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*
Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia; and
Biomolecular Research Institute, Parkville, Victoria, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-2Kb 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 Kb in the ER of murine RMA-S cells lacking a functional TAP complex (25). More recently, the thermolabile Kb 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 Kb 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-2Kb 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 Kb on the surface of RMA-S transfected with human ß2m (25).
Accordingly, we reasoned that these properties of Kb 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 Kb 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 Kb molecule on the tapasin-deficient .220 human cell line. Furthermore, in the absence of an interaction with tapasin, Kb 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 Kb hc with calreticulin and TAP. Despite these findings, there was a significant degree of endogenous H-2Kb peptide loading in the absence of tapasin as evidenced by functional Ag presentation, the repertoire of eluted peptides recovered from Kb 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).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-2b thymoma EL4 (32) and the Kb/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 pKb plasmid containing a genomic construct encoding the H-2Kb 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 Kb.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-Kb-specific mAb Y-3 (35) and the Kb/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 Kb-specific mAb Y-3 or hyperimmune rabbit anti-peptide 8 serum were used to immunoprecipitate Kb 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 Kb molecule was used to immunoprecipitate Kb 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-Kb-specific mAb Y-3 (35) and the Kb/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 Kb.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-2Kb and peptide elution
Kb.220- and Kb.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 Kb-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 Kb-peptide complexes were eluted with 0.2 N acetic acid, which also facilitates dissociation of Kb-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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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H-2Kb 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-2Kb in
human and murine cells may reflect, in part, greater stabilization of
Kb complexed to human ß2m
relative to Kb complexed to murine
ß2m. Therefore, we anticipated that
H-2Kb would also be expressed on the surface of
human cells deficient in tapasin even though
H-2Kb 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-2Kb molecules in the human tapasin-deficient
cell line .220. Genomic DNA encoding the Kb hc
was independently transfected into both the .220 cell line and .220
cells already expressing human tapasin, giving rise to the
Kb.220 and Kb.220.hTsn cell
lines, respectively. Several clones arising from each transfection were
screened for Kb surface expression by indirect
immunofluorescence and flow cytometry. All clones screened demonstrated
high levels of surface expression of H-2Kb
regardless of tapasin expression. Clones of
Kb.220 and Kb.220.hTsn that
were matched in their level of Kb surface
expression were selected for further analysis (Fig. 1
A). Expression of human
tapasin by the Kb.220.hTsn cell line was
confirmed by Western blot (data not shown).
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B) and Y-3.
The results were essentially identical for both the rabbit polyclonal
anti-sera and the mAb (data not shown). In the presence of tapasin,
the majority of Kb hc remained sensitive to endo
H digestion up to 120 min, reflecting retention of these molecules in
the ER of these APC over this period. This rate of class I maturation
is similar to that observed for HLA B8 expressed in .220 cells
cotransfected with human tapasin (42). Conversely, in the
absence of tapasin, the majority of Kb hc had
acquired resistance to endo H by 60 min. This indicates that in the
absence of tapasin Kb molecules exit the ER more
rapidly, and it suggests that tapasin plays a role in ER retention of
Kb molecules. Our observation in human APC is
consistent with observations of tapasin-mediated retention of murine
H-2 molecules in reconstituted insect cells (10).
To achieve equivalent surface Kb levels, the
tapasin-deficient Kb.220 cells appear to
biosynthesize greater quantities of H-2Kb
compared with Kb.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
Kb 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 Kb 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 Kb 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
Kb molecules in tapasin-deficient .220 cells
raises the possibility that in the absence of tapasin,
Kb molecules may be impaired in their ability to
acquire optimized and presumably high-affinity peptides. Thus,
Kb 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
Kb molecules could be occupied by the
immunodominant Kb-binding peptide from OVA,
OVA257–264 or SIINFEKL. The
Kb/SIINFEKL-specific mAb 25-D1.16
(36) was used to detect formation of surface
Kb/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, Kb/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
Kb-binding control peptide (46) from
the human La (SS-B) protein,
51IMIKFNRL58 (data not shown).
Moreover, the level of expression of Kb/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).
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B). The Kb
molecules on the surface of tapasin-deficient .220 cells were much more
receptive to exogenous peptide (>90% maximum occupancy at 50 µM
SIINFEKL) than those on the surface of tapasin-reconstituted .220
cells. These tapasin-reconstituted cells displayed similar peptide
receptivity to that of wild-type Kb molecules
expressed on the surface of EL-4 cells (40–50% maximum occupancy).
The marked receptivity of Kb molecules expressed
on tapasin-deficient .220 cells suggested that these molecules are
"empty" or complexed with suboptimal peptides easily exchanged for
the high-affinity SIINFEKL determinant (47). A prediction
of this hypothesis is that suboptimal peptide ligands would dissociate
more rapidly from Kb.220 at 37°C than for the
tapasin-expressing Kb.220.hTsn cells. Because
empty Kb molecules are less stable at 37°C,
loss of peptide ligand would result in progressive loss of serological
Kb determinants from the cell surface. Therefore,
we studied the decay of surface Kb expression on
Kb.220 and Kb.220.hTsn
under conditions where transport of newly synthesized
Kb molecules to the cell surface was arrested by
brefeldin A (BFA). As shown in Fig. 2C, in the presence of
BFA, the Kb molecules on tapasin-deficient .220
cells decay more rapidly than those on tapasin-competent .220 cells,
which is consistent with the presence of a greater proportion of
suboptimal, low-affinity peptides loaded into Kb
on the surface of these cells. H-2Kb molecules in tapasin-deficient cells fail to interact with calreticulin and TAP
To assess the requirement of tapasin for the association of
Kb with components of the peptide loading
complex, we analyzed immunoprecipitates of Kb
molecules from tapasin-deficient and tapasin-competent .220 cells for
the presence of coprecipitating calreticulin and TAP proteins. To
capture all Kb molecules we used anti-peptide
8 polyclonal Ab, which is directed against the exon 8-encoded
intracytoplasmic region of the Kb molecule and
therefore is able to detect both free and
ß2m-associated hc (37). Digitonin
lysates of .220, Kb.220, and
Kb.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 Kb 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
Kb.220.hTsn) as reported previously
(9). These results indicate that Kb
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.
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We next determined whether the absence of complex formation
between Kb, calreticulin, tapasin, and TAP
affected the ability of Kb 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, Kb.220.SIINFEKL
transfectants were effective in their ability to stimulate a T
hybridoma, GA4.2, specific for this determinant. Thus,
Kb 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/Kb/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
Kb.220 cells infected with a rVV construct that
expresses an ER-targeted form of SIINFEKL has also been reported
(48).
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To more directly analyze the peptides bound to
Kb, we immunoaffinity purified
Kb molecules from tapasin-deficient and
tapasin-competent .220 cells that express equivalent amounts of cell
surface H-2Kb as detected by flow cytometry (the
same cloned cell lines shown in Fig. 1A). The bound peptides
were eluted from the purified Kb complexes and
analyzed by a combination of reversed phase HPLC, MALDI-TOF mass
spectrometry, and Edman sequencing. Kb molecules
were immunoaffinity purified from 5 x 109
Kb.220 and Kb.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
Kb 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 Kb-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 Kb-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 Kb-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-2Kb-binding
motif are not relaxed in the absence of tapasin.
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). A high degree of spectral overlap was
observed in the eluates from H-2Kb from
tapasin-deficient and -proficient cell lines, which is consistent with
our observations with HLA B27 bound peptides.6 In
addition, some species were recovered differentially from peptide pools
derived from cells with and without tapasin. The differential recovery
of peptides sharing distinct m/z values was consistent with previous
observations of the HLA B27 repertoire in tapasin-negative
APC.6 Although the majority of endogenous
peptides are still loaded onto Kb in the absence
of tapasin, these data suggest that not all species are affected
equivalently by tapasin deficiency, reflecting a potential
tapasin-dependent editing mechanism for both HLA B27 and
H-2Kb.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-2Kb 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-2Kb molecules on the surface of tapasin-deficient .220 cells. Our results indicate that tapasin is involved in the retention of H-2Kb 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-2Kb 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, Kb 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-2Ld expressed in tapasin-deficient .220 cells. A moderate decrease in the yield of Kb-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 Kb 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-2Kb peptide
loading occurs in the absence of tapasin, as revealed by functional Ag
presentation, the repertoire of eluted peptides recovered from
Kb molecules, and their retention of the
canonical Kb-binding motif. Closer comparison of
the eluted peptides associated with Kb 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 Kb.220 and presence
of some species at higher levels in peptide eluates of
Kb.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-2Kb in mutant human cell lines is enigmatic. On one hand, there is evidence that Kb binds low-affinity peptides in RMA-S cells grown at 26°C (26), yet in T2.Kb similar assays have failed to reveal the presence of endogenous peptides bound to surface Kb molecules (27). These results imply that Kb molecules are "empty" in T2.Kb but were probably stabilized initially by low-affinity peptides. Also contributing to the stable expression of Kb in human TAP and tapasin-deficient cells is the nature of the Kb-human ß2m interaction (28, 29). In RMA-S cells transfected with human ß2m, H-2Kb 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 Kb 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-2Kb 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.
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Footnotes |
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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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),
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 (
), EL-4 (•), and SIINFEKL-pulsed EL-4
(