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Tapasin-Mediated Retention and Optimization of Peptide Ligands During the Assembly of Class I Molecules¹

Megan J. Barnden^2,3,*, Anthony W. Purcell^3,*, Jeffrey J. Gorman and James McCluskey^4,*

^* Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia; and Biomolecular Research Institute, Parkville, Victoria, Australia

	Abstract

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The murine class I H-2K^b molecule achieves high level surface expression in tapasin-deficient 721.220 human cells. Compared with their behavior in wild-type cells, K^b molecules expressed on 721.220 cells are more receptive to exogenous peptide, undergo more rapid surface decay, and fail to form macromolecular peptide loading complexes. As a result, they are rapidly transported to the cell surface, reflecting a failure of endoplasmic reticulum retention mechanisms in the absence of loading complex formation. Despite the failure of K^b molecules to colocalize to the TAP and their rapid egress to the cell surface, K^b is still capable of presenting TAP-dependent peptides in the absence of tapasin. Furthermore, pool sequencing of peptides eluted from these molecules revealed strict conservation of their canonical H-2K^b-binding motif. There was a reduction in the total recovery of peptides associated with K^b molecules purified from the surface of tapasin-deficient cells. Comparison of the peptides bound to K^b in the presence and absence of tapasin revealed considerable overlap in peptide repertoire. These results indicate that in the absence of an interaction with tapasin, K^b molecules fail to assemble with calreticulin and TAP, yet they are still capable of acquiring a diverse array of peptides. However, a significant proportion of these peptides appear to be suboptimal, resulting in reduced cell surface stability of K^b complexes. Taken together, the findings indicate that tapasin plays an essential role in the formation of the class I loading complex, which retains class I heterodimers in the endoplasmic reticulum until optimal ligand selection is completed.

	Introduction

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Recent studies have revealed the intricacy of molecular interactions that take place within the endoplasmic reticulum (ER),⁵ culminating in MHC class I-peptide assembly (recently reviewed by Cresswell et al. (1)). This highly orchestrated process involves molecules dedicated solely to the assembly of class I-peptide complexes together with more generic molecular chaperones. Newly synthesized class I heavy chains (hc) are translocated into the ER lumen where they associate with the resident membrane-bound chaperone calnexin. Upon binding of ß₂-microglobulin (ß₂m), the class I heterodimer dissociates from calnexin, and a new complex is formed comprising class I hc, ß₂m, calreticulin, ERp57, and tapasin (1). In murine cells the use of calreticulin and calnexin seems to be partially redundant such that class I heterodimers are observed complexed to both calnexin and calreticulin (2, 3). The multicomponent "loading complex" of class I heterodimer and associated ER-resident chaperones also includes the TAP heterodimer, bringing into close proximity peptide receptive class I molecules, the peptide translocation machinery, and auxiliary molecules that may contribute to peptide loading such as tapasin and ERp57 (1, 4). Upon peptide binding, class I-peptide complexes dissociate from all auxiliary molecules and are transported to the cell surface for recognition by CD8⁺ T cells.

Tapasin is a 48-kDa, MHC-encoded, type I transmembrane glycoprotein that is resident in the ER (4, 5). It functions to bridge peptide receptive class I loading complexes with the TAP (4, 6) such that up to four loading complexes associate with each TAP heterodimer (7). Tapasin expression also enhances TAP expression, leading to increased peptide translocation into the ER (8, 9). This suggests that tapasin increases overall peptide transport from the cytosol into the lumen of the ER, but it does not appear to affect the rate of peptide translocation (8). In addition, tapasin retains class I molecules that fail to bind peptide in the ER of insect cells (10) and may also be involved in the retention of peptide-bound class I molecules during a peptide optimization step in mammalian cells (11, 12). Paradoxically, most class I molecules expressed in human cells lacking functional tapasin are retained in the ER and are inefficiently expressed at the cell surface (4, 7, 9, 13, 14, 15, 16). Introduction of functional tapasin into these cells restores normal class I expression and functional Ag presentation (7, 9, 13, 14, 15, 16). These multiple functions of tapasin have led to speculation that it may be involved in peptide editing of class I bound ligands (10, 16, 17, 18, 19, 20, 21) in a manner similar to that of HLA-DM in class II Ag presentation (22). More recent studies have provided biochemical evidence for a specific peptide-editing function for tapasin (9).⁶ Furthermore, transfection studies introducing several HLA-A and -B alleles into the 721.220 (.220) cell line have revealed a spectrum of dependence on tapasin for surface expression and Ag presentation (14, 15, 16, 23) reflecting polymorphism in allelic requirements for tapasin in Ag presentation.

MHC class I molecules generally function normally across the human-mouse species barrier. Thus, murine class I molecules are expressed at high levels when transfected into human APC, whereas some HLA molecules require cotransfection of human ß₂m to permit high levels of surface expression (24). However, in mutant APC there are certain anomalies that remain unexplained. For example, the murine H-2K^b molecule is expressed on the cell surface of the TAP-deficient human cell line T2, despite failure of TAP-dependent peptide loading (24). This contrasts markedly with the retention of K^b in the ER of murine RMA-S cells lacking a functional TAP complex (25). More recently, the thermolabile K^b molecules expressed on RMA-S at 26°C were shown to be associated with low-affinity peptides, suggesting that these molecules are initially stabilized by suboptimal ligands, which dissociate rapidly at the cell surface when the temperature is returned to 37°C (26). Despite stable cell surface expression of K^b on the surface of T2, no peptides were isolated during radiolabeled peptide elution experiments at 37°C (27), although it seems likely that these molecules are also initially stabilized by low-affinity peptides. The enhanced stability of H-2K^b molecules in human cells may also in part relate to the high affinity of this class I molecule for human ß₂m (28, 29), which results in stable expression of K^b on the surface of RMA-S transfected with human ß₂m (25).

Accordingly, we reasoned that these properties of K^b molecules expressed in human cells might result in stable cell surface expression on human tapasin-deficient APC. These cells express functional TAP heterodimers and are therefore capable of translocating peptides from the cytosol into the ER; however, if tapasin is necessary for ER retention of empty K^b molecules, they would be expected to rapidly egress to the cell surface, possibly in association with suboptimal peptides. Moreover, such a phenotype would allow us to directly compare peptide presentation at the cell surface under tapasin-competent and tapasin-deficient conditions. We demonstrate efficient surface expression of the murine K^b molecule on the tapasin-deficient .220 human cell line. Furthermore, in the absence of an interaction with tapasin, K^b molecules traffic to the cell surface more rapidly, are rendered more receptive to exogenous peptide, and undergo more rapid surface decay kinetics. In addition, our data show that the absence of tapasin abrogates association of the K^b hc with calreticulin and TAP. Despite these findings, there was a significant degree of endogenous H-2K^b peptide loading in the absence of tapasin as evidenced by functional Ag presentation, the repertoire of eluted peptides recovered from K^b molecules, and their retention of the canonical binding motif. The data indicate that tapasin has evolved to optimize the basic process of class I-peptide loading and that this is achieved by increased ER retention through tapasin-dependent formation of a loading complex that includes calreticulin, TAP, class I hc/ß₂m, tapasin, and ERp57 (1).

	Materials and Methods

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Cell lines and transfection

The human B lymphoblastoid cell line .220 is a gamma irradiation mutant that lacks both HLA-A and -B genes and expresses a truncated and nonfunctional tapasin protein (30, 31). Cells were grown in RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 2 mM L-glutamine, 50 mM 2-ME, nonessential amino acids, antibiotics, and 10% FCS (Life Technologies). The murine H-2^b thymoma EL4 (32) and the K^b/SIINFEKL-specific T hybridoma GA4.2 (33) were grown in DMEM supplemented in the same manner as RPMI 1640. Cells grown in the miniPERM bioreactors (Heraeus Instruments, Hanau, Germany) were sequentially transferred into hybridoma serum-free medium (Life Technologies) supplemented in the same manner as RPMI 1640 but containing only 1% FCS.

The pMCFR.puro plasmid containing a cDNA construct encoding human tapasin has been previously described (7). This cDNA construct was transfected into the .220 cell line by electroporation (210 V and 960 µF). Transfected cells were selected with 2 µg/ml puromycin (Sigma, St. Louis, MO). The pK^b plasmid containing a genomic construct encoding the H-2K^b hc has been previously described (34). This genomic construct was transfected into the .220 and .220.hTsn cell lines by electroporation. Transfected cells were selected with 0.5 mg/ml geneticin (Life Technologies). The pRSV.5 (hygro) plasmid (35) containing a SIINFEKL minigene construct was transfected by electroporation into the K^b.220 cell line. Transfected cells were selected with 200 µg/ml hygromycin B (Life Technologies).

Flow cytometry

Cell surface staining by indirect immunofluorescence was performed as follows. The anti-K^b-specific mAb Y-3 (35) and the K^b/SIINFEKL-specific mAb 25-D1.16 (36) were used as primary Abs under saturating conditions before FITC-conjugated sheep anti-mouse Ig (Silenus Laboratories, Melbourne, Australia). Data was collected on 10⁴ viable cells using a Becton Dickinson (Mountain View, CA) FACSort equipped with CELLQuest software.

Immunoprecipitation and endoglycosidase H digestion

Cells were starved in methionine- and cysteine-free medium for 50 min, pulsed for 5 min with 150 µCi/ml [³⁵S]methionine, and chased in medium containing excess methionine (1 mM) and cystine (2 mM). At the indicated time points, 5 x 10⁶ cells were collected, washed in cold PBS, and lysed for 30 min at 4°C in TBS (10 mM Tris (pH 7.4), 150 mM NaCl) containing 0.5% Nonidet P-40 and Complete protease inhibitor cocktail (Roche, Mannheim, Germany). After centrifugation to remove nuclei, the supernatant was precleared with protein A-Sepharose (Amersham, Buckinghamshire, U.K.). The K^b-specific mAb Y-3 or hyperimmune rabbit anti-peptide 8 serum were used to immunoprecipitate K^b molecules (37) and the immune complexes isolated on protein A-Sepharose. Beads were washed twice in buffer containing 450 mM NaCl, 50 mM Tris (pH 7.4), 5 mM EDTA, and 0.05% Nonidet P-40 and once in 10 mM Tris (pH 7.4). Beads were boiled in 50 µl 0.1 M sodium phosphate (pH 6.5) containing 1% SDS and 2 mM DTT. Nine volumes of water was added to the supernatant, and samples were divided in two; one half was treated with 5 mU endoglycosidase H (endo H; Sigma), and the other half was left untreated. After overnight incubation at 37°C, an equal volume of 30% trichloroacetic acid was added to precipitate the proteins. After washing in cold methanol, proteins were separated by SDS-PAGE. Gels were fixed, amplified, dried, and exposed to film for up to 7 days.

Immunoprecipitations and Western blot

Cells (5 x 10⁶) were lysed for 1 h at 4°C in TBS containing 0.5% digitonin (Fluka, Buchs, Switzerland) and Complete protease inhibitor cocktail. After centrifugation to remove nuclei, the supernatant was precleared overnight with normal rabbit serum and protein A-Sepharose. Anti-peptide 8 hyperimmune serum (37) directed against the exon 8-encoded intracytoplasmic region of the K^b molecule was used to immunoprecipitate K^b heavy chains for 1 h at 4°C before protein A-Sepharose was added. Beads were washed twice in TBS containing 0.1% digitonin; once in buffer containing 10 mM Tris (pH 7.4), 450 mM NaCl, and 0.1% digitonin; and once in 10 mM Tris (pH 7.4). Beads were boiled in SDS, and the proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membrane (NEN Life Science, Boston, MA). The membrane was blocked using 3% skimmed milk in PBS containing 0.2% Tween 20 (BDH, Poole, England), and the membranes were probed with Ab to human calreticulin (FMC 75) (38) and the human TAP1 molecule (148.3) (39). HRP-conjugated sheep anti-mouse Ig (Silenus Laboratories) was used as a secondary Ab, and proteins were visualized using Renaissance chemiluminescence substrate (NEN Life Science). This conjugate did not react with the denatured rabbit Ig under the conditions used.

Peptide receptivity assay

Cells were washed twice in serum-free RPMI 1640 and then were added to a 96-well V-bottom plate (2 x 10⁵/well; Greiner, Frickenhausen, Germany). Serial dilutions of SIINFEKL peptide were added to the wells before the addition of cells. After incubation for 1 h at 37°C, the plate was transferred to 4°C and cells were stained for flow cytometric analysis. The anti-K^b-specific mAb Y-3 (35) and the K^b/SIINFEKL-specific mAb 25-D1.16 (36) were used as primary Abs before FITC-conjugated sheep anti-mouse Ig. Mean fluorescence intensity = 100 x [(25.D1.16 staining - secondary Ab staining)/(Y3 staining - secondary Ab staining)].

Ag presentation assay

GA4.2 hybridoma cells (1 x 10⁵/well) were added to a 96-well flat-bottom tissue culture plate (Greiner) in the presence of various densities of K^b.220.SIINFEKL transfectants. After 24-h culture at 37°C in 5% CO₂, supernatant (50 µl/well) was harvested into fresh 96-well plates, and Ag-specific IL-2 production by the GA4.2 hybridoma was determined by quantitation of [³H]thymidine incorporation into the IL-2-dependent CTLL cell line. Briefly, CTLL cells (5 x 10³/well) were cultured for 18 h in one to four diluted supernatants. Cells were then pulsed for 6 h with 1 µCi/well [methyl ³H]thymidine (ICN Pharmaceuticals, Costa Mesa, CA) and harvested, and thymidine incorporation was determined using a Matrix 9600 Direct Beta Counter (Packard, Meriden, CT).

Purification of H-2K^b and peptide elution

K^b.220- and K^b.220.hTsn-transfected cells were grown in miniPERM bioreactors in hybridoma serum-free medium supplemented with 1% FCS. For accurate comparison, cells were examined in tandem, and 5 x 10⁹ cells were lysed at 4°C in 0.1% Nonidet P-40, 20 mM Tris (pH 7.4), and 150 mM NaCl with Complete protease inhibitor cocktail. Cell lysates were clarified by two rounds of centrifugation, and the supernatant was filtered and passed over a Sephadex G50 pre-column (Pharmacia Biotech, Uppsala, Sweden) and then a protein A-Sepharose column onto which a combination of the K^b-specific Y-3 and 20.8.4 (40) mAbs had been chemically cross-linked.⁶ The column was extensively washed in 0.005% Nonidet P-40, 50 mM Tris (pH 8.0), and 150 mM NaCl, then in 50 mM Tris (pH 8.0) and 450 mM NaCl, and finally in 10 mM Tris (pH 8.0). Bound K^b-peptide complexes were eluted with 0.2 N acetic acid, which also facilitates dissociation of K^b-associated peptides. The eluate was then passed over a Centricon 3 membrane (Millipore, Bedford, MA), and the flow-through was concentrated by vacuum centrifugation to a final volume of 300 µl. The concentrated flow-through was separated on a SMART system HPLC (Pharmacia Biotech) with a µRPC C2/C18 2.1 mm internal diameter x 10 cm column (Pharmacia Biotech). This fractionated material was then subjected to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and pool Edman sequencing. MALDI-TOF mass spectrometry was performed using a Bruker Reflex instrument (Bruker-Franzen Analytik, Bremen, Germany) operated exclusively in the reflectron mode. N-terminal automated Edman sequencing was performed on a Hewlett Packard G1000A protein sequencer (Hewlett-Packard, Palo Alto, CA) using standard Edman chemistries.

	Results

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High-level surface expression of H-2K^b class I molecules on tapasin-deficient cells is associated with premature release from the ER

H-2K^b is expressed at high levels on the surface of the human TAP-deficient cell line T2, yet it is retained in the ER of the TAP-deficient mouse cell line RMA-S (24, 25). This discrepant behavior of H-2K^b in human and murine cells may reflect, in part, greater stabilization of K^b complexed to human ß₂m relative to K^b complexed to murine ß₂m. Therefore, we anticipated that H-2K^b would also be expressed on the surface of human cells deficient in tapasin even though H-2K^b is expressed at low levels (15% of wild-type levels) in tapasin knockout mice (A. G. Grandea, unpublished observations; and Ref. 41). Therefore, we examined the cell surface expression of murine H-2K^b molecules in the human tapasin-deficient cell line .220. Genomic DNA encoding the K^b hc was independently transfected into both the .220 cell line and .220 cells already expressing human tapasin, giving rise to the K^b.220 and K^b.220.hTsn cell lines, respectively. Several clones arising from each transfection were screened for K^b surface expression by indirect immunofluorescence and flow cytometry. All clones screened demonstrated high levels of surface expression of H-2K^b regardless of tapasin expression. Clones of K^b.220 and K^b.220.hTsn that were matched in their level of K^b surface expression were selected for further analysis (Fig. 1A). Expression of human tapasin by the K^b.220.hTsn cell line was confirmed by Western blot (data not shown).

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Premature release of H-2Kb molecules from the ER in the tapasin-deficient human cell line .220. A, The cell lines .220 and .220.hTsn were independently transfected with DNA encoding the H-2Kb heavy chain and stained by indirect immunofluorescence using the Kb-specific mAb Y-3 and were analyzed by flow cytometry. Histograms of Kb.220 (fine line) and Kb.220.hTsn (solid line) transfected cells are shown. The solid histogram indicates staining of the parental .220 and .220.hTsn cell lines. B, The transfected cells Kb.220 and Kb.220.hTsn were pulse-labeled with [35S]methionine for 5 min and chased in excess cold methionine for 30, 60, and 120 min. H-2Kb molecules were immunoprecipitated from cell lysates with anti-peptide 8 rabbit serum, which recognizes the C terminus of H-2Kb hc. At each time point, immunoprecipitates were divided in half and were either digested with endo H (+) or mock digested (-) overnight. Proteins were separated by SDS-PAGE and visualized by autoradiography. Endo H-sensitive (S) and -resistant (R) protein bands are indicated by arrowheads. Migration positions of molecular mass marker proteins are indicated at the right (kDa).

To achieve equivalent surface K^b levels, the tapasin-deficient K^b.220 cells appear to biosynthesize greater quantities of H-2K^b compared with K^b.220.hTsn cells (2- to 5-fold; Fig. 1B and data not shown); however, the steady-state levels of properly conformed class I molecules on the surface of both cells are similar (Fig. 1A). This suggests that the K^b complexes in the tapasin-deficient cells are more rapidly turned over. This turnover does not appear to result from ER retention of poorly conformed complexes. Given the equivalent yields of anti-peptide 8-precipitated labeled K^b hc at t = 0 and t = 90, it seems likely that surface-mediated degradation contributes to the lower levels of conformed complexes at the cell surface of the tapasin-deficient APC.

Enhanced peptide receptivity and rapid loss of cell surface K^b molecules on tapasin-deficient .220 cells

We and others have postulated the existence of peptide editing during class I assembly (9, 10, 12, 16, 17, 18, 19, 20, 43, 44, 45). The most compelling evidence that tapasin functions either directly or indirectly in peptide editing comes from our studies showing differences in the repertoire of peptides presented by HLA B*2705 in matched tapasin-deficient vs tapasin-proficient APC.⁶ The rapid egress of K^b molecules in tapasin-deficient .220 cells raises the possibility that in the absence of tapasin, K^b molecules may be impaired in their ability to acquire optimized and presumably high-affinity peptides. Thus, K^b molecules at the cell surface of tapasin-deficient cells might contain low affinity or suboptimal peptides. To test this hypothesis, we assessed the ease with which exogenous, high-affinity peptide could replace endogenously loaded ligands. We tested the extent to which cell surface K^b molecules could be occupied by the immunodominant K^b-binding peptide from OVA, OVA_257–264 or SIINFEKL. The K^b/SIINFEKL-specific mAb 25-D1.16 (36) was used to detect formation of surface K^b/SIINFEKL complexes after addition of graded amounts of exogenous SIINFEKL peptide. As shown in Fig. 2A, when SIINFEKL was added to the cells and incubated at 37°C, K^b/SIINFEKL complexes were evident on the surface of tapasin-deficient and tapasin-reconstituted .220 cells as well as the murine thymoma EL-4. There was no detectable 25-D1.16 staining with a K^b-binding control peptide (46) from the human La (SS-B) protein, ⁵¹IMIKFNRL⁵⁸ (data not shown). Moreover, the level of expression of K^b/SIINFEKL complexes detected by 25-D1.16 displayed dose responsiveness to exogenous peptide (Fig. 2, A and B). These complexes were also recognized by the Ag-specific T hybridoma GA4.2 (33), but the hybridoma did not discriminate between these different APC at such high levels of peptide occupancy (data not shown).

View larger version (30K): [in this window] [in a new window]   FIGURE 2. Surface H-2Kb molecules are less stable and contain suboptimal ligands in the absence of tapasin. A, The cell lines Kb.220, Kb.220.hTsn, and EL-4 were cultured in serum-free RPMI 1640 for 1 h in the presence of varying concentrations of SIINFEKL peptide, were stained by indirect immunofluorescence using the Kb-SIINFEKL-specific mAb 25-D1.16, and were analyzed by flow cytometry. Histograms of cells pulsed with 5 nM (dotted line), 0.3 µM (fine line), and 30 µM (thick line) SIINFEKL peptide are shown. The solid histograms indicate staining of cells without addition of exogenous SIINFEKL. The data shown are representative of three independent experiments. B, Differential loading of H-2Kb molecules with exogenous peptide. The cell lines Kb.220 (), Kb.220.hTsn (•), and EL-4 () were pulsed with serial concentrations of SIINFEKL peptide, and the Kb-SIINFEKL (25-D1.16) staining is shown as a proportion of total Kb (Y-3) staining based on mean channel fluorescence. C, The decay of H-2Kb molecules on the surface of Kb.220 () and Kb.220.hTsn (•) cells. Cells were cultured at 37°C in RPMI 1640 supplemented with 10 µg/ml BFA for varying lengths of time. The level of cell surface Kb was determined by indirect immunofluorescence using the Kb-specific mAb Y-3 and was analyzed by flow cytometry.

H-2K^b molecules in tapasin-deficient cells fail to interact with calreticulin and TAP

To assess the requirement of tapasin for the association of K^b with components of the peptide loading complex, we analyzed immunoprecipitates of K^b molecules from tapasin-deficient and tapasin-competent .220 cells for the presence of coprecipitating calreticulin and TAP proteins. To capture all K^b molecules we used anti-peptide 8 polyclonal Ab, which is directed against the exon 8-encoded intracytoplasmic region of the K^b molecule and therefore is able to detect both free and ß₂m-associated hc (37). Digitonin lysates of .220, K^b.220, and K^b.220.hTsn cells were immunoprecipitated with anti-peptide 8 serum, and the captured proteins were immunoblotted with anti-calreticulin or anti-TAP1 mAbs. As shown in Fig. 3, calreticulin was detected in the immunoprecipitate of K^b from tapasin-competent but not from tapasin-deficient cells, despite similar levels of calreticulin detected in immunoblots of the whole cell lysates from each cell line. Similarly, TAP association was detected in tapasin-competent but not in tapasin-deficient cells, despite ample TAP1 signal in immunoblots of all the whole cell lysates. Interestingly, the introduction of tapasin led to an increase in TAP1 expression (Fig. 3, asterisked band in the whole cell lysate of K^b.220.hTsn) as reported previously (9). These results indicate that K^b is capable of interacting with human calreticulin and human TAP but that in the absence of human tapasin it fails to associate with these molecules or to form a macromolecular loading complex.

View larger version (42K): [in this window] [in a new window]   FIGURE 3. H-2Kb molecules fail to interact with calreticulin and TAP in tapasin-deficient cells. H-2Kb hc were immunoprecipitated from .220 (control), Kb.220, and Kb.220.hTsn cells using anti-peptide 8 hyperimmune rabbit serum. In each case, 5 x 106 cells were lysed in 0.5% digitonin. Coprecipitating proteins were then denatured in SDS and DTT, separated by SDS-PAGE, and immunoblotted for the presence of calreticulin or TAP1. As a loading control, a portion of each whole cell lysate used for immunoprecipitation was also separated by SDS-PAGE and blotted for the total amount of calreticulin and TAP1 present. Arrows indicate calreticulin and TAP1 protein bands. Migration positions of molecular mass marker proteins are indicated at the right (kDa). *, Increase in TAP expression correlating with the introduction of the human tapasin gene.

We next determined whether the absence of complex formation between K^b, calreticulin, tapasin, and TAP affected the ability of K^b to present TAP-dependent peptide Ags to T cells. To remove any contribution of species incompatibility in determinant generation by the proteasome, we examined the presentation of the SIINFEKL determinant encoded as a peptide "minigene" lacking any signal sequence that therefore is dependent upon TAP for ER translocation. As shown in Fig. 4, K^b.220.SIINFEKL transfectants were effective in their ability to stimulate a T hybridoma, GA4.2, specific for this determinant. Thus, K^b molecules expressed in the absence of tapasin are capable of binding endogenous SIINFEKL peptide supplied via the TAP and forming a target structure that can be recognized by T cells. Moreover, the calreticulin/K^b/tapasin-TAP interaction is not absolutely essential for Ag presentation of TAP-dependent peptides to T cells. Consistent with our findings, the TAP-independent presentation of the SIINFEKL determinant in K^b.220 cells infected with a rVV construct that expresses an ER-targeted form of SIINFEKL has also been reported (48).

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Presentation of a TAP-dependent peptide by H-2Kb in tapasin-deficient cells. The GA4.2 T hybridoma was cocultured with varying densities of Kb.220 (), Kb.220.SIINFEKL (), EL-4 (•), and SIINFEKL-pulsed EL-4 () cells. After 24-h culture, Ag-specific IL-2 production was determined by performing a CTLL proliferative assay (46 ). Results are shown as [3H]thymidine incorporation and are an average of triplicate samples from one experiment. The data shown are representative of three independent clones arising from the same transfection of Kb.220 () with the SIINFEKL minigene.

To more directly analyze the peptides bound to K^b, we immunoaffinity purified K^b molecules from tapasin-deficient and tapasin-competent .220 cells that express equivalent amounts of cell surface H-2K^b as detected by flow cytometry (the same cloned cell lines shown in Fig. 1A). The bound peptides were eluted from the purified K^b complexes and analyzed by a combination of reversed phase HPLC, MALDI-TOF mass spectrometry, and Edman sequencing. K^b molecules were immunoaffinity purified from 5 x 10⁹ K^b.220 and K^b.220.hTsn cells using a combination of Y-3 and 20.8.4 mAbs chemically cross-linked to protein A-Sepharose. Peptides were eluted, ultrafiltered, and fractionated by reversed phase HPLC to yield a peptidic fraction with no traces of Nonidet P-40 detergent or ß₂m. Consistent with their equivalent level of K^b surface expression, equivalent yields of class I hc and ß₂m were obtained from both cell types as determined by SDS-PAGE analysis of the Centricon 3 filter retentates (data not shown).

The canonical K^b-binding motif consists of a phenylalanine or tyrosine residue at position 5 (P5) and a leucine residue at P8 of the peptide (49, 50). To examine the maintenance of this K^b-binding motif in tapasin-deficient cells, the eluted peptides were subjected to pool Edman sequencing (Table I). The assignment of anchor residues and auxiliary anchor residues were based on the methods of Rammensee and colleagues (50, 51). Only amino acids that increased in yield at least 100% from previous cycle or following cycle were considered significant. Anchor residues are in bold type and represent the dominant amino acid in the cycle; auxiliary anchor residues represent abundant amino acids in the same cycle. Other listed residues were abundant but less prominent compared with anchor residues. The majority of K^b-eluted peptides from both tapasin-deficient and tapasin-competent .220 cells possess the archetypal P5 phenylalanine or tyrosine residue and the canonical P8 leucine residue. The maintenance of a tyrosine auxiliary anchor residue at P3 is also consistent with the previously published motif (50). Therefore, the structural constraints conferring the P3, P5, and P8 specificities in the H-2K^b-binding motif are not relaxed in the absence of tapasin.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Overlap in the tapasin-independent and -dependent repertoire of H-2Kb-associated peptides. MALDI-TOF mass spectra of peptide eluates derived from the cell lines Kb.220 (spectra of negative polarity) and Kb.220.hTsn (spectra of positive polarity), indicating the high degree of spectral overlap and some differential recovery of peptide species with distinct m/z values in peptide eluates from these cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The multitude of related functions attributable to tapasin in class I assembly has led to proposals that this molecule, which has evolved as a class I MHC-specific chaperone, may be involved in editing the peptide repertoire. We have demonstrated that HLA B27 is expressed at "normal" levels in tapasin-deficient cells (16); however, a proportion of the ligands bound to B27 under these conditions are suboptimal (43). Despite this finding, a very high overlap in the eluted B27-peptide repertoire was observed in matched APC that expressed surface B27 in the presence and absence of functional tapasin.⁶ Moreover, some peptides appear to be "edited" out of the B27 repertoire when tapasin is introduced into .220 cells already transfected with B27, resulting in a subtly changed peptide repertoire and changes in B27-restricted CTL recognition of these APC. In this paper we have examined the expression of the murine class I molecule H-2K^b in .220 cells with and without coexpression of human tapasin. The ability of some H-2 class I molecules to be expressed in mutant human cells but not in their murine counterparts was exploited as an additional reporter system of the influence of tapasin on class I assembly and peptide repertoire. We have demonstrated efficient surface expression of the H-2K^b molecules on the surface of tapasin-deficient .220 cells. Our results indicate that tapasin is involved in the retention of H-2K^b molecules in the ER of mammalian cells, and they are consistent with reports of tapasin-mediated ER retention of these molecules expressed in insect cells (10). Tapasin is also critical for the formation of a peptide loading complex, and its deficiency ablates the interaction of H-2K^b with calreticulin and TAP. This is consistent with a number of studies of class I molecules expressed in tapasin-deficient cells (6, 7, 9, 13, 14, 15, 16) and demonstrates that in human cells H-2 molecules are normally loaded via a peptide loading complex, similarly to the way their human counterparts, HLA molecules, are loaded. Importantly, K^b still loads with TAP-dependent peptides as demonstrated by functional presentation of minigene-encoded SIINFEKL determinants and by the elution of a spectrum of endogenous peptides. However, these peptides are more easily displaced with exogenous peptide (up to 90% of surface molecules can be loaded), and the complexes formed in the absence of exogenous peptides demonstrate more rapid surface decay. These data are consistent with work of Solheim and colleagues (20), who show a similar increase in receptivity to exogenous peptide for H-2L^d expressed in tapasin-deficient .220 cells. A moderate decrease in the yield of K^b-restricted peptides recovered from tapasin-deficient cells was observed during peptide elution experiments, despite very similar yields in class I hc and ß₂m. Collectively, these results are consistent with the loading of a proportion of suboptimal ligands under tapasin-deficient conditions. We have made similar observations with B27 peptides eluted from tapasin-deficient and tapasin-proficient cells.⁶ Likewise, the mutant HLA A2 molecule T134K also fails to form a peptide loading complex, is rapidly transported to the cell surface, and fails to load with optimal ligands (19). The loading of suboptimal peptides by K^b molecules expressed in the absence of tapasin probably reflects both impaired ER retention and the loss of tapasin-facilitated peptide loading.

Despite the failure in formation of a peptide loading complex, a significant degree of endogenous H-2K^b peptide loading occurs in the absence of tapasin, as revealed by functional Ag presentation, the repertoire of eluted peptides recovered from K^b molecules, and their retention of the canonical K^b-binding motif. Closer comparison of the eluted peptides associated with K^b purified from tapasin-positive and tapasin-negative APC reveals the presence of a number of shared species (Fig. 5). However, the recovery of these species was differentially affected by the tapasin deficiency. For example, shared species at m/z of 1322.7, 905.6, 1074.5, and 1107.6 Da were all more abundantly recovered from the tapasin-positive APC. Species of m/z 860.9, 987.5, and 876.9 were recovered with equal efficiency from both cell lines. The relative instability of some complexes on the surface of K^b.220 and presence of some species at higher levels in peptide eluates of K^b.220.hTsn suggest that tapasin retains complexes in the ER until a suitable peptide is loaded. There is also evidence for this role of tapasin in ligand optimization for HLA A2 and HLA B27 expressed in tapasin-deficient cells (15, 19, 43).⁶

The expression of H-2K^b in mutant human cell lines is enigmatic. On one hand, there is evidence that K^b binds low-affinity peptides in RMA-S cells grown at 26°C (26), yet in T2.K^b similar assays have failed to reveal the presence of endogenous peptides bound to surface K^b molecules (27). These results imply that K^b molecules are "empty" in T2.K^b but were probably stabilized initially by low-affinity peptides. Also contributing to the stable expression of K^b in human TAP and tapasin-deficient cells is the nature of the K^b-human ß₂m interaction (28, 29). In RMA-S cells transfected with human ß₂m, H-2K^b is expressed stably on the cell surface (25), whereas no expression is observed in RMA-S expressing only endogenous murine ß₂m. We propose that human ß₂m may provide critical stabilization of the K^b heterodimer in tapasin-deficient cells. Thus, in the absence of calreticulin and tapasin-mediated stabilization of the folding class I complexes, human ß₂m may provide sufficient stabilization to allow these molecules to acquire peptide. In the presence of tapasin, these molecules are retained in the ER, preventing rapid exit and allowing further optimization of the peptide ligands. Taken together, the data indicate that peptide loading per se is not critically dependent upon the tapasin molecule. Rather, tapasin is essential through its roles in the formation of the loading complex, which retains suboptimal class I molecules in the ER until optimal ligand selection is completed.

The majority of class I alleles introduced into tapasin-deficient APC fail to be expressed on the cell surface, despite the absence of tapasin-mediated retention. This most likely reflects the inability of these molecules to acquire a suitable endogenous peptide capable of stabilizing the nascent class I heterodimer and thus avoiding additional more generic ER-retention mechanisms or degradation pathways. We believe that this is reflected in the proportion of unstable H-2K^b class I molecules that are expressed on the surface of tapasin-deficient APC and that are apparently devoid of high-affinity endogenous peptides. These molecules were most likely initially stabilized by low-affinity, suboptimal ligands as seen in other mutant cell lines (19, 26). In the absence of a tapasin-regulated quality control system, these poorly loaded yet quasi-stable class I molecules fail to associate with ER-resident chaperones (19) and, as a result, they rapidly egress to the cell surface without the opportunity for further ligand optimization. A proportion of these molecules that egress prematurely from the ER are thermolabile and exhibit low cell surface stability. Why are some alleles able to egress to the cell surface in the absence of tapasin? We believe that tapasin-independent alleles are able to compensate for the lack of the peptide loading complex by a variety of allele-specific traits. These may include the ability to access alternative pathways of peptide acquisition, a high lumenal abundance of appropriate endogenous peptides that may counteract the loss of facilitated loading, and the intrinsic stability of the "empty" heterodimer. It remains to be elucidated whether all tapasin-independent alleles will share specific properties or whether different alleles have evolved alternative pathways to avoid abrogations in normal Ag presentation.

	Acknowledgments

 
We thank those who provided us with reagents: Robert DeMars (.220 cell line), Peter Cresswell (Rgp48N serum), Angel Porgador (25-D1.16 mAb), Weisan Chen (anti-peptide 8 serum), Tom Gordon (FMC 75 ascites), and Rajiv Khanna (pRSV.5(hygro)-SIINFEKL vector). We also thank Jeanne Butler for technical assistance; Bill Heath, Nihay Laham, and Chen Au Peh for helpful suggestions; and Dr. G. Talbo and Prof. E. Reynolds for assistance with Edman sequencing.

	Footnotes

 
¹ This work was supported by the National Health and Medical Research Council of Australia.

² Current address: Immunology and Molecular Biology R&D Division, Commonwealth Serum Laboratories, 45 Poplar Road, Parkville, Victoria 3052, Australia.

³ M.J.B. and A.W.P. contributed equally to this work.

⁴ Address correspondence and reprint requests to Dr. James McCluskey, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3052, Australia.

⁵ Abbreviations used in this paper: ER, endoplasmic reticulum; hc, heavy chain; ß₂m, ß₂-microglobulin; .220, 721.220; hTsn, human tapasin; endo H, endoglycosidase H; MALDI-TOF, matrix assisted laser desorption/ionization time-of-flight; BFA, brefeldin A; P5, position 5; m/z, mass-to-charge ratio.

⁶ A. W. Purcell, J. J. Gorman, M. Garcia-Peydró, A. Paradela, G. H. Talbo, S. R. Burrows, N. Laham, C. A. Peh, E. C. Reynolds, J. A. López de Castro, and J. McCluskey. Quantitative and qualitative influence of tapasin on the class I peptide repertoire. Submitted for publication.

⁷ A. W. Purcell and J. J. Gorman. The use of post source decay in matrix assisted laser desorption-ionisation mass spectrometry to delineate T cell determinants. Submitted for publication.

Received for publication February 17, 2000. Accepted for publication April 20, 2000.

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