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Processing requirements for representation of antigens to T lymphocytes
Processing requirements -for presentation of antigens to T lymphocytes E.O. Long Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA Current
Opinion
in Immunology
Introduction One of the most exciting recent developments in immunology has been the demonstration that molecules encoded in the major histocompatibility complex (MHC) bind peptide fragments derived from foreign antigens and present these peptides to T lymphocytes. In order to be recognized by T lymphocytes, most antigens require processing so that immunogenic epitopes can be revealed and bound to MHC molecules. The interactions between processed peptides and MHC molecules have begun to be defined at the molecular level. In contrast, much less is known about how these peptides are generated from foreign antigens and where they interact with MHC molecules. The requirement for processing was first recognized in the case of soluble antigens presented to helper T cells by antigen-presenting cells (APC). These exogenous antigens are presented to T cells in the context of class II MHC molecules. It was recognized very recently that the presentation of endogenous antigens to cytotoxic T lymphocytes (CTL) also involved processing. Endogenous antigens are presented to CTL in the context of class I molecules. The function of class I and class II MHC molecules is, therefore, very similar: both bind peptides derived from processed antigens and present them to T cells. The functional difference is that class I antigens interact mainly with CD&positive T cells, whereas class II antigens interact primarily with CD4positive T cells. Whether these two classes of MHC molecules are also linked to specific processing pathways is an issue of great interest.
Nature of the processed peptides Binding of peptides to MHC molecules can be reproduced in vitro either by direct binding to purified class II molecules (Babbit et al., Nature 1985, 317:359)
1988,
1:98-102
or by sensitizing target cells expressing the appropriate class I or class II specificity for T cell recognition. Direct binding to class I molecules has not been reported yet, but the co-purification of a peptide or a mixture of peptides with crystallized human leukocyte antigen (HI&-A2 has provided compelling evidence for a peptide binding site on class I molecules [l]. Studies on peptide interactions with MHC molecules (reviewed by Fox in this issue, pp 103-106) have revealed the minimal requirements for binding; most antigenic peptides can be trimmed down to a length of 10-15 amino acids and still trigger T cells. What remains largely unknown, however, is the nature of the peptide bound to MHC molecules in vivo. For certain antigens, proteolytic cleavages are necessary for presentation, while for others, unfolding may be sufficient (Streicher et al., Proc Nat1 Acud Sci USA 1984, 81:6831). Fibrinogen and some proteins from L&e&z monocytogenes seem to be presented in the absence of processing [2,3]. T-cell determinants can thus be classified, as proposed by Allen [4], into type I requiring no processing, type II requiring unfolding, and type III requiring cleavage of the native antigen. Types I and II are compatible with the mechanism depicted in Fig. la, where exposed epitopes bind to MHC molecules prior to fragmentation of the antigen. The peptide in the MHC binding site may be protected from proteolysis. Type III suggests another mechanism (Fig. lb) whereby specific proteolytic cleavages would generate peptides that can subsequently interact with MHC molecules. These two mechanisms are not mutually exclusive and could even operate for different determinants on the same antigen. In conclusion, it appears that cleavage of antigens into peptides is not necessarily a prerequisite for binding to MHC’molecules, and that T cell recognition may occur when a large molecule is attached to the antigenic peptide; however, it is possible that antigens bound to MHC in tivo are trimmed by non-specific proteolysis so that cell surface MHC molecules present mostly short peptides.
Abbreviations T lymphocytes; AK-antigen-presenting cells; CTL-cytotoxic HLA-human leukocyte antigen; MHC-major histocompatibility complex.
98
@
Current Science Ltd ISSN 0952-7915
Presentation
Small antigen fragments can also be processed. A synthetic peptide recognized by T cells in the absence of processing was, nevertheless, processed by murine spleen cells so that its association with MHC molecules was altered [5]. The possibility that protein modifications, in addition to cleavages, may play a role in presentation must be considered. The 18 amino acid natural peptide apamin can be processed by different mechanisms into distinct T cell epitopes
[61. Different cells do not process antigens identically, i.e. L cells transfected with class II genes are unable to present certain epitopes (Shastri et al., Proc Nat/ Acad Sci USA 1985, 82:5885) [6]. This result reflects either the inability of L cells to process certain antigens, or the possibility that the peptides generated by proteolytic cleavages in L cells were different from those generated in B cells.
Pathways of antigen processing There are at least three pathways ing exist (Fig. 2), and each one
of antigen processmay be subdivided
of antigens to T lymphocytes
Long
further into separate compartments. First, exogenous antigens are taken up by APC through endocytosis. Because endocytosed transferrin was shown to intersect the pathway taken by newly synthesized class II MHC molecules to the cell surface (Cresswell, Proc Natl Acad Sci USA 1985, 82:8188), it seems likely that antigens processed in endosomes can interact with class II molecules. Exogenous antigens comprise soluble proteins but also include inactivated viral particles or even those intact viruses that enter host cells via endocytosis and pH-dependent fusion. This exogenous-endocytic processing pathway, sensitive to lysosomotropic agents, functions only in conjunction with class II-restricted antigen presentation [7]. The generation in viva of class I-restricted CTL to ovalbumin [B] can be explained by extracellular degradation of ovalbumin or by the existence of a putative specialized cell that could process exogenous antigens for class I-restricted presentation. Second, cell surface antigens can be processed. Evidence that an intact molecule anchored at the cell surface was not required for T cell recognition came from mutated influenza virus hemagglutinin et molecules lacking the signal sequence (Townsend al., Nature 1986, 324:575) or the transmembrane an-
(a)
(b)
Fig. 1.Mechanisms of antigen processing. (a) The immunogenic epitope (wavy line) binds to a major histocompatibility complex (MHC) molecule when exposed by unfolding of the anti en (Ag). The epitope bound to the MHC molecule is protected from . antigen by specific proteases and can then bind to an MHC proteolytic degradation. (b) The epitope is cleaved from t7l e native molecule.
99
100
Antigen
recognition
char [9]. Class I-restricted presentation of a secreted form of hemagglutinin [9] strongly suggests that processing occurs during transport to the cell surface, rather than by recycling through endosomes, because the exogenous pathway is not used for presentation by class I antigens [6]. Degradation of newly synthesized proteins in the endoplasmic reticulum [lo] may be a source of peptides that could bind to the MHC molecule. Finally, Townsend et al. (Cell 1986, 44:959) showed that cytoplasmic viral antigens were processed and presented by class I MHC molecules. This pathway contains an inherent difficulty in that cytoplasmic proteins and the binding site on MHC molecules are separated by a membrane: how, then, do immunogenic antigens reach their destination? It is not known whether this transport involves intact antigen or processed peptides, but it has been suggested that it may be linked to ubiquitin-mediated protein degradation [ 111. Protein synthesis is not necessary for cytoplasmic processing, as demonstrated by fusion of inactive influenza virus with target cells treated with emetine and cycloheximide [12]. CTL recognition of antigens processed in the cytoplasm was more efficient with internal viral proteins than with hemagglutinin [13]. It is possible that processing of hemagglutinin in membranes and in the cytoplasm generated different peptides. The influenza virus hemagglutinin processed by the exogenous pathway was recognized only by class IIrestricted CTL, whereas endogenous processing of the same antigen led to recognition by class I-restricted CTL only (Morrison et al, J Z3p Med 1986, 163:903); [7]. The distinct requirements for class I- and class II-restricted presentation led to the proposal that separate processing pathways operate for the two classes of MHC antigens (Germain, Nature 1986, 322:687) [7]. An alternative interpretation is that different peptides could be generated through the various processing pathways so that specific effector cells may recognize the antigen only if it was processed as it was during the initial immunization. If CTL do not recognize a target cell in vitro, either the processing pathway is missing or the peptides generated are different from those initially recognized in vivo The invariant chain associated intracellularly with class II MHC molecules was thought to be important for their function. Class II-restricted presentation of measles virus antigens to specific CTL occurred in transfected cells expressing class II molecules in the absence of the invariant chain [14]. The invariant chain is thus not required for processing of measles virus antigens. Because of the multiple processing pathways, it remains possible that the invariant chain plays an important role in a specific pathway not used by measles virus antigens. The cell surface of APC may be an important component of antigen processing and presentation. Processed antigen can be removed from APCs by treat-
ment with phospholipases [15]. Modification of peptides by lipids was suggested by the inhibition of antigen presentation by cerulenin, and antibiotic that blocks post-translational lipid modification of proteins
[161.
Discrimination
of self and non-self
Because self peptides appear to be bound to self MHC molecules [l], the distinction of non-self is probably not made by selective binding of foreign peptides to MHC. It is likely that MHC molecules are constantly occupied by peptides generated from normal protein degradation and that foreign peptides must compete for binding (Kourilsky and Claverie, Ann Imt Pusteur Zmmunol 1986, 137D:3). The immune system can distinguish non-self by becoming tolerant to self peptides bound to self MHC; this has important implications for allorecognition. As proposed more than 10 years ago, alloreactive T cells may recognize foreign MHC molecules in association with a multitude of self determinants (Matzinger and Bevan, Cell Zmmunol 1977, 29:l). The possibility that peptides derived from processed MHC molecules could be presented by intact surface MHC antigens was first demonstrated by Maryanski et al. (Nature 1986, 324:578), who studied a murine xenogeneic response to HLA class I. That alloreactive T cells can recognize MHC-derived peptides in the context of MHC molecules has now been demonstrated [17,18]. The second pathway of antigen processing (Fig. 2) would be a logical source of MHC-derived peptides.
n MHC
*.:.::~..~.~..*,.‘:.‘.’
:
.‘.‘:::,.:.~.~.~.._
~~~
.
.
. .
.
.Golgi
.
.
.
.
. .
.
.
.
.
.
I
.. Cytoplasm
1.-l’.
.._
. ’
Fig. 2. Pathways of antigen processing exist for exogenous antigens (I), cell surface antigens (2) and cytoplasmic antigens (3). MHC, major histocompatability complex; ER, endoplasmic reticulum.
Presentation
Conclusions The next major advances, after the realization that antigen processing may be a universal requirement for both class I- and class II-restricted presentation, will await new technological developments. The main stumbling block is that no standard reagent is available to follow the fate of processed antigen, to trace when epitopes are revealed and where peptide-MHC interactions occur within the cell. Special probes need to be developed for these purposes. The goal is to define the biochemical events and the cellular compartments that control antigen processing. The task is large, because multiple pathways exist.
of antigens to T lymphocytes
Long
The processing requirements for presentation of apamin, an 18 amino acid peptide with two disulfide bonds, were tested with I6 specific T hybridoma cells. All 16 required processing of apamin. Unfolding of apamin was sufficient for recognition by 8 T cells, but further processing was necessary for the remaining 8. Thus, even small antigens can be processed into distinct T-cell epitopes. BRACIALETJ, MORRISONLA, SWEE~~ERMT, SAMBR~~K J, GETHINGM-J, BRACLALE VL: Antigen presentation pathways to class I and class II MHC-restricted T lymphocytes. Immutwl Rev 1987, 98:95-114. Influenza virus hemagglutinin processed through the exogenous pathway was presented only to class II-restricted GIL, whereas the same antigen processed endogenously in infected cells was presented only to class I-restricted CTL. The correlation between MHC class restriction of T lymphocytes, seen at the effector level, and antigen processing pathway also held at the level of T lymphocyte activation in viva. 7. .
8. 0
Annotated reading 0 bb 1. ??@
references
and recommended
Of interest Of outstanding interest BJORKMAN PJ,SAPERMA, SAMRAOUI B, BENNETT WS, STROMINGER JL, Ww DC: Stru~e of the human class I histocom-
patibutty antigen, HLA-A2. Nature 1987, 329:50&511. The presence of a peptide or a mixture of peptides in the antigen binding site of the crystallized HLk42 molecule strongly suggests that surface class I molecules are occupied by peptides most of the time. These peptides may be derived from endogenous self proteins. ._ 2. .
LEE P, MATXIEDAGR, ALLENPM: T cell recognition of fibrinogen: a dete rminant on the A a chain does not require processing. J Immunol 1988, 140:1063-1068. A T cell epitope on intact fibrinogen has been identified in the carboxy portion of the a chain that can be presented by pre-lixed macrophages. The epitope is presumably located in a conformationally flexible area of the molecule. This finding implies that cleavage into peptides is not a prerequisite for binding to MHC molecules, and that large proteins may extend out of the binding site without preventing recognition by T cells. Differential requirements for the processing and presentation of soluble and particulate bacterial antigens by macrophages. Eur J Immunol 1987, 17:1287-1296. Soluble proteins of List& monoqtogenes were presented to T cells by fixed APC Such experiments do not formally rule out undetectable amounts of degraded antigen that were sufficient (see [2]) for presentation. 3. .
ZIEGLER HK, ORLIN CA, CLUFFCW
4. 0
AUEN PM: Antigen
processing at the molecuku level. Immurwl Today 1987, 8:27*273. A recent, concise and clear review of antigen processing. Many of the chains that constitute part of the T cell receptor are synthesized in great excess over the amount assembled into the mature complex. These excess chains are rapidly degraded after biosynthesis. Intracellular degradation of transmembrane proteins would be a good source of peptides for the second pathway of antigen processing (Fig. 2). Fox BS, CARBONE FR, GERMAINRN, PATERSON Y, SCHWARTZ RH: Processing of a minimal antlgenic pepride alters its interaction with MIiC molecules. Nuture 1988, 331:538-540. A synthetic peptide that does not require processing to be recognized by T cells can nevertheless be processed, both in virro and in vi&o, leading to alterations of its association with class II MHC molecules. 5. .
6. 0
REGNIER-VIGOURO~X A, EL AYEBM, DEFENDINI M-L, GRANIERC, PIERRES M: Processing by accessory cells for presenta-
tion to murlne T cells of apamin, a -de-bonded 18 amino acid peptide. J Immunol 1988, 140:106~1075.
STAERZ UD, KARAXNAMA H, GARNERAM: YCytotoxic T lymphocytes against a soluble protein. Nafure 1987, 329:449451. Mice immunized with soluble ovalbumin generated class I-restricted cytotoxic T cells specific for ovalbumin fragments. Processing of exogenous ovalbumin in viva occurred either extracellularly or within a putative antigen-presenting cell specialized in class I-restricted CTL inducrion.
BFXIALE TJ, BRACLALE VL, WINKLERM, STROYNOWSKI I, Hook L, SAMBR~K J, GETHINGM-J: On the role of the transmembrane anchor sequence of intluenza hemagglutinin in target cell recogoition by class I MHC-restricted, hemagglutinln-spedtic cytolytic T lymphocytes. J Fxp Med 1987, 166:678-692. Target cells expressing secreted forms of the influenza virus hemagglutinin, engineered by truncation of the transmembrane region, were still recognized by the majority of class I-restriced hemagglutinin-specific cytotoxic T lymphocytes. Membrane anchoring is not required for processing and presentation of cell surface proteins. These experiments identified the second processing pathway (Fig. 2). 9.
00
10. .
MINAMIY, WEISSMANAM, SAMEL~~NLE, KL.UJSNER D: Building a multichain receptor synthesis degradation and assembly of the T-cell antigen receptor. Proc Nafl Acud Sci USA 1987, 84~2688-2692.
11. TOWNSEND ARM: Recognition of iniluenza virus proteins . by cytotoxic T lymphocytes. Immunol Res 1987,6:80-100. An excellent review on CTL recognition of influenza virus antigens. 12. .
YEWDEKJW, BENNINKJR, HOSAKAY: Cells process arogenous proteins for recognition by cytotoxic T lymphocytes. Science 1988, 239637-640. Target cells fused with intact, non-infectious influenza virus in the absence of protein synthesis were recognized by class I-restricted influenza virus-specific cytotoxic T lymphocytes. Presence of viral antigens in the cytoplasm is sufficient for processing and presentation. HOSAKAY, SA.SAOF, YAMANAKAK, BENNINKJR, YEXQELLJW: Recognition of noninfectious h&wnza vlrus by class I-rest&ted murlne cytotoxic T lymphocytes. J Immunol 1988, 140:6~10. Target cells sensitized with inactivated but fusion-competent influenza virus were recognized by all T-cell clones specific for internal proteins and by one of 6 clones specific for hemagglutinin. Both internal and integral membrane proteins can be processed in the cytoplasm and presented by class I MHC molecules, but the processing of biosynthesized hemagglutinin may follow a different pathway. 13. 0
14. 0
SEKUY RP, JACOBSONS, RICHERT JR, TONNELLI! C, MCFARLAND HF, LONG EO: Antigen presentation to HLA class II-re-
stricted measles virus-specific T-cell clones can occur in the absence of the invariant chain. Proc Nat1 Acad Sci USA 1988, 85:1209-1212. The invariant chain associated intracellularly with MHC class II antigens was thought to be important for their function. This study shows that the invariant chain is dispensable in class II-restricted presentation of measles virus antigens to CK
101
102
Antigen
recognition
15.
FALO LD, HABER SI, HERRMANN S, BENACERJW B, RICK KL Vtion of antigen association with accessory cells: specific removal of pracessed antigens from the cell sudace by phospholipases. Proc Nat1 AC& Sci USA 1987, 84~522-526. Treatment of antigen-exposed APC, with phospholipase ~2 or with phospholipase C abolished their ability to stimulate antigen-specific T hybridomas, but not alloreactive hybridomas specific for the same class II molecule. These studies suggest that some processed antigenie peptides can be removed from the cell surface of AFC by phospholipase. ??
FALO LD JR, BENACERIW B, ROTHSTEINL, RICK KL: Cerulenin . is a potent inhibitor of antigen processing by antigenpresenting cell. J Immunol 1987, 139:391&3923. An antibiotic that inhibits lipid synthesis and blocks lipid modification of proteins strongly inhibited the ability of accessory cells to present 16.
antigen step. 17. 0
to T cells. The block was shown
to be at some processing
CLAVRERGER C, PARHAMP, ROTHBARDJ, LUDWIG DS, SCHO~LNIK GK, KRENSKYAM: IUA-AZ peptides can regulate cytoly-
allogeneic T lymphocytes. Nature 1987, sisbyhuman 330~763-765. Ailospecific qtotoxic T cells can recognize HL4-derived peptides in the context of class I MHC molecules. 18.
SONG ES, LINSK R, OLSON CA, MCMUAN M, G~KIDENOW RS
0
Allospec&
: cytotoxic T lymphocytes recognize an H-2
peptide in the context of a murine major histocompatibility complex class I molecule. Proc Nat1 Acad Sci USA 1988, 85:1927-1931. Many ailoreactive cytotoxic T cells specific for the class I MHC molecule H-2Ld also recognize an H-2Ld peptide in association with H-2k restriction elements.
Opinion
in Immunology
Introduction One of the most exciting recent developments in immunology has been the demonstration that molecules encoded in the major histocompatibility complex (MHC) bind peptide fragments derived from foreign antigens and present these peptides to T lymphocytes. In order to be recognized by T lymphocytes, most antigens require processing so that immunogenic epitopes can be revealed and bound to MHC molecules. The interactions between processed peptides and MHC molecules have begun to be defined at the molecular level. In contrast, much less is known about how these peptides are generated from foreign antigens and where they interact with MHC molecules. The requirement for processing was first recognized in the case of soluble antigens presented to helper T cells by antigen-presenting cells (APC). These exogenous antigens are presented to T cells in the context of class II MHC molecules. It was recognized very recently that the presentation of endogenous antigens to cytotoxic T lymphocytes (CTL) also involved processing. Endogenous antigens are presented to CTL in the context of class I molecules. The function of class I and class II MHC molecules is, therefore, very similar: both bind peptides derived from processed antigens and present them to T cells. The functional difference is that class I antigens interact mainly with CD&positive T cells, whereas class II antigens interact primarily with CD4positive T cells. Whether these two classes of MHC molecules are also linked to specific processing pathways is an issue of great interest.
Nature of the processed peptides Binding of peptides to MHC molecules can be reproduced in vitro either by direct binding to purified class II molecules (Babbit et al., Nature 1985, 317:359)
1988,
1:98-102
or by sensitizing target cells expressing the appropriate class I or class II specificity for T cell recognition. Direct binding to class I molecules has not been reported yet, but the co-purification of a peptide or a mixture of peptides with crystallized human leukocyte antigen (HI&-A2 has provided compelling evidence for a peptide binding site on class I molecules [l]. Studies on peptide interactions with MHC molecules (reviewed by Fox in this issue, pp 103-106) have revealed the minimal requirements for binding; most antigenic peptides can be trimmed down to a length of 10-15 amino acids and still trigger T cells. What remains largely unknown, however, is the nature of the peptide bound to MHC molecules in vivo. For certain antigens, proteolytic cleavages are necessary for presentation, while for others, unfolding may be sufficient (Streicher et al., Proc Nat1 Acud Sci USA 1984, 81:6831). Fibrinogen and some proteins from L&e&z monocytogenes seem to be presented in the absence of processing [2,3]. T-cell determinants can thus be classified, as proposed by Allen [4], into type I requiring no processing, type II requiring unfolding, and type III requiring cleavage of the native antigen. Types I and II are compatible with the mechanism depicted in Fig. la, where exposed epitopes bind to MHC molecules prior to fragmentation of the antigen. The peptide in the MHC binding site may be protected from proteolysis. Type III suggests another mechanism (Fig. lb) whereby specific proteolytic cleavages would generate peptides that can subsequently interact with MHC molecules. These two mechanisms are not mutually exclusive and could even operate for different determinants on the same antigen. In conclusion, it appears that cleavage of antigens into peptides is not necessarily a prerequisite for binding to MHC’molecules, and that T cell recognition may occur when a large molecule is attached to the antigenic peptide; however, it is possible that antigens bound to MHC in tivo are trimmed by non-specific proteolysis so that cell surface MHC molecules present mostly short peptides.
Abbreviations T lymphocytes; AK-antigen-presenting cells; CTL-cytotoxic HLA-human leukocyte antigen; MHC-major histocompatibility complex.
98
@
Current Science Ltd ISSN 0952-7915
Presentation
Small antigen fragments can also be processed. A synthetic peptide recognized by T cells in the absence of processing was, nevertheless, processed by murine spleen cells so that its association with MHC molecules was altered [5]. The possibility that protein modifications, in addition to cleavages, may play a role in presentation must be considered. The 18 amino acid natural peptide apamin can be processed by different mechanisms into distinct T cell epitopes
[61. Different cells do not process antigens identically, i.e. L cells transfected with class II genes are unable to present certain epitopes (Shastri et al., Proc Nat/ Acad Sci USA 1985, 82:5885) [6]. This result reflects either the inability of L cells to process certain antigens, or the possibility that the peptides generated by proteolytic cleavages in L cells were different from those generated in B cells.
Pathways of antigen processing There are at least three pathways ing exist (Fig. 2), and each one
of antigen processmay be subdivided
of antigens to T lymphocytes
Long
further into separate compartments. First, exogenous antigens are taken up by APC through endocytosis. Because endocytosed transferrin was shown to intersect the pathway taken by newly synthesized class II MHC molecules to the cell surface (Cresswell, Proc Natl Acad Sci USA 1985, 82:8188), it seems likely that antigens processed in endosomes can interact with class II molecules. Exogenous antigens comprise soluble proteins but also include inactivated viral particles or even those intact viruses that enter host cells via endocytosis and pH-dependent fusion. This exogenous-endocytic processing pathway, sensitive to lysosomotropic agents, functions only in conjunction with class II-restricted antigen presentation [7]. The generation in viva of class I-restricted CTL to ovalbumin [B] can be explained by extracellular degradation of ovalbumin or by the existence of a putative specialized cell that could process exogenous antigens for class I-restricted presentation. Second, cell surface antigens can be processed. Evidence that an intact molecule anchored at the cell surface was not required for T cell recognition came from mutated influenza virus hemagglutinin et molecules lacking the signal sequence (Townsend al., Nature 1986, 324:575) or the transmembrane an-
(a)
(b)
Fig. 1.Mechanisms of antigen processing. (a) The immunogenic epitope (wavy line) binds to a major histocompatibility complex (MHC) molecule when exposed by unfolding of the anti en (Ag). The epitope bound to the MHC molecule is protected from . antigen by specific proteases and can then bind to an MHC proteolytic degradation. (b) The epitope is cleaved from t7l e native molecule.
99
100
Antigen
recognition
char [9]. Class I-restricted presentation of a secreted form of hemagglutinin [9] strongly suggests that processing occurs during transport to the cell surface, rather than by recycling through endosomes, because the exogenous pathway is not used for presentation by class I antigens [6]. Degradation of newly synthesized proteins in the endoplasmic reticulum [lo] may be a source of peptides that could bind to the MHC molecule. Finally, Townsend et al. (Cell 1986, 44:959) showed that cytoplasmic viral antigens were processed and presented by class I MHC molecules. This pathway contains an inherent difficulty in that cytoplasmic proteins and the binding site on MHC molecules are separated by a membrane: how, then, do immunogenic antigens reach their destination? It is not known whether this transport involves intact antigen or processed peptides, but it has been suggested that it may be linked to ubiquitin-mediated protein degradation [ 111. Protein synthesis is not necessary for cytoplasmic processing, as demonstrated by fusion of inactive influenza virus with target cells treated with emetine and cycloheximide [12]. CTL recognition of antigens processed in the cytoplasm was more efficient with internal viral proteins than with hemagglutinin [13]. It is possible that processing of hemagglutinin in membranes and in the cytoplasm generated different peptides. The influenza virus hemagglutinin processed by the exogenous pathway was recognized only by class IIrestricted CTL, whereas endogenous processing of the same antigen led to recognition by class I-restricted CTL only (Morrison et al, J Z3p Med 1986, 163:903); [7]. The distinct requirements for class I- and class II-restricted presentation led to the proposal that separate processing pathways operate for the two classes of MHC antigens (Germain, Nature 1986, 322:687) [7]. An alternative interpretation is that different peptides could be generated through the various processing pathways so that specific effector cells may recognize the antigen only if it was processed as it was during the initial immunization. If CTL do not recognize a target cell in vitro, either the processing pathway is missing or the peptides generated are different from those initially recognized in vivo The invariant chain associated intracellularly with class II MHC molecules was thought to be important for their function. Class II-restricted presentation of measles virus antigens to specific CTL occurred in transfected cells expressing class II molecules in the absence of the invariant chain [14]. The invariant chain is thus not required for processing of measles virus antigens. Because of the multiple processing pathways, it remains possible that the invariant chain plays an important role in a specific pathway not used by measles virus antigens. The cell surface of APC may be an important component of antigen processing and presentation. Processed antigen can be removed from APCs by treat-
ment with phospholipases [15]. Modification of peptides by lipids was suggested by the inhibition of antigen presentation by cerulenin, and antibiotic that blocks post-translational lipid modification of proteins
[161.
Discrimination
of self and non-self
Because self peptides appear to be bound to self MHC molecules [l], the distinction of non-self is probably not made by selective binding of foreign peptides to MHC. It is likely that MHC molecules are constantly occupied by peptides generated from normal protein degradation and that foreign peptides must compete for binding (Kourilsky and Claverie, Ann Imt Pusteur Zmmunol 1986, 137D:3). The immune system can distinguish non-self by becoming tolerant to self peptides bound to self MHC; this has important implications for allorecognition. As proposed more than 10 years ago, alloreactive T cells may recognize foreign MHC molecules in association with a multitude of self determinants (Matzinger and Bevan, Cell Zmmunol 1977, 29:l). The possibility that peptides derived from processed MHC molecules could be presented by intact surface MHC antigens was first demonstrated by Maryanski et al. (Nature 1986, 324:578), who studied a murine xenogeneic response to HLA class I. That alloreactive T cells can recognize MHC-derived peptides in the context of MHC molecules has now been demonstrated [17,18]. The second pathway of antigen processing (Fig. 2) would be a logical source of MHC-derived peptides.
n MHC
*.:.::~..~.~..*,.‘:.‘.’
:
.‘.‘:::,.:.~.~.~.._
~~~
.
.
. .
.
.Golgi
.
.
.
.
. .
.
.
.
.
.
I
.. Cytoplasm
1.-l’.
.._
. ’
Fig. 2. Pathways of antigen processing exist for exogenous antigens (I), cell surface antigens (2) and cytoplasmic antigens (3). MHC, major histocompatability complex; ER, endoplasmic reticulum.
Presentation
Conclusions The next major advances, after the realization that antigen processing may be a universal requirement for both class I- and class II-restricted presentation, will await new technological developments. The main stumbling block is that no standard reagent is available to follow the fate of processed antigen, to trace when epitopes are revealed and where peptide-MHC interactions occur within the cell. Special probes need to be developed for these purposes. The goal is to define the biochemical events and the cellular compartments that control antigen processing. The task is large, because multiple pathways exist.
of antigens to T lymphocytes
Long
The processing requirements for presentation of apamin, an 18 amino acid peptide with two disulfide bonds, were tested with I6 specific T hybridoma cells. All 16 required processing of apamin. Unfolding of apamin was sufficient for recognition by 8 T cells, but further processing was necessary for the remaining 8. Thus, even small antigens can be processed into distinct T-cell epitopes. BRACIALETJ, MORRISONLA, SWEE~~ERMT, SAMBR~~K J, GETHINGM-J, BRACLALE VL: Antigen presentation pathways to class I and class II MHC-restricted T lymphocytes. Immutwl Rev 1987, 98:95-114. Influenza virus hemagglutinin processed through the exogenous pathway was presented only to class II-restricted GIL, whereas the same antigen processed endogenously in infected cells was presented only to class I-restricted CTL. The correlation between MHC class restriction of T lymphocytes, seen at the effector level, and antigen processing pathway also held at the level of T lymphocyte activation in viva. 7. .
8. 0
Annotated reading 0 bb 1. ??@
references
and recommended
Of interest Of outstanding interest BJORKMAN PJ,SAPERMA, SAMRAOUI B, BENNETT WS, STROMINGER JL, Ww DC: Stru~e of the human class I histocom-
patibutty antigen, HLA-A2. Nature 1987, 329:50&511. The presence of a peptide or a mixture of peptides in the antigen binding site of the crystallized HLk42 molecule strongly suggests that surface class I molecules are occupied by peptides most of the time. These peptides may be derived from endogenous self proteins. ._ 2. .
LEE P, MATXIEDAGR, ALLENPM: T cell recognition of fibrinogen: a dete rminant on the A a chain does not require processing. J Immunol 1988, 140:1063-1068. A T cell epitope on intact fibrinogen has been identified in the carboxy portion of the a chain that can be presented by pre-lixed macrophages. The epitope is presumably located in a conformationally flexible area of the molecule. This finding implies that cleavage into peptides is not a prerequisite for binding to MHC molecules, and that large proteins may extend out of the binding site without preventing recognition by T cells. Differential requirements for the processing and presentation of soluble and particulate bacterial antigens by macrophages. Eur J Immunol 1987, 17:1287-1296. Soluble proteins of List& monoqtogenes were presented to T cells by fixed APC Such experiments do not formally rule out undetectable amounts of degraded antigen that were sufficient (see [2]) for presentation. 3. .
ZIEGLER HK, ORLIN CA, CLUFFCW
4. 0
AUEN PM: Antigen
processing at the molecuku level. Immurwl Today 1987, 8:27*273. A recent, concise and clear review of antigen processing. Many of the chains that constitute part of the T cell receptor are synthesized in great excess over the amount assembled into the mature complex. These excess chains are rapidly degraded after biosynthesis. Intracellular degradation of transmembrane proteins would be a good source of peptides for the second pathway of antigen processing (Fig. 2). Fox BS, CARBONE FR, GERMAINRN, PATERSON Y, SCHWARTZ RH: Processing of a minimal antlgenic pepride alters its interaction with MIiC molecules. Nuture 1988, 331:538-540. A synthetic peptide that does not require processing to be recognized by T cells can nevertheless be processed, both in virro and in vi&o, leading to alterations of its association with class II MHC molecules. 5. .
6. 0
REGNIER-VIGOURO~X A, EL AYEBM, DEFENDINI M-L, GRANIERC, PIERRES M: Processing by accessory cells for presenta-
tion to murlne T cells of apamin, a -de-bonded 18 amino acid peptide. J Immunol 1988, 140:106~1075.
STAERZ UD, KARAXNAMA H, GARNERAM: YCytotoxic T lymphocytes against a soluble protein. Nafure 1987, 329:449451. Mice immunized with soluble ovalbumin generated class I-restricted cytotoxic T cells specific for ovalbumin fragments. Processing of exogenous ovalbumin in viva occurred either extracellularly or within a putative antigen-presenting cell specialized in class I-restricted CTL inducrion.
BFXIALE TJ, BRACLALE VL, WINKLERM, STROYNOWSKI I, Hook L, SAMBR~K J, GETHINGM-J: On the role of the transmembrane anchor sequence of intluenza hemagglutinin in target cell recogoition by class I MHC-restricted, hemagglutinln-spedtic cytolytic T lymphocytes. J Fxp Med 1987, 166:678-692. Target cells expressing secreted forms of the influenza virus hemagglutinin, engineered by truncation of the transmembrane region, were still recognized by the majority of class I-restriced hemagglutinin-specific cytotoxic T lymphocytes. Membrane anchoring is not required for processing and presentation of cell surface proteins. These experiments identified the second processing pathway (Fig. 2). 9.
00
10. .
MINAMIY, WEISSMANAM, SAMEL~~NLE, KL.UJSNER D: Building a multichain receptor synthesis degradation and assembly of the T-cell antigen receptor. Proc Nafl Acud Sci USA 1987, 84~2688-2692.
11. TOWNSEND ARM: Recognition of iniluenza virus proteins . by cytotoxic T lymphocytes. Immunol Res 1987,6:80-100. An excellent review on CTL recognition of influenza virus antigens. 12. .
YEWDEKJW, BENNINKJR, HOSAKAY: Cells process arogenous proteins for recognition by cytotoxic T lymphocytes. Science 1988, 239637-640. Target cells fused with intact, non-infectious influenza virus in the absence of protein synthesis were recognized by class I-restricted influenza virus-specific cytotoxic T lymphocytes. Presence of viral antigens in the cytoplasm is sufficient for processing and presentation. HOSAKAY, SA.SAOF, YAMANAKAK, BENNINKJR, YEXQELLJW: Recognition of noninfectious h&wnza vlrus by class I-rest&ted murlne cytotoxic T lymphocytes. J Immunol 1988, 140:6~10. Target cells sensitized with inactivated but fusion-competent influenza virus were recognized by all T-cell clones specific for internal proteins and by one of 6 clones specific for hemagglutinin. Both internal and integral membrane proteins can be processed in the cytoplasm and presented by class I MHC molecules, but the processing of biosynthesized hemagglutinin may follow a different pathway. 13. 0
14. 0
SEKUY RP, JACOBSONS, RICHERT JR, TONNELLI! C, MCFARLAND HF, LONG EO: Antigen presentation to HLA class II-re-
stricted measles virus-specific T-cell clones can occur in the absence of the invariant chain. Proc Nat1 Acad Sci USA 1988, 85:1209-1212. The invariant chain associated intracellularly with MHC class II antigens was thought to be important for their function. This study shows that the invariant chain is dispensable in class II-restricted presentation of measles virus antigens to CK
101
102
Antigen
recognition
15.
FALO LD, HABER SI, HERRMANN S, BENACERJW B, RICK KL Vtion of antigen association with accessory cells: specific removal of pracessed antigens from the cell sudace by phospholipases. Proc Nat1 AC& Sci USA 1987, 84~522-526. Treatment of antigen-exposed APC, with phospholipase ~2 or with phospholipase C abolished their ability to stimulate antigen-specific T hybridomas, but not alloreactive hybridomas specific for the same class II molecule. These studies suggest that some processed antigenie peptides can be removed from the cell surface of AFC by phospholipase. ??
FALO LD JR, BENACERIW B, ROTHSTEINL, RICK KL: Cerulenin . is a potent inhibitor of antigen processing by antigenpresenting cell. J Immunol 1987, 139:391&3923. An antibiotic that inhibits lipid synthesis and blocks lipid modification of proteins strongly inhibited the ability of accessory cells to present 16.
antigen step. 17. 0
to T cells. The block was shown
to be at some processing
CLAVRERGER C, PARHAMP, ROTHBARDJ, LUDWIG DS, SCHO~LNIK GK, KRENSKYAM: IUA-AZ peptides can regulate cytoly-
allogeneic T lymphocytes. Nature 1987, sisbyhuman 330~763-765. Ailospecific qtotoxic T cells can recognize HL4-derived peptides in the context of class I MHC molecules. 18.
SONG ES, LINSK R, OLSON CA, MCMUAN M, G~KIDENOW RS
0
Allospec&
: cytotoxic T lymphocytes recognize an H-2
peptide in the context of a murine major histocompatibility complex class I molecule. Proc Nat1 Acad Sci USA 1988, 85:1927-1931. Many ailoreactive cytotoxic T cells specific for the class I MHC molecule H-2Ld also recognize an H-2Ld peptide in association with H-2k restriction elements.