- Email: [email protected]
Genes in the MHC that may affect antigen processing
Genes in the MHC that may affect antigen processing John J. Monaco Virginia
Commonwealth
University,
Richmond,
Virginia,
USA
Recent evidence indicates that genes required for antigen processing are present in the class II region of the MHC. Two new classes of MHC genes that may encode much of the machinery required in the class I (endogenous) antigen-processing pathway have been discovered. There is evidence
that
genes
required
in the
class II pathway
also reside
in this
region.
Current
Opinion
in Immunology
these class I-deficient mutants to CTL lysis and induce increased MIX class I surface expression [l-3 1.
Introduction T-cell recognition of antigen requires that the antigen first be ‘processed’ by an appropriate antigen-presenting cell. Peptide fragments derived from this antigenprocessing event are bound by MHC class I or MHC class II molecules. As has been reviewed elsewhere in this volume (Unanue, this issue, pp 63-69 and Bra&ale, this issue, pp 5!9-62), the source and the characteristics (i.e. sequence and length) of the peptides found associated with class I and class II molecules differs. This reflects the existence of two largely or completely separate antigen-processing pathways within the cell. Peptides derived from cytoplasmic antigens (endogenous antigens) feed class I molecules, whereas peptides derived from antigens taken up from the extracellular fluid (exogenous antigens) feed class II molecules. However, the exact molecular mechanisms underlying the proteolytic and subsequent peptide-handling events in either pathway are at present largely unknown.
The class I (endogenous)
1992, 4170-73.
antigen-processing
pathway
The fact that peptides can reverse the class I defect in
mutant cells suggests that peptide is an indispensable component of stable class I molecules, and that the phenotypic defect in these mutants results fi;om an inability to provide the MHC class I molecules with appropriate peptides. This phenotype is consistent with a defect in either the ability to generate appropriate peptides from native antigen, or in the ability to allow the extracellular portion of the class I molecule access to the peptide (i.e. a defect in the ability to transport peptides from the cytoplasm into the secretory pathway). Further analysis of these mutants indicated that such defects in the ability to provide peptide to MHC class I molecules result in the formation of class I heterodimers of heavy chain plus pz microglobulin that are unstable at 37°C (but not at 26°C) [4-8]. At the cell surface, these dimers dissociate, pz microglobulin is released into the medium, and the heavy chain undergoes a rapid conformational change resulting in loss of reactivity with anti-class I antibodies. Addition of appropriate peptides and an exogenous source of p2 microglobulin allows spontaneous assembly of stable peptide-containing class I molecules.
Studies of class I antigen processing have been greatly
facilitated by the availability of mutant cell lines with defects in this pathway. A number of cell lines with greatly reduced surface expression of MHC class I antigens have been isolated. In some of these lines, the class I genes are wild type in sequence and are transcribed normally. Furthermore, the resulting mRNA is translated efficiently, but stable class I molecules fail to be expressed at the cell surface. When infected by virus, these cells fail to serve as targets for virus-specific cytotoxic T lymphocytes (Cl%). Interestingly, however, appropriate synthetic peptides derived from viral protein sequences can both sensitize
MHC-linked
transport
protein
genes
Some of the cell lines with the antigen-processing defect described above have known deletions in the class II region of the MHC [4,5]. This portion of the MHC of mouse [!?*I, rat [lo*] and human [11*,12**] has recently been shown to contain two genes that belong to a superfamily of genes whose products mediate transport of a wide variety of substances across biological
Abbreviations ABC-ATP-binding casette; CT--cytotoxic T lymphocyte; ER-endoplasmic reticulum; hsp-heat-shock protein; WI-low molecular weight polypeptide; MHC-major histocompatibility complex; SRP--signal recognition particle. 70
@ Current Biology Ltd ISSN 0952-7915
Genes in the MHC that may affect antigen processing Monaco
membranes. In the mouse, these two genes have been named Hum-l and Hum-2. This superfamily is sometimes called the ‘ABC’(ATP-binding cassette> family because of the presence of a pair of highly conserved sequence motifs believed to form a protein domain capable of binding ATP. A large number of different genes belonging to the ABC family have been identified in both eukaryotic and prokaryotic organisms. Different members of the family are Involved in the transPort of molecules ranging in size from metal ions and small sugars to large (> 1OOkD) proteins. Other mam malian members of the ABC family include the multidrug resistance (mdr) genes, which protect cells from the actions of chemotherapeutic drugs by transporting the drugs out of the cell, and the cystic fibrosis (CFlip) gene (which transports chloride ions out of cells). A cloned cDNA derived from the human homologue of Ham-2 (called Y3 or PSF-I), restores MHC class I surface expression to a class I-deficient mutant cell line [ 13**], and Ham-2 cDNA corrects the defect in another such line [ 14**]. These data indicate that a defect in either Ham- 1 or Ham-2 alone results in a defect in antigen processing and loss of class I surface expression. The simplest interpretation of these results is that the products of the Ham-l and Hum-2 genes form a heterodimer that serves to transport peptide from the cytoplasm Into, presumably, the lumen of the endoplasmic reticulum (ER), where it associates with newly syntheSupport for this interpresized class I molecules [l&l. tation comes from studies in which a minigene corresponding to an epitope of influenza virus matrix protein was introduced Into wild-type (Tl) and the class I-deficient derivative (T2) cells [ 15**]. T2 cells contain a homozygous deletion of the region containing the two transporter genes, Both cell lines present the influenza peptide to class I-restricted CTLs if a classic leader sequence is engineered into the minigene immediately upstream of the influenza peptide. Thus, both cell lines possess all of the machinery required to present this peptide when it is introduced directly into the ER lumen (in this case the peptide is transported via the normal signal recognition particle (SRP)-dependent secretory pathway). However, when the minigene lacks the signal sequence, and hence the peptide is produced in the cytoplasm, Tl but not T2 cells present the peptide to CTL. This indicates that cytoplasmic peptides can in fact be transported into the 50
0
100
Mouse
Pb
Ma
Human
DPB
DMA
Mb.2
DMB
Mb1
secretory pathway in normal cells, independently of SRP, and that this transport is dependent on genes in the class II region of the MI-X.
MHC-linked
protease genes
The region of the MHC that contains the Hum-2 and Ham-2 genes also contains the genes for at least two of the subunits of a large cytoplasmic structure called the low molecular weight polypeptide (IMP) complex [l&18]. This structure has recently been shown to be closely related to a structure possessing multiple proteolytic activities, called the multicatalytic proteinase complex, or proteasome [lp*]. The genes encoding both of these subunits have recently been cloned (CK Martinez, JJ Monaco, unpublished data) [20*-22*]. One of these genes lies immediately upstream of each of the two transporter genes (see Fig. 1). Although there is currently no functional evidence linking the IMP complex to class I antigen processing, the fact that it resides in the cy toplasm, possesses proteolytic activity, and at least two of its subunits map to a region of DNA known to be Important for antigen processing, strongly suggests that this structure may be responsible for the conversion of native antigen Into the peptides that are transported into the ER for association with class I molecules. We have previously presented other arguments for the possible involvement of the LMP complex in antigen processing [ 19**,21*]. The unusual structure of the complex, coupled with in vitro analysis of the proteolytic function of the purified complex, led us to propose that this structure may be spectically designed to produce peptides tailored to the peptide binding site of MHC class I molecules [ 19.01.
The class II (exogenous) antigen-processing pathway It seems unlikely that any of the genes discussed above would be required in the class II antigen processing path way. In this pathway, antigen enters the cell within endocytic vesicles, and is presumed to travel to its ultimate 150
200
250
Eb
Ea
DRB
DRA
Fig. 1. Organization of the class II region of the mouse MHC. Class II genes are shown as light grey boxes, transporter genes are shown as dark grey boxes, and low molecular weight polypeptide (LMP) genes are shown as black boxes. The names given to the human homologues of each of the genes shown is indicated below the name of the mouse gene. The LMP gene positioned between the two transporter genes probably corresponds to the LMP-7 subunit, but this has not been proven. Map is approximately to scale.
71
72
Antigen recognition
destination by vesicular transport. It thus remains topologically outside the cell during processing, and there would be no reason to require transporters such as the Hum-l and Ham-2 gene products. Because processing in this pathway is believed to occur within the acidified environment of endosomal or lysosomal compartments (and is sensitive to agents that raise the pH of these compartments), it also seems unlikely that the IMP complex, with its cytoplasmic location and neutral pH optimum, would be used for antigen degradation in this pathway. However, there is evidence that genes required in the class II antigen processing pathway map to the MHC.
Class II antigen-processing-defective
cell lines
A number of mutated human B-cell lines, derived by negative selection with an anti-DR3 monoclonal antibocty plus complement, have been shown to have a class II antigen presentation defect [ 231. The phenotype of these cells closely parallels the phenotype of the class I mutants described above; they fail to present native antigens to class II-restricted T cells, but efficiently present the corresponding peptides. The class II heterodimer on the surface of these cells has lost the immunoselecting antigenic determinant, and is demonstrably less stable than its wild-type counterpart, even though the sequences of the HL4-DR CL-and P-chains are identical to those in the wildtype parental cell line. These results suggest that the defect resides in an inability to provide the class II molecule with peptides, analogous to the class I mutants. Recently, the defect in these cells was mapped to the class II region of the MI-K [ 241, the region that contains the Hum and Lmp genes. However, class I expression on these cells is normal and this fact, taken together with the arguments above, strongly suggests that the gene (or genes) responsible for the class II defect is linked to, but Merent from, any of the genes involved in the class I antigen processing pathway. The identity of the MHC-linked gene required for class II-restricted antigen processing is still unknown.
MHC-linked
Conclusion The past year has witnessed a resurgence of Interest in the MHC resulting from a growing realization that much of the machinery required for antigen processing may be encoded by genes within the MIX. Analysis of these genes has allowed the construction of what may be a fairly complete model of class I-restricted antigen processing [ 29”], and there are preliminary indications that many of the mysteries of the class II antigen processing pathway will yield to the same type of genetic approach.
Acknowledgements The author is supportedby grants from the National Institute of General Medical Sciences and the National Institute of AUergy and Infectious Diseases.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: . of special interest of outstanding interest .. 1.
TOWNSEND A, OH&N C, BASTINJ, LJUNGGREN HG, FOSTERL, K&RE K: Association of Class I Major Histocompatibility Heavy and Light Chains Induced by Viral Peptides. Nature 1989, 34Od43-448.
2.
HOSKENNA, BEVANMJ: Defective
3.
CERUNDOL~ V, ALEXANDER J, ANDERSON K, LAMBC, CRESSWELL P, MCMICHAEL A, GOTCH F, TOWNSEND A: Presentation of Viral Antigen Controlled by a Gene in the Major HistocompatibilIty Complex. Nature 1990, 345:44!k452.
4.
TOWNSEND A, EILIOTTT, CERUNDOLO V, FOSTERL, BARBERB, TSE A Assembly of MHC Class I Molecules Analyzed In Vitro. Cell 1990, 62~285.295.
5.
LJUNGGREN HG, SAM NJ, OH&N C, NEEF’J!XS JJ, HOGLUNDP, HEEME~S MT, BASTINJ, SCHUMACHER TNM, TOWNSEND A, KUm K, PLOEGHHL: Empty MHC Class I Molecules Come out ln
heat shock protein genes
Presentation of Endogenous Antigen by a Cell Line Expressing Class I Molecules. Science 1990, 248:367-370.
the Cold. Nature 1990, 346:476-W.
The MHC contains at least two heat shock protein (hsp) genes [ 251. Also linked to, but outside of, the MHC is an hsp84 gene [26]. Some hsp can bind peptides, and the peptidebinding domain of hsp70 can be modeled into a structure essentially identical to the known structure of the peptide-binding domain of HIA class I molecules [27]. Evidence exists for the involvement of members of the hsp70 family in class II-restricted antigen presentation [28] : Thus, it is possible that MHC-encoded hsp may be involved in the transport and/or protection from further degradation of peptides destined for class II molecules. It is also conceivable that MHC-encoded hsp could perform the same function(s) in the class I antigen processing pathway, as many of the prokaryotic members of the ABC transporter superfamily are dependent on soluble substrate binding proteins for the delivery of substrate to the transporter.
6.
SCHUMACHER TNM, HEEMEL~ MT, NEEFJEZS JJ, KAsT WM, MELIEF CJM, PLOEGHHL: Direct Binding of Peptide to Empty MHC
Class I Molecules on Intact CeIIs and In Vitro. Cefl 1990, 62:563-567. 7.
ELUO’ITT, CERUNDOL~V, ELVINJ, TOWNSEND A: Peptide-induced Conformational Change of the Class I Heavy Chain. Nature 1991, 351:402dl6.
8.
RICK KL, GRWM C, BENACERRAF B: Low Temperature and Peptides Favor the Formation of Class I Heterodimers on RMA-S Cells at the Cell Surfhce. Proc Nat1 Acud Sci U S A 1991, 8842OOA204.
9. ..
MONACO JJ, CHO S, AITAYAM: Transport protein genes in the murine MHC: Possible Implications for Antigen Processing. Science 1990, 250:1723-1726. Reports the identication of two genes (Hum- 1 and Hum-Z) in the class II region of the mouse MHC that, by sequence analysis, belong to a superfamily of genes involved in the transport of soluble molecules across cell membranes.
Genes in the MHC that may affectantigen processingMonaco 10. .
DEVERSONEv, Gow IR, COAD~EU WJ, MONACO JJ, BUTCHER GW, HOWARDJC: MHC Class II Region Encoding Proteins Related to the Multidrug Resistance Family of Transmembrane Transporters. Nature 1990, 348:7%741. The class Il region of the rat MHC contains two genes homologous fo the mouse Ham- 1 and Ham-2 transport protein genes. 11. .
TROW~DAIEJ, HANSON I, MOCKRIDGEI, BECK S, TOWNSENDA, KELLYA: Sequences Encoded in the Class II Region of the MHC Related to the ‘ABC’Superfamily of Transporters. Nature 1990, 348741-744. Reports the mapping and sequence of the RING4 gene, the human homologue of the mouse Hum- 1 gene. 12. ..
SPIEST, BRESNAHAN M, BAHRAMS, ARNOLDD, B~ANCKG, MELLINS
E, PIOUS D, DEMARSR: A Gene in the Human Major Histocompatibility Complex Class II Region Controlling the Class I Ant&n Presentation Pathway. Nature 1990, 3&:744-747. Describes the identification of a number of new genes in the class II region of the human MHC, including the two transporter genes. Also shows that a class I-deficient cell line speciiically lacks expression of the Y3, or PSFl, gene (the human homologue of the mouse Ham-l gene). SPIEST, DEMARSR: Restored Expression of Major Histocompatlbility Class I Molecules by Gene Transfer of a Putative Peptide Transporter. Nature 1991, 351:32%324. A cloned cDNA copy of the PSFl (Hum- 1) gene restores class I surface expression to the class I-deficient ,134 cell line. 13. ..
14. ..
ATTAYAM, JAMESONS, MARTINEZCK, HERMEL E, ALDRICH C, FORMANJ, FISCHERLINDAHLK, BEVANMJ, MONACOJJ: Hum-2 Corrects the Class I Antigen Processing Defect in RMA-S Cells. Nature 1992, 355647-649. A cloned cDNA copy of the Hum-2 gene restores class I surface expression and sensitivity to class I-restricted qtotoxic T lymphocytes in the RMAS cell line. Antigen processing for both classic (H-2K and D> and non-classic (Qal and HMT) class I molecules is restored.
J: GLYNNER, POXVISSH, BECK S, KELLYA, KERRL, TROW~DAW A Proteasome-related Gene Between the two ABC Transporter Loci ln the Class II Region of the Human MHC. Nature 1991, 353~357-360. Reports the cloning of a gene from the human MHC with sequence similarity to proteasome subunits.
20. .
MARTE%? CK, MONACO JJ: Homology of Proteasome Subunits to a Major Histocompatibillty Complex-linked LMP Gene. Nature 1991, 353:664X%7. Reports the cloning of the gene corresponding to one of the polymerphic LMP subunits (LMP-2) from the mouse MHC. The sequence of the LMP-2 cDNA matches a published rat proteasome peptide sequence. 21. .
KELLYA, Pow~s SH, GLYNNER, RAD~EYE, BECK S, TROW~DALE J: Second Proteasome-related Gene in the Human MHC Class II Region. Nature 1991, 353~667-668. Reports the cloning of the human homologue of the mouse Lmp2 22. .
iwe 23.
&tEUINS E, SMXH L, ARP B, COTN!ZRT, CELIS E, PIOUS D: Defective Processing and Presentation of Exogenous Antigens in Mutants with Normal HLA Class II Genes. Nuture 1990, 343:71-74.
24.
MEANS E, KEMPIN S, SMITH L, MONJI T, PIOUS D: Mutations Mapping to the Major Histocompatlbility Complex Affect Class II Rest&ted Antigen Presentation and the Surface Abundance of Class I Molecules. J Eap Med 1991, 174:1607-1615.
25.
SARGENT CA, DUNHAM I, TROWSDALEJ, CAMPBE~ RD: Human Major Hlstocompatlbility Complex Contains Genes for the Major Heat Shock Protein HSP70. Proc Nutl Acad Sci CJS A 1989, 86:196%-1972.
26.
ROMANOJW, SELDIN MF, APPELIA E: Linkage of the Mouse Hsp84 Heat Shock Protein Structural Gene to the H-2 Complex. Immunogenetics 1989, 29142-144.
27.
RIPPMANNF, TAYLORWR, RO~IBARD JB, GREEN NM: A Hypo thetical Model for the Peptide Binding Domain of hsp70 Based on tbe Peptide Binding Domain of HLA. EMBO J 1991, 10:1053-1059.
28.
VANBUSKIRK A, CRUMPBL, MARGOLIASH E, PIERCESK: A Peptide Binding Protein Having a Role in Antigen Presentation is a Member of the hsp70 Heat Shock Family. J E@ Med 1989, 170:1799-1809.
ANDERSON
K, CRESSXVELL P, GAMMON M, HERMESJ, WILUAM~ON A, ZWEEIUNKH: Endogenously Synthesized Peptide with an Endoplasmlc Reticulum Signal Sequence Sensitizes Antigen Processing Mutant Cells to Class I-restricted Cell-mediated Lysls. J E3Q) Med 1991, 1?4:48%92. Reports that normal cells can translocate preformed peptide from the cytoplasm into the secretory pathway for association with MHC class I molecules, and that this function requires genes in the class II region of the MHC. 15. ..
The MHC-linked LMP complex is closely related to a structure called the proteasome, which possesses multiple distinct proteolytic activities. Suggests that the LMP complex may be ideally suited to producing peptides for class I MHC molecules.
16.
MONACOJJ, McD~vrrr HO: Identification of a Fourth Class of Proteins Linked to tbe Murine Major Histocompatibility Complex. Proc Nat1 Acud Sci Cl S A 1982, 79:300-3005.
17.
MONACOJJ, McDm HO: H-2-linked Low-molecular Weight Assemble Into an Unusual MacroPolypeptide Antigens molecular Complex. Nature 1984, 309:797-799.
18.
MONACOJJ, McD~vrrr HO: The LMP antigens: A Stable MHCcontrolled Multisubunit Protein Complex. Hum Immunol 1986, 15:416426.
19. ..
BROWN MG, DRISCOLLJ, MONACO JJ: Structural and Serological Slmllarity of MHC-linked LMP and Proteasome (Multicatalytic Protelnase) Complexes. Nature 1991, 353:355-357.
MONACO JJ: A Molecular Model of MHC Class I-restricted 29. .. Antigen Processing. Immunol Toady 1992, in press. Presents a comprehensive molecular model of the events occurring during antigen processing for class I-restricted T-cell responses.
JJ Monaco, Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Box 678 MCV Station, Richmond, Virginia 23298.0678, USA
73
Commonwealth
University,
Richmond,
Virginia,
USA
Recent evidence indicates that genes required for antigen processing are present in the class II region of the MHC. Two new classes of MHC genes that may encode much of the machinery required in the class I (endogenous) antigen-processing pathway have been discovered. There is evidence
that
genes
required
in the
class II pathway
also reside
in this
region.
Current
Opinion
in Immunology
these class I-deficient mutants to CTL lysis and induce increased MIX class I surface expression [l-3 1.
Introduction T-cell recognition of antigen requires that the antigen first be ‘processed’ by an appropriate antigen-presenting cell. Peptide fragments derived from this antigenprocessing event are bound by MHC class I or MHC class II molecules. As has been reviewed elsewhere in this volume (Unanue, this issue, pp 63-69 and Bra&ale, this issue, pp 5!9-62), the source and the characteristics (i.e. sequence and length) of the peptides found associated with class I and class II molecules differs. This reflects the existence of two largely or completely separate antigen-processing pathways within the cell. Peptides derived from cytoplasmic antigens (endogenous antigens) feed class I molecules, whereas peptides derived from antigens taken up from the extracellular fluid (exogenous antigens) feed class II molecules. However, the exact molecular mechanisms underlying the proteolytic and subsequent peptide-handling events in either pathway are at present largely unknown.
The class I (endogenous)
1992, 4170-73.
antigen-processing
pathway
The fact that peptides can reverse the class I defect in
mutant cells suggests that peptide is an indispensable component of stable class I molecules, and that the phenotypic defect in these mutants results fi;om an inability to provide the MHC class I molecules with appropriate peptides. This phenotype is consistent with a defect in either the ability to generate appropriate peptides from native antigen, or in the ability to allow the extracellular portion of the class I molecule access to the peptide (i.e. a defect in the ability to transport peptides from the cytoplasm into the secretory pathway). Further analysis of these mutants indicated that such defects in the ability to provide peptide to MHC class I molecules result in the formation of class I heterodimers of heavy chain plus pz microglobulin that are unstable at 37°C (but not at 26°C) [4-8]. At the cell surface, these dimers dissociate, pz microglobulin is released into the medium, and the heavy chain undergoes a rapid conformational change resulting in loss of reactivity with anti-class I antibodies. Addition of appropriate peptides and an exogenous source of p2 microglobulin allows spontaneous assembly of stable peptide-containing class I molecules.
Studies of class I antigen processing have been greatly
facilitated by the availability of mutant cell lines with defects in this pathway. A number of cell lines with greatly reduced surface expression of MHC class I antigens have been isolated. In some of these lines, the class I genes are wild type in sequence and are transcribed normally. Furthermore, the resulting mRNA is translated efficiently, but stable class I molecules fail to be expressed at the cell surface. When infected by virus, these cells fail to serve as targets for virus-specific cytotoxic T lymphocytes (Cl%). Interestingly, however, appropriate synthetic peptides derived from viral protein sequences can both sensitize
MHC-linked
transport
protein
genes
Some of the cell lines with the antigen-processing defect described above have known deletions in the class II region of the MHC [4,5]. This portion of the MHC of mouse [!?*I, rat [lo*] and human [11*,12**] has recently been shown to contain two genes that belong to a superfamily of genes whose products mediate transport of a wide variety of substances across biological
Abbreviations ABC-ATP-binding casette; CT--cytotoxic T lymphocyte; ER-endoplasmic reticulum; hsp-heat-shock protein; WI-low molecular weight polypeptide; MHC-major histocompatibility complex; SRP--signal recognition particle. 70
@ Current Biology Ltd ISSN 0952-7915
Genes in the MHC that may affect antigen processing Monaco
membranes. In the mouse, these two genes have been named Hum-l and Hum-2. This superfamily is sometimes called the ‘ABC’(ATP-binding cassette> family because of the presence of a pair of highly conserved sequence motifs believed to form a protein domain capable of binding ATP. A large number of different genes belonging to the ABC family have been identified in both eukaryotic and prokaryotic organisms. Different members of the family are Involved in the transPort of molecules ranging in size from metal ions and small sugars to large (> 1OOkD) proteins. Other mam malian members of the ABC family include the multidrug resistance (mdr) genes, which protect cells from the actions of chemotherapeutic drugs by transporting the drugs out of the cell, and the cystic fibrosis (CFlip) gene (which transports chloride ions out of cells). A cloned cDNA derived from the human homologue of Ham-2 (called Y3 or PSF-I), restores MHC class I surface expression to a class I-deficient mutant cell line [ 13**], and Ham-2 cDNA corrects the defect in another such line [ 14**]. These data indicate that a defect in either Ham- 1 or Ham-2 alone results in a defect in antigen processing and loss of class I surface expression. The simplest interpretation of these results is that the products of the Ham-l and Hum-2 genes form a heterodimer that serves to transport peptide from the cytoplasm Into, presumably, the lumen of the endoplasmic reticulum (ER), where it associates with newly syntheSupport for this interpresized class I molecules [l&l. tation comes from studies in which a minigene corresponding to an epitope of influenza virus matrix protein was introduced Into wild-type (Tl) and the class I-deficient derivative (T2) cells [ 15**]. T2 cells contain a homozygous deletion of the region containing the two transporter genes, Both cell lines present the influenza peptide to class I-restricted CTLs if a classic leader sequence is engineered into the minigene immediately upstream of the influenza peptide. Thus, both cell lines possess all of the machinery required to present this peptide when it is introduced directly into the ER lumen (in this case the peptide is transported via the normal signal recognition particle (SRP)-dependent secretory pathway). However, when the minigene lacks the signal sequence, and hence the peptide is produced in the cytoplasm, Tl but not T2 cells present the peptide to CTL. This indicates that cytoplasmic peptides can in fact be transported into the 50
0
100
Mouse
Pb
Ma
Human
DPB
DMA
Mb.2
DMB
Mb1
secretory pathway in normal cells, independently of SRP, and that this transport is dependent on genes in the class II region of the MI-X.
MHC-linked
protease genes
The region of the MHC that contains the Hum-2 and Ham-2 genes also contains the genes for at least two of the subunits of a large cytoplasmic structure called the low molecular weight polypeptide (IMP) complex [l&18]. This structure has recently been shown to be closely related to a structure possessing multiple proteolytic activities, called the multicatalytic proteinase complex, or proteasome [lp*]. The genes encoding both of these subunits have recently been cloned (CK Martinez, JJ Monaco, unpublished data) [20*-22*]. One of these genes lies immediately upstream of each of the two transporter genes (see Fig. 1). Although there is currently no functional evidence linking the IMP complex to class I antigen processing, the fact that it resides in the cy toplasm, possesses proteolytic activity, and at least two of its subunits map to a region of DNA known to be Important for antigen processing, strongly suggests that this structure may be responsible for the conversion of native antigen Into the peptides that are transported into the ER for association with class I molecules. We have previously presented other arguments for the possible involvement of the LMP complex in antigen processing [ 19**,21*]. The unusual structure of the complex, coupled with in vitro analysis of the proteolytic function of the purified complex, led us to propose that this structure may be spectically designed to produce peptides tailored to the peptide binding site of MHC class I molecules [ 19.01.
The class II (exogenous) antigen-processing pathway It seems unlikely that any of the genes discussed above would be required in the class II antigen processing path way. In this pathway, antigen enters the cell within endocytic vesicles, and is presumed to travel to its ultimate 150
200
250
Eb
Ea
DRB
DRA
Fig. 1. Organization of the class II region of the mouse MHC. Class II genes are shown as light grey boxes, transporter genes are shown as dark grey boxes, and low molecular weight polypeptide (LMP) genes are shown as black boxes. The names given to the human homologues of each of the genes shown is indicated below the name of the mouse gene. The LMP gene positioned between the two transporter genes probably corresponds to the LMP-7 subunit, but this has not been proven. Map is approximately to scale.
71
72
Antigen recognition
destination by vesicular transport. It thus remains topologically outside the cell during processing, and there would be no reason to require transporters such as the Hum-l and Ham-2 gene products. Because processing in this pathway is believed to occur within the acidified environment of endosomal or lysosomal compartments (and is sensitive to agents that raise the pH of these compartments), it also seems unlikely that the IMP complex, with its cytoplasmic location and neutral pH optimum, would be used for antigen degradation in this pathway. However, there is evidence that genes required in the class II antigen processing pathway map to the MHC.
Class II antigen-processing-defective
cell lines
A number of mutated human B-cell lines, derived by negative selection with an anti-DR3 monoclonal antibocty plus complement, have been shown to have a class II antigen presentation defect [ 231. The phenotype of these cells closely parallels the phenotype of the class I mutants described above; they fail to present native antigens to class II-restricted T cells, but efficiently present the corresponding peptides. The class II heterodimer on the surface of these cells has lost the immunoselecting antigenic determinant, and is demonstrably less stable than its wild-type counterpart, even though the sequences of the HL4-DR CL-and P-chains are identical to those in the wildtype parental cell line. These results suggest that the defect resides in an inability to provide the class II molecule with peptides, analogous to the class I mutants. Recently, the defect in these cells was mapped to the class II region of the MI-K [ 241, the region that contains the Hum and Lmp genes. However, class I expression on these cells is normal and this fact, taken together with the arguments above, strongly suggests that the gene (or genes) responsible for the class II defect is linked to, but Merent from, any of the genes involved in the class I antigen processing pathway. The identity of the MHC-linked gene required for class II-restricted antigen processing is still unknown.
MHC-linked
Conclusion The past year has witnessed a resurgence of Interest in the MHC resulting from a growing realization that much of the machinery required for antigen processing may be encoded by genes within the MIX. Analysis of these genes has allowed the construction of what may be a fairly complete model of class I-restricted antigen processing [ 29”], and there are preliminary indications that many of the mysteries of the class II antigen processing pathway will yield to the same type of genetic approach.
Acknowledgements The author is supportedby grants from the National Institute of General Medical Sciences and the National Institute of AUergy and Infectious Diseases.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: . of special interest of outstanding interest .. 1.
TOWNSEND A, OH&N C, BASTINJ, LJUNGGREN HG, FOSTERL, K&RE K: Association of Class I Major Histocompatibility Heavy and Light Chains Induced by Viral Peptides. Nature 1989, 34Od43-448.
2.
HOSKENNA, BEVANMJ: Defective
3.
CERUNDOL~ V, ALEXANDER J, ANDERSON K, LAMBC, CRESSWELL P, MCMICHAEL A, GOTCH F, TOWNSEND A: Presentation of Viral Antigen Controlled by a Gene in the Major HistocompatibilIty Complex. Nature 1990, 345:44!k452.
4.
TOWNSEND A, EILIOTTT, CERUNDOLO V, FOSTERL, BARBERB, TSE A Assembly of MHC Class I Molecules Analyzed In Vitro. Cell 1990, 62~285.295.
5.
LJUNGGREN HG, SAM NJ, OH&N C, NEEF’J!XS JJ, HOGLUNDP, HEEME~S MT, BASTINJ, SCHUMACHER TNM, TOWNSEND A, KUm K, PLOEGHHL: Empty MHC Class I Molecules Come out ln
heat shock protein genes
Presentation of Endogenous Antigen by a Cell Line Expressing Class I Molecules. Science 1990, 248:367-370.
the Cold. Nature 1990, 346:476-W.
The MHC contains at least two heat shock protein (hsp) genes [ 251. Also linked to, but outside of, the MHC is an hsp84 gene [26]. Some hsp can bind peptides, and the peptidebinding domain of hsp70 can be modeled into a structure essentially identical to the known structure of the peptide-binding domain of HIA class I molecules [27]. Evidence exists for the involvement of members of the hsp70 family in class II-restricted antigen presentation [28] : Thus, it is possible that MHC-encoded hsp may be involved in the transport and/or protection from further degradation of peptides destined for class II molecules. It is also conceivable that MHC-encoded hsp could perform the same function(s) in the class I antigen processing pathway, as many of the prokaryotic members of the ABC transporter superfamily are dependent on soluble substrate binding proteins for the delivery of substrate to the transporter.
6.
SCHUMACHER TNM, HEEMEL~ MT, NEEFJEZS JJ, KAsT WM, MELIEF CJM, PLOEGHHL: Direct Binding of Peptide to Empty MHC
Class I Molecules on Intact CeIIs and In Vitro. Cefl 1990, 62:563-567. 7.
ELUO’ITT, CERUNDOL~V, ELVINJ, TOWNSEND A: Peptide-induced Conformational Change of the Class I Heavy Chain. Nature 1991, 351:402dl6.
8.
RICK KL, GRWM C, BENACERRAF B: Low Temperature and Peptides Favor the Formation of Class I Heterodimers on RMA-S Cells at the Cell Surfhce. Proc Nat1 Acud Sci U S A 1991, 8842OOA204.
9. ..
MONACO JJ, CHO S, AITAYAM: Transport protein genes in the murine MHC: Possible Implications for Antigen Processing. Science 1990, 250:1723-1726. Reports the identication of two genes (Hum- 1 and Hum-Z) in the class II region of the mouse MHC that, by sequence analysis, belong to a superfamily of genes involved in the transport of soluble molecules across cell membranes.
Genes in the MHC that may affectantigen processingMonaco 10. .
DEVERSONEv, Gow IR, COAD~EU WJ, MONACO JJ, BUTCHER GW, HOWARDJC: MHC Class II Region Encoding Proteins Related to the Multidrug Resistance Family of Transmembrane Transporters. Nature 1990, 348:7%741. The class Il region of the rat MHC contains two genes homologous fo the mouse Ham- 1 and Ham-2 transport protein genes. 11. .
TROW~DAIEJ, HANSON I, MOCKRIDGEI, BECK S, TOWNSENDA, KELLYA: Sequences Encoded in the Class II Region of the MHC Related to the ‘ABC’Superfamily of Transporters. Nature 1990, 348741-744. Reports the mapping and sequence of the RING4 gene, the human homologue of the mouse Hum- 1 gene. 12. ..
SPIEST, BRESNAHAN M, BAHRAMS, ARNOLDD, B~ANCKG, MELLINS
E, PIOUS D, DEMARSR: A Gene in the Human Major Histocompatibility Complex Class II Region Controlling the Class I Ant&n Presentation Pathway. Nature 1990, 3&:744-747. Describes the identification of a number of new genes in the class II region of the human MHC, including the two transporter genes. Also shows that a class I-deficient cell line speciiically lacks expression of the Y3, or PSFl, gene (the human homologue of the mouse Ham-l gene). SPIEST, DEMARSR: Restored Expression of Major Histocompatlbility Class I Molecules by Gene Transfer of a Putative Peptide Transporter. Nature 1991, 351:32%324. A cloned cDNA copy of the PSFl (Hum- 1) gene restores class I surface expression to the class I-deficient ,134 cell line. 13. ..
14. ..
ATTAYAM, JAMESONS, MARTINEZCK, HERMEL E, ALDRICH C, FORMANJ, FISCHERLINDAHLK, BEVANMJ, MONACOJJ: Hum-2 Corrects the Class I Antigen Processing Defect in RMA-S Cells. Nature 1992, 355647-649. A cloned cDNA copy of the Hum-2 gene restores class I surface expression and sensitivity to class I-restricted qtotoxic T lymphocytes in the RMAS cell line. Antigen processing for both classic (H-2K and D> and non-classic (Qal and HMT) class I molecules is restored.
J: GLYNNER, POXVISSH, BECK S, KELLYA, KERRL, TROW~DAW A Proteasome-related Gene Between the two ABC Transporter Loci ln the Class II Region of the Human MHC. Nature 1991, 353~357-360. Reports the cloning of a gene from the human MHC with sequence similarity to proteasome subunits.
20. .
MARTE%? CK, MONACO JJ: Homology of Proteasome Subunits to a Major Histocompatibillty Complex-linked LMP Gene. Nature 1991, 353:664X%7. Reports the cloning of the gene corresponding to one of the polymerphic LMP subunits (LMP-2) from the mouse MHC. The sequence of the LMP-2 cDNA matches a published rat proteasome peptide sequence. 21. .
KELLYA, Pow~s SH, GLYNNER, RAD~EYE, BECK S, TROW~DALE J: Second Proteasome-related Gene in the Human MHC Class II Region. Nature 1991, 353~667-668. Reports the cloning of the human homologue of the mouse Lmp2 22. .
iwe 23.
&tEUINS E, SMXH L, ARP B, COTN!ZRT, CELIS E, PIOUS D: Defective Processing and Presentation of Exogenous Antigens in Mutants with Normal HLA Class II Genes. Nuture 1990, 343:71-74.
24.
MEANS E, KEMPIN S, SMITH L, MONJI T, PIOUS D: Mutations Mapping to the Major Histocompatlbility Complex Affect Class II Rest&ted Antigen Presentation and the Surface Abundance of Class I Molecules. J Eap Med 1991, 174:1607-1615.
25.
SARGENT CA, DUNHAM I, TROWSDALEJ, CAMPBE~ RD: Human Major Hlstocompatlbility Complex Contains Genes for the Major Heat Shock Protein HSP70. Proc Nutl Acad Sci CJS A 1989, 86:196%-1972.
26.
ROMANOJW, SELDIN MF, APPELIA E: Linkage of the Mouse Hsp84 Heat Shock Protein Structural Gene to the H-2 Complex. Immunogenetics 1989, 29142-144.
27.
RIPPMANNF, TAYLORWR, RO~IBARD JB, GREEN NM: A Hypo thetical Model for the Peptide Binding Domain of hsp70 Based on tbe Peptide Binding Domain of HLA. EMBO J 1991, 10:1053-1059.
28.
VANBUSKIRK A, CRUMPBL, MARGOLIASH E, PIERCESK: A Peptide Binding Protein Having a Role in Antigen Presentation is a Member of the hsp70 Heat Shock Family. J E@ Med 1989, 170:1799-1809.
ANDERSON
K, CRESSXVELL P, GAMMON M, HERMESJ, WILUAM~ON A, ZWEEIUNKH: Endogenously Synthesized Peptide with an Endoplasmlc Reticulum Signal Sequence Sensitizes Antigen Processing Mutant Cells to Class I-restricted Cell-mediated Lysls. J E3Q) Med 1991, 1?4:48%92. Reports that normal cells can translocate preformed peptide from the cytoplasm into the secretory pathway for association with MHC class I molecules, and that this function requires genes in the class II region of the MHC. 15. ..
The MHC-linked LMP complex is closely related to a structure called the proteasome, which possesses multiple distinct proteolytic activities. Suggests that the LMP complex may be ideally suited to producing peptides for class I MHC molecules.
16.
MONACOJJ, McD~vrrr HO: Identification of a Fourth Class of Proteins Linked to tbe Murine Major Histocompatibility Complex. Proc Nat1 Acud Sci Cl S A 1982, 79:300-3005.
17.
MONACOJJ, McDm HO: H-2-linked Low-molecular Weight Assemble Into an Unusual MacroPolypeptide Antigens molecular Complex. Nature 1984, 309:797-799.
18.
MONACOJJ, McD~vrrr HO: The LMP antigens: A Stable MHCcontrolled Multisubunit Protein Complex. Hum Immunol 1986, 15:416426.
19. ..
BROWN MG, DRISCOLLJ, MONACO JJ: Structural and Serological Slmllarity of MHC-linked LMP and Proteasome (Multicatalytic Protelnase) Complexes. Nature 1991, 353:355-357.
MONACO JJ: A Molecular Model of MHC Class I-restricted 29. .. Antigen Processing. Immunol Toady 1992, in press. Presents a comprehensive molecular model of the events occurring during antigen processing for class I-restricted T-cell responses.
JJ Monaco, Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Box 678 MCV Station, Richmond, Virginia 23298.0678, USA
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