From the Department of Cell Research and Immunology,
The George S. Wise Faculty of Life Sciences, Tel Aviv University,
Tel Aviv 69978, Israel and the § Laboratory of Molecular
Structure, NIAID, National Institutes of Health,
Bethesda, Maryland 20892
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ABSTRACT |
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In primary embryonal fibroblasts from transgenic mice expressing H-2b genes and a miniature swine class I transgene (PD1), transformation with adenovirus 12 results in suppression of assembly and cell surface expression of all class I complexes. Cell surface expression of PD1 can be recovered by transfecting the cells with peptide transporter genes. However, reconstitution of the H-2Kb gene expression requires, in addition, a 2-fold increase in the steady state level of the H-2Kb mRNA that can be attained by treatment of the cells with interferons or by transfecting them with the H-2Kb gene. A detailed analyses of the biogenesis of class I molecules has revealed the steady state expression of free class I heavy chains that are not converted into conformed complexes even when peptide transporter genes are overexpressed. The fact that class I complex assembly seems to be highly inefficient in certain cell lines might be a major in vivo obstacle for the elimination of transformed or virus-infected cells by cytotoxic T lymphocytes, especially in view of the fact that the level of class I gene transcription is often down-regulated in cancer cells and/or that assembly of class I major histocompatibility complexes can be subverted by virus-encoded proteins.
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INTRODUCTION |
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Major histocompatibility complex (MHC)1 class I molecules are polymorphic, integral membrane proteins that bind a diverse group of peptides derived from endogenous antigens and display these peptides for recognition by cytotoxic T lymphocytes (CTL) (1). This mechanism enables the immune system to control infectious diseases and the growth of tumor cells (2-4).
The biochemistry and cell biology of antigen processing and
presentation by MHC class I molecules has been analyzed in detail in
the recent years (5, 6). It is well established that the efficient
transport of class I molecules to the cell surface depends on the
assembly of the heavy chain/2m dimer with peptides, most
of which are generated by cleavage of proteins in the cytosol and are
actively transported into the ER by a heterodimeric complex, known as
transporter associated with antigen presentation (TAP). The trimeric
complex is then transported though the Golgi apparatus to the cell
surface. En route to complex formation, the nascent heavy chain
transiently associates with the ER resident proteins calreticulin and
calnexin, as well as with the adaptor molecule tapasin and with TAP
(7-9).
Mutant cell lines (5, 6, 10, 11), "knockout" mice (12, 13), and
tumor cells (4, 14, 15), which do not express TAP genes and/or
2m (16-18), are generally devoid of cell surface MHC
class I molecules. We have previously shown that in cell lines derived
from primary embryonal fibroblasts from transgenic mice expressing both
the endogenous H-2 genes and a miniature swine class I transgene (PD1),
transformation with the highly oncogenic Ad12 results in a significant
reduction in peptide transporter (TAP1 and TAP2) and in
proteasome-associated (LMP2 and LMP7) gene expression, and consequently
in suppression of cell surface expression of all class I antigens (15,
19-21). The mRNA levels encoded by class I heavy chain and
2m genes in most of these cell lines are reduced by only
2-3-fold (15, 19). Expression of these genes is either normal or
up-regulated in cells transformed by the non-oncogenic virus Ad5.
Re-expression of TAP and LMP gene products by stable transfection of an
Ad12-transformed cell line completely reconstituted the cell surface
expression and assembly of PD1 but did not affect the expression of
H-2Kb molecules (21). Enhanced expression of
2m in the cells further induced the assembly and
expression of PD1 but did not affect the cell surface expression of H-2
molecules (22). These data raised the possibilities that a modest
reduction in H-2 mRNA levels critically affects the efficiency of
assembly of H-2 complexes, that the H-2 heavy chains are mutated,
and/or that Ad12-transformed cells are deficient in a factor(s) that
facilitates the assembly of
2m with a particular set of
class I heavy chains. We now show that a modest enhancement in the
steady state level of class I mRNA, above a threshold level,
results in complete reconstitution of assembly and cell surface
expression of fully conformed class I complexes. These studies brought
to light the existence of a large pool of biologically inactive, free
heavy chains in both "normal" and transformed cells that we
characterized and whose biological significance we discuss.
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EXPERIMENTAL PROCEDURES |
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Cell Cultures-- The Ad12-transformed (VAD12.79, VAD12.42), E1Ad5-transformed (A5O5), TAP-transfected cell line (VAD12.79/TAP), and the "normal" cell line M1 have been previously described (19-21). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 10% fetal calf serum, penicillin, streptomycin, gentamycin, and amphotericin B at the recommended concentrations (23). Media and supplements were purchased from Biological Industries (Bet Ha'emek, Israel).
Cell lines were treated with 600 units/ml of IFN-Stable Transfection-- VAD12.79 and VAD12.79/TAP were transfected by the calcium phosphate-DNA coprecipitation method (21). The transfection mixture contained 10 µg of plasmid DNA (the H-2Kb cDNA expressed from a CMV promoter in pcDNAI (Invitrogen)), 1 µg of plasmid DNA containing the puromycin gene (pBabe encoding the puromycin resistance gene was a kind gift from P. Murray, Whitehead Institute, Boston), and 5 µg of carrier DNA (sheared salmon sperm DNA, Sigma). 24 h after transfection, the cells were washed with phosphate-buffered saline and fresh medium was added; after 24 h, the medium was supplemented with 110 units/ml puromycin (Sigma). Following selection of puromycin-resistant cells, individual cell colonies were isolated and expanded in culture.
Antibodies--
The following antibodies were routinely used for
FACS analyses and immunoprecipitations: 20.8.4S (recognizes the
1/
2 epitope on H-2Kb) (24), 27.11.13 (recognizes an
1 epitope on H-2Db) (24), PT85A (recognizes a public
determinant on swine lymphocyte antigens) (25), and rabbit antibodies
directed against H-2Kb peptide 8 (26). Rabbit antibodies
directed against free heavy chains (H-2Kb + H-2Db) (27) were a kind gift from Prof. H. Ploegh (MIT).
Other antibodies are cited in the text.
FACS Analysis-- Cells were harvested by mild trypsinization, followed by washes in media supplemented with 5% fetal calf serum and 0.01% sodium azide. About 106 cells were incubated at 4 °C with the appropriate concentration of the first antibody for 60 min, washed, and then incubated in the dark for another 45 min with the second antibody. Cells were washed with phosphate-buffered saline, and fluorescence intensity was analyzed with a Becton-Dickinson cell sorter (Becton Dickinson, Mountain View, CA).
Metabolic Labeling, Protease Inhibitors, and Immunoprecipitation-- Cells were grown to 80% confluence and starved for 60 min in methionine-free medium. They were then labeled in methionine-free medium containing 150 µCi/ml [35S]methionine (Amersham International, Little Chalfort, UK) for 60 min unless otherwise indicated, washed with phosphate-buffered saline, and chased for the indicated intervals. The cells were lysed with buffer containing 0.5% Triton X-100, 50 mM Tris (pH 7.5), and 150 mM NaCl. The immunoprecipitates were washed with buffer containing 0.1% Triton X-100, 50 mM Tris (pH 7.5), and 150 mM NaCl. For Endo H treatment, immunoprecipitates were eluted by adding 25 µl containing 50 mM Tris (pH 8), 1% SDS and boiled for 5 min. The samples were centrifuged, and the supernatant was added to 25 µl of 50 mM citrate buffer (pH 5.5) containing 1000 units of Endo H (New England Biolabs, Beverly, MA). The samples were incubated at 37 °C for 18 h, followed by the addition of sample buffer. All the buffers contained the following protease inhibitors: 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin (Sigma). Protein concentration was determined by Bradford reagent (Sigma), and equivalent protein amounts were loaded on the gels.
The protease inhibitor MG-132 at a concentration of 10 µM (Calbiochem) was added to the chase media as indicated. The immunoprecipitates were fractionated on 10 or 15% SDS-polyacrylamide gel electrophoresis and X-OMAT AR x-ray films (Eastman Kodak Co.) were exposed to the dried gels.Probes and Plasmids--
The following probes were previously
described (19-21). The PD1 specific probe was a
SacI-BamHI genomic fragment containing exons 2-7
of the PD1 gene; the H-2 probe was an
EcoRI-HindIII fragment derived from
pH-2d33 (H-2Kd); the 2m probe
was a PstI-PstI fragment from
2m
cDNA; ribosomal RNA probe (rRNA) was a
HindIII-HindIII fragment derived from pWES. A
plasmid containing H-2Kb cDNA was a kind gift from Dr.
L. Eisenbach (The Weizmann Institute of Science, Rehovot, Israel).
Hybridizations-- The hybridization solution contained 4× SSC, 50% formamide, 0.2% SDS, 0.1% polyvinylpyrrolidone, and 100 µg/ml sheared salmon sperm DNA. Hybridizations were carried out at 42 °C, followed by washes with 2× SSC, 0.1% SDS at room temperature, and 0.2× SSC at temperatures ranging between 55 and 65 °C. After stripping with a boiling solution of 0.1% SDS, the blots were used for additional hybridizations.
RNA Analysis--
Cytoplasmic RNA was prepared using a
modification of the White and Bancroft method (28) as described
previously (21). RNA was denatured and fractionated on a 1.2%
formaldehyde/formamide agarose gel, blotted onto a Hybond-N membrane
(Amersham International, Little Chalfort, UK), and hybridized with the
appropriate probe, which had been labeled with
[-32P]dCTP (Rotem Industries, Dimona, Israel), using a
random priming labeling kit (United States Biochemical Corp.,
Cleveland, OH).
Quantitation of Radioactive Signals-- Dried radioactive gels or blots were exposed to a phosphoimager screen and analyzed with a phosphoimager (Fuji BAS1000, Tokyo). The data are presented as phospho-stimulated luminescence (PSL) units.
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RESULTS |
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IFN-/
and -
Induce Cell Surface Expression of Class I
Molecules in Ad12-transformed Cells--
The reconstitution of cell
surface expression of the miniature swine transgene product PD1 but not
of H-2Kb class I complexes in Ad12-transformed cells that
have been reconstituted for TAP and LMP expression (15, 21) raised the
possibility that mutation(s) in the endogenous H-2 genes (in particular
in the H-2Kb gene), but not in the transgene, result in
their aberrant conformation such that they are not recognized by
conformation-dependent antibodies. To test this
possibility, we have treated the cells with either
/
- or
-IFNs
that induce the expression of most genes that are associated with
antigen presentation and compared the resulting cell surface expression
with that of the "normal" cell line M1. The results of a
representative FACS analysis are summarized in Fig.
1. All the cell lines were derived from
mouse embryonic fibroblasts expressing H-2Kb,
H-2Db, and PD1 genes. The data demonstrate that the
Ad12-transformed cells (VAD12.79, VAD12.42) can be induced to express
all the class I gene products (Fig. 1, A-C). The enhancing
effects of IFN treatment on the cell surface expression of class I
molecules by the "normal" cell line M1 and the E1Ad5-transformed
cell line A5O5, which expresses high levels of these molecules, are
minimal (data not shown). Both types of IFNs induced more efficiently
the expression of PD1 (15-fold induction by IFN-
and 25-fold
induction by IFNs-
/
) then the expression of H-2Db
molecules (5-10-fold induction) or H-2Kb molecules, whose
expression is less enhanced then H-2Db. The same results
were obtained with a large panel of Ad12-transformed cell lines and
with seven monoclonal antibodies that recognize different
H-2Kb epitopes (20.8.4S, Y3, B8.24.3, 5F1, 28.8.6S,
28.13.3S, and AF6 88.5.3). These data, as well as the fact that direct
sequencing of short polymerase chain reaction fragments derived from
the extracellular domains of the H-2Kb gene did not reveal
the existence of nucleotide substitutions, show that the
H-2Kb gene is not mutated in Ad12-transformed cells, and
when all the essential factors for assembly and transport of class I
complexes are expressed as by IFN induction, the cell surface level of
H-2 molecules reaches normal or higher levels (Fig. 1, compare
panels A-C with panel D).
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IFN Treatment of Ad12-transformed Cells Results in Only 1.5-Fold
Increase in the Steady State Level of Class I mRNA--
To examine
if increased class I expression was due to increase in mRNA steady
state levels, the effect of IFNs on class I heavy chain,
2m, and peptide transporter genes was tested for Ad12-transformed cell lines and compared with that of M1 and A5O5 cells. A representative experiment is shown in Fig.
2A, and the results of the
individual experiments are summarized in Fig. 2B. In Fig.
2B, the PSL values for each mRNA have been normalized to
those of rRNA, and the expression of H-2, PD1, and
2m
genes in untreated Ad12-transformed cells has been arbitrarily defined as 100% (Fig. 2B (panels a and c)).
Because we could not detect a hybridization signal for TAP genes in
untreated cells (see also Ref. 15), the hybridization signal values for
TAP1 and TAP2 in treated cells were normalized to those of rRNA and are
presented as 100 × PSL units (Fig. 2B (panels
b and c)). The figure shows that the expression of
2m following treatment with
-IFN is induced by 2-fold
(a and c) and the expressions of TAP1 and TAP2
are induced from zero to levels that are equal or higher then those
expressed by M1 and A5O5 cells (compare panels b and
c to panel d), whereas the expression of H-2
class I heavy chain mRNA increases by a maximum of 1.5-fold, and
the expression of PD1 is not induced at all (a and
c). Quantitative reverse transcriptase-polymerase chain
reaction was utilized to distinguish H-Db from
H-2Kb mRNA (22) and demonstrated that both were
comparably induced (data not shown). The compiled data demonstrate that
the transcription of
2m and TAP genes is more
effectively induced by IFN-
then by IFN-
/
and that both IFNs
affect to the same extent (1.5-fold induction) the steady state level
of H-2 heavy chain mRNA.
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2-Fold Increase in the Steady State Level of Class I Heavy Chain mRNA Increases Dramatically the Cell Surface Expression of Class I Complexes-- Previous analyses of several individual Ad12-transformed cell lines revealed the following: (a) there is only 1.5-3-fold decrease in the H-2 steady state mRNA levels of Ad12-transformed cells as compared with the "normal" or E1Ad5-transformed cell lines, yet Ad12-transformed cells did not express any cell surface H-2 complexes (15, 19, 21, 22; see also Figs. 1-3); and (b) TAP-transfected Ad12-transformed cells express normal levels of PD1 but very low or undetectable levels of H-2 molecules, whereas the steady state level of PD1 is only 2-fold higher than that of H-2 mRNA (15, 21, 22; see also Fig. 3). These observations, in addition to the dramatic effects of IFNs on the cell surface expression of H-2 complexes relative to the weak enhancement in class I heavy chain mRNA levels, suggested that a small increase (1.5-2-fold) in the level of class I heavy chain mRNA can dramatically affect the cell surface expression of class I complexes. To directly test this hypothesis, an Ad12-transformed cell line (VAD12.79) and TAP1+TAP2 transfected VAD12.79 cells (VAD12.79/TAP) were stably transfected with a vector expressing H-2Kb from a CMV promoter. Individual clones that express 1.5-2-fold higher levels of H-2Kb mRNA than the parental VAD12.79 cells, and thus comparable levels to the "normal" cell line M1 or to the E1Ad5-transformed cells line A5O5, have been expanded in culture and analyzed. Fig. 3 shows a representative FACS analysis of control and transfected cells. The E1Ad5-transformed cell line A5O5 expresses all class I molecules (the data for H-2Db are not shown), whereas VAD12.79 expresses neither, as was demonstrated before (15, 21, 22). VAD12.79/TAP cells are reconstituted for the expression of PD1 but do not express H-2Kb complexes as was described previously (15, 21, 22). The re-expression of normal levels of H-2Kb mRNA induces high cell surface expression of H-2Kb complexes only in VAD12.79/TAP cells (VAD12.79/TAP/Kb). Hence, nearly complete reconstitution of H-2 expression in Ad12-transformed cells can be achieved by re-expression of peptide transporters coupled with a 2-fold induction in H-2Kb mRNA level. Thus, the level of cell surface expression of class I complexes increases exponentially when mRNA steady state levels are enhanced beyond a certain threshold. These data clearly show that class I heavy chain mRNA must exceed a certain threshold to generate efficient assembly of class I complexes for cell surface expression. The potential significance of this phenomenon for antigen presentation by tumor cells and virus-infected cells in vivo led us to perform a detailed analysis of class I heavy chain biogenesis in "normal" and transformed cells.
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Turnover Rate of Conformed and Free Heavy Chains in TAP-expressing and TAP-deficient Cells-- Cell lysates prepared from the cell lines described above were immunoprecipitated with the conformation-dependent antibody 20.8.4S and the anti-heavy chain antiserum (anti-HC) that immunoprecipitates only H-2Kb and H-2Db free heavy chains and does not react with conformed heavy chains. Two experiments are shown in Fig. 4, A and B, demonstrating that the "normal" cell line M1 (Fig. 4A) and the E1Ad5-transformed cell line A5O5 (Fig. 4B) express, in addition to the fully conformed H-2Kb complexes, a large pool of free heavy chains. VAD12.79 cells (Fig. 4, A and B) do not express a significant level of conformed complexes but express a small amount of free heavy chains. The level of free heavy chains depends both on the amount of newly synthesized class I heavy chains and on the expression of TAP genes, as both VAD12.79/Kb (Fig. 4A) and especially VAD12.79/TAP (Fig. 4B) express higher levels of free heavy chains than the parental non-transfected cells (VAD12.79). VAD12.79/TAP/Kb cells (Fig. 4B) express more conformed H-2Kb complexes than VAD12.79/Kb, as expected, and these cells also express very high levels of free class I heavy chains. Because VAD12.79/TAP/Kb cells over-express TAP1 and TAP2 (15), the data suggest that there is a stable pool of free heavy chains in the cells even when peptide transporter genes are over-expressed; thus, presumably, a sufficient amount of class I binding peptides exists in the ER.
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DISCUSSION |
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The expression of MHC class I molecules is essential in the immune
response against viruses and tumor cells because they present antigenic
peptides to CTL. In accordance with this key role in antigen
presentation, MHC class I molecules are expressed in most cells, and
their basal level of expression can be induced by a number of cytokines
(29-31). The IFN-stimulated response element is the DNA binding site
for factors of the IFN regulatory factor family, and it mediates the
induction of MHC class I expression by type I and type II IFNs (32). In
particular, IFN-, a product of activated T lymphocytes and natural
killer cells, regulates the expression of different components in the
pathway of MHC class I-restricted antigen presentation and is a potent
inducer of class I heavy chain,
2m, TAP1, TAP2, LMP2,
LMP7, and the regulator PA28 genes (33-37).
Certain cells, such as some tumor and normal cells (e.g. trophoblast cells), are down-regulated for the expression of either class I heavy chain genes or peptide transporter and LMP genes or both (2, 4, 38-44); consequently, these cells cannot be targets for killing mediated by CTL (45). IFNs can often enhance the cell surface expression of MHC class I complexes in such cells; however, despite the extensive information about IFNs and their functions, little information exists as how this is accomplished. Specifically, it is surprising that although IFNs dramatically stimulate class I expression, previous studies (44, 46) revealed only a 1.5-2-fold increase in the steady state mRNA levels encoded by class I heavy chain genes. This moderate effect of IFNs on class I heavy chain mRNA levels agrees with chloramphenicol acetyltransferase-transient transfection assays (47, 48) that showed a small effect on heavy chain gene transcription. Because IFN-induced class I expression is accompanied by much larger increases in TAP and LMP in comparison with class I heavy chain gene expression, it has been assumed that the availability of class I binding peptides is the limiting factor for assembly of class I complexes (49).
We describe a set of "normal" E1Ad5- and Ad12-transformed cell
lines that respond to IFN treatment. Ad12-transformed cells are
down-regulated for the cell surface expression of class I molecules,
but this expression can be induced by IFNs such that it reaches normal
levels for all class I molecules (Fig. 1, compare panels A-C
with panel D). A detailed analysis of mRNA steady state levels encoded by class I heavy chain, 2m, TAP1, and
TAP2 genes revealed that although class I heavy chain gene expression
levels are induced by a maximum of 1.5-fold, the induction of peptide transporter gene expression is more dramatic going from zero to nearly
normal levels (Fig. 2B, compare panels a and
b). Previous studies had shown that the protein expression
of LMP subunits was also induced to nearly normal levels (21). The
compiled data are in accordance with the concept that the most
significant effect of IFNs, and in particular of IFN-
, is on peptide
production and their transport to the ER. Consequently, we anticipated
that the reexpression of TAP genes in Ad12-transformed cells would reconstitute the expression of class I complexes. However, in Ad12-transformed cells that have been stably transfected with TAP genes
or with TAP+LMP genes, the assembly and cell surface expression of the
transgene product (PD1) is reconstituted, whereas the expression of
H-2Kb remains undetectable or very low (15, 21, 22). One
possible explanation for this could be that in Ad12-transformed cells
the moderate suppression in the steady state level of H-2Kb
mRNA and, consequently, in the amount of newly synthesized class I
heavy chains (21, 22) significantly reduces the efficiency of assembly
of H-2Kb complexes. This explanation is in accordance with
the fact that the expression of PD1 mRNA in all the cells
(including VAD12.79/TAP cells in which PD1 cell surface expression, but
not H-2, is reconstituted) is 2-3-fold higher than that of the H-2
genes.
To directly test this hypothesis, an Ad12-transformed cell line
(VAD12.79) and the TAP-reconstituted VAD12.79 cell line (VAD12.79/TAP) were stably transfected with a vector expressing H-2Kb from
a CMV promoter. Individual clones that expressed H-2Kb
mRNA in comparable levels with those of the "normal" (M1 cells) or E1Ad5-transformed (A5O5) cells were isolated. The FACS analyses of
the transfected cells demonstrated that indeed VAD12.79 cells expressing peptide transporters coupled with 2-fold higher levels of
H-2Kb mRNA (VAD12.79/TAP/Kb) are completely
reconstituted for the cell surface expression of H-2Kb
(Fig. 3). Collectively, the data show that when peptide transporters are expressed, and thus, presumably, peptides are available for assembly, a 1.5-2-fold induction in the amount of newly synthesized class I heavy chains results in a dramatic increase in cell surface expression of class I complexes. Thus, assembly of class I molecules and consequently expression seems to occur once a threshold level of
class I heavy chain expression is surpasseda phenomenon that might
significantly impact upon the ability of cells (especially non-lymphoid
cells that normally express low levels of class I molecules) to
function in peptide presentation in vivo. Specific examples
are primary human tumors and metastases of both human and murine tumors
that demonstrate down-regulation of class I heavy chain gene expression
(2-4) and cells infected with adenoviruses of subclass C or with human
CMV that express viral proteins such as the gp19 (adenoviruses) and US3
(human CMV) that bind class I heavy chains and thus interfere with
assembly of class I complexes (50, 51). In these cases, a modest
increase in the amount of newly synthesized class I molecules might
reverse significantly host immune responses in vivo.
To delineate the factors that might enhance assembly of class I
complexes, we have analyzed the synthesis and maturation of class I
heavy chains at steady state levels in "normal" cells and tumor
cells that have been reconstituted for the expression of either peptide
transporter or class I heavy chain genes or both, with and without
treatment with IFN-. Our data demonstrate that assembly of class I
complexes is inefficient in fibroblast-derived cell lines and that a
large and relatively stable pool of free heavy chains that directly
depends on the expression level of both class I heavy chain and TAP
genes exists in these cells (Fig. 4, compare the signal obtained in
VAD12.79 with that obtained in VAD12.79/Kb and
VAD12.79/TAP). For instance, VAD12.79/TAP cells express a significant
amount of free heavy chains but only low levels of conformed
H-2Kb molecules. The latter are probably highly unstable,
as (a) associated
2m molecules are not
detectable in coimmunoprecipitates (see Fig. 4B, 12.79/TAP)
and (b) these cells do not express cell surface H-2Kb molecules (Fig. 3). A significant amount of conformed
H-2Kb complexes can be detected only when the expression of
both peptide transporter and class I heavy chain genes is reconstituted
(Fig. 4B, compare VAD12.79/TAP and
VAD12.79/TAP/Kb). The VAD12.79/TAP/Kb cells are
similar to the "normal" and E1Ad5-transformed cells in that they
express not only conformed H-2Kb complexes but a large pool
of free heavy chains. These data suggest that the expression of peptide
transporters is essential not only for the assembly of conformed class
I complexes but also for the maintenance of free heavy chains. Whether
the physical presence of peptide transporters is important for the
stabilization of associated free heavy chains or the transported
peptides can bind free heavy chains in the absence of associated
2m and stabilize them has yet to be determined. Solheim
et al. (7) demonstrated that free human heavy chains can be
found associated with peptide transporters in
2m-deficient cell lines, supporting the possibility that
the direct association of free heavy chains with TAP might stabilize at
least some of the free heavy chains.
In view of the data described above, one could speculate that a pool of
free heavy chains needs to be established in the cells prior to the
assembly of the heavy chains into conformed complexes. In accordance
with this hypothesis are data demonstrating the appearance of fully
conformed heavy chains in parallel to a rapid decrease in free heavy
chains in RMA cells following a 1-min pulse with [35S]Met
(26), suggesting that free heavy chains are directly converted to
conformed complexes. In contrast, our pulse-chase experiments performed
with E1Ad5-transformed cells that express high levels of class I
molecules on the cell surface showed that the free heavy chains
observed at steady state are relatively stable with a half-life of
60-100 min (Fig. 5D) and that they remain Endo H sensitive
throughout the chase period (Fig. 5B). They are slowly degraded within 3 h (Fig. 6) and are not converted into fully conformed class I complexes. Hence, our results support a model in
which a variable percentage of newly synthesized heavy chains are not
assembled into class I complexes and are eliminated from the cells as
part of a quality control mechanism. The relative percentage of free
heavy chains does not change even in cells pretreated with IFN-
(Fig. 7). In fibroblast-derived cell lines, the biologically inactive
heavy chain pool might be larger and more stable then in lymphoid cells
because of reduced amounts of another molecule(s), such as tapasin,
which assists assembly of class I complexes, or from a slower
degradation process of misfolded molecules.
In summary, our data suggest that class I complex formation in
fibroblasts and probably in other non-professional antigen-presenting cells is highly inefficient and, indeed, does not occur to a
significant degree until a threshold level of expression of peptide
transporter and class I heavy chain proteins is exceeded. Even in the
presence of optimal levels of peptide transporter and class I heavy
chain molecules, about half of the newly synthesized class I heavy
chains are not assembled into mature class I complexes and are degraded intracellularly. In cells where heavy chains are not limiting, IFN-
treatment dramatically enhances the expression of both conformed class
I complexes and free heavy chains. It is likely that the induction
results from the enhanced transcription of the LMP and peptide
transporter genes and, consequently, the increase in class I binding
peptides in the ER. It will be interesting to determine whether free
heavy chains are incapable of binding
2m and peptides as
was previously suggested (52-54) or if they are located in a subcompartment that is inaccessible to peptides and/or
2m or both. Studies are currently underway to answer
these questions and to determine what factors can facilitate assembly
of class I complexes in both normal and transformed cells.
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ACKNOWLEDGEMENT |
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We are grateful to Prof. H. Ploegh (MIT) for the anti-heavy chain antibodies and for the productive discussions.
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FOOTNOTES |
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* This work was supported by the US-Israel Binational Science Fund (BSF) and by a grant from the Ela Kodesz Institute for Research on Cancer Development Prevention.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 972-3-6409238; Fax: 972-3-6422046.
1 The abbreviations used are: MHC, major histocompatibility complex; CTL, cytotoxic T lymphocytes; TAP, transporter associated with antigen presentation; ER, endoplasmic reticulum; IFN, interferon; CMV, cytomegalovirus; PSL, phospho-stimulated luminescence.
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REFERENCES |
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