molecules that modify antigen recognition

Molecules that modify antigen recognition

Norbert Koch and Brigitta Stockinger

Institute for Zoology, University of Bonn, FRC and Basei institute for Immunology, Switzerland

MHC class II molecules assemble in the presence of invariant chains.

These probably serve not only to protect the peptide-binding site on MHC class II molecules from endogenous peptides, but also to sort MHC class II molecules from the Colgi complex to endosomes and there to retain

the class II polypeptides to allow binding of peptides generated from exogenous antigens.

Current Opinion in Immunology 1991, 331s15

Introduction

Unlike B cells, T cells do not recognize native, unmodi fied proteins but instead recognize processed fragments of proteins bound to a major histocompatibility corn- plex (MHC) molecule on the surface of antigen-presenting cells @PCs). The pathways leading to antigen recognition by class I and class II MHC-restricted T cells are distinguishable. Peptide fragments of endogenously synthesized antigens bind to newly synthesized MHC class I molecules in the endoplasmic reticulum (ER) from where they are transported to the cell surface [ 1,2 1. By contrast, class II-restricted T cells are usually stimulated by exogenous antigens that need first to be taken up by an antigen- presenting cell [3**], a process that involves fluid-phase pinocytosis, receptor-mediated endocytosis or phagocy tosis. Ingested antigen is then proteolytically degraded to peptides in an endosomal compartment [4]. There they meet MHC class II molecules which, in association with invariant chain (Ii) molecules, are en route to the cell surface [5*]. Dissociation of Ii from class II molecules in a post-Go@ compartment [6] allows binding of the fragments of peptides to the class II molecules. The peptideclass II complex subsequently travels to the cell surface where it is recognized by the T-cell receptor.

The biosynthetic pathway of class I and class II polypeptides encounters the processing pathway of antigen. The catabolism of cellular proteins, as well as the import and degradation of exogenous proteins, provides a gen- eral source of peptides [7-l. The immune system ex- ploits what are essentially by~products of normal cellular metabolism in allowing the MHC transporting pathway to intersect with the protein processing pathway, so that MHC molecules can serve as peptide receptors. Binding of MHC molecules rescues peptides from further degradation and allows their exposure on the cell surface for recognition by T cells [8].

A number of accessory molecules are involved in the processes of MHC-antigen complex formation, transport and presentation. This article will summarize recent knowledge of these accessory molecules, focusing on ad- vances, particularly in the past year, in understanding of the role of Ii in antigen presentation by class II molecules.

Accessory molecules involved in antigen recognition

The specific interaction of T cells and APCs is supported by cell-surface receptors which regulate adherence. Multi- ple properties of these receptors coordinate antigen-specific communication of T cells and APCs 191. The contact of a T cell and an APC is augmented by the interaction of CD4 and CD8 molecules expressed on T cells with the class II and class I polypeptides on APCs [ 101. Ad- ditionally, interaction of T cells and ApCs are likely to be reinforced by adhesion molecules such as intercellu- lar adhesion molecule-l with lymphocyte function-associated antigen (LPA)-l and CD2 with LFA-3, which increase avidity and provide a co-stimulatory signal for activation of resting T cells [ 1 l-131. Among other candidates for adhesion receptors are the cell-surface molecules, very late antigen-4 and the T-cell surface glycoprotein E2. An tibodies to both have been shown to interfere with T-cell adhesion [ 14,151.

Biosynthesis and transport of MHC molecules

Intracellular events that coordinate antigen processing and transport of MHC class I and class II molecules pre- cede presentation of antigen on the cell surface. Intracel- lular transport and correct folding of class I heavy chain

10

Abbreviations APC-antigen-presenting cell; fl,m-&-microglobulin; ER--endoplasmic reticulum; Ii-invariant chain;

LFA-lymphocyte function-associated antigen; MHC-major histocompatibility complex.

@ Current Biology Ltd ISSN 0952-7915

Molecules that modify antigen recognition Koch and Stockinger 11

molecules requires their association with &microglobulin (&m> [ 16-1, However, the correct assembly of class I heavy chain molecules and &rn is dependent on the presence of a binding peptide [17,18*,19*]. On the ba- sis of this observation, it can be inferred that charging of class I molecules with peptide occurs during assembly of class I in the ER. There is evidence that any peptide provided by the metabolism of the cell has the potential to be presented by class I molecules. Extensive protein degradation in the ER itself supplies some of the peptides [ 201 while others are delivered from the cytoplasm [ 171.

MHC class II assembly and transport follows slightly different rules. Class II cz- and /3-dimers within cells are associated with Ii molecules. Although the genes coding for class II and Ii molecules are located on different chromo- somes and their coding sequences are not related, their transcriptional activation by interferon-y, tumour necrosis factor-u or interleukin-4 is co-regulated. Binding of nu- clear factors to consen& promoter regions accounts for this co-regulation [ 21-241.

The oligomeric complex of class II 01 and p chains and Ii polypeptides is formed soon after insertion of these proteins into the lipid bilayer of the ER. A great proportion of the Ii polypeptides is retained in the ER and presumably metabolised. An as: yet undefined signal gives rise to the export of Ii polypeptides and Ii complexed with class II, from the ER to Golgi compartments. Recent experiments suggest that association of Ii with class II is required for export of Ii from the ER [ 251. However, this contradicts earlier results that demonstrated efficient transport of Ii in the absence of class II [ 261. Transfection of the Ii gene into different class II-negative cell types has revealed that whether Ii is retained in the ER or efficiently transported to the trunsGolgi complex depends on the cell type (unpublished data). Beyond the ER, class II and Ii molecules are mod&d by the binding of palmitic acid to a cysteine residue in either the cytoplasmic or membrane domains [27,28]. This modification could result in an important cytoplasmic recognition site and may be used for fusion of transport vesicles. In addition, N- and O-linked carbo- hydrates modify the class II and Ii polypeptides and these glycan side chains are sialylated in trans_Golgi compart ments [29]. After leaving the Golgi stack, Ii and class II are separated from the exocytic route and enter the endocytic pathway. Upon immigration into endosomes, the fate of class II and Ii polypeptides diverges. A signal provided by degradation of Ii chain initiates export of class II to the cell surface [6].

It is conceivable that Ii promotes the assembly of class II polypeptides although its presence is less stringently required than Pam for class I assembly as shown by the surface expression of class II a/p complex in the absence of Ii [30,31]. However, preliminary results from several lab- oratories (unpublished data) on the transfection of class II and Ii genes suggest that Ii has an auxilliary effect on the assembly of class II molecules. One could speculate that, in cells devoid of Ii, an internal peptide fulfills this role in analogy to the assembly of class I heavy chain, &rn and peptide. Recent data [32*] suggest conforma-

tional differences in class II molecules expressed in tmns- fected fibroblasts in the presence or absence of Ii. This further supports the theory that Ii contributes to class II assembly.

Functional dissection of the class II antigen processing pathway

Antigen taken up by ARCS very rapidly becomes localized to endosome-like vesicles. Class II MHC and Ii molecules, but not class I MI-K are co-localized with the antigen [33*]. Compartmentalization of ingested antigens with class II MHC provides the rational explanation for their selective association in presentation to class II MHC- restricted T cells. The association of class II MHC with invariant chains led to the proposal that Ii also plays a role in antigen presentation [34]. Chloroquine, a potent inhibitor of the class II presentation pathway, interferes with the dissociation of Ii from class II molecules and further transport of class II to the cell surface [35]. It seems reasonable to propose that dissociation of Ii from class II molecules in an endosome-like compartment and association of processed protein fragments with class II molecules are functionally connected. One might therefore assume that class II molecules, in contrast with class I molecules, are loaded with peptides from a different source at a much later point on their route to the cell surface.

As class II and Ii are usually co-expressed in cells, a dissection of the function of Ii has not been possible with conventional ARCS. Class II transfection of L-cell fibroblasts, which only express vety low levels of endogenous Ii, provided the first clues that Ii is needed for processing of an intact protein antigen. These class II-express- ing fibroblasts showed a defect in presentation of native proteins but were effective presenters of peptide fragments [36]. Supertransfection of such class II-bearing L cells with the Ii gene converted them to ARCS that were much more potent than even standard ARC populations such as spleen cells or B lymphoma cells for very small doses of antigen [ 371.

Recent unpublished work in our laboratory has shown that the requirement for Ii in processing of native antigens may be overcome by high antigen doses. It is conceivable that in conditions of antigen excess, and espe- cially when peptide fragments are generated which have high binding alhnity for class II, Ii is not required for optimal presentation. In this situation, one could envisage that class II molecules of fibroblasts with low Ii which are presumably occupied by endogenous peptides, easily ex- change these peptides for high alfinity peptides from an exogenous antigen source.

Interestingly, we lind that presentation of high doses of hen-egg lysosome by fibroblasts not supertransfected with Ii is completely abrogated by chloroquine. This sug- gests that the interaction between peptides and class II still takes place in a chloroquine-sensitive, possibly en-

12 Antigen recognition

dosomal, compartment. Considering the fact that L-cell fibroblasts express low levels of endogenous Ii, however, it is not possible at present to conclude unequivocally that class II can be transported to endosomes indepen- dently of Ii. In addition, it has become clear recently that an R-dependent endosomal pathway is not the only way peptides can get access to class II molecules. An exam- ple is provided by class II-restricted T-killer cells specific for measles virus antigen which kill Ii-negative fibroblasts infected with the virus or transfected with expressible cDNAs [ 391. This killing event is not inhibited by chloroquine, indicating that peptide generation does not occur in an endosomal compartment. Similarly, cells infected with live inlluenza virus can be recognized by both class I and class II-restricted T cells [ 40.1. The T-cell response was not inhibited by chloroquine but was totally abrogated by the fungal antibiotic brefeldin A, which had previously been shown to inhibit class I antigen presenta tion by blocking transport of peptide-loaded MHC class I molecules to the surface [41-l. These data imply that loading of MHC class II molecules with endogenously derived peptides takes place at an early point in biosyn thesis, possibly within the ER. Peptides may be delivered by peptide transporters or by diffusion of peptides from the cytoplasm into the ER, and a peptide-binding protein with a role in class II antigen presentation has recently been identified as a member of the heat-shock protein (HSP)70 family [ 380 ]

It is interesting to note that the non-endosomal Ii-independent pathway of class II presentation has only been described for viral antigens. Conceivably, under these conditions, which probably supply high doses of anti- genie peptides, Ii is dispensable; whereas in situations where only low amounts of exogenous antigen are available Ii may be crucially important to ensure optimal access of peptides to the class II MHC molecules.

Molecular and cellular models of class II

antigen processing

More than 10 years after the discovety of Ii as a compo- nent of MHC class II immunoprecipitates [42], it has become generally accepted that it is involved in the mecha- nism of class II antigen processing and presentation. Sev eral molecular models have been put forward in the past year in an attempt to explain more precisely the molecular role of Ii in these events,

Targeting model

Along their biosynthetic route class II and associated Ii molecules are sorted from the constitutive secretory path way into the endocytic route [43]. Intracellular sorting of polypeptides usually requires a signal sequence which guides the transport of a protein to a cellular compart ment. Thus it can be suggested that the class II/Ii complex bears a signal sequence that directs its transport to endosomes. A recent report by Bakke and Dobber-

stein [44**], in fact indicates that the amino-terminus of Ii contains a sorting signal to post-Golgi vesicles. Bakke and Dobberstein show that in the absence of class II molecules, Ii is sorted to post-Go@ compartments. How- ever, when the first 15 amino acids of the amino-terminus of Ii are deleted this sorting signal is lost and Ii ap- pears on the cell surface. The post-Golgi compartment where Ii was localized is possibly the endocytic compart ment where MHC class II molecules interact with processed antigen. At this stage the transport of the class II/Ii complex is delayed, possibly by means of a retention signal on Ii, and Ii is detached from class II. The delay of transport could additionally provide the time required for binding of the peptide to the class II cleft; subsequently, class II molecules loaded with peptide continue their route to the cell surface.

Protection model

Invariant chain stabilizes the conformation of class II a- and P-chains and possibly interacts with the peptide binding cleft of class II [45]. The presence of associated Ii in the ER could prevent binding of endogenous peptides derived from normal biosynthesis activities of the cell and postpone charging of the cleft until exogenous peptides become available [46]. Biochemical evidence for this model has recently been provided by Roche and Cresswell [47**]. They show that class II molecules with associated Ii have low capacity to bind peptides, while binding increases dramatically after dissociation of Ii. In cells devoid of Ii, class II molecules acquire the capabil- ity to bind and potentially present endogenous peptides which do not bind in the presence of Ii.

Additionally, it is conceivable that Ii contributes to the formation of stable a- and P-dimers and protects class II molecules against proteolytic digestion. This becomes important when class II complexes traverse proteolytic antigen-processing compartments. In these compartments Ii, but not class II polypeptides, is degraded by proteolysis and class II a/P-peptide complexes escape to the cell surface.

Targeting of recycled class II molecules

At present it is a controversial issue whether presentation of endocytosed antigen requires freshly biosynthesized class II molecules or whether class II recycled from the cell surface is available to bind further peptides. The extent to which class II molecules appear to be internalized and/or recycled to the cell surface may depend on the cell type studied or on the technique employed [ 5’ ,48**,49,50*]. Recent experiments [51**] with a cleav able surface labeling reagent indicated that class II rapidly recycles from a primaquine-sensitive intracellular compartment in a human lymphoblastoid line. The short half life of the internal pool of class II may explain why recycling had not previously been demonstrated. Calcula- tion of this recycling traflic reveals that it exceeds that of biosynthesized class II by more than 60-fold. On the other hand, recent antigen presentation studies 152.1


showed inhibition of presentation of exogenous antigen to class II-restricted T cells by brefeldin A applied to B- lymphoma ARCS as well as macrophages and class II- transfected libroblasts. Apparently brefeldin A prevents the encounter of newly synthesized class II with peptides generated in an endosomal compartment by retarding class II in the ER. This result points to an important role for newly synthesized class II in antigen presentation. Ob- viously, MHC class II u-a& and its relevance for antigen presentation still poses a number of unsolved problems

Recently, we obtained evidence that Ii can be detected on the surface of human B-lymphoma cells [53]. This surface Ii is clearly distinct from the proteoglycan form which has been described before [ 541. In B-lymphoma cells, Ii molecules reach the cell surface on a chloroquine- insensitive pathway and thus bypass degradation. On the cell surface a proportion of Ii is associated with class II molecules (unpublished result). It is conceivable that those class II molecules that are associated with Ii are tar- geted upon recycling to compartments where they will encounter processed antigen.

Concluding remarks

Under physiological conditions in uiuo, all MHC class II positive-cells also express intracellular Ii. It is therefore likely that Ii has an essential role in their antigen presentation function. Class R-transfected cells devoid of Ii provide a crucial in vitro system for investigation of the molecular function of Ii.

Experimental evidence now supports the hypothesis that Ii serves to protect the class II peptide-binding cleft from endogenously derived peptides before class II encounters processed antigen fragments from the exogenous source. It is worth noting that there may not be an abso- lute requirement for Ii in presentation of all exogenous antigens and that the presence of Ii does not necessar- ily preclude the presentation of some endogenous mate- rial. High concentrations of available antigenic peptides or high affinities for class 11 will affect the equilibrium of the interaction between class II and Ii. Recent results indicate a sorting and targeting function of Ii. This supports the hypothesis that Ii directs the transport of class II to specialized processing compartments and this son- ing event is possibly mediated by an endosomal receptor that binds Ii and carries it from the Golgi complex to endosomes. Additionally, Ii may be involved in retention of MHC class II molecules in endosomes to allow optimal loading with peptides before the complex travels to the cell surface.

Acknowledgements

We gratefully acknowledge Bruno Kyewski and David Gray for critical reading of the manuscript This work was supported by a grant from

the Deutsche Forschungsgemeinschaft (Ko 810/4-l). The Base1 Insti- tute for Immunology was founded and is supported by F Hoffman Ia Roche and Co. Ltd., Basel, Switzerland.

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53. WRAIGHT CH, LIPP J, MOLLER P, VAN ENDERT P, LING N, MCLENNAN I, KOCH N, MOLDENHAUER G: Human MHC Class II Invariant Chain is Expressed on the Cell Surface. J Bid Chem 1990, 265~5787-5792.

54. SANT AJ, CU~N SE, SCHWARTZ BD: Biosynthetic Relationship of the Chondroitiu Sulfate Proteoglycan with Ia and Invari- ant Chain Glycoproteins. J Immunol 1985, 135:416422.

N Koch, Department of Immunology, Institute for Zoology, University of Bonn, D-53 Bonn, RbmerstmJ3e 164, 7 Etage, FRG.

B Stockinger, Base1 Institute for Immunology, C44058 Basel, Gren- zachenstra8e 487, Switzerland.

molecules that modify antigen recognition

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