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,¶
*
Department of Immunology, Allergy and Arthritis, Flinders University of South Australia, Bedford Park, South Australia, Australia;
Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia;
Queensland Institute of Medical Research, The Bancroft Centre, Brisbane, Queensland, Australia;
§
Department of Immunology, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China; and
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Victorian Transplantation and Immunogenetics Service, Australian Red Cross Blood Service-Victoria, South Melbourne, 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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In this paper, we show that constitutively expressed murine tapasin does not support normal surface expression of the human class I molecule HLA-B*4402 (B*4402) in mouse cells expressing normal levels of H-2Kk and H-2Dk. Although constitutive expression of murine tapasin in these mouse APC is sufficient to mediate a stable and prolonged association between TAP and B*4402 molecules, this alone is insufficient for efficient loading of B*4402 molecules. Expression of membrane-anchored murine tapasin at high levels fully restores the surface expression of B*4402 in mouse APC without augmenting the level of B*4402-TAP association. Furthermore, overexpression of soluble forms of either murine or human tapasin (sol-mTapasin or sol-hTapasin) also rescued surface expression of B*4402 molecules, revealing a critical role for tapasin in peptide loading of class I molecules independent of TAP-class I bridging, increased peptide translocation, or enhanced TAP expression. These data also reveal a degree of incompatibility between human B*4402 molecules and components of the murine Ag presentation machinery, and they suggest an important role for structural class I polymorphism in controlling the efficiency of interactions with components of the peptide-loading complex. The findings imply that the use of HLA class I transgenic mice as human models of specific immunity might be conceptually flawed if species incompatibility in tapasin interaction leads to presentation of an altered peptide repertoire.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J26 (ATCC CRL-1802) of C3H/A background is derived from murine L cells by transfection with the human ß2m gene; Jurkat (ATCC TIB-152) is a human T cell leukemic line; 721.220 is derived from mutagenesis of a human lymphoblastoid cell line (LCL) in which the HLA-A, HLA-B, and tapasin genes have been deleted but HLA-C remains intact on one chromosome (8); and EL4 (ATCC TIB-39) of C57BL/6 background is a mouse T lymphoma; 50125 is an EBV-transformed B*4402 homozygous human B LCL (a gift from Sue Lester, Red Cross Blood Bank, Adelaide, Australia). Cells were grown in either RPMI 1640 or DMEM supplemented with 10% FCS (CSL, Melbourne, Australia), 2 mM glutamine, and antibiotics. EBV-specific CTL clone SE15 was generated from EBV-sero+ donor SE (B*4403) as described (9). CTL clone SE recognizes the epitope VEITPYKPTW from EBNA4 in association with either B*4403 or B*4402.
W6/32 (10) is a conformation-dependent mouse mAb specific for all HLA class I molecules; mouse mAb 116.5.28 recognizes the HLA-Bw4 epitope (Saxon Europe, Suffolk, U.K.); mouse mAbs 148.3 and 429.3 recognize the human TAP1 (hTAP1) and hTAP2 molecules, respectively (11); HC-10 is a mouse mAb which recognizes free HLA class I heavy chains (12); mouse mAb 28.14.8 recognizes H-2Db (13); mouse mAb 16.1.2 recognizes H-2Kk and H-2Dk (13); Rgp48N is a rabbit Ab against the N terminus of human tapasin (2); Ra#2223 is a rabbit Ab against the N terminus of murine tapasin (a gift from Ted Hansen, Washington University School of Medicine, St. Louis, MO); and anti-HAM1 is a rabbit Ab which recognizes mouse TAP1 (14).
Gene constructs and DNA-mediated gene transfer
Genomic B*4402 was subcloned from pSV2neo.B*4402 (15) into the pUC13 vector. Genomic B*2705 was also contained in pUC13 vector (16). cDNAs encoding hTAP1 and hTAP2 were contained in pHßApr-1-neo vector (17). The cDNAs encoding the transmembrane-anchored (6) and soluble forms (18) of human tapasin were cloned into pMCFR.puromycin (6). The wild-type murine tapasin cDNA (courtesy of Bodo Ortmann, Universitaet zu Koeln, Cologne, Germany) was also subcloned into pMCFR. puromycin. A cDNA (pMCFR.sol-mTapasin.puromycin) was constructed to encode sol-mTapasin using standard PCR methods analogous to those described for generating sol-hTapasin (18). Mouse J26 cells were transfected by the calcium phosphate-DNA precipitation method (19) with either pUC13.B*4402 or pUC13.B*2705 and the selectable marker gene pSV2neo. In other transfections, pHßApr-1-neo.hTAP1 and/or pHßApr-1-neo.hTAP2 were cotransfected with a genomic B*4402. A clone of J26.B*4402 was subsequently cotransfected with either pMCFR.hTapasin.puromycin or pMCFR.mTapasin.puromycin. J26.B*2705 transfectants were also cotransfected with pMCFR.hTapasin.puromycin. Jurkat was transfected with linearized pSV2neo.B*4402 plasmid by electroporation at 220 V and 960 µF. The same electroporation settings were used to transfect EL4 with either pUC13.B*4402 or pUC13.B*2705 and pSV2neo, and 721.220 with pUC13.B*4402 with pSV2neo. 721.220.B*4402 transfectants were subsequently cotransfected with either pMCFR.puromycin vector alone, pMCFR.hTapasin.puromycin, pMCFR.sol-hTapasin.puromycin, pMCFR.mTapasin.puromycin, or pMCFR.sol-mTapasin.puromycin. J26 transfectants were selected with 0.5 mg/ml G418 ± 4 µg/ml puromycin. Jurkat transfectants were selected with 2.0 mg/ml G418 initially. EL4 transfectants were selected with 0.4 mg/ml G418. 721.220 transfectants were selected with 0.6 mg/ml G418 ± 0.5 µg/ml puromycin. Where possible, positive transfectants were sorted for peak channel expression of HLA molecules using magnetic beads that had been coated with an appropriate mAb.
Flow cytometry
Cell surface staining for HLA expression was performed by indirect immunofluorescence on cells cultured overnight at 37°C, unless indicated otherwise. mAbs W6/32, 116.5.28, 28.14.8, and 16.1.2 were used as primary Abs followed by goat anti-mouse Ig conjugated to FITC (Silenus, Melbourne, Australia). 104 cells were analyzed for each histogram using a FACS analyzer (Becton Dickinson, Mountain View, CA) with logarithmic gain.
Western blot analysis
For anti-hTAP immunoblots, cells were lysed in SDS buffer and then boiled for 5 min. Proteins were separated by SDS-PAGE in a 10% gel, transferred onto nitrocellulose paper, blocked with 5% skim milk in PBS, probed with either anti-hTAP1 mAb 148.3 or anti-hTAP2 mAb 429.3 for 1 h, washed extensively with 5% skim milk in PBS, and incubated with sheep anti-mouse Ig conjugated to HRP for 1 h (Silenus). For anti-tapasin immunoblots, cells were lysed in 1% Triton X-100 in TBS (10 mM Tris and 150 mM NaCl (pH 7.4)) and then centrifuged to remove nuclei. Extracts from 50,000 cells per lane were boiled in SDS sample buffer and separated by SDS-PAGE in a 10% gel. After transfer onto nitrocellulose paper, proteins were probed with either anti-human tapasin Rgp48N Ab (courtesy of P. Cresswell, Yale University Medical School, New Haven, CT) or anti-murine tapasin Ra#2223 Ab (courtesy of T. Hansen, Washington University School of Medicine, St. Louis, MO), and subsequently sheep anti-rabbit Ig conjugated to HRP (Silenus). For anti-tapasin immunoblots, the entire procedure was conducted in the presence of 0.2% Tween 20. The immunoblots were developed with chemiluminescence agent (NEN, Boston, MA) and exposed to radiographic film.
Immunoprecipitation
Cells (4 x 106) were starved of methionine and then labeled with [35S]methionine for 15 min and lysed in 0.5% digitonin buffer in TBS containing PMSF, leupeptin, and aprotinin for 30 min at 4°C. After centrifugation to remove cell nuclei, samples were precleared overnight with normal rabbit serum and protein A-Sepharose. Primary immunoprecipitation was conducted with anti-HAM1 (anti-murine TAP1) Ab before protein A-Sepharose beads were added. Beads were washed twice in 0.1% digitonin buffer containing 450 mM NaCl and 10 mM Tris (pH 7.4), and once in 10 mM Tris buffer. Immune complexes were dissociated by incubation in 50 µl of 0.2% SDS in TNE (50 mM Tris, 150 mM NaCl, and 5 mM EDTA (pH 7.4)) for 1 h at 37°C. Supernatants were equilibrated with nine volumes of 1% Nonidet P-40 and 0.5% nonanoyl-N-methyl-glucamide (Sigma, St.Louis, MO) in TNE. Secondary immunoprecipitation was conducted with mAb HC-10, protein A-Sepharose beads were added, and the beads were washed twice with 0.5% Nonidet P-40 in TNE. Beads were boiled in SDS sample buffer, proteins were separated by SDS-PAGE in a 10% gel, and the gel was then fixed, amplified, dried, and exposed to radiographic film for 5 days. For pulse-chase immunoprecipitation, aliquots of 4 x 106 cells were radiolabeled for 15 min and chased in medium containing excess methionine (0.5 mM) and cystine (0.5 mM). At the indicated time points, cells were collected, washed in cold PBS, and then treated as outlined above.
Cytotoxicity assay
Target cells were tested for lysis in duplicate in a standard 5-h chromium release assay. Synthetic peptide was added to some target cells during chromium labeling (10 µM for 1 h), and unbound peptide was washed off target cells before effector CTL was added. A topcount microplate ß scintillation counter (Packard Instrument, Meriden, CT) was used to measure 51Cr levels in assay supernatant samples. The mean spontaneous lysis for target cells in culture medium was <20%, and the variation from the mean specific lysis was <10%. Peptides were synthesized by Chiron Mimotopes (Chiron, Emeryville, CA) using pin technology (20).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously observed poor surface expression of B*4402 in
murine J26 cells (L cells expressing human ß2m)
after transfection with either B*4402 cDNA or a genomic construct
(21). Poor surface expression was evident with mAbs
recognizing both the W6/32 and Bw4 epitopes and was associated with
high levels of intracellular B*4402 heavy chains (21).
Moreover, the low level surface expression of B*4402 could be greatly
improved by overnight incubation of these transfectants at 26°C,
indicating that B*4402 complexes could not load efficiently with
peptides in murine cells (Fig. 1
A). Low surface expression of
B*4402 was also evident in another commonly studied murine cell line,
EL4 (Fig. 1B). After gene transfection, the surface
expression of B*4402 was low in murine cells, whereas the surface
expression of other HLA class I molecules such as HLA-B8 and B*2705 was
much higher, thus revealing allelic variation in the efficiency of
assembly of HLA class I molecules in murine cells (Ref.
21 ; Fig. 1, A and B). Significantly,
in contrast to the poor surface expression of B*4402 in murine cells,
the surface expression of B*4402 was normal in both human B LCL and
human Jurkat cells transfected with B*4402 (Fig. 1C).
Furthermore, expression of endogenous mouse class I molecules was
normal in the murine transfectants that expressed B*4402 poorly (Fig. 1, D and E). Therefore, we tested whether the
behavior of B*4402 molecules in murine cells might be due to poor
cross-species compatibility between human B*4402 and components of the
murine Ag presentation pathway.
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Because coexpression of human tapasin fully restored the
intracellular maturation and surface expression of B*4402 complexes in
murine cells (21), we reasoned that B*4402 molecules may
not interact properly with endogenous murine tapasin to achieve
assembly with peptides in murine cells. This possibility was consistent
with the observation that human and murine tapasin protein sequences
shared only 75% identity (7). Therefore, the genes
encoding B*4402, and either human or murine tapasin were transfected
into tapasin-defective human 721.220 cells. 721.220 is a HLA-A and -B
negative, EBV-transformed human cell line which is defective in tapasin
expression as a result of a mRNA splicing error (8, 22).
B*4402 molecules are critically dependent upon human tapasin for
surface expression and Ag presentation by these cells
(21). Surprisingly, introduction of a murine tapasin cDNA
restored the surface expression of B*4402 in 721.220 transfectants to
the same degree as did a human tapasin cDNA (Fig. 2A). Furthermore, B*4402
complexes assembled in the murine or human tapasin transfectants were
equally efficient at presenting endogenous EBV peptides to an
EBV-specific, HLA-B44-restricted CTL clone (Fig. 2B).
Together, these findings suggest that after gene transfection, murine
tapasin could facilitate peptide loading of B*4402 complexes, but also
raises the question of why B*4402 was poorly expressed on the surface
of murine cells expressing endogenous tapasin.
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The poor surface expression of B*4402 in murine cells might have
been due to low or absent expression of endogenous murine tapasin in
these cells even though assembly of murine MHC class I complexes was
normal in both J26 and EL4 cells. Therefore, we co-transfected cloned
murine J26.B*4402 cells with either murine or human tapasin cDNAs and
correlated the surface expression of B*4402 with the level of tapasin
expression (Fig. 3). Western blots of
cell lysates showed that before tapasin transfection, J26.B*4402 cells
expressed readily detectable endogenous murine tapasin (Fig. 3B, lower panel, lane 4). Therefore,
the poor surface expression of B*4402 in J26 cells was not due to an
absolute lack of murine tapasin. Co-transfection of the murine tapasin
cDNA into J26.B*4402 cells (J26.B*4402.mTapasin) increased the total
level of expression of murine tapasin (Fig. 3B, lower
panel, lane 6), and this correlated with significantly
improved surface expression of B*4402 (Fig. 3A, filled grey
and broken-line histograms).
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B, upper
panel, lanes 2 and 5) even though both cells expressed
high surface amounts of B*4402 (Figs. 1C and 3A).
Transfection of the human tapasin cDNA into J26.B*4402 cells
(J26.B*4402.hTapasin) also restored the normal surface expression
of B*4402 (Ref. 21 ; Fig. 3, A and
B (upper panels)). When cell extracts obtained
from J26.B*4402.hTapasin cells were separated by gel
electrophoresis and immunoblotted using an Ab with specificity for
murine tapasin, a doublet of 
48 kDa was detected (Fig. 3B, lower panel, lane 5), which was
more evident in films exposed for a longer time (data not shown). The
upper and lower bands of this doublet probably correspond to endogenous
murine tapasin and human tapasin, respectively, consistent with murine
tapasin containing an extra 14 residues in its cytoplasmic tail
(7). Evidently, the high levels of human tapasin expressed
after gene transfection reveal cross-reactivity of this protein with
the anti-murine tapasin Ab. Also evident from this experiment is
the reduced level of mouse tapasin detected in J26.B*4402 cells
expressing human tapasin. This might simply reflect clonal variation in
transfectants but could also indicate competition between the two forms
of tapasin with preference for human tapasin leading to premature
degradation of the mouse tapasin molecules.
To examine the effect of increased tapasin expression upon the surface
levels of murine MHC class I molecules, the expression of
endogenous H-2Kk and
H-2Dk in J26.B*4402, J26.B*4402.hTapasin, and
J26.B*4402.mTapasin transfectants was examined by staining with mAb
16.1.2 (Fig. 3C). Interestingly, the surface expression of
endogenous MHC class I molecules was significantly increased after the
overexpression of either murine or human tapasin. These findings
indicate that the constitutive surface expression of MHC class I
molecules in mouse APCs is modulated by the limiting expression of
murine tapasin, implying a role for tapasin in the quantitative
regulation of MHC class I ligands expressed at the cell surface.
Constitutively expressed murine tapasin mediates a prolonged and stable association between B*4402 and TAP without augmenting peptide loading
Besides class I-TAP bridging, other mechanisms have been proposed
to account for the role of tapasin in class I Ag presentation. For
instance, tapasin increases the expression of the TAP complex and hence
may increase the rate of peptide translocation (18),
perhaps by stabilizing the TAP1/TAP2 heterodimer (23). To
test whether increased TAP expression alone could restore surface
expression of B*4402 in J26 cells, human TAP1 and TAP2 cDNAs were
co-transfected into J26.B*4402. Expression of these proteins was
verified by immunoblots of transfected cell lysates. As shown in Fig. 4, A and B,
overexpression of human TAP in murine cells did not improve the poor
surface expression of B*4402, suggesting that increased TAP expression
by itself is insufficient to restore B*4402 expression at the cell
surface. The data also suggest that poor B*4402 surface expression was
not due to either functional incompatibility with the murine TAP
protein or delivery of peptide ligands unsuitable for binding to the
B*4402 molecule.
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C, upper panel), but
proteins reactive with mAb HC-10 were not detected in wild-type murine
J26 cells (Fig. 4C, lower panel). In transfected
cells, bands comprising HLA class I heavy chains were detected after
dissociation of bound immune complexes and sequential reprecipitation
with mAb HC-10 (Fig. 4C, lower panel, lanes
2–6). Significantly, the recovery of B*4402 from the murine TAP
complex was indistinguishable in cells expressing only constitutive
endogenous tapasin vs transfected cells expressing much higher levels
of murine or human tapasin (Fig. 4C, lower panel,
lanes 4–6). Thus, endogenous murine tapasin expressed at
constitutive levels facilitates the binding of B*4402 to murine TAP but
does not facilitate its normal surface expression. Notably, B*2705
molecules were also associated with constitutively expressed murine
TAP, and this association was not significantly different from that
observed with B*4402 or in human tapasin transfectants (Fig. 4C). The findings suggest that TAP-class I bridging alone
does not correlate with full tapasin function.
Notwithstanding the apparently normal association between B*4402 and
murine TAP, it was possible that complexes of endogenous mouse tapasin,
B*4402, and TAP were unstable or too short-lived to permit proper
peptide loading of B*4402. Therefore, we examined the stability of
these complexes in a pulse-chase experiment using the same approach as
in Fig. 4C. TAP complexes were recovered by
immunoprecipitation at various time intervals after a 15-min pulse
labeling, and the presence of B*4402 molecules in these complexes was
ascertained by a second immunoprecipitation with HC-10 mAb. As shown in
Fig. 4D, B*4402 heavy chains remained stably associated with
TAP proteins for at least 2 h in J26 cells expressing only
endogenous mouse tapasin. However, the introduction of human tapasin
into these cells (J26.B*4402.hTapasin) resulted in a more rapid
dissociation of B*4402-TAP complexes consistent with more efficient
peptide loading of B*4402 molecules and their transport to the cell
surface (21). These results confirm that the physical
interaction between TAP and HLA class I complexes in itself does not
necessarily augment peptide loading and indicates that tapasin has an
additional function in the loading of peptides into peptide-receptive
HLA class I complexes.
High levels of sol-mTapasin restore surface expression of B*4402 in human tapasin-deficient 721.220 cells
The restoration of B*4402 surface expression in J26 mouse cells
after overexpression of mouse tapasin did not appear to be the result
of enhanced association between TAP and B*4402, which was already
demonstrable with endogenously expressed mouse tapasin. However,
improved B*4402 expression could still reflect enhanced peptide
translocation associated with increased TAP expression or improved
TAP-class I-tapasin oligomerization (18, 24). Soluble
tapasin lacking the transmembrane and cytoplasmic domains still
restores surface expression and Ag presentation by HLA class I
molecules in the tapasin-deficient 721.220 cell line despite a lack of
detectable TAP-class I association due to loss of the C-terminal region
which mediates TAP association (25). Therefore, we tested
whether sol-mTapasin and sol-hTapasin could correct the impaired
surface expression of B*4402 in these tapasin-deficient human cells. As
shown in Fig. 5A, the
introduction of genes encoding either sol-hTapasin or sol-mTapasin into
721.220.B*4402 cells significantly restores surface expression of
B*4402 by these cells. Western blots of detergent lysates from
transfected cells confirmed the overexpression of sol-mTapasin in
721.220.B*4402 cells (Fig. 5B, lane 4) relative
to the transmembrane-anchored form of mouse tapasin expressed
endogenously by J26.B*4402 (Fig. 5B, lane 6) or
after transfection of 721.220 cells (Fig. 5B, lane
5). Because soluble tapasin does not associate efficiently with
TAP or enhance TAP expression or peptide translocation, the findings
reveal a critical function of tapasin which can be separated from its
other roles associated with TAP-class I bridging
(18).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observation that overexpression of murine tapasin overcomes the poor surface expression of B*4402 in murine cells suggests an underlying structural incompatibility in the interaction between B*4402 and murine tapasin. Presumably, high concentrations of murine tapasin can compensate for suboptimal interactions between these molecules. However, the possibility of an incompatible interaction between B*4402 and some other component of the murine Ag processing and presentation pathway cannot be entirely discounted. For instance, species-specific generation of peptide epitopes has been implicated as an explanation of differential presentation of a HLA-A3-restricted antigenic epitope from influenza A nucleoprotein by mouse and human cells (28). There are several reasons why it is unlikely that differences in cytosolic processing of proteins between rodent and human APC account for the low expression of B*4402 in mouse cells. First, expression of peptide-loaded B*4402 molecules on the surface of mouse cells is partially restored by culture of cells at 26°C, which is known to stabilize class I molecules and facilitate the capture of peptides (21). Second, expression of both human and mouse tapasin restores intracellular assembly of B*4402 peptide complexes, which rescues normal maturation, transport, and surface expression of B*4402 in mouse cells (21). Together these observations suggest that appropriate ligands for binding to B*4402 are generated normally in mouse cells. Impaired translocation of suitable peptides into the ER lumen by mouse TAP also seems improbable because coexpression of human TAP failed to restore the surface expression of B*4402 in murine cells. Incompatibility with either murine calnexin or murine calreticulin is unlikely to contribute significantly to the poor surface expression of B*4402 in murine cells because class I molecules assemble properly even in calnexin-deficient cells (29, 30, 31), and murine calreticulin shares a high degree of identity with human calreticulin (>95%). More recently, the thioreductase ER-60 has also been detected in the MHC class I peptide-loading complex (3, 4, 5). It remains to be seen whether species-specific differences in ER-60 may influence the assembly of human HLA class I molecules in murine cells.
Recently, it has been reported from studies of MHC class I assembly in
Drosophila cells that tapasin may have a role in ER
retention of empty MHC class I complexes until they have been loaded
with peptides (23). Failure of retention of mutant HLA-A2
molecules (with a T134K substitution in the 
2-domain) by molecular
chaperones may also account for impaired association with TAP,
inefficient peptide loading, and faster rates of egress from the ER
compared with wild-type HLA-A2 complexes (32). However,
failure of retention by tapasin evidently cannot solely account for the
inefficient assembly of B*4402 in murine cells because these
complexes are already retained for much longer than those
molecules assembled in the presence of excess tapasin (Fig. 4 and Ref.
21).
Although it seems clear that tapasin occupies a pivotal role in mediating the association of MHC class I peptide-loading complexes to TAP, it is unclear how tapasin functions to enhance the peptide-loading process. Lehner and colleagues (18) observed that soluble tapasin lacking the transmembrane and cytoplasmic domains restored peptide loading and surface expression of HLA-B8 without restoring HLA-B8-TAP binding. Our data confirm that sol-hTapasin and sol-mTapasin also restore surface expression of B*4402 in the 721.220 cell line. Thus, TAP-class I bridging is not essential for enhanced peptide loading. An important corollary to this observation is our finding that the stable association of class I molecules with TAP is not sufficient for enhanced peptide loading. This indicates that the most important function of tapasin can be exerted independently of bridging MHC class I to TAP.
We hypothesize that tapasin may play a vital role in stabilizing and transforming the conformation of the empty MHC class I complex into a peptide-receptive state. The impaired assembly of B*4402 in normal murine cells indicates that constitutive levels of endogenous murine tapasin are insufficient for this function. Indeed, it is unclear how well various HLA, or even murine H-2 class I, molecules interact with murine tapasin for assembly in murine cells. For example, in transgenic mice expressing HLA class I molecules, inefficient interactions between these molecules and components of the murine Ag presentation machinery (such as tapasin) may modify the spectrum of peptide Ags presented at the cell surface and may contribute to difficulties encountered in generating HLA-restricted CTL (33).
Little is known about the structural determinants controlling
interactions between TAP, tapasin, and class I molecules (24, 34, 35). The 3 domain of class I molecules might be involved
either directly or indirectly in binding to the TAP complex (24, 34), and the N-terminal domain of tapasin is required to
stabilize the class I loading complex (25). Moreover,
evidence also suggests that tapasin might interact directly with
ß2m (36). Thus, it is tempting to
speculate that the N-terminal region of tapasin might interact directly
with the class I heavy chain 3 domain/ß2m
complex. However, the relative species incompatibility of mouse tapasin
for B*4402 does not seem to be controlled solely by the 3
domain of the MHC class I heavy chain because replacement of this
region of B*4402 with a mouse equivalent does not improve expression of
chimeric B*4402 molecules in mouse cells (data not shown).
Nonetheless, it is still possible that in mice transgenic for other
chimeric HLA class I heavy chain/murine 3-domain molecules
(37) improved CTL activity may be partially due to
improved interactions between these chimeric molecules and murine
TAP/tapasin complex.
The C-terminal domain of tapasin mediates TAP-class I bridging
(25). Because the cytosolic domain of mouse tapasin is 14
amino acids longer than the human analogue at the C terminus, it has
been suggested this region might determine species specificity of
interaction with class I or TAP1/2 (35). However, it does
not seem likely that the C-terminus of tapasin alone controls species
specificity of TAP-class I interactions (35) given that
B*4402 associates stably with mouse TAP in J26 cells (Fig. 4, C and D) and that high levels of sol-mTapasin
restore B*4402 expression in 721.220 transfectants (Fig. 5).
How essential is interaction with tapasin/TAP for peptide loading of MHC class I molecules? Whereas some MHC class I molecules such as B*4402 and HLA-B8 are highly dependent upon tapasin (6, 21), others like B*2705, HLA-A2, murine H-2Kb, and H-2Dd (E222K) may still retain residual capability to assemble and present endogenous peptide Ags in the absence of TAP bridging by tapasin (21, 24, 38, 39). The complexes which form under these circumstances may be suboptimal in quantity and in binding affinity, thus creating poor ligands for T cell recognition (Refs. 21, 24 ; footnote 4). Even so, the existence of an active tapasin-independent loading mechanism for some class I alleles might be advantageous in the presence of viral immune evasion mechanisms which depend upon tapasin inhibition (40) or where there is competition between class I molecules in the ER for access to the TAP/tapasin complexes (41). Furthermore, the quantitative dependence of B*4402 surface expression upon the level of murine tapasin might indicate that constitutive expression of endogenous tapasin is normally finely tuned to exert sensitive control over the peptide supply to nascent class I molecules. This notion is consistent with the expression of some "empty" class I molecules on the surface of normal cells (42) and with the inducibility of tapasin by cytokines such as GM-CSF (43). Such sensitive tuning could effectively render peptide supply functionally limiting under basal conditions but would allow a rapid response of the Ag presentation machinery to new ligands generated upon infection or Ag uptake.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James McCluskey, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3052, Australia. E-mail address:
3 Abbreviations used in this paper: ß2m, ß2-microglobulin; B*4402, HLA-B*4402; hTAP, human TAP; ER, endoplasmic reticulum; sol-hTapasin, soluble human tapasin; sol-mTapasin, soluble murine tapasin; LCL, lymphoblastoid cell line.
4 A. W. Purcell, J. J. Gorman, M. Garcia-Peydró, N. Laham, C. A. Peh, J. A. L. de Castro, and J. McCluskey. Evidence for an editing role for the ER glycoprotein tapasin in the assembly of class I molecules with peptide ligands. Submitted for publication.
Received for publication August 11, 1999. Accepted for publication October 13, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 domain. J. Immunol. 162:1530.3 domain with the transporter associated with antigen presentation. J. Exp. Med. 187:865.This article has been cited by other articles:
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  M. C. Verweij, D. Koppers-Lalic, S. Loch, F. Klauschies, H. de la Salle, E. Quinten, P. J. Lehner, A. Mulder, M. R. Knittler, R. Tampe, et al. The Varicellovirus UL49.5 Protein Blocks the Transporter Associated with Antigen Processing (TAP) by Inhibiting Essential Conformational Transitions in the 6+6 Transmembrane TAP Core Complex J. Immunol., October 1, 2008; 181(7): 4894 - 4907. [Abstract] [Full Text] [PDF]
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 G. Roder, L. Geironson, I. Bressendorff, and K. Paulsson Viral Proteins Interfering with Antigen Presentation Target the Major Histocompatibility Complex Class I Peptide-Loading Complex J. Virol., September 1, 2008; 82(17): 8246 - 8252. [Full Text] [PDF]
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V. Montserrat, B. Galocha, M. Marcilla, M. Vazquez, and J. A. Lopez de Castro HLA-B*2704, an Allotype Associated with Ankylosing Spondylitis, Is Critically Dependent on Transporter Associated with Antigen Processing and Relatively Independent of Tapasin and Immunoproteasome for Maturation, Surface Expression, and T Cell Recognition: Relationship to B*2705 and B*2706 J. Immunol., November 15, 2006; 177(10): 7015 - 7023. [Abstract] [Full Text] [PDF]
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L. Sesma, B. Galocha, M. Vazquez, A. W. Purcell, M. Marcilla, J. McCluskey, and J. A. L. de Castro Qualitative and Quantitative Differences in Peptides Bound to HLA-B27 in the Presence of Mouse versus Human Tapasin Define a Role for Tapasin as a Size-Dependent Peptide Editor J. Immunol., June 15, 2005; 174(12): 7833 - 7844. [Abstract] [Full Text] [PDF]
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 D. Zernich, A. W. Purcell, W. A. Macdonald, L. Kjer-Nielsen, L. K. Ely, N. Laham, T. Crockford, N. A. Mifsud, M. Bharadwaj, L. Chang, et al. Natural HLA Class I Polymorphism Controls the Pathway of Antigen Presentation and Susceptibility to Viral Evasion J. Exp. Med., November 8, 2004; (2004) jem.20031680. [Abstract] [Full Text] [PDF]
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L. Li, B. A. Sullivan, C. J. Aldrich, M. J. Soloski, J. Forman, A. G. Grandea III, P. E. Jensen, and L. Van Kaer Differential Requirement for Tapasin in the Presentation of Leader- and Insulin-Derived Peptide Antigens to Qa-1b-Restricted CTLs J. Immunol., September 15, 2004; 173(6): 3707 - 3715. [Abstract] [Full Text] [PDF]
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 J. Koch, R. Guntrum, S. Heintke, C. Kyritsis, and R. Tampe Functional Dissection of the Transmembrane Domains of the Transporter Associated with Antigen Processing (TAP) J. Biol. Chem., March 12, 2004; 279(11): 10142 - 10147. [Abstract] [Full Text] [PDF]
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L. Sesma, I. Alvarez, M. Marcilla, A. Paradela, and J. A. L. de Castro Species-specific Differences in Proteasomal Processing and Tapasin-mediated Loading Influence Peptide Presentation by HLA-B27 in Murine Cells J. Biol. Chem., November 21, 2003; 278(47): 46461 - 46472. [Abstract] [Full Text] [PDF]
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S. Tourdot and K. G. Gould Competition Between MHC Class I Alleles for Cell Surface Expression Alters CTL Responses to Influenza A Virus J. Immunol., November 15, 2002; 169(10): 5615 - 5621. [Abstract] [Full Text] [PDF]
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 H. Firat, M. Cochet, P.-S. Rohrlich, F. Garcia-Pons, S. Darche, O. Danos, F. A. Lemonnier, and P. Langlade-Demoyen Comparative analysis of the CD8+ T cell repertoires of H-2 class I wild-type/HLA-A2.1 and H-2 class I knockout/HLA-A2.1 transgenic mice Int. Immunol., August 1, 2002; 14(8): 925 - 934. [Abstract] [Full Text] [PDF]
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P. Tan, H. Kropshofer, O. Mandelboim, N. Bulbuc, G. J. Hammerling, and F. Momburg Recruitment of MHC Class I Molecules by Tapasin into the Transporter Associated with Antigen Processing-Associated Complex Is Essential for Optimal Peptide Loading J. Immunol., February 15, 2002; 168(4): 1950 - 1960. [Abstract] [Full Text] [PDF]
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D. G. Kavanagh, M. C. Gold, M. Wagner, U. H. Koszinowski, and A. B. Hill The Multiple Immune-evasion Genes of Murine Cytomegalovirus Are Not Redundant: m4 and m152 Inhibit Antigen Presentation In a Complementary and Cooperative Fashion J. Exp. Med., October 1, 2001; 194(7): 967 - 978. [Abstract] [Full Text] [PDF]
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M. R. Harris, L. Lybarger, N. B. Myers, C. Hilbert, J. C. Solheim, T. H. Hansen, and Y. Y. L. Yu Interactions of HLA-B27 with the peptide loading complex as revealed by heavy chain mutations Int. Immunol., October 1, 2001; 13(10): 1275 - 1282. [Abstract] [Full Text] [PDF]
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L. Lybarger, Y. Y. L. Yu, T. Chun, C.-R. Wang, A. G. Grandea III, L. Van Kaer, and T. H. Hansen Tapasin Enhances Peptide-Induced Expression of H2-M3 Molecules, but Is Not Required for the Retention of Open Conformers J. Immunol., August 15, 2001; 167(4): 2097 - 2105. [Abstract] [Full Text] [PDF]
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T. Chun, A. G. Grandea III, L. Lybarger, J. Forman, L. Van Kaer, and C.-R. Wang Functional Roles of TAP and Tapasin in the Assembly of M3-N-Formylated Peptide Complexes J. Immunol., August 1, 2001; 167(3): 1507 - 1514. [Abstract] [Full Text] [PDF]
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A. W. Purcell, J. J. Gorman, M. Garcia-Peydro, A. Paradela, S. R. Burrows, G. H. Talbo, N. Laham, C. A. Peh, E. C. Reynolds, J. A. Lopez de Castro, et al. Quantitative and Qualitative Influences of Tapasin on the Class I Peptide Repertoire J. Immunol., January 15, 2001; 166(2): 1016 - 1027. [Abstract] [Full Text] [PDF]
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M. J. Barnden, A. W. Purcell, J. J. Gorman, and J. McCluskey Tapasin-Mediated Retention and Optimization of Peptide Ligands During the Assembly of Class I Molecules J. Immunol., July 1, 2000; 165(1): 322 - 330. [Abstract] [Full Text] [PDF]
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