The assembly of functional ß2-microglobulin-free MHC class I molecules that interact with peptides and CD8+ T lymphocytes
Todd D. Schell1,
Lawrence M. Mylin1,3,
Satvir S. Tevethia1 and
Sebastian Joyce2
1 Department of Microbiology and Immunology, Pennsylvania State University, College of Medicine, Hershey, PA 17033, USA 2 Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA 3 Present address: Messiah College, Grantham, PA 17027, USA
Correspondence to: S. Joyce; E-mail: sebastian.joyce{at}vanderbilt.edu
Transmitting editor: L. L. Lanier
 |
Abstract
|
---|
Functional MHC class I molecules are expressed on the cell surface in the absence ofß2-microglobulin (ß2m) light chain that can interact with CD8+ T lymphocytes. Whether their assembly requires peptide binding and whether their recognition by CD8+ T lymphocytes involves the presentation of peptide epitopes remains unknown. We show that ß2m-free H-2Db assembles with short peptides that are
9 amino acid residues in length, akin to ligands associated with completely assembled ß2m+ H-2Db. Remarkably, a subset of the peptides associated with the ß2m-free H-2Db has an altered anchor motif. However, they also include peptides that contain a ß2m+H-2Db binding anchor motif. Further, the H-2Kb- and H-2Db-restricted peptide epitopes derived from SV-40 T antigen also assemble with H-2b class I in ß2m-deficient cells and are recognized by epitope-specific CD8+ T lymphocytes. Taken together our data reveal that functional MHC class I molecules assemble in the absence of ß2m with peptides and form CD8+ T lymphocyte epitopes.
Keywords: ß2-microglobulin, antigen processing and presentation, CD8+ T lymphocytes, MHC class I, SV-40 T antigen epitope
 |
Introduction
|
---|
MHC-encoded class I molecules play a central role in the presentation of cytosolic antigens to CD8+ cytotoxic T lymphocytes (CTL) [reviewed in (1)]. A functional class I molecule consists of a 45-kDa membrane anchored heavy chain non-covalently associated with a 11.5-kDa light chain ß2-microglobulin (ß2m) and a peptide of 811 amino acid residues [see references in (2)]. The membrane distal
1 and
2 domains of the heavy chain form a superdomain containing the peptide antigen-binding groove (3,4). Because of its central role in CTL-mediated acquired immunity, the biochemistry of class I assembly and intracellular traffic have been studied extensively [reviewed in (5,6)]. From these studies the following model emerges. The assembly of class I begins as the heavy chain co-translationally inserts into the endoplasmic reticulum (ER); it binds calnexin, which assists its folding (79) presumably by preventing the aggregation of nascent heavy chains (10). Upon partial folding, the heavy chain is receptive to ß2m. Calnexin within the initial heavy chain ß2m complex rapidly exchanges for another ER chaperone, calreticulin. Calreticulin assists the heavy chain ß2m complex to associate with the peptide loading and assembly complex (11), which consists of ERp57, tapasin and the transporters associated with antigen presentation, TAP1 and TAP2. ERp57, a thiol-dependent reductase, interacts with the non-disulfide-bonded class I heavy chain. This heavy chain ERp57 interaction facilitates disulfide bond formation (1214). The assembly complex facilitates peptide loading onto class I molecules, subsequent to which the components of the assembly complex dissociate from the completely assembled class I molecule allowing their egress from the ER through the Golgi apparatus to the plasma membrane (1522). Thus native class I molecules are an ensemble of a heavy chain, ß2m and peptide.
Phenotypic studies have shown that class I molecules such as H-2Db and H-2Ld are stably expressed at the surface of ß2m-deficient cells (2325). Additionally, functional studies have demonstrated that ß2m-free H-2b class I molecules can interact with conventional
ß T lymphocytes because, albeit poorly, they engage in positive selection of CD8+ T lymphocytes, mediate allograft immunity and promote tumor rejection in ß2m-deficient mice (2533). Thus the resulting CTL are capable of responding to native syngeneic as well as allogeneic H-2 class I molecules (2630).
We have previously reported that ß2m-free H-2Kb molecules associate with unusually long peptides; these peptides bind H-2Kb without a discernable binding motif (34). The unique properties of peptides bound to ß2m-free H-2Kb differ from ligands associated with native ß2m-containing H-2Kb, which are short, predominantly octameric peptides composed of a tyrosine or phenylalanine residue at position (P) 5 and an aliphatic, hydrophobic residue at the C-terminus (P
) of the peptide (34). The function of peptide-associated ß2m-free H-2Kb and how they assemble in cells remain unknown.
Herein we report that ß2m-free H-2Db associates with peptides of
9 amino acid residues, a subset of which has an altered class I binding motif. Additional data reveal that the peptides associated with ß2m-free H-2Db and H-2Kb form epitopes for specific CD8+ T lymphocytes. Thus, ß2m-independent assembly of heavy chain with peptides is a generic property of class I molecules.
 |
Methods
|
---|
Metabolic labeling and chase of labeled proteins
Kb-high and Db-high cells, which express wild type H-2Kb and H-2Db respectively, are described (35). The methods used for metabolic labeling of cells with [35S]methionine and [35S]cysteine (NEN Life Science Products, Boston, MA), pulse labeling and chase, immune precipitation, and endoglycosidase H (Endo H) digestion have been described (35). Antibodies used for immune precipitation included Y3, a native conformation-dependent H-2Kb-specific mAb (36), B22-249, a native conformation-dependent H-2Db-specific mAb, and 28-14-8s, a ß2m-free H-2Db-specific mAb (37) as well as the rabbit polyclonal heteroantiserum (hAs),
X8 directed against the tail end of K locus products (34). Immune complexes were separated by 15% SDSPAGE, fixed, soaked in Amplify (Amersham Pharmacia Biotechnology, Piscataway, NJ) and detected by autoradiography.
Immune affinity purification of MHC class I, peptide isolation and characterization
The procedures adopted for the purification of ß2m-free and ß2m+ class I molecules using antibody (listed above)-coupled Protein ASepharose, the isolation of associated peptides, and amino acid sequence analyses are described in detail elsewhere (34).
Generation and maintenance of antigen-specific CTL and 51Cr-release assay
SV-40 T antigen-specific CTL clones against SV-40 T antigen-derived epitope I, epitope II/III, epitope IV and epitope V have been described previously (3842). Conditions for culture and use of these CTL clones have been described previously (39,40). 51Cr-release assays using target cells expressing endogenous antigens were performed according to standard protocols and the data are represented as percent specific lysis (39,40).
 |
Results
|
---|
H-2Db is expressed in ß2m-associated and ß2m-free forms within cells
We previously reported that cell lines express two immunochemically distinct forms of H-2Kb. One form is associated with ß2m (ß2m+ class I) and the second (ß2m-free) is weakly or not at all associated with ß2m (34). Additionally, ß2m-free H-2Db and H-2Ld are stably expressed at the surface of cell lines as well as of freshly isolated cells derived from ß2m-deficient mice (2325). Therefore, to study the relationship between ß2m+ and ß2m-free H-2Db, NS0 plasmacytoma (H-2d) that overexpresses the class I molecule (Db-high) was generated (35). Biochemical characterization of Db-high cell lines revealed that akin to Kb-high, they express two immunochemically distinct forms of H-2Db that can be distinguished by sequential precipitation with specific antibodies. B22-249 (a conformation-dependent mAb) immune precipitates ß2m+ H-2Db from detergent lysates of metabolically radiolabeled Db-high cells (Fig. 1, lanes 24 in left panel). However, subsequent precipitation of the B22-249 cleared, labeled lysates with an
3 domain-specific mAb 28-14-8s reveals (ß2m-free H-2Db) a form of H-2Db that is weakly or not at all associated with the light chain (Fig. 1, lanes 24 in right panel). We have previously shown that these cells contain an excess of light chains and, hence, it should not be limiting in the assembly of ß2m+ H-2Db (35). Immunochemical analyses of normal cell lines, such as EL-4 (Fig. 1, lane 1 in both panels), also reveal the presence of both ß2m+ and ß2m-free forms of H-2Db. Thus ß2m-free class I is stably expressed in cell lines.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 1. Cells express ß2m-free H-2Db. One-hour [35S]methionine-labeled cell lysates of 5 x 105 Db-high clones 13 along with 10 times as many EL-4 were immune precipitated with conformation-dependent mAb specific for H-2Db (B22-249) followed by immune precipitation of ß2m-free H-2Db with 28-14-8s mAb. Immune complexes were separated by 15% SDSPAGE and visualized by autoradiography. H = heavy chain. The right panel was previously reported in (35).
|
|
As ß2m-free class I molecules are rapidly degraded in the ER (43), the biosynthetic fate of the ß2m-free H-2Db and H-2Kb were determined. A representative Db-high cell line and Kb-high, a cell line that overexpresses H-2Kb, were pulse-labeled and chased for the indicated times, solubilized in detergent, and ß2m+ and ß2m-free class I were immune precipitated successively with specific antibodies as described above. The immune precipitates were digested with Endo H to determine the rate of egress of class I from the ER. The data reveal that
50% of the ß2m-free H-2Db and H-2Kb egress from the ER and turn over with the same kinetics as the ß2m+ form of the respective class I molecule (Fig. 2). Further, both the Endo H-sensitive and the Endo H-resistant ß2m-free H-2Db turn over more rapidly than the ß2m+ form of this class I molecule (Fig. 2). Thus
50% ß2m-free class I escapes the architectural editing mechanism within the ER and arrives into the Golgi apparatus and beyond.

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 2. ß2m-free H-2Db that negotiate the secretory pathway. Cells were pulse-labeled with [35S]methionine for 10 min and chased for the indicated periods of time at 37°C. ß2m+ and ß2m-free H-2Db and H-2Kb were immune precipitated from post-nuclear lysates with B22-249 or Y3 and 28-14-8s or X8 respectively. The immune complexes were Endo H-digested (r = resistant, s = sensitive), separated by SDSPAGE and detected by autoradiography. The traffic of ß2m+ class I was reported in (35).
|
|
ß2m-free H-2Db is associated with peptides that contain an altered binding motif
The discovery of a pool of ß2m-free class I molecules that egress from the ER akin to the completely assembled ß2m+ form raises the question as to whether the former associates with peptides. Biochemical analysis of ß2m-free H-2Kb revealed that it is associated with peptides whose properties were distinct from ß2m+ H-2Kb bound ligands (34). Thus peptides associated with the two forms of H-2Db were isolated and a fraction of the peptide pool was subjected to microsequence analysis. This analysis reveals that peptides associated with the native ß2m+ H-2Db are
9 amino acid residues long and contain the dominant H-2Db binding anchor residues: asparagine at position 5 (P5) and methionine at P
(Table 1A). Thus, as described previously (44), native ß2m+ H-2Db is complexed with canonical short peptides containing the appropriate anchor motif.
Akin to the features of peptides eluted from the native ß2m+ H-2Db, pool sequence analysis of an aliquot of the unfractionated peptides associated with ß2m-free H-2Db molecules reveals that they are also short, averaging
9 amino acid residues in length (Table 1B). However, in striking contrast, in the ß2m-free H-2Db-derived peptide pool, the expected H-2Db anchor residue P5 asparagine is present only in a subset of peptides (Table 1B). Moreover, histidine and tyrosine are also represented at the P5 dominant anchor position (Table 1B). Further, the expected hydrophobic residue methionine at P
of H-2Db binding peptides was not present, but contained isoleucine, valine and leucine at this position (Table 1B). Two controls were set-up to monitor non-specific binding of peptides to 28-14-8s mAb. In one control, detergent lysates of Db-high cells were passed over the
X8 [a H-2K locus encoded product-specific, conformation-independent hAs (34)] column. In the second control, detergent lysates of Kb-high cells were passed over the 28-14-8s column. The non-specifically bound material was eluted and treated in an identical manner as ligands isolated from ß2m+ and ß2m-free H-2Db. The two control preparations did not contain peptides (data not shown). Thus peptides associated with the ß2m-free class I molecules are not contaminants that co-purify during affinity chromatography. The finding that ß2m-free class I are associated with peptides suggests that the H-2Db heavy chain peptide interactions form stable complexes and may explain their expression on the surface of ß2m-deficient cells.
Functional peptide epitopes are presented by ß2m-free H-2Kb and H-2Db to specific CD8+ T lymphocyte clones
The presence of ß2m-free class I in cells and their ability to associate with peptides raises the question whether this form of class I molecule has any functional significance. Thus we determined whether SV-40 T antigen-transformed renal fibroblasts derived from ß2m-deficient mice are recognized by a panel of well characterized H-2Kb- and H-2Db-restricted CD8+ T lymphocyte (CTL) clones (3842). The data reveal that the H-2Db -restricted T antigen-derived epitope I is presented by the ß2m-deficient fibroblasts (Fig. 3, top left panel), as are epitope II/III and, although less efficiently, epitope V (Fig. 3, right panels). On the contrary, the H-2Kb-restricted epitope IV is not presented by the ß2m-deficient fibroblast to the CTL clone Y4 (Fig. 3, bottom left panel). The addition of human ß2m (huß2m) alone did not sensitize ß2m-deficient fibroblasts to any SV-40 T antigen-specific CTL clones (Fig. 3). Further, the addition of H-2Db-restricted peptide epitopes II/III and V in addition to huß2m failed to increase the efficiency of target cell lysis by CTL clones K19 and H1 respectively (Fig. 3). In contrast, addition of exogenous peptide epitope I, in the presence or absence of huß2m, significantly enhanced lysis of ß2m-deficient fibroblasts by the CTL clone K11. Significant enhancement of Y4 CTL clone-mediated lysis required the addition of huß2m (Fig. 3, bottom left panel). These data suggest that in cells, ß2m-free H-2Db assembles with peptide epitopes, which form CTL antigens.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 3. ß2m-free H-2Db but not ß2m-free H-2Kb present antigen to specific CTL. SV-40 T antigen-transformed ß2m-deficient fibroblast was tested for their ability to display H-2Db-restricted (epitope I, top left; epitope II/III, top right; epitope V, bottom right) and H-2Kb-restricted (epitope IV, bottom left) SV-40 T antigen-derived peptide epitopes to specific CTL. CTL epitope display was monitored either without any manipulation of ß2m-deficient targets or in the presence of synthetic peptide epitopes I or IV and/or huß2m.
|
|
Two key questions arise from the above findings. First, where do the H-2Db-restricted epitopes assemble: intracellularly or at the cell surface? Second, does the recognition of the H-2Db-restricted epitopes depend on ß2m? In the above experiment, the ß2m-free H-2Db could associate with bovine ß2m present in FBS used to maintain cells in culture. To address these questions, ß2m-deficient and ß2m+ wild-type SV-40 T antigen-transformed fibroblasts along with ß2m+ fibroblasts transformed with a T antigen variant which lacks all four epitopes were grown in dialyzed or normal FBS. Dialyzed FBS should not contain the 11.5-kDa bovine ß2m because it would be lost during dialysis; note that dialysis of serum supplied by J. R. H. Biosciences was performed with a 10-kDa cut-off membrane. The ability of targets grown under these serum conditions were tested for their ability to present H-2b class I-restricted SV-40 T antigen epitopes to specific CTL clones.
The data reveal that epitope I- and epitope II/III-specific CTL recognize H-2Db expressed by wild-type SV-40 T antigen-transformed ß2m-deficient fibroblasts. The growth of these cells in dialyzed FBS did not alter CTL recognition of the specific epitopes (Fig. 4A, top and middle rows). As expected, the recognition of ß2m+ fibroblast was 2- to 3-fold better than the ß2m-deficient targets. Additionally, ß2m+ fibroblasts transformed with all four epitope-loss variant of SV-40 T antigen was not recognized by any of the CTL clones (Fig. 4A, left and middle panels). Curiously, in contrast to the results presented in Fig. 3, epitope V was not recognized in this experiment by CTL clone Y5 (Fig. 4A, bottom panels). The epitope V-specific CTL clones used in the two experiments were different. The sensitivities of the two epitope V-specific CTL clones may be different and, hence, may explain the contrasting recognition pattern in the two experiments. Because the two distinct CTL clones against epitope V behave differently, we tested whether CTL clones other than epitope IV-specific Y4 recognize antigen in the absence of ß2m. The H-2Kb-restricted CTL clone SV2168T specific for epitope IV, in contrast to Y4 (also a H-2Kb-restricted CTL clone specific for epitope IV), recognized epitope IV assembled and presented in the absence of ß2m (Fig. 4B, left panel). Addition of huß2m in the absence of epitope IV peptide did not enhance antigen recognition by SV2168T (Fig. 4B, left panel). Thus, epitope I, epitope II/III and epitope IV assemble in cells with H-2b class I molecules in the absence of ß2m, whose display at the cell surface form targets for specific CTL clones.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 4. T cells recognize ß2m-free MHC class I molecules. SV-40 T antigen-transformed ß2m-deficient (ß2m wt T), ß2m+ (ß2m+ wt T) fibroblasts as well as ß2m+ fibroblasts transformed with all four epitope-loss variant of SV-40 T antigen (ß2m+ epi T) grown in dialyzed (10-kDa cut-off; left panels) or normal (middle panels) FBS were tested for their ability to display H-2Db-restricted (A; epitope I, top row; epitope II/III, middle row; epitope V, bottom row) and H-2Kb-restricted (B; epitope IV) SV-40 T antigen-derived peptide epitopes to specific CTL. Right panels show the specificity of the CTL clones, which recognize their cognate peptide epitopes when provided exogenously to otherwise SV-40 T antigen-devoid RMA cells (A) or epitope-loss mutant T antigen-transformed ß2m+ fibroblasts (B). A herpes simplex virus Is glycoprotein B-derived H-2Kb-restricted epitope [gB (59)] was used as a negative control (B).
|
|
 |
Discussion
|
---|
The current model for class I assembly suggests that incomplete molecules, either lacking ß2m and/or peptide, undergo architectural editing in the ER and are lost to cytosolic degradation by the proteasomes (43). However, numerous reports have demonstrated that ß2m-free H-2Db and H-2Ld are expressed on the cell surface (2325,45). Such ß2m-free class I permits CD8+ T lymphocyte development (28,30). These CTL elicit a lytic response and mediate tumor-specific immunity as well as allograft rejection in ß2m-deficient mice (2527,29,30,32,33). These data suggest an interaction between the antigen-specific TCR and the ß2m-free class I molecule. Whether the ß2m-free class I assembles with peptides and whether their recognition by T cells depends on peptides, remains unanswered questions.
The results presented herein provide direct evidence that endogenous peptides assemble functionally competent complexes with ß2m-free class I. Two criteria were used to detect the association of peptides with ß2m-free class I: (i) direct isolation and amino acid sequence analysis of the ligands associated with the ß2m-free class I, and (ii) presentation of CTL epitopes to antigen-specific T lymphocytes. We previously described the characterization of peptides associated with ß2m-free H-2Kb. The associated peptides had unusual characteristics in that they were longer than any known ß2m+ H-2Kb binding ligands and did not contain the H-2Kb binding anchor motif (34). In striking contrast, we show here that ß2m-free H-2Db-associated peptides were the expected 9 amino acid residues long. Many contained a discernable binding motif, but only a subset contained the dominant P5 asparagine, which serves as the dominant H-2Db binding anchor. The remainder contained histidine or tryrosine at P5. Strikingly, none of the associated peptides contained P
methionine, the C-terminal dominant anchor. Alteration in the binding motif, albeit relatively uncommon, is observed in bone fide CTL epitopes. For example, the H-2Db-restricted H13-derived CTL epitope contains a glycine residue at P5. Despite the altered P5 anchor, the H13-derived CTL epitope binds strongly to H-2Db (46). Similarly, numerous examples of altered P5 anchor residues have been reported for H-2Kb-restricted CTL epitopes (2). Whether epitopes with an altered anchor motif associate with ß2m+ or ß2m-free class I remains to be determined. However, our data would suggest that endogenous peptides with altered binding motifs have the potential to assemble with ß2m-free class I molecules.
The above interpretation is limited by the fact that the ß2m-free class I may have resulted from the dissociation of the light chain following assembly of ß2m+ class I. Thus we recognize that the isolation and characterization of class I-associated ligands should be performed with ß2m-deficient cells expressing ß2m-free class I molecules to obtain accurate information regarding the associated peptides. However, such an analysis is not technically feasible. First, it would take
10,000 ß2m-deficient mouse spleens and thymi to obtain sufficient amounts of ß2m-free class I molecules to perform Edman sequence analysis of peptides eluted from them. Alternatively it would take >1013 ß2m-deficient cells maintained in serum-free conditions to purify sufficient amounts of heavy chain to analyze peptides associated with them. Thus the analyses described herein was performed on peptides isolated from ß2m-free class I remaining following the purification of almost all ß2m+ class I. That the ß2m-free class I molecules indeed assemble in the absence of the light chain comes from functional studies discussed below.
Cells deficient in ß2m present all four endogenously derived H-2b class I-restricted SV-40 T antigen-derived epitopes to specific CD8+ T lymphocyte clones. Even ß2m-free H-2Kb presents epitope IV to a select CTL clone. Considering that ß2m-free H-2Kb binds peptides that deviate considerably in length and side chains from the native class I-binding ligands, it is surprising that epitope IV is presented by ß2m-free H-2Kb. Two different epitope V-specific CTL clones, H1 and Y5, were used to detect the presentation of this epitope by ß2m-free H-2Db. CTL clone H-1 recognizes epitope V presented by ß2m-free H-2Db, whereas clone Y-5 does not. One plausible explanation for the difference in epitope V recognition may be that Y-5 is sensitive to conformational difference caused by the lack of ß2m, while H-1 is insensitive to such alterations. Alternatively, epitope V being a poor binder to H-2Db (47) may be readily lost in the absence of ß2m. Therefore, it is conceivable that H-1 is highly reactive so that a few molecules of the epitope trigger its activity, while Y-5 may require the presentation of more epitope V to elicit activity. A similar argument could be responsible for the differential recognition of H-2Kb-restricted epitope IV by the two distinct reactive clones Y4 and SV2168T. Peptide specificity and the differential sensitivity of different CTL clones to the same epitope has been recognized previously (28). Thus, all the SV-40 T antigen-derived epitopes assemble in cells with H-2b class I molecules in the absence of ß2m, whose display at the cell surface form targets for specific CTL clones. Additionally, the CTL clones can recognize, albeit less efficiently, class I in the absence of ß2m.
The recognition of ß2m-free H-2Db by SV-40-specific CTL may be due to residual bovine ß2m present in dialyzed FCS or from mouse ß2m released from the CTL during the assay. Whether dialyzed bovine serum contained residual ß2m could not be ascertained empirically because neither an antibody nor a biochemical method to monitor this light chain currently exists. However, we do know that the dialysis of an MHC class I-like molecule against water across a 10-kDa membrane cut-off results in complete loss of associated ß2m (S. Joyce, unpublished data). Regarding mouse ß2m in the assay, note that at the highest E:T ratio there are 1 x 105 CTL/well. Cell lines such as RMA and EL4 express
50,000 class I molecules/cell. Although lymphoblasts express fewer class I molecules per cell compared to cell lines, but assuming that there are as many class I per CTL, there are 5 x 109 ß2m molecules/well. Even the presence of 5.2 x 1013 molecules of huß2m added exogenously to the assay did not alter the recognition of ß2m-free H-2Db by SV-40 T antigen-derived epitope-specific CTL. Therefore, it is less likely that even if all ß2m were released from the CTL in the assay, it would have altered the presentation of CTL epitopes by ß2m-free class I. Also note that mouse class I heavy chains have greater affinity for huß2m than for its own or bovine ß2m. Thus, most importantly, the fact remains that the CTL epitopes assemble with class I molecules in the absence of ß2m in the ER during biosynthetic assembly.
The mechanism by which ß2m-free class I assemble in cells remains unknown. Although free heavy chain is edited in the ER and lost to cytosolic degradation, a few molecules could fold with peptides pumped into the lumen by TAP with or without the assistance of the class I assembly and loading complex. The CTL epitopes presented by ß2m-free class I molecules contain the canonical H-2Kb and H-2Db binding motifs. Therefore, we predict that their assembly may have occurred through transient associations of the ß2m-free heavy chain with the class I assembly complex in an alternate as yet unidentified pathway. Alternatively, free class I might escape the ER editing mechanism, negotiate the secretory pathway and acquire peptides in the Golgi apparatus, at the cell surface or within the recycling compartment. Interestingly, MHC class I molecules, albeit inefficiently, can acquire antigenic peptides from the endosomal/lysosomal compartment (4855). Indeed, SV-40 T antigen-derived epitope I and epitope II/III can be presented to specific CTL by TAP-deficient cells with the assistance of a cytosolic chaperone, hsp73 (49). Further evidence suggests that their assembly occurs in a post-Golgi recycling compartment (49). Thus the assembly of ß2m-free class I with peptides may adopt the hitherto less well-characterized, non-canonical post-ER antigen processing and presentation mechanism (56).
Taken together, the results reported herein indicate that ß2m-free MHC class I molecules assemble with peptides and some of the ß2m-free class I molecules associated with peptides form CTL antigens. However, whether peptides lacking the canonical class I-binding motif that assemble with ß2m-free class I egress from the ER and form CTL epitopes at the cell surface remain to be determined. It is possible that the peptides associated with ß2m-free class I may function as mini-chaperones, protecting the free heavy chain from editing and degradation within the ER. In this manner the ß2m-free class I may have retained the fold and function of class I-like MIC-A, MIC-B and zinc-binding proteins, which have a MHC class I-like fold but do not utilize ß2m as part of their structure (57,58).
 |
Acknowledgements
|
---|
We thank W. Ajayi, C. Aiken, A.K. Stanic, D. Unutmaz, R. Yadav and members of the Joyce Laboratory for critical evaluation of the data, comments on the manuscript and support. Supported by grants from the NIH [CA25000 (S. S. T.) and HL54977 (S. J.)] and the Childrens Miracle Network (S. J.). T. D. S. was funded by the Four Diamonds Fund for Cancer Research and S. J. was a recipient of American Cancer Societys Junior Faculty Research Award.
 |
Abbreviations
|
---|
ß2mß2-microglobulin
CTLcytotoxic T lymphocyte
Endo Hendoglycosidase H
ERendoplasmic reticulum
hAsheteroantiserum
PTHphenylthiohydantoin
 |
References
|
---|
- Townsend, A. R. M. and Bodmer, H. 1989. Antigen recognition by class I restricted T lymphocytes. Annu. Rev. Immunol. 7:601.[ISI][Medline]
- Rammensee, H. G., Bachmann, J. and Stevanovic, S. 1998. MHC Ligands and Peptide Motifs. Landes Bioscience, Austin, TX.
- Bjorkman, P. J., Saper, M. A., Samraoui, B., Bennett, W. S., Strominger, J. L. and Wiley, D. C. 1987. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329:512.[ISI][Medline]
- Matsumura, M., Fremont, D. H., Peterson, P. and Wilson, I. A. 1992. Emerging principles for the recognition of peptide antigens by MHC class I molecules. Science 257:927.[ISI][Medline]
- Cresswell, P., Bangia, N., Dick, T. and Diedrich, G. 1999. The nature of the MHC class I peptide loading complex. Immunol. Rev. 172:21.[ISI][Medline]
- Grandea, A. G., III and Van Kaer, L. 2001. Tapasin: an ER chaperone that controls MHC class I assembly with peptide. Trends Immunol. 22:194.[ISI][Medline]
- Degan, E. and Williams, D. 1991. Participation of a novel 88 kD protein in the biogenesis of the murine class I histocompatibility molecules. J. Cell Biol. 112:1099.[Abstract]
- Rajagopalan, S. and Brenner, M. B. 1994. Calnexin retains unassembled major histocompatibility complex class I free heavy chains in the endoplasmic reticulum. J. Exp. Med. 180:407.[Abstract]
- Vassilakos, A., Cohen-Doyle, M. F., Peterson, P. A., Jackson, M. R. and Williams, D. R. 1996. The molecular chaperone calnexin facilitates folding and assembly of class I histocompatibility molecules. EMBO J. 15:1495.[Abstract]
- Ou, W.-J., Cameron, P. H., Thomas, D. Y. and Bergeron, J. J. M. 1993. Association of folding intermediates of glycoproteins with calnexin during protein maturation. Nature 364:771.[ISI][Medline]
- Gao, B., Adhikari, R., Howarth, M., Nakamura, K., Gold, M. C., Hill, A. B., Knee, R., Michalak, M. and Elliott, T. 2002. Assembly and antigen-presenting function of MHC class I molecules in cells lacking the ER chaperone calreticulin. Immunity 16:99.[ISI][Medline]
- Dick, T. P., Bangia, N., Peaper, D. R. and Cresswell, P. 2002. Disulfide bond isomerization and the assembly of MHC class Ipeptide complexes. Immunity 16:87.[ISI][Medline]
- Farmery, M. R., Allen, S., Allen, A. J. and Bulleid, N. J. 2000. The role of ERp57 in disulphide bond formation during the assembly of major histocompatibility complex class I in a synchronized semipermiabilized cell translation system. J. Biol. Chem. 275:14933.[Abstract/Free Full Text]
- Lindquist, J. A., Jensen, O. N., Mann, M. and Hammerling, G. J. 1998. ER-60, a chaperone with thiol-dependent reductase activity involved in MHC class I assembly. EMBO J. 17:2186.[Abstract/Free Full Text]
- Suh, W., Cohen-Doyle, M., Fruh, K., Wang, K., Peterson, P. A. and Williams, D. 1994. Interaction of MHC class I molecules with the transporter associated with antigen processing. Science 264:1322.[ISI][Medline]
- Sadasivan, B., Lehner, P. J., Ortmann, B., Spies, T. and Cresswell, P. 1996. Roles for calreticulin and a novel glycoprotein, tapasin, in the interaction of MHC class I molecules and TAP. Immunity 5:11.
- Ortmann, B., Androlewicz, M. J. and Cresswell, P. 1994. MHC class I/ß2-microglobulin complexes associate with TAP transporters before peptide binding. Nature 368:864.[ISI][Medline]
- Ortmann, B., Copeman, J., Lehner, P. J., Sadasivan, B., Herberg, J. A., Grandea, A. G., Riddell, S. R., Tampe, R., Spies, T., Trowsdale, J. and Cresswell, P. 1997. A critical role for tapasin in the assembly and function of multimeric MHC class I-TAP complexes. Science 277:1306.[Abstract/Free Full Text]
- Suh, W.-K., Mitchell, E. K., Yang, Y., Peterson, P. A., Waneck, G. L. and Williams, D. B. 1996. MHC class I molecules form ternary complexes with calnexin and TAP and undergo peptide regulated interaction with TAP via their extracellular domains. J. Exp. Med. 184:337.[Abstract]
- Grandea, A. G., III, Androlewicz, M. J., Athwal, R., Geraghty, D. and Spies, T. 1995. Dependence of peptide binding by MHC class I molecules on their interaction with TAP. Science 270:105.[Abstract]
- Grandea III, A. G., Golovina, T. N., Hamilton, S. E., Sriram, V., Spies, T., Brutkiewicz, R. R., Harty, J. T., Eisenlohr, L. C. and Van Kaer, L. 2000. Impaired assembly yet normal trafficking of MHC class I molecules in Tapasin mutant mice. Immunity 13:213.[ISI][Medline]
- Garbi, N., Tan, P., Diehl, A. D., Chambers, B. J., Ljunggren, H.-G., Momberg, F. and Hammerling, G. J. 2000. Impaired immune responses and altered peptide repertoire in tapasin-deficient mice. Nat. Immunol. 1:234.[ISI][Medline]
- Allen, H., Fraser, J., Flyer, D., Calvin, S. and Flavell, R. A. 1986. ß2-microglobulin is not required for the cell surface expression of the murine class I histocompatibility antigen H-2Db or of a truncated H-2Db. Proc. Natl Acad. Sci. USA 83:7447.[Abstract]
- Smith, J. D., Myers, N. B., Gorka, J. and Hansen, T. H. 1993. Model for the in vivo assembly of nascent Ld class I molecules and for the expression of unfolded Ld molecules at the cell surface. J. Exp. Med. 178:2035.[Abstract]
- Bix, M. and Raulet, D. 1992. Functionally conformed free class I heavy chains exist on the surface of ß2-microglobulin negative cells. J. Exp. Med. 176:829.[Abstract]
- Apasov, S. and Sitkovsky. 1993. Highly lytic CD8+,
ß T-cell receptor cytotoxic T cells with major histocompatibility complex (MHC) class I antigen-directed cytotoxicity in ß2-microglobulin, MHC class I-deficient mice. Proc. Natl Acad. Sci. USA 90:2837.[Abstract]
- Apasov, S. G. and Sitkovsky, M. V. 1994. Development and antigen specificity of CD8+ cytotoxic T lymphocytes in ß2-microglobulin-negative, MHC class I-deficient mice in response to immunization with tumor cells. J. Immunol. 152:2087.[Abstract/Free Full Text]
- Cook, J. R., Solheim, J. C., Connolly, J. M. and Hansen, T. H. 1995. Induction of peptide-specific CD8+ CTL clones in ß2-microglobulin-deficient mice. J. Immunol. 154:47.[Abstract/Free Full Text]
- Freland, S., Chambers, B. J., Anderson, M., Van Kaer, L. and Ljunggren, H.-G. 1998. Rejection of allogeneic and syngeneic but not MHC class I-deficient tumor grafts by MHC class I-deficient mice. J. Immunol. 160:572.[Abstract/Free Full Text]
- Glas, R., Ohlen, C., Hogland, P. and Karre, K. 1994. The CD8+ T cell repertoire in ß2-microglobulin-deficient mice is biased towards reactivity against self-major histocompatibility class I. J. Exp. Med. 179:661.[Abstract]
- Lamouse-Smith, E., Clements, V. K. and Ostrand-Rosenberg, S. 1993. ß2m/ knockout mice contain low levels of CD8+ cytotoxic T lymphocyte that mediate specific tumor rejection. J. Immunol. 151:6283.[Abstract/Free Full Text]
- Nesic, D., Santori, F. R. and Vukmanovic, S. 2000.
ß TCR+ cells are a minimal fraction of peripheral CD8+ pool in MHC class I-deficient mice. J. Immunol. 165:1896.[Abstract/Free Full Text]
- Zilstra, M., Auchincloss, H., Jr, Loring, J. M., Chase, C. M., Russell, P. S. and Jaenisch, R. 1992. Skin graft rejection by ß2-microglobulin-deficient mice. J. Exp. Med. 175:885.[Abstract]
- Joyce, S., Kuzushima, K., Kepecs, G., Angeletti, R. H. and Nathenson, S. G. 1994. Characterization of an incompletely assembled major histocompatibility class I molecule (H-2Kb) associated with unusually long peptides: implications for antigen processing and presentation. Proc. Natl Acad. Sci. USA 91:4145.[Abstract]
- Joyce, S. 1997. Traffic control of completely assembled MHC class I molecules beyond the endoplasmic reticulum. J. Mol. Biol. 267:993.
- Ajitkumar, P., Geier, S. S., Kesari, K. V., Borriello, F., Nakagawa, M., Bluestone, J. A., Saper, M. A., Wiley, D. C. and Nathenson, S. G. 1988. Evidence that multiple residues on both the
-helices of the class I MHC molecule are simultaneously recognized by the T cell receptor. Cell 54:47.[ISI][Medline]
- Allen, H., Wraith, D., Pala, P., Askonas, B. and Flavell, R. A. 1984. Domain interactions of H-2 class I antigens alter cytotoxic T-cell recognition. Nature 309:279.[ISI][Medline]
- Campbell, A. E., Foley, F. L. and Tevethia, S. S. 1983. Demonstration of multiple antigenic sites of the SV-40 transplantation rejection antigen by using cytotoxic T lymphocyte clones. J. Immunol. 130:490.[Abstract/Free Full Text]
- Mylin, L. M., Bonneau, R. H., Lipolis, J. D. and Tevethia, S. S. 1995. Hierarchy among multiple H-2b restricted cytotoxic T lymphocyte epitopes within simian virus 40 T antigen. J. Virol. 69:6665.[Abstract]
- Schell, T. D. and Tevethia, S. S. 2001. Control of advanced choroid plexus tumors in SV-40 T antigen transgenic mice following priming of donor CD8+ T lymphocytes by the endogenous tumor antigen. J. Immunol. 167:6947.[Abstract/Free Full Text]
- Tanaka, Y., Tevethia, M. J., Kalderon, D., Smith, A. E. and Tevethia, S. S. 1988. Clustering of antigenic sites recognized by cytotoxic T lymphocyte clones in the amino terminal half of SV-40 T antigen. Virology 162:427.[ISI][Medline]
- Tanaka, Y., Anderson, R. W., Maloy, W. L. and Tevethia, S. S. 1989. Localization of an immunorecessive epitope on SV-40 t antigen by H-2Db-restricted cytotoxic T-lymphocyte clones and T synthetic peptide. Virology 171:205.[ISI][Medline]
- Hughes, E. A., Hammond, C. and Cresswell, P. 1997. Misfolded major histocompatibility complex class I heavy chains are translocated into the cytoplasm and degraded by the proteasome. Proc. Natl Acad. Sci. USA 94:1896.[Abstract/Free Full Text]
- Falk, K., Rotzschke, O., Stevanovic, S., Jung, G. and Rammensee, H.-G. 1991. Allele-specific motifs revealed by sequencing of self peptides eluted from MHC molecules. Nature 351:290.[ISI][Medline]
- Machold, R. P., Andree, S., Van Kaer, L., Ljunggren, H.-G. and Ploegh, H. L. 1995. Peptide influences the folding and intracellular transport of free major histocompatibility complex class I heavy chains. J. Exp. Med. 181:1111.[Abstract]
- Mendoza, L. M., Paz, P., Zuberi, A., Christianson, G., Roopenian, D. and Shastri, N. 1997. Minors held by majors: the H13 minor histocompatibility locus defined as a peptide/MHC class I complex. Immunity 7:461.[ISI][Medline]
- Fu, T.-M., Mylin, L. M., Schell, T. D., Bacik, I., Russ, G., Yewdell, J. W., Bennink, J. R. and Tevethia, S. S. 1998. An endoplasmic reticulum-targeting signal sequence enhances the immunogenicity of an immunosuppressive simian virus 40 large T antigen cytotoxic T-lymphocyte epitope. J. Virol. 72:1469.[Abstract/Free Full Text]
- Campbell, D. J., Serwold, T. and Shastri, N. 2000. Bacterial proteins can be processed by macrophages in a transporter associated with antigen processing-independent, cysteine protease-dependent manner for presentation by MHC class I molecules. J. Immunol. 164:168.[Abstract/Free Full Text]
- Schirmbeck, R. and Reimann, J. 1994. Peptide transporter-independent, stress protein-mediated endosomal processing of endogenous protein antigens for major histocompatibility complex class I presentation. Eur. J. Immunol. 24:1478.[ISI][Medline]
- Schirmbeck, R., Melber, K. and Reimann, J. 1995. Hepatitis B virus small surface antigen particles are processed in a novel endosomal pathway for major histocompatibility complex class I-restricted epitope presentation. Eur. J. Immunol. 25:1063.[ISI][Medline]
- Schirmbeck, R. and Reimann, J. 1996. Empty Ld molecules capture peptides from endocytosed hepatitis B surface antigen particles for major histocompatibility complex class I-restricted presentation. Eur. J. Immunol. 26:2812.[ISI][Medline]
- Schirmbeck, R., Thoma, S. and Reimann, J. 1997. Processing of exogenous hepatitis B surface antigen particles for Ld-restricted epitope presentation depends on exogenous ß2-microglobulin. Eur. J. Immunol. 27:3471.[ISI][Medline]
- Schirmbeck, R., Wild, J. and Reimann, J. 1998. Similar as well as distinct MHC class I-binding peptides are generated by exogenous and endogenous processing of hepatitis B virus surface antigen. Eur. J. Immunol. 28:4149.[ISI][Medline]
- Song, R. and Harding, C. V. 1996. Roles of proteasomes, transporters for antigen presentation (TAP) and ß2-microglobulin in the processing of bacterial or particulate antigens via an alternate class I MHC processing pathway. J. Immunol. 156:4182.[Abstract]
- Song, R., Porgador, A. and Harding, C. V. 1999. Peptide-receptive class I major histocompatibility complex molecules on TAP-deficient and wild-type cells and their roles in the processing of exogenous antigens. Immunology 97:316.[ISI][Medline]
- Reimann, J. and Schirmbeck, R. 1999. Alternative pathways for processing exogenous and endogenous antigens that can generate peptides for MHC class I-restricted presentation. Immunol. Rev. 172:131.[ISI][Medline]
- Sanchez, L. M., Chirino, A. J. and Bjorkman, P. 1999. Crystal structure of human ZAG, a fat-depleting factor related to MHC molecules. Science 283:1914.[Abstract/Free Full Text]
- Li, P., Willie, S. T., Bauer, S., Morris, D., Spies, T. and Strong, R. K. 1999. Crystal structure of the MHC class I homolog MIC-A, a

T cell ligand. Immunity 10:577.[ISI][Medline]
- Bonneau, R. H., Salvucci, L. A., Johnson, D. C. and Tevethia, S. S. 1993. Epitope specificity of H-2Kb-restricted, HSV-1-, and HSV-2-cross-reactive cytotoxic T lymphocyte clones. Virology 195:62.[ISI][Medline]