and E, are polymorphic and differ considerably between different class I MHC molecules [28]. For each class I molecule, these pockets determine the shared (anchor) residues [29 "~ of the epitope peptides, because they can accommodate their side chains. For instance, HLA B27 allows only an arginine side chain in its B pocket [26".], whereas the HLA A2 B pocket can accomodate leucine or isoleucine [29-o1. In the latter case, this requirement has been confirmed by analysis of a very large number of eluted peptides by mass spectroscopy [30"]. Close to the F pocket is a critical amino acid at position 116 that plays an important role in determining the nature of the side chain of the carboxyl amino acid [27"]; for instance, residue 116 is aspartate in B27 and the commonest carboxyl terminal amino acid is arginine or lysine [31"], residue 116 is tyrosine in HLA A2 and the last residue of the peptide is valine or leucine [29~176Thus, it becomes clear how the structure of the groove selects the peptide epitope and it should soon be possible to predict peptides that bind at relatively high affinity with some accuracy. The peptide is an integral part of the class I molecule. The mutant cell lines that lack functional transporters do not fill their class I molecules and they may be retained in the ER or appear on the cell surface in an unstable, and probably empty, form [32]. Townsend's group [9,33~ -] has shown that the association between a newly synthesized class I molecule and peptide occurs in the ER and is a critical step in the maturation of the class I molecule. The peptides that bind to class I molecules have been shown to be remarkably consistent in their length, nine +/-one, amino acids [36,37]. It has been found that the epitope peptides tend to bind to class I molecules with relatively high affinity [37] and nonamer peptides bind with greater avidity than longer or shorter peptides [34",35"]. However, not all peptides (made synthetically) that bind with high affinity under in vitro conditions are necessarily epitopes [35"]. Naturally processed high affinity binding peptides may not be epitopes because they are cross-reactive with self peptides (and therefore tolerize reactive T cells). Also, not all theoretical nonamers may be generated by the antigen-processing system. In support of the latter, various groups have demonstrated that flanking sequences in a protein can affect epitope generation [38%39",40]. Another likely factor is competition between self and virus peptides in the ER or for the transporters. Analysis of peptides, eluted from class I molecules, by mass spectroscopy suggests that over 200 different peptides may be present in HLA A2 purified from a single cell line [30"]. The threshold for antigen recognition by CTL has been estimated to be 200 molecules per cell [41]; therefore, to be antigenic peptides have to bind to > 0.2% of surface class I molecules; affinity and concentration are likely to be im-
Antigen presentation in virus infection Murray and McMichael portant in this competitive process. Finally, as discussed below there may be differences between antigenicity and immunogenicity dependent on cell type or state (e.g., activation) and this will also determine whether a particular peptide sequence stimulates a CTL response.
over several months. This difference could be accounted for by dosage effects if the transgene peptides mix with those derived from cellular proteins and compete for transport into the ER and subsequent binding. However, when Oldstone et al. injected CTL clones specific for the relevant class I molecules plus virus peptide into the transgenic animals they showed that these CTLs were localized to the pancreatic islets in the transgenics (and not in controls), indicating that in the absence of virus infection or other inflammatory stimulus at least some of the cells in the islets were expressing class I complexes containing transgene peptide. It is worth making the piont here that in the presence of inflammation cytokines are released, particularly IFN-7, that induce the upregulation of MHC class I expression by cells, thereby increasing the number and variety of peptide-class I complexes available to T cells; concievably this might occasionally lead to presentation of self peptides not normally seen, resulting in autoimmune reactions.
Immunogenicity in M H C class I restricted T-cell responses Assembly of a trimolecular complex of heavy chain, ~2-microglobulin and peptide and its delivery to the cell surface for interaction with the T-cell receptor creates a structure which is antigenic, but not necessarily immunogenic. That is, the presence of antigenic cellsurface class-I peptide complexes does not guarantee T-cell recognition and activation i n v i v a Recent experiments with transgenic mice have made this point clearly [42.-44..].
Experiments with mice transgenic for both a virus protein under a tissue specific promoter and for a T-cell receptor specific for host MHC class I together with a peptide from that protein, have demonstrated that there can be circulating T cells with high affinity for the anti genic class I complex without tissue damage [43"~ In the allo-reactive systems, where expression is relatively high, peripheral anergy seems to operate, implying some stimulus to the T cells, albeit negative. In the islet cell experiments, the T cells appear to be 'ignorant', demonstrating that the presence of an antigenic complex capable of a biologically relevant interaction with circulating T cells is on its own not sufl~cient for T-cell activation.
Ohashi e t aL [42 "~ created mice transgenic for lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) under the rat insulin promoter and showed that the protein was expressed only in pancreatic islet cells. These H-2b mice were phenotypicaUy normal and did not develop diabetes, even when crossed with mice expressing a transgenic T-cell receptor known to recognize a peptide from LCMV GP in the context of H-2D b. However, when the mice were infected with LCMV they developed fatal diabetes rapidly, over 9-11 days in the single transgenics, and 4-5 days in the double transgenics. Oldstone e t al. [43 ~ made very similar transgenic animals using LCMV GP or LCMV NP, but in their system expression may have been at a lower level, in that immunohistochemical staining of the pancreas for the virus proteins was negative in contrast to the transgenic mice generated by Ohahsi e t aL [42~ Nevertheless, northern blots revealed transgene mRNA which was confined to the pancreas. The majority of these mice (94%) did not progress to diabetes, unless infected with LCMV, when the time course of disease onset was very different from that seen by Ohashi e t al. - - the disease appeared only
Cytosol Virus protein
Endoplasmic reticulum
Peptides t
")~
Golgi
What are the requirements for presentation of class I MHC and virus antigen in immunogenic form? This issue has rarely been addressed but we can extrapolate from studies involving allogeneic responses. The most potent of the so called 'professional APCs' is the dendritic cell (DC); cell fractionation experiments have shown that for allo responses the ability to induce CTL responses lies almost exclusively in this population [45]. It has been possible to prime CTL responses to influenza virus i n
Infected cell membrane
CTL membrane
] ,
Proteolysis
Transport /
MHC I peptide
Fig. 1. The diagram shows the pathway within the cell by which virus antigens are processed and peptides presented at the cell surface by class I MHC molecules. MHC encoded genes may contribute to the proteolysis (proteasome complex) and peptide transport (TAP-1 and TAP-2). Viral peptides and unfolded HLA class I heavy and light ([32-microglobulin) chains associate in the endoplasmic reticulum. Accessory molecules and their ligands, including CD8, lymphocyte function associated antigen (LFA)-I and CD2, are necessary for the interaction with cytotoxic T lymphocytes (CTLs) leading to lysis. Additional accessory molecules, such as CD28 interacting with B7, are probably necessary to activate CTL precursors and to initiate primary responses.
I•A•ccessory
//
activators 1
403
404
Immunityto infection vitro by exposing T cells to infected DCs [46]; similar in vitro primary CTL responses have also been made to human immunodeficiency virus [47"]. In vitro priming
has been achieved by pulsing DCs with epitope peptides [46,47"]; however, similarly pulsed macrophages or spleen cells did not prime CTL responses to influenza virus [46]. Culture of mouse spleen cells at high density with peptide initiated anti-influenza responses [48], but the cell types involved were not studied. Fewer experiments have been carried out in vivo, Kast et al. [49] have used virus-infected DCs to prime CTLs in non-responsive mice. There have been many attempts to prime for CTLs with non-infectious vaccines with varying success, but the cells involved have not been analyzed and detailed discussion of vaccines is beyond the scope of this review. There could be several reasons for the special effectiveness of DCs in CTL priming. The high level of MHC class II expression may not be relevant because virus-specific CTLs can be induced in mice depleted of CD4 + T cells [50]. Boog et al. [51] showed that DCs express MHC class I with a much lower level of sialation (i.e. glycosylation with negatively charged sialic acid residues) than other cells, and that treatment of monocytes with neuraminidase conferred the capacity to induce class I specific allo responses in vitro. Decreased glycosylation reduces surface negative charge and this may facilitate the interaction between T cells and their targets. DCs also have very high levels of adhesion molecules [52], which again may increase the affinity of interactions between these APCs and T cells compared with normal cells. Notwithstanding this potentially increased affinity, evidence from MHC class II based systems strongly suggests that the interactions between APCs and responder T cells are in fact qualitatively different from those with other cells [53-56]. Engagement of the CD28 surface receptor (present on around 85% of CD4 + cells and 50% of CD8 + cells) results in specific intracellular events that are different from, but complementary to, those due to signalling from the T-cell receptor. Cytokine mRNA is specifically stabilized, and these effects are not blocked by cyclosporin (whereas intracellular events arising from CD3 complex activation are) [53-56]. The ligand for CD28 has been identified as B7 [57",58], a molecule found on activated B cells; its distribution among other APCs is not clear. Transfection of B7 into a B7-negative Burkitts lymphoma cell line, which is incapable of inducing class I specific allo responses, reconstituted the ability to stimulate alloantigen-induced proliferation [59"]. Furthermore, the presence of CD28 has been shown to be sufficient to prevent induction of anergy in T-cell clones [60"]. B ceils have not been investigated for their ability to prime virus-specific CTL responses, but might take up virus following specific recognition by surface antibody, even when they do not express the normal ligand for viral entry. It is interesting to note that the molecule CTLA-4, which was defined by subtraction cloning from cDNA from a mouse CTL line, has strong homology with CD28, and in fact appears to bind with a higher affinity to B7 than does CD28 [61.-]. Other T-cell molecules that may also be involved in cell--cell signalling in antigen presentation include CD5 [62,63] (the ligand for which, CD72,
seems to be confined to B cells [64..]); CD2 (the ligand for which is lymphocyte function associated antigen (LFA)-3 [65]); and the ligand for heat stable antigen (recently defined as a costimulatory molecule in CD4 + Tcell responses [66"]). Molecules such as CD8 (which binds the ~z 3 domain of class I [67]) and LFA-1 (interacts with intercellular adhesion molecule (ICAM)-I and -2 [65]), whose ligands are not confined to specialized APCs are also capable of delivering intracellular signals, but in view of the ubiquity of ICAM-1 these sig nals cannot be sufficient to confer immunogenicity upon peptide-class I complex. tafferty in 1983 [68] argued that transplantation rejection was initiated by passenger APCs of a haemopoietic lineage, and that without effective antigen presentation a mismatched transplant remained unseen by an immune system perfectly capable of rejecting it. Similarly, workers on a number of tumor models have shown that a tumor may be manipulated into acting as its own APC, in general by transfection of the gene for one of an increasing number of cytokines, including interleukin (IL)-2 [69"*,70], IL4 [71], IL-7 [72], IFN-y [73,74], tumor necrosis factor-a [75,76], and granulocyte-macrophage colony stimulating factor [77]; despite this, tumors are in general 'potentially antigenic' rather than immunogenic. As an example, one might argue that viral warts, where multiple warts will often disappear simultaneously, are antigenic but unseen by the immune system until some triggering event (viral uptake by a Langerhans cell?) occurs.
Conclusions There is clearly a processing pathway for presentation of peptide breakdown products of vires (and cellular) proteins that is common to all cells that express class I MHC; although the processing machinery is likely to be 'idling' at very low turnover levels in uninfected, non-immune system cells in the absence of IFNs or other cytokines. However, only certain cells, specialized APCs, are likely to be able to activate T cells to induce effector function, although once this activation step is taken the resulting CTL is capable of interacting with any cell bearing the appropriate class I molecule in combination with peptide. This provides a mechanism by which some self proteins, or rather their derivative peptides, can become antigenic by cross-reacting with an immunogenic antigen, such as a virus. It could explain why potentially antigenic tumour cells escape T-cell surveillance. On the other hand, it is less clear how viruses which do not infect specialist APCs initiate CTL responses; all virus infections so far examined stimulate CTL responses. Possibly low levels of infection are sufficient, particularly in the environment of a lymph node. Alternatively, perhaps DCs or B cells can process non-infectious antigen into the class I MHC antigen processing pathway.
Acknowledgements We are grateful to the Imperial Cancer Research Fund (N.M) and Medical Research Council (A.M.) for support.
A n t i g e n p r e s e n t a t i o n in v i r u s i n f e c t i o n M u r r a y a n d M c M i c h a e l
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N Murray and A McMichael, Molecular Immunology Group, Insitute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK.
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