(Received for publication, July 28, 1994)
From the
Self or foreign cellular proteins provide peptides for
presentation by major histocompatibility complex (MHC) class I
molecules on the surface of antigen presenting cells (APC).
Surprisingly, several studies have shown that T-cells can recognize APC
transfected with antigen genes that were not present in the appropriate
translational context. To understand the basis of this phenomenon, APC
were transfected with DNA constructs encoding the OVA257-264
(SL8) peptide, but with varying translation initiation codons. We
report that, in addition to ATG, 6 other codons (ATT, ACG, CTG, GCG,
TGG, GAT) also allowed presentation to SL8K
-specific
T-cells. Significantly, this set includes 3 of 4 known non-ATG
translation initiation codons strongly suggesting that cryptic
translation accounts for this phenomenon. Although expression of the
SL8
K
complex was readily detected by T-cell
activation, the amount of processed peptides was below detection limit
(<30 copies/cell) in cell extracts. Thus, the fortuitous presence of
these cryptic translation initiation sites in transcribed genes can
explain how peptide
MHC complexes were obtained in sufficient
amounts for T-cell activation. The translation initiation codons
identified here could also be useful for identifying potential open
reading frames that possess biological and/or immunological activities.
Major histocompatibility complex (MHC) ()class I
molecules display peptides on the antigen presenting cell (APC)
surface. CD8+ cytolytic T-cells probe these peptide
MHC
complexes to identify and to subsequently eliminate cells that express
foreign peptides. The fact that these peptides are derived from
cellular proteins allows T-cells to detect intracellular pathogens or
transformation events due to expression of new proteins (reviewed in (1, 2, 3) ).
The thousands of distinct
peptides that are constitutively displayed by MHC depends upon the
corresponding diversity of donor proteins that provide these
peptides(4, 5, 6, 7) . The mechanism
by which cellular proteins are targeted to the processing pathway
remains poorly understood. Some studies have proposed that these
peptides are generated as by-products of normal turnover of cellular
proteins(8, 9) . The generality of this mechanism is,
however, not clear because differences in antigen presentation activity
often do not correlate with differences in stability of donor
proteins(8, 10, 11) . As an alternate
mechansim, it has been proposed that these peptides are generated via a
specialized pathway that does not depend upon protein turnover but
instead involves novel transcriptional and/or translational mechanisms.
Evidence in support of this latter mechanism, referred to as the
``pepton hypothesis''(12) , comes from several
independent studies that peptideMHC complexes are generated in
APC transfected with antigen genes despite the absence of obvious
promoters or translation initiation
sites(11, 13, 14, 15) . Again, which
one or more of the several possible mechanisms that can account for
this phenomenon has not been yet determined.
Our previous study
confirmed that presentation of either ovalbumin (OVA)K
or influenza nucleoprotein (NP)
D
complexes did
indeed occur in APC transfected with OVA or NP genes that were in
inappropriate expression context (15) . By contrast to other
studies, our experiments suggested that presentation of
peptide
MHC complexes was related to the translational mechanism.
First, the relative differences in presentation activity correlated
with particular translational reading frames of the peptide coding
sequence. Second, and more significantly, the T-cell stimulating
activity was completely abrogated by placing translational stops
immediately upstream of the peptide coding sequence. How translation
was obtained in the absence of the ATG translation initiation codon was
not ascertained.
In this report, we have analyzed the mechanism that
accounts for the generation of these endogenous peptideMHC
complexes. We identified the cryptic translation initiation codons by
screening a random set of codons that allowed presentation of the
OVA257-264 (SL8)
K
complex to T-cells.
Interestingly, the seven codons thus identified include ATG, as well as
three of four known non-ATG translational initiation codons.
Furthermore, we show that despite the ability of these non-ATG codons
to allow peptide/MHC expression, the amount of processed peptides in
the cells remained below biochemical detection limits estimated to be
less than 30 copies/cell. These findings explain previously surprising
results and are important for understanding the mechanism of the
endogenous peptide/MHC presentation pathway.
Figure 2:
Nucleotide sequence of XGSL8 expression
constructs for generating the variable translation initiation
``NNN'' codon (a) and expression of the
SL8K
complex in transiently transfected
K
-COS cells (b and c). See
``Materials and Methods'' for details of construct
preparation and the legend to Fig. 1for details of DNA
transfections and measurement of peptide
MHC expression using B3Z
T-cells.
Figure 1:
Stimulation of
SL8K
-specific B3Z T-cell response by transiently
transfected K
-COS cells. DNA constructs encoded the SL8
residues in appropriate (Met-Gly-SL8, MG-SL8), inappropriate (RF-SL8),
or without a translational initiation codon (*RF-SL8). The RF-SL8 and
*RF-SL8 constructs, respectively, contain the SL8 codons preceded by
either no in-frame ATG or with a stop codon in each of the three
translational reading frames (see Fig. 2a)(15) . MG-SL8 is identical with
*RF-SL8, but with an in-frame ATG codon. Varying concentrations of
indicated CsCl-purified plasmids were transfected into 2
10
K
-COS cells by the DEAE-dextran procedure.
Two days later, transfected cells were co-cultured with 1
10
SL8
K
-specific B3Z T-cells. After
overnight culture, antigen-specific lacZ activity induced in
B3Z T-cells was determined by measuring conversion of the lacZ substrate chlorophenol red
-galactoside (CPRG) to
chlorophenol red at 595 nm as described(15, 17) .
Responses shown are means of duplicate
cultures.
In an earlier study, we showed that the ovalbumin
(OVA)K
MHC or the influenza nucleoprotein
(NP)
D
MHC complexes were expressed in cells
transfected with DNA constructs encoding the minimal OVA257-264
(SL8) or Flu NP366-374 (NP) peptides(15) . As in other
studies(11, 14) , we also obtained significant T-cell
stimulation by APC transfected with DNA constructs in which the codons
for SL8 or NP peptides were ``out-of-frame'' relative to the
ATG-defined translation reading frame. This antigen presentation
activity of APC transfectants was, however, completely abrogated by
placing stop codons directly upstream of the SL8 coding sequence,
suggesting that cryptic translation of the SL8 residues could explain
the presence of the SL8
K
complex (Fig. 1).
Accordingly, the loss of presentation activity was completely restored
by inserting the ATG codon between the termination and the SL8 codons
yielding the Met-Gly-SL8 precursor peptide (Fig. 1). The
restoration of presentation activity by insertion of the ATG codon
suggested the strategy for identifying the cryptic translation
initiation codons by their ability to restore presentation of
SL8
K
complex to T-cells.
A panel of DNA constructs
(referred to as XGSL8) was prepared with synthetic oligonucleotides
encoding SL8 octapeptide but preceded by varying translational
initiation codons (Fig. 2a). The degenerate initiation
codon was preceded by translational stop codons and followed by a
glycine codon (GGA) to provide the essential features of the Kozak
consensus sequence (RYYRYY ATGG) except for the ATG
codon(19, 20) . Plasmid DNA from randomly picked
bacterial colonies was transfected into K-COS cells as APC.
Two days later, transfected cells were assayed for the presence of the
peptide
MHC complex by their ability to stimulate the
SL8
K
-specific B3Z T-cells. By this assay,
10% of
the 367 recombinant plasmids tested scored positive (>2
over
background), and 20 with the highest activity were selected for further
analysis. Nucleotide sequences of these recombinants revealed seven
different codons at the X(NNN) position, ATG (7) ,
CTG(5) , ATT(3) , TGG(2) , ACG(1) ,
GCG(1) , and GAT (1) (Fig. 1b). The
frequency (7/367 = 1:53) of recombinants containing ATG was
close to the theoretical frequency (1:64 codons) and thus validates the
random representation of X codons tested in this screen. In
addition, four other randomly picked recombinants that did not score
positive in the screen were selected as negative controls.
The
relative efficiency of generating the SL8K
complex
from these seven constructs was determined by transfecting DNA into
K
-COS cells and then measuring their ability to stimulate
SL8
K
-specific B3Z T-cells. Among these recombinants,
the construct containing ATG as the translation initiation codon was
clearly the most efficient (Fig. 2b). When compared at
the optimal DNA concentration (100-300 ng/well), the T-cell
stimulatory activity generated by the non-ATG constructs was within
2-10-fold that of the ATG construct and was within
10-100-fold when compared at half-maximal response. By contrast
to these seven constructs, negligible T-cell stimulation was obtained
with constructs containing CTA, TCT, GAA, or TTG as the X translation initiation codons (Fig. 2, b and c). These results clearly demonstrate that the usage of
initiation codons in translation was not random. The seven codons
identified by this assay included, besides ATG, 3 of the 4 (CTG, ATT,
ACG, GTG) known non-ATG translational initiation
codons(20, 21) . To rule out the possibility that the
fourth known initiator codon, GTG, could have been used but may have
been missed among the recombinants screened, a construct encoding GTG
at the X position was prepared with synthetic oligonucleotides (Fig. 2a). This construct was transfected into
K
-COS cells, but, like the other negative control
constructs, it was also inactive, further validating the screening
strategy and emphasizing the non-random usage of X codons (Fig. 2c). Moreover, this result also indicates that
relative efficiency differences exist among the non-ATG translation
initiation codons. We conclude that K
-MHC can present
endogenous SL8 peptide synthesized via cryptic non-ATG translation
initiation codons.
We next assessed whether the translation
initiation codons identified by the transient expression assay were
also active when the DNAs were stably integrated. Stable transfectants
were generated with murine K L-cells with these seven
constructs. To control for possible differences in gene expression due
to variable integration sites, pools (
50-100 colonies) of
G418-resistant cells were tested for their ability to stimulate
SL8
K
-specific B3Z T-cells. As a further internal
control, the same transfectants were also tested in the same experiment
with another T-cell hybrid, 27.5Z, that recognizes an endogenous and
constitutively expressed peptide presented by
K
-MHC(17, 18) . Transfectants with each of
the seven constructs generated the SL8
K
complex,
while the parental Ltk
and K
L-cells were
incapable of stimulating B3Z T-cells (Fig. 3, a and b). Again, transfectants with the ATG construct were the most
stimulatory, and relative differences in presentation with
transfectants expressing the non-ATG constructs were similar to those
obtained in transient transfections. The reasons for the somewhat
different hierarchy of the non-ATG codons in stable versus transient transfectants are not clear, but may be due to species
(murine versus simian) or to cell type (fibroblast versus kidney epithelium) differences. These relative differences in
presentation efficiency were, however, limited to expression of the
SL8
K
complex, because the same transfectant cells
yielded superimposable response profiles with K
-restricted
27.5Z T-cells (Fig. 3, c and d). This result
confirmed that K
-MHC can present cryptic translation
products derived from either transiently or stably expressed genes. The
fortuitous presence of one or more of these alternate initiation codons
upstream of the antigen coding sequences can also now satisfactorily
explain how antigen presentation activity could have been obtained in
our own (15) and in other previous studies (11, 13, 14) .
Figure 3:
Mouse K L-cells, stably
transfected with indicated XGSL8 constructs, stimulate
SL8
K
-specific B3Z T-cells. The nucleotide triplets
shown represent the X translational initiation codon. Varying
numbers of parental Ltk
, K
L-cells (K89),
or G418
cells obtained by transfecting K
L-cells with the indicated XGSL8 plasmids, were co-cultured
overnight with 1
10
B3Z (a and b)
or unknown peptide
K
-specific 27.5Z (c and d) T-cells. Antigen-specific response was measured by the
induced lacZ activity in the T-cells as in the legend to Fig. 1.
The high SL8K
expression in APC transfected with non-ATG constructs was
surprising. It was possible that, despite predominance of ATG as the
most efficient translation initiation codon
known(19, 20) , significant amounts of processed SL8
peptide may have been generated in cells expressing the non-ATG
constructs. To directly measure the amount of processed peptides in
cells, we assayed trifluoroacetic acid extracts prepared from the
stable L-cell transfectants (Fig. 4). High levels of processed
peptide were present in extracts of transfectants expressing the ATG
construct. However, extracts of transfectants expressing either of the
six non-ATG constructs were completely inactive (>300-fold lower
yield). This result directly shows that the amounts of the SL8 gene
product present in cells was profoundly influenced by the identity of
the translation initiation codon, with ATG being by far the most
effective. Nevertheless, despite the lack of detectable peptide in the
extracts, APC transfected with non-ATG constructs presented the
SL8
K
complex in amounts sufficient to stimulate
T-cells (Fig. 3). Due to the complexity of these trifluoroacetic
acid extracts (4, 5, 6) , the exact peptide
amounts are difficult to assess accurately. However, using synthetic
SL8 peptide as a standard (sensitivity
1-3
pM)(15) , and assuming quantitative recovery, we
estimate that the amount of processed SL8 peptide obtained from non-ATG
constructs was equal to or less than 30 SL8 copies/cell. This estimate
sets the threshold of peptide
MHC required for T-cell stimulation
within a magnitude of the theoretical limit of one peptide
MHC
complex on the APC surface. Furthermore, this result directly
demonstrates that T-cell assay is an extraordinarily sensitive measure
of gene expression in APC.
Figure 4:
Quantitation of processed peptides
extracted from stable K L-cells transfected with XGSL8
constructs. Processed SL8 peptides are detected only in extracts of
cells expressing the ATG construct. Serial dilutions of trifluoroacetic
acid extracts were prepared as described under ``Materials and
Methods'' and were assayed using B3Z T-cells and untransfected
K
L-cells as APC.
Why do cells initiate translation at non-ATG codons? Note that although the fidelity of translation initiation in vitro is often less stringent and was the basis for the discovery of the genetic code, translation initiation in living cells virtually always occurs at the ATG codon(22) . Examples of rare exceptions to this rule that allowed the discovery of non-ATG codons include transcription and growth factors, as well as viral proteins of unknown function(21, 23, 24, 25, 26) . One possible explanation proposed is that these genes use non-ATG initiation codons, despite their inefficient translation capacity(21) , because expression of these potent gene products must be kept extremely low(20) . Thus, while the occurrence of cryptic translation is firmly established, its mechanism and its significance for biological regulation is yet to be clarified. By contrast to these normal genes where usage of the non-ATG codons is apparently promoted by stable RNA hairpin structures that cause stalling of the ribosome(21) , no such RNA structures were apparent in the constructs used by us and by others. Thus, it is unlikely that our artificial constructs were fortuitously favored for cryptic translation. Rather, a more attractive possibility is that cryptic translation could have resulted as a consequence of the high levels of constitutive transcription from viral promoters widely used in expression vectors. Whether similar conditions are possible for normal genes and whether any of the thousands of peptides displayed by MHC include cryptic translation products is not yet known. Conceivably, expression of viral genes in infected cells could be similar to the conditions tested in our experiments.
Regardless of the mechanism of
cryptic translation, the ability of MHC molecules to present
vanishingly small amounts of endogenous peptides is relevant to the
efficiency of the immune surveillance mechanism. Ideal surveillance of
viral infection or of transformation events within cells requires that all cellular proteins be available as peptideMHC for
recognition by T-cells. However, MHC are constrained to present only
consensus motif-bearing peptides(3) , of which only a small
subset are apparently used(27) . Thus, the presentation of rare
cryptic translation products by MHC molecules could be rationalized as
a property of the antigen processing mechanism whose function is to
ensure that the largest possible set of peptides is made available for
survey by the T-cell repertoire. Indeed, the mechanism of cryptic
translation and the capture of these small amounts of precursors by the
antigen presentation pathway remain to be elucidated. Regardless of the
mechanism, our results suggest that the identity of antigenic peptides
for candidate vaccines (28, 29) may not be limited
only to those found within the most abundant ATG defined open reading
frames. The alternate translational initiation codons identified here
could prove useful in these pursuits.