©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Major Histocompatibility Class I Molecules Can Present Cryptic Translation Products to T-cells (*)

(Received for publication, July 28, 1994)

Nilabh Shastri (§) Vu Nguyen Federico Gonzalez

From the Division of Immunology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 SL8bulletK^b-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 SL8bulletK^b 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 peptidebulletMHC 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.


INTRODUCTION

Major histocompatibility complex (MHC) (^1)class I molecules display peptides on the antigen presenting cell (APC) surface. CD8+ cytolytic T-cells probe these peptidebulletMHC 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 peptidebulletMHC 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)bulletK^b or influenza nucleoprotein (NP)bulletD^b 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 peptidebulletMHC 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 peptidebulletMHC complexes. We identified the cryptic translation initiation codons by screening a random set of codons that allowed presentation of the OVA257-264 (SL8)bulletK^b 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.


MATERIALS AND METHODS

Plasmid DNA Constructs

The XGSL8 DNA constructs encoded the SL8 (SIINFEKL) residues that were preceded by a translational stop codon in each of three reading frames, a degenerate codon X, and a glycine codon (Fig. 2a). The constructs were prepared using oligonucleotides synthesized with a mixture of equal parts of A, T, C, and G nucleotides at the codon shown as NNN in Fig. 2a. The oligonucleotides contained complementary overhangs for insertion into the corresponding 5` HindIII and 3` XbaI sites in the pcDNAI/neo vector (Invitrogen). Transcription of cDNAs in this vector is regulated by the constitutive cytomegalovirus promoter. The GTG construct was prepared using synthetic oligonucleotides with GTG as the X codon. Preparation of MGSL8, RF-SL8, and *RF-SL8 constructs in the same pcDNAI/neo vector has been described(15) . Initial screens were carried out with miniprep DNAs, and all subsequent sequencing and functional analysis was carried out with CsCl-purified plasmids as described below.


Figure 2: Nucleotide sequence of XGSL8 expression constructs for generating the variable translation initiation ``NNN'' codon (a) and expression of the SL8bulletK^b complex in transiently transfected K^b-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 peptidebulletMHC expression using B3Z T-cells.




Figure 1: Stimulation of SL8bulletK^b-specific B3Z T-cell response by transiently transfected K^b-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 times 10^4 K^b-COS cells by the DEAE-dextran procedure. Two days later, transfected cells were co-cultured with 1 times 10^5 SL8bulletK^b-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 beta-galactoside (CPRG) to chlorophenol red at 595 nm as described(15, 17) . Responses shown are means of duplicate cultures.



Transfections and T-cell Activation Assays

Varying concentrations of indicated CsCl-purified plasmids were transfected into 2 times 10^4 K^b-COS cells/well of a 96-well plate, by the DEAE-dextran procedure described earlier(15) . Two days later, transfected cells were co-cultured with 1 times 10^5 SL8bulletK^b-specific B3Z T-cells(15, 16) . After overnight culture, antigen-specific lacZ activity induced in B3Z T-cells was measured by the conversion of the lacZ substrate chlorophenol red beta-galactoside to chlorophenol red at 595 nm as described(15, 17) . Responses shown are means of duplicate cultures with standard deviations less than 10%. Stable XGSL8bulletK^b-expressing L-cell transfectants were generated by electroporation with 50 µg of plasmid DNA as described earlier (15) . Pools of 50-100 colonies selected in 1 mg/ml G418 were tested for their T-cell stimulation activity as above.

Analysis of Processed Peptides

10 times 10^6 cells were extracted with 0.1% trifluoroacetic acid according to the protocol described by Rotzschke et al.(18) . Extracts were centrifuged for 30 min at 20,000 times g and passed through a 10-kDa cutoff filter (UltraFree, Millipore). The filtrate was dried in a vacuum centrifuge, resuspended in complete medium, and assayed for the presence of processed peptides with K^b L-cells as APC and 1 times 10^5 B3Z T-cells. The amount of processed SL8 peptide recovered was estimated by comparison with synthetic SL8 peptide as a standard.


RESULTS AND DISCUSSION

In an earlier study, we showed that the ovalbumin (OVA)bulletK^b MHC or the influenza nucleoprotein (NP)bulletD^b 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 SL8bulletK^b 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 SL8bulletK^b 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^b-COS cells as APC. Two days later, transfected cells were assayed for the presence of the peptidebulletMHC complex by their ability to stimulate the SL8bulletK^b-specific B3Z T-cells. By this assay, 10% of the 367 recombinant plasmids tested scored positive (>2times 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 SL8bulletK^b complex from these seven constructs was determined by transfecting DNA into K^b-COS cells and then measuring their ability to stimulate SL8bulletK^b-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^b-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^b-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^b 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 SL8bulletK^b-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^b-MHC(17, 18) . Transfectants with each of the seven constructs generated the SL8bulletK^b complex, while the parental Ltk and K^b 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 SL8bulletK^b complex, because the same transfectant cells yielded superimposable response profiles with K^b-restricted 27.5Z T-cells (Fig. 3, c and d). This result confirmed that K^b-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^b L-cells, stably transfected with indicated XGSL8 constructs, stimulate SL8bulletK^b-specific B3Z T-cells. The nucleotide triplets shown represent the X translational initiation codon. Varying numbers of parental Ltk, K^b L-cells (K89), or G418^r cells obtained by transfecting K^b L-cells with the indicated XGSL8 plasmids, were co-cultured overnight with 1 times 10^5 B3Z (a and b) or unknown peptidebulletK^b-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 SL8bulletK^b 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 SL8bulletK^b 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 peptidebulletMHC required for T-cell stimulation within a magnitude of the theoretical limit of one peptidebulletMHC 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^b 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^b 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 peptidebulletMHC 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.


FOOTNOTES

*
This work was supported by grants from the National Institutes of Health (to N. S.) and the Tobacco Related Disease Research Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: LSA 421, Dept MCB, University of California, Berkeley, CA 94720-3200. Tel.: 510-643-9197; Fax: 510-643-9230.

(^1)
The abbreviations used are: MHC, major histocompatibility complex; APC, antigen presenting cell; OVA, ovalbumin; NP, nucleoprotein.


ACKNOWLEDGEMENTS

We thank Dr. M. Kozak for discussions and Dr. A. Winoto for comments on the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.