The assembly of functional b 2 -microglobulin-free MHC class I molecules that interact with peptides and CD8 + T lymphocytes

Transcription

1 International Immunology, Vol. 14, No. 7, pp. 775±782 ã 2002 The Japanese Society for Immunology The assembly of functional b 2 -microglobulin-free MHC class I molecules that interact with peptides and CD8 + T lymphocytes Todd D. Schell 1, Lawrence M. Mylin 1,3, Satvir S. Tevethia 1 and Sebastian Joyce 2 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 Keywords: b 2 -microglobulin, antigen processing and presentation, CD8 + T lymphocytes, MHC class I, SV- 40 T antigen epitope Abstract Functional MHC class I molecules are expressed on the cell surface in the absence of b 2 -microglobulin (b 2 m) 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 b 2 m-free H-2D b assembles with short peptides that are ~9 amino acid residues in length, akin to ligands associated with completely assembled b 2 m + H-2D b. Remarkably, a subset of the peptides associated with the b 2 m- free H-2D b has an altered anchor motif. However, they also include peptides that contain a b 2 m + H-2D b binding anchor motif. Further, the H-2K b - and H-2D b -restricted peptide epitopes derived from SV-40 T antigen also assemble with H-2 b class I in b 2 m-de cient cells and are recognized by epitope-speci c CD8 + T lymphocytes. Taken together our data reveal that functional MHC class I molecules assemble in the absence of b 2 m with peptides and form CD8 + T lymphocyte epitopes. 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 b 2 -microglobulin (b 2 m) and a peptide of 8±11 amino acid residues [see references in (2)]. The membrane distal a 1 and a 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 traf c 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 cotranslationally inserts into the endoplasmic reticulum (ER); it binds calnexin, which assists its folding (7±9) presumably by preventing the aggregation of nascent heavy chains (10). Upon partial folding, the heavy chain is receptive to b 2 m. Calnexin within the initial heavy chain b 2 m complex rapidly exchanges for another ER chaperone, calreticulin. Calreticulin assists the heavy chain b 2 m 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-disul de-bonded class I heavy chain. This heavy chain ERp57 interaction facilitates disul de bond formation (12±14). 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 Correspondence to: S. Joyce; sebastian.joyce@vanderbilt.edu Transmitting editor: L. L. Lanier Received 21 November 2001, accepted 12 April 2002

2 776 Assembling functional b 2 m-free MHC class I molecules in vivo plasma membrane (15±22). Thus native class I molecules are an ensemble of a heavy chain, b 2 m and peptide. Phenotypic studies have shown that class I molecules such as H-2D b and H-2L d are stably expressed at the surface of b 2 m-de cient cells (23±25). Additionally, functional studies have demonstrated that b 2 m-free H-2 b class I molecules can interact with conventional ab T lymphocytes because, albeit poorly, they engage in positive selection of CD8 + T lymphocytes, mediate allograft immunity and promote tumor rejection in b 2 m-de cient mice (25±33). Thus the resulting CTL are capable of responding to native syngeneic as well as allogeneic H-2 class I molecules (26±30). We have previously reported that b 2 m-free H-2K b molecules associate with unusually long peptides; these peptides bind H-2K b without a discernable binding motif (34). The unique properties of peptides bound to b 2 m-free H-2K b differ from ligands associated with native b 2 m-containing H-2K b, 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 (PW) of the peptide (34). The function of peptide-associated b 2 m-free H- 2K b and how they assemble in cells remain unknown. Herein we report that b 2 m-free H-2D b 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 b 2 m-free H-2D b and H-2K b form epitopes for speci c CD8 + T lymphocytes. Thus, b 2 m-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-2K b and H-2D b respectively, are described (35). The methods used for metabolic labeling of cells with [ 35 S]methionine and Fig. 1. Cells express b 2 m-free H-2D b. One-hour [ 35 S]methioninelabeled cell lysates of Db-high clones 1±3 along with 10 times as many EL-4 were immune precipitated with conformationdependent mab speci c for H-2D b (B22-249) followed by immune precipitation of b 2 m-free H-2D b with s mab. Immune complexes were separated by 15% SDS±PAGE and visualized by autoradiography. H = heavy chain. The right panel was previously reported in (35). [ 35 S]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-2K b -speci c mab (36), B22-249, a native conformation-dependent H-2D b -speci c mab, and s, a b 2 m-free H-2D b -speci c mab (37) as well as the rabbit polyclonal heteroantiserum (has), ax8 directed against the tail end of K locus products (34). Immune complexes were separated by 15% SDS±PAGE, xed, soaked in Amplify (Amersham Pharmacia Biotechnology, Piscataway, NJ) and detected by autoradiography. Immune af nity puri cation of MHC class I, peptide isolation and characterization The procedures adopted for the puri cation of b 2 m-free and b 2 m + class I molecules using antibody (listed above)-coupled Protein A±Sepharose, the isolation of associated peptides, and amino acid sequence analyses are described in detail elsewhere (34). Generation and maintenance of antigen-speci c CTL and 51 Cr-release assay SV-40 T antigen-speci c CTL clones against SV-40 T antigenderived epitope I, epitope II/III, epitope IV and epitope V have been described previously (38±42). Conditions for culture and use of these CTL clones have been described previously (39,40). 51 Cr-release assays using target cells expressing endogenous antigens were performed according to standard protocols and the data are represented as percent speci c lysis (39,40). Results H-2D b is expressed in b 2 m-associated and b 2 m-free forms within cells We previously reported that cell lines express two immunochemically distinct forms of H-2K b. One form is associated with b 2 m (b 2 m + class I) and the second (b 2 m-free) is weakly or not at all associated with b 2 m (34). Additionally, b 2 m-free H-2D b and H-2L d are stably expressed at the surface of cell lines as well as of freshly isolated cells derived from b 2 m-de cient mice (23±25). Therefore, to study the relationship between b 2 m + and b 2 m-free H-2D b, NS0 plasmacytoma (H-2 d ) 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-2D b that can be distinguished by sequential precipitation with speci c antibodies. B (a conformation-dependent mab) immune precipitates b 2 m + H-2D b from detergent lysates of metabolically radiolabeled Db-high cells (Fig. 1, lanes 2±4 in left panel). However, subsequent precipitation of the B cleared, labeled lysates with an a 3 domain-speci c mab s reveals (b 2 m-free H-2D b ) a form of H-2D b that is weakly or not at all associated with the light chain (Fig. 1, lanes 2±4 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 b 2 m + H-2D b (35). Immunochemical analyses of

3 Assembling functional b 2 m-free MHC class I molecules in vivo 777 normal cell lines, such as EL-4 (Fig. 1, lane 1 in both panels), also reveal the presence of both b 2 m + and b 2 m-free forms of H-2D b. Thus b 2 m-free class I is stably expressed in cell lines. As b 2 m-free class I molecules are rapidly degraded in the ER (43), the biosynthetic fate of the b 2 m-free H-2D b and H-2K b were determined. A representative Db-high cell line and Kbhigh, a cell line that overexpresses H-2K b, were pulse-labeled and chased for the indicated times, solubilized in detergent, and b 2 m + and b 2 m-free class I were immune precipitated Fig. 2. b 2 m-free H-2D b that negotiate the secretory pathway. Cells were pulse-labeled with [ 35 S]methionine for 10 min and chased for the indicated periods of time at 37 C. b 2 m + and b 2 m-free H-2D b and H-2K b were immune precipitated from post-nuclear lysates with B or Y3 and s or ax8 respectively. The immune complexes were Endo H-digested (r = resistant, s = sensitive), separated by SDS±PAGE and detected by autoradiography. The traf c of b 2 m + class I was reported in (35). successively with speci c 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 b 2 m-free H-2D b and H-2K b egress from the ER and turn over with the same kinetics as the b 2 m + form of the respective class I molecule (Fig. 2). Further, both the Endo H- sensitive and the Endo H-resistant b 2 m-free H-2D b turn over more rapidly than the b 2 m + form of this class I molecule (Fig. 2). Thus ~50% b 2 m-free class I escapes the architectural editing mechanism within the ER and arrives into the Golgi apparatus and beyond. b 2 m-free H-2D b is associated with peptides that contain an altered binding motif The discovery of a pool of b 2 m-free class I molecules that egress from the ER akin to the completely assembled b 2 m + form raises the question as to whether the former associates with peptides. Biochemical analysis of b 2 m-free H-2K b revealed that it is associated with peptides whose properties were distinct from b 2 m + H-2K b bound ligands (34). Thus peptides associated with the two forms of H-2D b were isolated and a fraction of the peptide pool was subjected to microsequence analysis. This analysis reveals that peptides associated with the native b 2 m + H-2D b are ~9 amino acid residues long and contain the dominant H-2D b binding anchor residues: asparagine at position 5 (P5) and methionine at PW (Table 1A). Thus, as described previously (44), native b 2 m + H- 2D b is complexed with canonical short peptides containing the appropriate anchor motif. Akin to the features of peptides eluted from the native b 2 m + H-2D b, pool sequence analysis of an aliquot of the unfractionated peptides associated with b 2 m-free H-2D b molecules reveals that they are also short, averaging ~9 amino acid Table 1. The binding motif of peptides assembled with b 2 m-associated and b 2 m-free H-2D b Form of class I mab (speci city) Position in peptide A: a Native, b2-m + B (a 1 ) A b A P R N c K H H M ± d ± ± F Q I E I E Y I Y M Y H Q T K L I G L Q F T V K L F T V S V B: b 2 m-free s (a 3 ) F A P K N R G Y I ± d ± ± I K L Q H Q Y K V Y R V V Y F F L V F R Q a Part (A) was previously reported in (35), which was a part of the experiment described in (B). b The assignment of residues at a given position (cycle) in the peptide is based on the decreasing order of their yields. c Residues in bold type indicate the D b -binding anchor motif. d Yields of phenylthiohydantoin (PTH) derivatized amino acids signi cantly decreased at and beyond the 10th Edman degradation cycle to barely detectable levels. b 2 m + (A) and b 2 m-free (B) H-2D b were af nity puri ed from detergent lysates of ~ Db-high cells, and the associated self-peptides were isolated and separated from the heavy and light chains by Centricon 3 ltration. Approximately 10% of the peptides in the Centricon 3 ltrate were subjected to pool sequencing by 12 automated cycles of Edman degradation using an ABI 477A sequencer (Applied Biosystems). Control peptide preparation included Centricon 3 ltrate of the material eluted from an anti-x8 column chromatography of Dbhigh detergent lysate as well as s-coupled column chromatography of Kb-high detergent lysate. These two preparations did not yield any signi cant amounts of PTH-amino acids upon Edman degradation.

4 778 Assembling functional b 2 m-free MHC class I molecules in vivo Fig. 3. b 2 m-free H-2D b but not b 2 m-free H-2K b present antigen to speci c CTL. SV-40 T antigen-transformed b 2 m-de cient broblast was tested for their ability to display H-2D b -restricted (epitope I, top left; epitope II/III, top right; epitope V, bottom right) and H-2K b - restricted (epitope IV, bottom left) SV-40 T antigen-derived peptide epitopes to speci c CTL. CTL epitope display was monitored either without any manipulation of b 2 m-de cient targets or in the presence of synthetic peptide epitopes I or IV and/or hub 2 m. residues in length (Table 1B). However, in striking contrast, in the b 2 m-free H-2D b -derived peptide pool, the expected H-2D b 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 PW of H-2D b binding peptides was not present, but contained isoleucine, valine and leucine at this position (Table 1B). Two controls were set-up to monitor non-speci c binding of peptides to s mab. In one control, detergent lysates of Db-high cells were passed over the ax8 [a H-2K locus encoded product-speci c, conformation-independent has (34)] column. In the second control, detergent lysates of Kbhigh cells were passed over the s column. The nonspeci cally bound material was eluted and treated in an identical manner as ligands isolated from b 2 m + and b 2 m-free H-2D b. The two control preparations did not contain peptides (data not shown). Thus peptides associated with the b 2 m-free class I molecules are not contaminants that co-purify during af nity chromatography. The nding that b 2 m-free class I are associated with peptides suggests that the H-2D b heavy chain peptide interactions form stable complexes and may explain their expression on the surface of b 2 m-de cient cells. Functional peptide epitopes are presented by b 2 m-free H- 2K b and H-2D b to speci c CD8 + T lymphocyte clones The presence of b 2 m-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 signi cance. Thus we determined whether SV-40 T antigen-transformed renal broblasts derived from b 2 m-de cient mice are recognized by a panel of well characterized H-2K b - and H-2D b -restricted CD8 + T lymphocyte (CTL) clones (38±42). The data reveal that the H-2D b -restricted T antigen-derived epitope I is presented by the b 2 m-de cient broblasts (Fig. 3, top left panel), as are epitope II/III and, although less ef ciently, epitope V (Fig. 3, right panels). On the contrary, the H-2K b -restricted epitope IV is not presented by the b 2 m-de cient broblast to the CTL clone Y4 (Fig. 3, bottom left panel). The addition of human b 2 m (hub 2 m) alone did not sensitize b 2 m-de cient broblasts to any SV-40 T antigen-speci c CTL clones (Fig. 3). Further, the addition of H-2D b -restricted peptide epitopes II/III and V in addition to hub 2 m failed to increase the ef ciency 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 hub 2 m, signi cantly enhanced lysis of b 2 m-de cient broblasts by the CTL clone K11. Signi cant enhancement of Y4 CTL clone-mediated lysis required the addition of hub 2 m (Fig. 3, bottom left panel). These data suggest that in cells, b 2 m-free H-2D b assembles with peptide epitopes, which form CTL antigens. Two key questions arise from the above ndings. First, where do the H-2D b -restricted epitopes assemble: intracellularly or at the cell surface? Second, does the recognition of the H-2D b -restricted epitopes depend on b 2 m? In the above experiment, the b 2 m-free H-2D b could associate with bovine b 2 m present in FBS used to maintain cells in culture. To address these questions, b 2 m-de cient and b 2 m + wild-type SV-40 T antigen-transformed broblasts along with b 2 m + broblasts 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 b 2 m 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-2 b class I-restricted SV-40 T antigen epitopes to speci c CTL clones. The data reveal that epitope I- and epitope II/III-speci c CTL recognize H-2D b expressed by wild-type SV-40 T antigentransformed b 2 m-de cient broblasts. The growth of these cells in dialyzed FBS did not alter CTL recognition of the speci c epitopes (Fig. 4A, top and middle rows). As expected, the recognition of b 2 m + broblast was 2- to 3-fold better than the b 2 m-de cient targets. Additionally, b 2 m + broblasts 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-speci c CTL clones used in the two experiments were different. The sensitivities of the two epitope V-speci c CTL clones may be

5 Assembling functional b 2 m-free MHC class I molecules in vivo 779 Fig. 4. T cells recognize b 2 m-free MHC class I molecules. SV-40 T antigen-transformed b 2 m-de cient (b 2 m ± wt T), b 2 m + (b 2 m + wt T) broblasts as well as b 2 m + broblasts transformed with all four epitope-loss variant of SV-40 T antigen (b 2 m + epi ± T) grown in dialyzed (10-kDa cut-off; left panels) or normal (middle panels) FBS were tested for their ability to display H-2D b -restricted (A; epitope I, top row; epitope II/III, middle row; epitope V, bottom row) and H-2K b -restricted (B; epitope IV) SV-40 T antigen-derived peptide epitopes to speci c CTL. Right panels show the speci city of the CTL clones, which recognize their cognate peptide epitopes when provided exogenously to otherwise SV-40 T antigendevoid RMA cells (A) or epitope-loss mutant T antigen-transformed b 2 m + broblasts (B). A herpes simplex virus I's glycoprotein B-derived H- 2K b -restricted epitope [gb (59)] was used as a negative control (B). 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-speci c Y4 recognize antigen in the absence of b 2 m. The H-2K b -restricted CTL clone SV2168T speci c for epitope IV, in contrast to Y4 (also a H-2K b -restricted CTL clone speci c for epitope IV), recognized epitope IV assembled and presented in the absence of b 2 m (Fig. 4B, left panel). Addition of hub 2 m 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-2 b class I molecules in the absence of b 2 m, whose display at the cell surface form targets for speci c CTL clones.

6 780 Assembling functional b 2 m-free MHC class I molecules in vivo Discussion The current model for class I assembly suggests that incomplete molecules, either lacking b 2 m 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 b 2 m-free H-2D b and H-2L d are expressed on the cell surface (23±25,45). Such b 2 m-free class I permits CD8 + T lymphocyte development (28,30). These CTL elicit a lytic response and mediate tumor-speci c immunity as well as allograft rejection in b 2 m-de cient mice (25± 27,29,30,32,33). These data suggest an interaction between the antigen-speci c TCR and the b 2 m-free class I molecule. Whether the b 2 m-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 b 2 m-free class I. Two criteria were used to detect the association of peptides with b 2 m-free class I: (i) direct isolation and amino acid sequence analysis of the ligands associated with the b 2 m-free class I, and (ii) presentation of CTL epitopes to antigen-speci c T lymphocytes. We previously described the characterization of peptides associated with b 2 m-free H-2K b. The associated peptides had unusual characteristics in that they were longer than any known b 2 m + H-2K b binding ligands and did not contain the H-2K b binding anchor motif (34). In striking contrast, we show here that b 2 m- free H-2D b -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-2D b binding anchor. The remainder contained histidine or tryrosine at P5. Strikingly, none of the associated peptides contained PW methionine, the C-terminal dominant anchor. Alteration in the binding motif, albeit relatively uncommon, is observed in bone de CTL epitopes. For example, the H-2D b -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- 2D b (46). Similarly, numerous examples of altered P5 anchor residues have been reported for H-2K b -restricted CTL epitopes (2). Whether epitopes with an altered anchor motif associate with b 2 m + or b 2 m-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 b 2 m-free class I molecules. The above interpretation is limited by the fact that the b 2 m- free class I may have resulted from the dissociation of the light chain following assembly of b 2 m + class I. Thus we recognize that the isolation and characterization of class I-associated ligands should be performed with b 2 m-de cient cells expressing b 2 m-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 b 2 m- de cient mouse spleens and thymi to obtain suf cient amounts of b 2 m-free class I molecules to perform Edman sequence analysis of peptides eluted from them. Alternatively it would take >10 13 b 2 m-de cient cells maintained in serumfree conditions to purify suf cient amounts of heavy chain to analyze peptides associated with them. Thus the analyses described herein was performed on peptides isolated from b 2 m-free class I remaining following the puri cation of almost all b 2 m + class I. That the b 2 m-free class I molecules indeed assemble in the absence of the light chain comes from functional studies discussed below. Cells de cient in b 2 m present all four endogenously derived H-2 b class I-restricted SV-40 T antigen-derived epitopes to speci c CD8 + T lymphocyte clones. Even b 2 m-free H-2K b presents epitope IV to a select CTL clone. Considering that b 2 m-free H-2K b 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 b 2 m-free H-2K b. Two different epitope V-speci c CTL clones, H1 and Y5, were used to detect the presentation of this epitope by b 2 m-free H- 2D b. CTL clone H-1 recognizes epitope V presented by b 2 m- free H-2D b, 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 b 2 m, while H-1 is insensitive to such alterations. Alternatively, epitope V being a poor binder to H-2D b (47) may be readily lost in the absence of b 2 m. 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-2K b -restricted epitope IV by the two distinct reactive clones Y4 and SV2168T. Peptide speci city 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-2 b class I molecules in the absence of b 2 m, whose display at the cell surface form targets for speci c CTL clones. Additionally, the CTL clones can recognize, albeit less ef ciently, class I in the absence of b 2 m. The recognition of b 2 m-free H-2D b by SV-40-speci c CTL may be due to residual bovine b 2 m present in dialyzed FCS or from mouse b 2 m released from the CTL during the assay. Whether dialyzed bovine serum contained residual b 2 m 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 cutoff results in complete loss of associated b 2 m (S. Joyce, unpublished data). Regarding mouse b 2 m in the assay, note that at the highest E:T ratio there are 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 b 2 m molecules/well. Even the presence of molecules of hub 2 m added exogenously to the assay did not alter the recognition of b 2 m-free H-2D b by SV-40 T antigen-derived epitope-speci c CTL. Therefore, it is less likely that even if all b 2 m were released from the CTL in the assay, it would have altered the presentation of CTL epitopes by b 2 m-free class I. Also note that mouse class I heavy chains have greater af nity for hub 2 m than for its own or bovine b 2 m. Thus, most importantly, the fact remains that the CTL epitopes assemble with class I molecules in the absence of b 2 m in the ER during biosynthetic assembly.

7 The mechanism by which b 2 m-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 b 2 m-free class I molecules contain the canonical H-2K b and H-2D b binding motifs. Therefore, we predict that their assembly may have occurred through transient associations of the b 2 m-free heavy chain with the class I assembly complex in an alternate as yet unidenti ed 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 inef ciently, can acquire antigenic peptides from the endosomal/lysosomal compartment (48±55). Indeed, SV-40 T antigen-derived epitope I and epitope II/III can be presented to speci c CTL by TAPde cient 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 b 2 m-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 b 2 m-free MHC class I molecules assemble with peptides and some of the b 2 m-free class I molecules associated with peptides form CTL antigens. However, whether peptides lacking the canonical class I-binding motif that assemble with b 2 m-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 b 2 m-free class I may function as mini-chaperones, protecting the free heavy chain from editing and degradation within the ER. In this manner the b 2 m-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 b 2 m 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 Children's 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 Society's Junior Faculty Research Award. Abbreviations b 2 m CTL Endo H ER has PTH References b 2 -microglobulin cytotoxic T lymphocyte endoglycosidase H endoplasmic reticulum heteroantiserum phenylthiohydantoin 1 Townsend, A. R. M. and Bodmer, H Antigen recognition by class I restricted T lymphocytes. Annu. Rev. Immunol. 7:601. Assembling functional b 2 m-free MHC class I molecules in vivo Rammensee, H. G., Bachmann, J. and Stevanovic, S MHC Ligands and Peptide Motifs. Landes Bioscience, Austin, TX. 3 Bjorkman, P. J., Saper, M. A., Samraoui, B., Bennett, W. S., Strominger, J. L. and Wiley, D. C The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329: Matsumura, M., Fremont, D. H., Peterson, P. and Wilson, I. A Emerging principles for the recognition of peptide antigens by MHC class I molecules. Science 257: Cresswell, P., Bangia, N., Dick, T. and Diedrich, G The nature of the MHC class I peptide loading complex. Immunol. Rev. 172:21. 6 Grandea, A. G., III and Van Kaer, L Tapasin: an ER chaperone that controls MHC class I assembly with peptide. Trends Immunol. 22: Degan, E. and Williams, D Participation of a novel 88 kd protein in the biogenesis of the murine class I histocompatibility molecules. J. Cell Biol. 112: Rajagopalan, S. and Brenner, M. B Calnexin retains unassembled major histocompatibility complex class I free heavy chains in the endoplasmic reticulum. J. Exp. Med. 180: Vassilakos, A., Cohen-Doyle, M. F., Peterson, P. A., Jackson, M. R. and Williams, D. R The molecular chaperone calnexin facilitates folding and assembly of class I histocompatibility molecules. EMBO J. 15: Ou, W.-J., Cameron, P. H., Thomas, D. Y. and Bergeron, J. J. M Association of folding intermediates of glycoproteins with calnexin during protein maturation. Nature 364: Gao, B., Adhikari, R., Howarth, M., Nakamura, K., Gold, M. C., Hill, A. B., Knee, R., Michalak, M. and Elliott, T Assembly and antigen-presenting function of MHC class I molecules in cells lacking the ER chaperone calreticulin. Immunity 16: Dick, T. P., Bangia, N., Peaper, D. R. and Cresswell, P Disul de bond isomerization and the assembly of MHC class I± peptide complexes. Immunity 16: Farmery, M. R., Allen, S., Allen, A. J. and Bulleid, N. J 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: Lindquist, J. A., Jensen, O. N., Mann, M. and Hammerling, G. J ER-60, a chaperone with thiol-dependent reductase activity involved in MHC class I assembly. EMBO J. 17: Suh, W., Cohen-Doyle, M., Fruh, K., Wang, K., Peterson, P. A. and Williams, D Interaction of MHC class I molecules with the transporter associated with antigen processing. Science 264: Sadasivan, B., Lehner, P. J., Ortmann, B., Spies, T. and Cresswell, P Roles for calreticulin and a novel glycoprotein, tapasin, in the interaction of MHC class I molecules and TAP. Immunity 5: Ortmann, B., Androlewicz, M. J. and Cresswell, P MHC class I/b 2 -microglobulin complexes associate with TAP transporters before peptide binding. Nature 368: 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 A critical role for tapasin in the assembly and function of multimeric MHC class I-TAP complexes. Science 277: Suh, W.-K., Mitchell, E. K., Yang, Y., Peterson, P. A., Waneck, G. L. and Williams, D. B 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: Grandea, A. G., III, Androlewicz, M. J., Athwal, R., Geraghty, D. and Spies, T Dependence of peptide binding by MHC class I molecules on their interaction with TAP. Science 270: 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 Impaired assembly yet normal traf cking of MHC class I molecules in Tapasin mutant mice. Immunity 13: Garbi, N., Tan, P., Diehl, A. D., Chambers, B. J., Ljunggren, H.-G., Momberg, F. and Hammerling, G. J Impaired immune

8 782 Assembling functional b 2 m-free MHC class I molecules in vivo responses and altered peptide repertoire in tapasin-de cient mice. Nat. Immunol. 1: Allen, H., Fraser, J., Flyer, D., Calvin, S. and Flavell, R. A b 2 - microglobulin is not required for the cell surface expression of the murine class I histocompatibility antigen H-2D b or of a truncated H-2D b. Proc. Natl Acad. Sci. USA 83: Smith, J. D., Myers, N. B., Gorka, J. and Hansen, T. H Model for the in vivo assembly of nascent L d class I molecules and for the expression of unfolded L d molecules at the cell surface. J. Exp. Med. 178: Bix, M. and Raulet, D Functionally conformed free class I heavy chains exist on the surface of b 2 -microglobulin negative cells. J. Exp. Med. 176: Apasov, S. and Sitkovsky Highly lytic CD8 +, ab T-cell receptor cytotoxic T cells with major histocompatibility complex (MHC) class I antigen-directed cytotoxicity in b 2 -microglobulin, MHC class I-de cient mice. Proc. Natl Acad. Sci. USA 90: Apasov, S. G. and Sitkovsky, M. V Development and antigen speci city of CD8 + cytotoxic T lymphocytes in b 2 - microglobulin-negative, MHC class I-de cient mice in response to immunization with tumor cells. J. Immunol. 152: Cook, J. R., Solheim, J. C., Connolly, J. M. and Hansen, T. H Induction of peptide-speci c CD8 + CTL clones in b 2 - microglobulin-de cient mice. J. Immunol. 154: Freland, S., Chambers, B. J., Anderson, M., Van Kaer, L. and Ljunggren, H.-G Rejection of allogeneic and syngeneic but not MHC class I-de cient tumor grafts by MHC class I-de cient mice. J. Immunol. 160: Glas, R., Ohlen, C., Hogland, P. and Karre, K The CD8 + T cell repertoire in b 2 -microglobulin-de cient mice is biased towards reactivity against self-major histocompatibility class I. J. Exp. Med. 179: Lamouse-Smith, E., Clements, V. K. and Ostrand-Rosenberg, S b 2 m ±/± knockout mice contain low levels of CD8 + cytotoxic T lymphocyte that mediate speci c tumor rejection. J. Immunol. 151: Nesic, D., Santori, F. R. and Vukmanovic, S ab TCR + cells are a minimal fraction of peripheral CD8 + pool in MHC class I- de cient mice. J. Immunol. 165: Zilstra, M., Auchincloss, H., Jr, Loring, J. M., Chase, C. M., Russell, P. S. and Jaenisch, R Skin graft rejection by b 2 - microglobulin-de cient mice. J. Exp. Med. 175: Joyce, S., Kuzushima, K., Kepecs, G., Angeletti, R. H. and Nathenson, S. G Characterization of an incompletely assembled major histocompatibility class I molecule (H-2K b ) associated with unusually long peptides: implications for antigen processing and presentation. Proc. Natl Acad. Sci. USA 91: Joyce, S Traf c control of completely assembled MHC class I molecules beyond the endoplasmic reticulum. J. Mol. Biol. 267: 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 Evidence that multiple residues on both the a-helices of the class I MHC molecule are simultaneously recognized by the T cell receptor. Cell 54: Allen, H., Wraith, D., Pala, P., Askonas, B. and Flavell, R. A Domain interactions of H-2 class I antigens alter cytotoxic T-cell recognition. Nature 309: Campbell, A. E., Foley, F. L. and Tevethia, S. S Demonstration of multiple antigenic sites of the SV-40 transplantation rejection antigen by using cytotoxic T lymphocyte clones. J. Immunol. 130: Mylin, L. M., Bonneau, R. H., Lipolis, J. D. and Tevethia, S. S Hierarchy among multiple H-2 b restricted cytotoxic T lymphocyte epitopes within simian virus 40 T antigen. J. Virol. 69: Schell, T. D. and Tevethia, S. S 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: Tanaka, Y., Tevethia, M. J., Kalderon, D., Smith, A. E. and Tevethia, S. S Clustering of antigenic sites recognized by cytotoxic T lymphocyte clones in the amino terminal half of SV-40 T antigen. Virology 162: Tanaka, Y., Anderson, R. W., Maloy, W. L. and Tevethia, S. S Localization of an immunorecessive epitope on SV-40 t antigen by H-2D b -restricted cytotoxic T-lymphocyte clones and T synthetic peptide. Virology 171: Hughes, E. A., Hammond, C. and Cresswell, P Misfolded major histocompatibility complex class I heavy chains are translocated into the cytoplasm and degraded by the proteasome. Proc. Natl Acad. Sci. USA 94: Falk, K., Rotzschke, O., Stevanovic, S., Jung, G. and Rammensee, H.-G Allele-speci c motifs revealed by sequencing of self peptides eluted from MHC molecules. Nature 351: Machold, R. P., Andree, S., Van Kaer, L., Ljunggren, H.-G. and Ploegh, H. L Peptide in uences the folding and intracellular transport of free major histocompatibility complex class I heavy chains. J. Exp. Med. 181: Mendoza, L. M., Paz, P., Zuberi, A., Christianson, G., Roopenian, D. and Shastri, N Minors held by majors: the H13 minor histocompatibility locus de ned as a peptide/mhc class I complex. Immunity 7: Fu, T.-M., Mylin, L. M., Schell, T. D., Bacik, I., Russ, G., Yewdell, J. W., Bennink, J. R. and Tevethia, S. S 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: Campbell, D. J., Serwold, T. and Shastri, N 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: Schirmbeck, R. and Reimann, J Peptide transporterindependent, stress protein-mediated endosomal processing of endogenous protein antigens for major histocompatibility complex class I presentation. Eur. J. Immunol. 24: Schirmbeck, R., Melber, K. and Reimann, J 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: Schirmbeck, R. and Reimann, J `Empty' L d molecules capture peptides from endocytosed hepatitis B surface antigen particles for major histocompatibility complex class I-restricted presentation. Eur. J. Immunol. 26: Schirmbeck, R., Thoma, S. and Reimann, J Processing of exogenous hepatitis B surface antigen particles for L d -restricted epitope presentation depends on exogenous b 2 -microglobulin. Eur. J. Immunol. 27: Schirmbeck, R., Wild, J. and Reimann, J 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: Song, R. and Harding, C. V Roles of proteasomes, transporters for antigen presentation (TAP) and b 2 -microglobulin in the processing of bacterial or particulate antigens via an alternate class I MHC processing pathway. J. Immunol. 156: Song, R., Porgador, A. and Harding, C. V Peptidereceptive class I major histocompatibility complex molecules on TAP-de cient and wild-type cells and their roles in the processing of exogenous antigens. Immunology 97: Reimann, J. and Schirmbeck, R Alternative pathways for processing exogenous and endogenous antigens that can generate peptides for MHC class I-restricted presentation. Immunol. Rev. 172: Sanchez, L. M., Chirino, A. J. and Bjorkman, P Crystal structure of human ZAG, a fat-depleting factor related to MHC molecules. Science 283: Li, P., Willie, S. T., Bauer, S., Morris, D., Spies, T. and Strong, R. K Crystal structure of the MHC class I homolog MIC-A, a gd T cell ligand. Immunity 10: Bonneau, R. H., Salvucci, L. A., Johnson, D. C. and Tevethia, S. S Epitope speci city of H-2K b -restricted, HSV-1-, and HSV-2- cross-reactive cytotoxic T lymphocyte clones. Virology 195:62.