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DM and DO shape the repertoire of peptide–MHC-class-II complexes
DM and DO shape the repertoire of peptide–MHC-class-II complexes Lars Karlsson The presentation of antigenic peptides by MHC class II molecules is essential for activation of CD4+ T cells. The formation of most peptide–MHC-class-II complexes is influenced by the actions of two specialized accessory proteins — DM and DO — located in the endosomal/ lysosomal system where peptide loading occurs. DM removes class-II-associated invariant-chain peptide (CLIP) from newly synthesized class II molecules, but by now it is clearly established that this is only a special case of the general peptide-editing function of DM. Recent data have begun to explain the molecular basis for the editing activity. The other accessory protein, DO, modulates DM activity in vitro, but the physiological importance of DO is unclear. New evidence from several laboratories has provided clues that may soon change this. Addresses Johnson & Johnson Pharmaceutical Research and Development, 3210 Merryfield Row, San Diego, CA 92121, USA Corresponding author: Karlsson, Lars ([email protected])
Current Opinion in Immunology 2005, 17:65–70 This review comes from a themed issue on Antigen processing and recognition Edited by Clifford V Harding and Jacques Neefjes Available online 8th December 2004
processing and assembly of peptide-loaded MHC class II molecules occur [1]. The endosomal/lysosomal system is rich in proteases, particularly cysteine proteases, and these enzymes are crucial both for the generation of peptides from antigenic proteins, and for the removal of Ii from class II molecules. CLIP is the final fragment of Ii and is generated by the protease cathepsin S [2]; CLIP is not spontaneously released from class II molecules in most cases, and exchange of this fragment for antigenic peptides is mediated by the accessory protein DM (HLA-DM in humans, H2-DM [previously termed H2-M] in the mouse) [3,4]. In B cells, but not in other types of APCs, a substantial fraction of DM is associated with another accessory protein, DO (HLA-DO in humans, H2-O in the mouse; here collectively referred to as DO, except when data suggest that species differences may exist) [5]. DO has been shown to modulate the function of DM in vitro, but there has been some confusion about how this affects the outcome of the peptide-loading process. DM and DO properties have been reviewed extensively previously [6–8] and here I will focus on recent research progress in this area.
DM — editor in chief
Abbreviations APC antigen-presenting cell BCR B cell receptor CLIP class-II-associated Ii peptide ER endoplasmic reticulum Ii invariant chain
DM was initially identified because cells that lacked this protein expressed high levels of CLIP–MHC-class-II complexes at their cell surface, and as a consequence had an altered reactivity with certain class II reactive monoclonal antibodies [9]. DM is mainly localized in the vesicles of the endosomal/lysosomal system of APCs, where it interacts with newly synthesized class II molecules, and catalyzes the release of CLIP from the peptidebinding groove. DM activity is not restricted to CLIP release, however, and it is now clear this is a special case of a more general ability of DM to edit the repertoire of class-II-associated peptides [10,11].
Introduction
Interaction with class II molecules and substrate specificity
MHC class II molecules associate with the invariant chain (Ii) in the endoplasmic reticulum (ER) of antigen-presenting cells (APCs), with the CLIP (class-II-associated Ii peptide) region of Ii occupying the peptide-binding groove of the class II a–b heterodimer. This association serves both to prevent peptide binding to the class II molecules within the ER and to direct class II molecules into the endosomal/lysosomal pathway, where antigen
How DM interacts with class II molecules is still not fully understood at the molecular level, and no crystal structure of the complex has been published to date. Initial observations of HLA-DR (DR) glycosylation mutants suggested that DM bound to the area around the P1 pocket, that is the area of the DR molecule where the amino termini of bound peptides reside [12,13]. Subsequent mutational studies have confirmed that this is
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Current Opinion in Immunology 2005, 17:65–70
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indeed the DR area facing DM [14], and the corresponding area on DM has been mapped using the same technique [15]. Further evidence that the area around the P1 pocket interacts with DM comes from a study where DM was directly coupled to a high affinity DR-binding peptide [16]. Only when DM was linked to the amino terminus of the peptide was there a striking increase in the rate of peptide release from the DR molecule, whereas carboxy-terminal fusions did not give this effect. In general, DM increases the rate of peptide exchange compared with the uncatalyzed state. Early experiments suggested that the rate of peptide dissociation from class II molecules in the presence of DM was directly proportional to the intrinsic rate of dissociation in the uncatalyzed state [11,17]. This conclusion was reached mainly using known class II ligand-peptides where the main chain was kept constant, while the side chains were being altered. More recent data from several laboratories [18,19,20] challenged the view that DM activity is independent of the peptide sequence, and it now appears that the degree to which DM increases peptide exchange is dependent on the structural features of the particular peptide–MHCclass-II complex [19,20]. Thus, Belmares et al. [19] analyzed the dissociation kinetics of 36 different DR– peptide complexes. They found large variations in DM susceptibility for different complexes and concluded that interactions throughout the peptide-binding groove contributed to these differences. Stratikos et al. [20] used variant peptides that lacked the capacity to form one or more of the conserved hydrogen bonds that are the basis for peptide binding to class II molecules. They also found that DM increased the dissociation rate of some complexes much more than of others, and related this to the inability to form hydrogen bonds between the class II molecule and the amino-terminal part of the peptide. DM also appears to differentially influence peptide association [19,21], but this is a less studied phenomenon than that of peptide dissociation. Practically, the lack of a direct correlation between DMcatalyzed and intrinsic peptide-exchange is likely to make useful predictions of which class-II-binding peptides are actually going to be presented at the surface of APCs even more difficult than previously thought.
Importance of DM for antigen presentation and immune responses Because CLIP release was the most apparent function of DM, it was initially thought that DM would have little effect on peptide loading of class II molecules that have low affinity for CLIP, and early experiments did indeed suggest this was the case [22], although DM had been shown to be able to influence both the release and loading Current Opinion in Immunology 2005, 17:65–70
of non-CLIP peptides in vitro [10]. DM-knockout mice, generated in H2-Ab mice, did not provide much clarity in this respect, since the affinity of H2-Ab for CLIP is so high that CLIP-free H2-Ab molecules are virtually undetectable in cells from these mice using biochemical methods, and the ability of these cells to present intracellularly processed antigen is minimal [23,24]. Somewhat surprisingly, DM deficient H2-Ab-expressing mice were still able to elicit IgG responses to some antigens, although antibody titers were lower than in wild-type littermates and affinity maturation did not seem to occur [24]. By contrast, transgenic expression of H2-Eb/d (which has lower affinity for CLIP than H2-Ab) in the DM-deficient mice largely restored the antibody responses, even though antigen presentation by H2-Eb/d was essentially undetectable. Recent data show that, also in the case of H2-Ab, removal of CLIP may be only the most apparent activity of DM. Expression of Ii transgenes where the CLIP region had either high or low affinity for H2-Ab resulted in very similar phenotypes with respect to antigen presentation in mice expressing DM, even though the low-affinity CLIP dissociated much more rapidly from the class II molecules than the high-affinity CLIP did [25]. In the absence of DM, the class II molecules in cells expressing Ii with lowaffinity CLIP had a more diverse peptide repertoire, but lower cell surface expression of class II molecules than the cells expressing high-affinity CLIP had. These data show that DM promotes the formation of long-lived H2-Ab– peptide complexes (resulting in higher expression at the cell surface), most likely by maintaining class II molecules in a peptide-receptive state after CLIP release. Low affinity for CLIP has been suggested to be a possible contributing factor for the development of autoimmunity, particularly in rheumatoid arthritis [26]. No evidence for increased autoimmunity was seen in the mice expressing low-affinity CLIP [25], but further studies will be necessary to exclude the possibility that CLIP affinity is correlated to disease, since genetic factors other than class II haplotype (and thus CLIP affinity) influence the development of autoimmunity. The importance of DM for peptide acquisition by other class II molecules was recently more directly addressed through the generation of DM-deficient H2d and H2k haplotype mice [27,28]. The data generated using these mice suggest that both H2-Ad (which binds CLIP relatively strongly) and H2-Ak (which has low affinity for CLIP) are dependent on DM for acquisition of endogenously processed peptides. By contrast, both H2-Ed and H2-Ek seem to be less affected by the absence of DM, suggesting that although DM clearly influences the peptide repertoire beyond CLIP removal, the relative importance for molding the normal peptide repertoire depends on which class II molecule is analyzed. www.sciencedirect.com
DM and DO shape pMHC II complexes Karlsson 67
Peptide immunization has been reported to result in the formation of two types of T cells, type A and type B respectively, which recognize distinct conformations of the same class-II–peptide complexes [29]. Type A cells recognize peptide–class-II complexes that have been formed after endogenous processing of whole antigen, whereas type B cells only recognize complexes containing exogenously provided peptide. A recent publication has demonstrated that resistance or sensitivity of the different class-II–peptide conformations to editing by DM explains the phenomenon [30]. The complexes recognized by type A T cells were formed in lysosome-like compartments, and have thus been subjected to (and survived) the editing activity of DM. By contrast, the complexes recognized by type B T cells were not formed (or did not survive) in lysosomes, and were sensitive to DM activity. The physiological implications of these findings are not yet clear, although the phenomenon of type A and type B T cell reactivity is likely to have implications for the development of peptide vaccines, and may be important for the development of autoimmunity, although this has not been tested experimentally. Altogether, presently available data show that although differences exist between different class II molecules, DM activity is important both for CLIP removal in the cases where CLIP–class-II affinity is high, and for the formation of the normal class-II-associated peptide repertoire displayed at the cell surface.
DO — coeditor While the effect of DM on antigen presentation is becoming increasingly clear, the function of DO has remained obscure. Like DM, DO is a nonpolymorphic intracellular class-II-like molecule that is evolutionary conserved. However, transcriptional control of DO expression is different from that of other class II molecules (including DM) and DO has a more restricted expression pattern, with expression being limited to B cells and thymic epithelial cells. The transcriptional basis for these differences is only partially understood, but it was previously assumed that expression of the DO b chain (but not the a chain) was independent of CIITA [31,32], the class II transcriptional transactivator that controls both class II and DM expression. Recent studies have explored this issue further and it is now clear that there is a basal DO b expression in B cells, but that CIITA can increase the expression level [33–35]. The factors that control basal DO b expression in B cells have not been identified. Mature DO protein is localized in endosomal/lysosomal compartments, but newly synthesized DO is unstable and normally requires physical association with DM to exit the ER [5]. The association between DM and DO is tight and it is not clear if the two molecules dissociate from each other under steady-state conditions even after the DM–DO complexes have reached endosomes or lysowww.sciencedirect.com
somes. To date it is also not clear whether DM–DO complexes directly interact with class II molecules (although this has been suggested [36]), or whether the complexes dissociate to release free DM and DO that may interact with class II molecules. In addition to the association with DM, a recent report shows that DO can associate with free MHC class I heavy chains to form complexes that can be detected in small amounts at the cell surface [37], but the function (if any) of this complex is unknown.
Effects of DO on antigen processing and presentation The function of DO at the molecular level has been debated; thus initial studies suggested that DO either inhibited [38–40] or enhanced [36] the DM-mediated exchange of class-II-associated peptides. The reasons for these differences are not entirely clear, but more recent studies generally support the notion that DO inhibits DM function. Increased H2-O expression (achieved by transfection) has been shown to decrease presentation of several antigenic epitopes from processed proteins [41,42], whereas decreased H2-O expression (achieved by transfection of an antisense ribozyme [41]) had the opposite effect. One of these studies also found that splenocytes from mice overexpressing H2-O under a class I promoter had a decreased ability to present epitopes from protein antigens [41]. Another study analyzed the phenotype of mice and cells from transgenic mice that expressed human HLA-DO under a dendritic-cell-specific promoter [43]. In this study the effect on antigen presentation was variable, but the surface expression of H2-Ab–CLIP was increased, while the expression of other peptide–class-II complexes analyzed was decreased. The increase in H2-Ab–CLIP expression in HLA-DO transgenic mice is somewhat unexpected, as surface H2Ab–CLIP levels are unchanged both in H2-O-transgenic mice and in H2-O-deficient mice [40,44]. The basis for this difference is unclear, but may be related to the expression level of DO, the fact that dendritic cells rather than B cells were analyzed, or to the possibility the human HLA-DO and mouse H2-O may interact differently with H2-DM. In human cells, in contrast to mouse cells, there appears to be very good correlation between the level of DO expression and the amount of CLIP–class-II complexes expressed at the cell surface [45–47]. Several studies have shown that the effect of DO on DM is pH dependent, so that DM-mediated peptideexchange activity in the presence of DO is relatively higher at low pH (corresponding to lysosomes) than at higher pH (corresponding to earlier endosomal compartments) [5,36,41,48]. This finding, together with early antigen presentation studies did lead to the suggestion that DO expression may promote presentation of antigen Current Opinion in Immunology 2005, 17:65–70
68 Antigen processing and recognition
internalized by the B cell receptor (BCR) over antigens internalized by fluid-phase uptake (see [6] for a review). The experimental data available so far only partially support the hypothesis that DO favors presentation of antigen taken up by the BCR. Thus, extended analysis of antigen processing using DO (i.e. H2-O) deficient mice showed that presentation of most antigenic epitopes tested after fluid-phase uptake of antigen was not influenced by the presence or absence of DO whether presented by H2-Ab or H2-Eb/d [49,50]. By contrast, antigens specifically internalized by transgenic hapten-specific BCRs were strongly affected by DO. Most epitopes from a given haptenated antigen were presented better in the presence of DO, but this was highly dependent on which receptor was internalizing the antigen, and some epitopes were actually better presented in the absence of DO [49]. Further studies with BCRs directed against protein antigens (rather than haptens) may be necessary to allow more generalized conclusions about how DO affects presentation after BCR-mediated antigen uptake.
Physiological function of DO The notion that DO function is related to antigen processing after antigen uptake by the BCR is indirectly supported by several recent reports showing that germinal center B cells have low levels of DO expression compared with naı¨ve and memory B cells [45–47]. Although DM expression is also decreased somewhat in the germinal center B cells compared with the naı¨ve and memory B cells, the DM:DO ratio is increased and results in decreased CLIP–class-II expression, suggesting that the antigen-processing capacity is increased in germinal center B cells relative to the other B cell types. The downmodulation of DO in germinal center B cells is likely to be a reflection of signaling events occurring during the germinal center reaction. Supporting this conclusion is the fact that several groups have reported that stimulation of naı¨ve B cells through CD40 [47,51] or the BCR [47,52], or by pharmacological treatment with a phorbol ester (PMA), or a protein kinase C (PKC) inhibitor [52], does result in decreased DO expression (although DM expression is also decreased to a lesser extent by some of these treatments). DO downregulation after stimulation occurs over several hours to overnight, and could in the case of the PKC inhibitor be inhibited by chloroquine [52], suggesting that degradation in endosomes or lysosomes is the reason for the stimulationinduced DO decrease. This conclusion is also supported by the finding that there was no decrease in DO gene transcription in germinal center B cells compared with naı¨ve B cells [45]. The B cell selection process that occurs in germinal centers is driven by BCR affinity for the relevant antigen and results in the development of memory B cells and Current Opinion in Immunology 2005, 17:65–70
plasma cells producing high-affinity antibodies [53]. Interaction with CD40-ligand-expressing CD4+ T cells is required for the germinal center reaction to occur, and it has been suggested that improved antigen presentation due to DO downregulation could favor the selection of high-affinity cells [45–47]. This is an attractive hypothesis, but it has not yet been confirmed experimentally. Initial analysis of DO (i.e. H2-O) deficient mice suggested that there were differences in affinity maturation of the antibody response between these mice and DOexpressing control animals, but further analyses have failed to confirm the early findings (L Karlsson, unpublished). These studies were done in mice with a C57BL/6 background and it is possible that a difference in affinity maturation could be detected in DO-deficient mice on a different background, or that class II alleles other than H2-Ab could give different results. Alternatively, the downmodulation of DO in germinal centers may not be directly correlated to the BCR affinity of the B cells surviving the germinal center reaction, but may alter some other quality of the selected cell repertoire that has not yet been identified.
Conclusions DM is by now established as a necessary component for normal peptide-loading of all class II molecules studied so far, whether these bind CLIP strongly or not. Exactly how DM interacts with class II molecules is not known and will require co-crystallization of DM and class II molecules. However, it is clear that DM interacts with the amino-terminal part of the class II peptide-binding pocket, most likely resulting in a structural change that facilitates peptide exchange. Data also show that the outcome of DM activity is more complex than initially thought; thus the rates of peptide release in the presence or absence of DM are not highly correlated. DO expression in B cells can clearly influence antigen processing, although this is more obvious in human than in mouse B cells. The downregulation of DO after activation and in germinal centers seems to result in an improved ability to process antigens (although this has only been demonstrated indirectly), but the functional consequences for the germinal center reaction and for physiological antibody responses are still unclear. Further study will undoubtedly address these questions.
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Current Opinion in Immunology 2005, 17:65–70
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Current Opinion in Immunology 2005, 17:65–70 This review comes from a themed issue on Antigen processing and recognition Edited by Clifford V Harding and Jacques Neefjes Available online 8th December 2004
processing and assembly of peptide-loaded MHC class II molecules occur [1]. The endosomal/lysosomal system is rich in proteases, particularly cysteine proteases, and these enzymes are crucial both for the generation of peptides from antigenic proteins, and for the removal of Ii from class II molecules. CLIP is the final fragment of Ii and is generated by the protease cathepsin S [2]; CLIP is not spontaneously released from class II molecules in most cases, and exchange of this fragment for antigenic peptides is mediated by the accessory protein DM (HLA-DM in humans, H2-DM [previously termed H2-M] in the mouse) [3,4]. In B cells, but not in other types of APCs, a substantial fraction of DM is associated with another accessory protein, DO (HLA-DO in humans, H2-O in the mouse; here collectively referred to as DO, except when data suggest that species differences may exist) [5]. DO has been shown to modulate the function of DM in vitro, but there has been some confusion about how this affects the outcome of the peptide-loading process. DM and DO properties have been reviewed extensively previously [6–8] and here I will focus on recent research progress in this area.
DM — editor in chief
Abbreviations APC antigen-presenting cell BCR B cell receptor CLIP class-II-associated Ii peptide ER endoplasmic reticulum Ii invariant chain
DM was initially identified because cells that lacked this protein expressed high levels of CLIP–MHC-class-II complexes at their cell surface, and as a consequence had an altered reactivity with certain class II reactive monoclonal antibodies [9]. DM is mainly localized in the vesicles of the endosomal/lysosomal system of APCs, where it interacts with newly synthesized class II molecules, and catalyzes the release of CLIP from the peptidebinding groove. DM activity is not restricted to CLIP release, however, and it is now clear this is a special case of a more general ability of DM to edit the repertoire of class-II-associated peptides [10,11].
Introduction
Interaction with class II molecules and substrate specificity
MHC class II molecules associate with the invariant chain (Ii) in the endoplasmic reticulum (ER) of antigen-presenting cells (APCs), with the CLIP (class-II-associated Ii peptide) region of Ii occupying the peptide-binding groove of the class II a–b heterodimer. This association serves both to prevent peptide binding to the class II molecules within the ER and to direct class II molecules into the endosomal/lysosomal pathway, where antigen
How DM interacts with class II molecules is still not fully understood at the molecular level, and no crystal structure of the complex has been published to date. Initial observations of HLA-DR (DR) glycosylation mutants suggested that DM bound to the area around the P1 pocket, that is the area of the DR molecule where the amino termini of bound peptides reside [12,13]. Subsequent mutational studies have confirmed that this is
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Current Opinion in Immunology 2005, 17:65–70
66 Antigen processing and recognition
indeed the DR area facing DM [14], and the corresponding area on DM has been mapped using the same technique [15]. Further evidence that the area around the P1 pocket interacts with DM comes from a study where DM was directly coupled to a high affinity DR-binding peptide [16]. Only when DM was linked to the amino terminus of the peptide was there a striking increase in the rate of peptide release from the DR molecule, whereas carboxy-terminal fusions did not give this effect. In general, DM increases the rate of peptide exchange compared with the uncatalyzed state. Early experiments suggested that the rate of peptide dissociation from class II molecules in the presence of DM was directly proportional to the intrinsic rate of dissociation in the uncatalyzed state [11,17]. This conclusion was reached mainly using known class II ligand-peptides where the main chain was kept constant, while the side chains were being altered. More recent data from several laboratories [18,19,20] challenged the view that DM activity is independent of the peptide sequence, and it now appears that the degree to which DM increases peptide exchange is dependent on the structural features of the particular peptide–MHCclass-II complex [19,20]. Thus, Belmares et al. [19] analyzed the dissociation kinetics of 36 different DR– peptide complexes. They found large variations in DM susceptibility for different complexes and concluded that interactions throughout the peptide-binding groove contributed to these differences. Stratikos et al. [20] used variant peptides that lacked the capacity to form one or more of the conserved hydrogen bonds that are the basis for peptide binding to class II molecules. They also found that DM increased the dissociation rate of some complexes much more than of others, and related this to the inability to form hydrogen bonds between the class II molecule and the amino-terminal part of the peptide. DM also appears to differentially influence peptide association [19,21], but this is a less studied phenomenon than that of peptide dissociation. Practically, the lack of a direct correlation between DMcatalyzed and intrinsic peptide-exchange is likely to make useful predictions of which class-II-binding peptides are actually going to be presented at the surface of APCs even more difficult than previously thought.
Importance of DM for antigen presentation and immune responses Because CLIP release was the most apparent function of DM, it was initially thought that DM would have little effect on peptide loading of class II molecules that have low affinity for CLIP, and early experiments did indeed suggest this was the case [22], although DM had been shown to be able to influence both the release and loading Current Opinion in Immunology 2005, 17:65–70
of non-CLIP peptides in vitro [10]. DM-knockout mice, generated in H2-Ab mice, did not provide much clarity in this respect, since the affinity of H2-Ab for CLIP is so high that CLIP-free H2-Ab molecules are virtually undetectable in cells from these mice using biochemical methods, and the ability of these cells to present intracellularly processed antigen is minimal [23,24]. Somewhat surprisingly, DM deficient H2-Ab-expressing mice were still able to elicit IgG responses to some antigens, although antibody titers were lower than in wild-type littermates and affinity maturation did not seem to occur [24]. By contrast, transgenic expression of H2-Eb/d (which has lower affinity for CLIP than H2-Ab) in the DM-deficient mice largely restored the antibody responses, even though antigen presentation by H2-Eb/d was essentially undetectable. Recent data show that, also in the case of H2-Ab, removal of CLIP may be only the most apparent activity of DM. Expression of Ii transgenes where the CLIP region had either high or low affinity for H2-Ab resulted in very similar phenotypes with respect to antigen presentation in mice expressing DM, even though the low-affinity CLIP dissociated much more rapidly from the class II molecules than the high-affinity CLIP did [25]. In the absence of DM, the class II molecules in cells expressing Ii with lowaffinity CLIP had a more diverse peptide repertoire, but lower cell surface expression of class II molecules than the cells expressing high-affinity CLIP had. These data show that DM promotes the formation of long-lived H2-Ab– peptide complexes (resulting in higher expression at the cell surface), most likely by maintaining class II molecules in a peptide-receptive state after CLIP release. Low affinity for CLIP has been suggested to be a possible contributing factor for the development of autoimmunity, particularly in rheumatoid arthritis [26]. No evidence for increased autoimmunity was seen in the mice expressing low-affinity CLIP [25], but further studies will be necessary to exclude the possibility that CLIP affinity is correlated to disease, since genetic factors other than class II haplotype (and thus CLIP affinity) influence the development of autoimmunity. The importance of DM for peptide acquisition by other class II molecules was recently more directly addressed through the generation of DM-deficient H2d and H2k haplotype mice [27,28]. The data generated using these mice suggest that both H2-Ad (which binds CLIP relatively strongly) and H2-Ak (which has low affinity for CLIP) are dependent on DM for acquisition of endogenously processed peptides. By contrast, both H2-Ed and H2-Ek seem to be less affected by the absence of DM, suggesting that although DM clearly influences the peptide repertoire beyond CLIP removal, the relative importance for molding the normal peptide repertoire depends on which class II molecule is analyzed. www.sciencedirect.com
DM and DO shape pMHC II complexes Karlsson 67
Peptide immunization has been reported to result in the formation of two types of T cells, type A and type B respectively, which recognize distinct conformations of the same class-II–peptide complexes [29]. Type A cells recognize peptide–class-II complexes that have been formed after endogenous processing of whole antigen, whereas type B cells only recognize complexes containing exogenously provided peptide. A recent publication has demonstrated that resistance or sensitivity of the different class-II–peptide conformations to editing by DM explains the phenomenon [30]. The complexes recognized by type A T cells were formed in lysosome-like compartments, and have thus been subjected to (and survived) the editing activity of DM. By contrast, the complexes recognized by type B T cells were not formed (or did not survive) in lysosomes, and were sensitive to DM activity. The physiological implications of these findings are not yet clear, although the phenomenon of type A and type B T cell reactivity is likely to have implications for the development of peptide vaccines, and may be important for the development of autoimmunity, although this has not been tested experimentally. Altogether, presently available data show that although differences exist between different class II molecules, DM activity is important both for CLIP removal in the cases where CLIP–class-II affinity is high, and for the formation of the normal class-II-associated peptide repertoire displayed at the cell surface.
DO — coeditor While the effect of DM on antigen presentation is becoming increasingly clear, the function of DO has remained obscure. Like DM, DO is a nonpolymorphic intracellular class-II-like molecule that is evolutionary conserved. However, transcriptional control of DO expression is different from that of other class II molecules (including DM) and DO has a more restricted expression pattern, with expression being limited to B cells and thymic epithelial cells. The transcriptional basis for these differences is only partially understood, but it was previously assumed that expression of the DO b chain (but not the a chain) was independent of CIITA [31,32], the class II transcriptional transactivator that controls both class II and DM expression. Recent studies have explored this issue further and it is now clear that there is a basal DO b expression in B cells, but that CIITA can increase the expression level [33–35]. The factors that control basal DO b expression in B cells have not been identified. Mature DO protein is localized in endosomal/lysosomal compartments, but newly synthesized DO is unstable and normally requires physical association with DM to exit the ER [5]. The association between DM and DO is tight and it is not clear if the two molecules dissociate from each other under steady-state conditions even after the DM–DO complexes have reached endosomes or lysowww.sciencedirect.com
somes. To date it is also not clear whether DM–DO complexes directly interact with class II molecules (although this has been suggested [36]), or whether the complexes dissociate to release free DM and DO that may interact with class II molecules. In addition to the association with DM, a recent report shows that DO can associate with free MHC class I heavy chains to form complexes that can be detected in small amounts at the cell surface [37], but the function (if any) of this complex is unknown.
Effects of DO on antigen processing and presentation The function of DO at the molecular level has been debated; thus initial studies suggested that DO either inhibited [38–40] or enhanced [36] the DM-mediated exchange of class-II-associated peptides. The reasons for these differences are not entirely clear, but more recent studies generally support the notion that DO inhibits DM function. Increased H2-O expression (achieved by transfection) has been shown to decrease presentation of several antigenic epitopes from processed proteins [41,42], whereas decreased H2-O expression (achieved by transfection of an antisense ribozyme [41]) had the opposite effect. One of these studies also found that splenocytes from mice overexpressing H2-O under a class I promoter had a decreased ability to present epitopes from protein antigens [41]. Another study analyzed the phenotype of mice and cells from transgenic mice that expressed human HLA-DO under a dendritic-cell-specific promoter [43]. In this study the effect on antigen presentation was variable, but the surface expression of H2-Ab–CLIP was increased, while the expression of other peptide–class-II complexes analyzed was decreased. The increase in H2-Ab–CLIP expression in HLA-DO transgenic mice is somewhat unexpected, as surface H2Ab–CLIP levels are unchanged both in H2-O-transgenic mice and in H2-O-deficient mice [40,44]. The basis for this difference is unclear, but may be related to the expression level of DO, the fact that dendritic cells rather than B cells were analyzed, or to the possibility the human HLA-DO and mouse H2-O may interact differently with H2-DM. In human cells, in contrast to mouse cells, there appears to be very good correlation between the level of DO expression and the amount of CLIP–class-II complexes expressed at the cell surface [45–47]. Several studies have shown that the effect of DO on DM is pH dependent, so that DM-mediated peptideexchange activity in the presence of DO is relatively higher at low pH (corresponding to lysosomes) than at higher pH (corresponding to earlier endosomal compartments) [5,36,41,48]. This finding, together with early antigen presentation studies did lead to the suggestion that DO expression may promote presentation of antigen Current Opinion in Immunology 2005, 17:65–70
68 Antigen processing and recognition
internalized by the B cell receptor (BCR) over antigens internalized by fluid-phase uptake (see [6] for a review). The experimental data available so far only partially support the hypothesis that DO favors presentation of antigen taken up by the BCR. Thus, extended analysis of antigen processing using DO (i.e. H2-O) deficient mice showed that presentation of most antigenic epitopes tested after fluid-phase uptake of antigen was not influenced by the presence or absence of DO whether presented by H2-Ab or H2-Eb/d [49,50]. By contrast, antigens specifically internalized by transgenic hapten-specific BCRs were strongly affected by DO. Most epitopes from a given haptenated antigen were presented better in the presence of DO, but this was highly dependent on which receptor was internalizing the antigen, and some epitopes were actually better presented in the absence of DO [49]. Further studies with BCRs directed against protein antigens (rather than haptens) may be necessary to allow more generalized conclusions about how DO affects presentation after BCR-mediated antigen uptake.
Physiological function of DO The notion that DO function is related to antigen processing after antigen uptake by the BCR is indirectly supported by several recent reports showing that germinal center B cells have low levels of DO expression compared with naı¨ve and memory B cells [45–47]. Although DM expression is also decreased somewhat in the germinal center B cells compared with the naı¨ve and memory B cells, the DM:DO ratio is increased and results in decreased CLIP–class-II expression, suggesting that the antigen-processing capacity is increased in germinal center B cells relative to the other B cell types. The downmodulation of DO in germinal center B cells is likely to be a reflection of signaling events occurring during the germinal center reaction. Supporting this conclusion is the fact that several groups have reported that stimulation of naı¨ve B cells through CD40 [47,51] or the BCR [47,52], or by pharmacological treatment with a phorbol ester (PMA), or a protein kinase C (PKC) inhibitor [52], does result in decreased DO expression (although DM expression is also decreased to a lesser extent by some of these treatments). DO downregulation after stimulation occurs over several hours to overnight, and could in the case of the PKC inhibitor be inhibited by chloroquine [52], suggesting that degradation in endosomes or lysosomes is the reason for the stimulationinduced DO decrease. This conclusion is also supported by the finding that there was no decrease in DO gene transcription in germinal center B cells compared with naı¨ve B cells [45]. The B cell selection process that occurs in germinal centers is driven by BCR affinity for the relevant antigen and results in the development of memory B cells and Current Opinion in Immunology 2005, 17:65–70
plasma cells producing high-affinity antibodies [53]. Interaction with CD40-ligand-expressing CD4+ T cells is required for the germinal center reaction to occur, and it has been suggested that improved antigen presentation due to DO downregulation could favor the selection of high-affinity cells [45–47]. This is an attractive hypothesis, but it has not yet been confirmed experimentally. Initial analysis of DO (i.e. H2-O) deficient mice suggested that there were differences in affinity maturation of the antibody response between these mice and DOexpressing control animals, but further analyses have failed to confirm the early findings (L Karlsson, unpublished). These studies were done in mice with a C57BL/6 background and it is possible that a difference in affinity maturation could be detected in DO-deficient mice on a different background, or that class II alleles other than H2-Ab could give different results. Alternatively, the downmodulation of DO in germinal centers may not be directly correlated to the BCR affinity of the B cells surviving the germinal center reaction, but may alter some other quality of the selected cell repertoire that has not yet been identified.
Conclusions DM is by now established as a necessary component for normal peptide-loading of all class II molecules studied so far, whether these bind CLIP strongly or not. Exactly how DM interacts with class II molecules is not known and will require co-crystallization of DM and class II molecules. However, it is clear that DM interacts with the amino-terminal part of the class II peptide-binding pocket, most likely resulting in a structural change that facilitates peptide exchange. Data also show that the outcome of DM activity is more complex than initially thought; thus the rates of peptide release in the presence or absence of DM are not highly correlated. DO expression in B cells can clearly influence antigen processing, although this is more obvious in human than in mouse B cells. The downregulation of DO after activation and in germinal centers seems to result in an improved ability to process antigens (although this has only been demonstrated indirectly), but the functional consequences for the germinal center reaction and for physiological antibody responses are still unclear. Further study will undoubtedly address these questions.
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