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*
Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark;
Department of Biochemistry and Nutrition, Technical University, Lyngby, Denmark;
Unit for Glycoconjugate Chemistry, CID-Consejo Superior de Investigaciones Cientificas, Barcelona, Spain;
§
The Netherlands Cancer Institute, Amsterdam, The Netherlands; and
¶
Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom
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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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Little is known about the effect on class I MHC-restricted Ag presentation of naturally occurring posttranslational modifications of peptide Ags in vivo. These modifications may affect Ag processing, MHC binding, and interaction with the TCR. Evidence that posttranslational modifications may play a role in Ag presentation and T cell recognition has been accumulating over the last few years. Recently, it has been described that class I (7, 8, 9, 10) and class II (11) MHC-restricted T cells can recognize posttranslationally modified peptides specifically (12, 13).
Phosphorylation is a tightly regulated posttranslational cytosolic event. However, inflammation, intracellular infection, cellular activation, and malignant transformation may cause deregulation of phosphorylation, perhaps leading to generation of sufficient amounts of phosphopeptides for class I MHC-restricted presentation to occur. For example, it is known that following chromosomal translocation in leukemia, certain protooncogenes acquire novel kinase activities leading to the appearance of new transformation-specific phosphorylation (14, 15). Similarly, novel phosphopeptide Ags could be derived from phosphorylated oncogenes exhibiting deregulated phosphorylation upon malignant transformation. Such proteins are phosphorylated on tyrosine, serine, and threonine residues by different cellular kinases, and up-regulation of phosphorylation at specific sites has been associated with transformation (16, 17, 18). Finally, a large number of phosphorylated viral proteins have been characterized, which also might generate phosphopeptide fragments for MHC-restricted presentation.
In this study, we have analyzed the effect of phosphorylation on the ability of peptides to be transported by TAP, binding to class I molecules, and CTL recognition. We conclude that posttranslationally modified cytosolic phosphopeptides could be presented to the immune system by class I MHC molecules and recognized by CTL in a phosphorylation-specific manner.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The TAP-deficient cell lines RMA-S and T2 were maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS and glutamine. T2 cells transfected with HLA-B*0702 (T2-B7) (19), HLA-B*3501 (T2-B35) (20), and HLA-B*2705 (T2-B27) (21) were kindly supplied by A. McMichael (Oxford University, Oxford, U.K.), and T2 cells transfected with H-2Kk (T2-Kk) were kindly supplied by P. Cresswell (Yale University, New Haven, CT). For the T2-B7 and T2-B27 cell lines, the medium was supplemented with 100 µg/ml hygromycin and 250 µg/ml G418, respectively.
EBV-transformed B lymphoblastoid cell lines (BCL)3 were established by coculturing PBMC with supernatant from the EBV-infected marmoset cell line B95.8 in the presence of cyclosporin A. Approximately 3 x 107 PBMC were resuspended in 5 ml R10 and 5 ml B95.8 supernatant and incubated for 4 h at 37°C. The cells were then resuspended in fresh R10 (8 ml), incubated at 37°C, and the following day cyclosporin A was added to the culture (1 µg/ml). Two EBV-transformed BCL lines were established: JS-BCL from the donor JS expressing HLA-A2, -A3, -B7, -B60, and -Cw3, and KIG-BCL from the donor KIG expressing HLA-A2, -A3, -B7, and -B15(62).
Synthetic peptides
The nonphosphorylated peptides were from Research Genetics (Huntsville, AL) and provided at >90% purity, as analyzed by HPLC and mass spectroscopy analysis.
Phosphopeptides containing phosphoserine or phosphotyrosine residues were synthesized using a phosphorylated Fmoc-serine or Fmoc-tyrosine building blocks (FmocSer(PO(Obzl)OH)OH or Fmoc-Tyr(PO(Obzl)OH)OH); Calbiochem, Lisztweg, Germany) in the manual solid-phase Fmoc synthesis of phosphopeptides. All phosphopeptides were HPLC purified and characterized by mass spectroscopy and nuclear magnetic resonance. In addition to oncogene- or virus protein-derived phosphopeptides, the following peptides were synthesized for the TAP assays: 417 [TVNKTERAY], and 417-S [TVNKTESAY], as well as the phosphopeptide 417-P [TVNKTES(-PO32-)AY]. Before use in the TAP assay, some peptides were radiolabeled with Na125I catalyzed by chloramine-T.
Assembly assay for peptide binding to class I MHC molecules
Assembly assays for binding of the synthetic peptides to class I MHC molecules metabolically labeled with [35S]methionine were conducted essentially as described (22, 23). This assay is based on the peptide-dependent stabilization of class I molecules after lysis of peptide transporter-deficient cell lines (T2 or RMA-S) and the subsequent detection of correctly folded, stable MHC heavy chains by immunoprecipitation with conformation-dependent Abs. To prevent dephosphorylation of peptides during the assembly assay, phosphatase inhibitors were added (1 mM Na3VO4 and 5 mM NaF). After electrophoresis, gels were exposed to PhosphorImager screens, and peptide binding was quantitated using the Imagequant PhosphorImager program (Molecular Dynamics, Sunnyvale, CA).
TAP-mediated transport of phosphopeptides across the ER membrane
Assays for the TAP-mediated translocation across the ER membrane of the radiolabeled peptide 417, the serine-substituted peptide 417-S, as well as a phosphorylated (417-P) peptide analogue in LCL721 cells (LCL; human) or RMA cells (murine) were performed as described (24, 25). T2 cells or RMA-S cells were used to demonstrate the TAP dependence of transport. Briefly, 5 x 106 cells per sample were washed in incubation buffer (130 mM KCl, 10 mM NaCl, 1 mM CaCl2, 2 mM EGTA, 2 mM MgCl2, and 5 mM HEPES, pH 7.3) before permeabilization with 2.5 IU/ml streptolysin O (Wellcome Diagnostics, Dartford, U.K.). The cell suspension was then added to 0.1 µg of iodinated peptide (sp. act., 30 µCi/µg), with or without ATP (Boehringer Mannheim, Mannheim, Germany) in a final volume of 100 µl. Translocation was performed at 37°C over 5 min before ice-cold lysis buffer (1% Triton X-100, 500 mM NaCl, 5 mM MgCl2, 50 mM Tris-HCl, pH 7.5) was added to each tube. After 30 min of lysis (4°C), the nuclei were removed by centrifugation (12,000 rpm, 10 min). Then the N-glycosylated peptides were precipitated by the addition of Con A-Sepharose beads (Pharmacia, Piscataway, NJ) and washed five times with ice-cold lysis buffer, and the counts were associated with the Con A-Sepharose pellet quantitated by gamma counting. For experiments with phosphorylated peptides, the phosphatase inhibitor NaF (10 mM) was included in all buffers after having established that this did not affect translocation of peptide 417.
Induction of murine CTL in vivo
Peptide-specific CTL lines were generated by immunization of C57BL/6 mice, as described (26). Briefly, RMA-S cells (108) were pulsed with the phosphopeptide K3-P (10 µM) at 26°C for 18 h. The cells were irradiated (60 Gy) and inoculated i.p. into C57BL/6 mice. Mice were sacrificed 3 wk later and the spleen cells were restimulated in vitro with syngeneic spleen cells (4 x 106) pulsed with K3-P for 5 days in RPMI 1640 supplemented with 10% FCS.
Generation of human CTL lines as primary responses in vitro
Peptide-specific CTL were established in vitro as primary
responses using autologous dendritic cells (DC) as APC. DC were
developed from PBMC from healthy donors, as described previously
(27). Briefly, PBMC were resuspended at 4 x
106 cells/ml in R10 medium in six-well plates
(Nunc, Naperville, IL) at 3 ml/well. After 2 h of incubation at
37°C, the nonadherent cells were washed away, and 3 ml R10 medium
supplemented with 500 U/ml GM-CSF (Leucomax, Sandoz, Germany) and 25
U/ml IL-4 (PeproTec, Boston, MA) was added to each well. Every third
day, 0.5 ml medium was replaced with fresh R10 medium containing GM-CSF
and IL-4 to a final concentration of 500 U/ml and 25 U/ml,
respectively. After 9 days, 50 U/ml IL-1
was added to the culture,
and DC were harvested the next day. DC were resuspended at 5 x
106 cells/ml in RPMI without serum containing 40
µg/ml peptide and 3 µg/ml human
ß2-microglobulin for 4 h and subsequently
irradiated (30 Gy). The peptide-pulsed, irradiated DC (2 x
105/well) were used to stimulate fresh
lymphocytes (5 x 106/well) in 24-well
plates (Costar) in 2 ml RPMI medium with 20% AIM V medium (Life
Technologies) and 5% human serum supplemented with 5 ng/ml IL-7
(PeproTech). On day 10, and weekly thereafter, the responder population
was restimulated. Irradiated, peptide-pulsed autologous PBMC were used
to restimulate the T cell line at a stimulator:responder ratio of 1:5,
with addition of IL-2 (40 U/ml; PeproTech) on the following day. CTL
clones were established from bulk cultures by limiting dilution in
96-well plates using peptide-pulsed, irradiated PBMC as feeder cells in
the presence of 40 U/ml IL-2 and 1 µg/ml PHA.
CTL cytotoxicity assay
Conventional 51Cr release assays for CTL-mediated cytotoxicity were used to test the specificity of CTL lines against peptide-pulsed target cells. Target cells were autologous EBV-transformed B cell lines (assays with human cells) or T2 cells transfected with H-2Kb or H-2Db (murine assays), as described (7). For a typical CTL assay, 106 target cells in 50 µl R10 were labeled with 51Cr (100 µCi; Amersham, Arlington Heights, IL) and in some cases pulsed with peptide (4 µg) in a round-bottom well of a 96-well plate at 37°C for 60 min. The washed target cells were plated out in 96-well plates with CTL at various E:T ratios and incubated at 37°C for 4 h, before 100 µl of medium was aspirated and 51Cr release counted in a gamma counter (Cobra 5005; Packard Instruments, Meriden, CT). The maximum 51Cr release was determined in separate wells by addition of 100 µl 10% Triton X-100, and spontaneous release was determined by the addition of 100 µl R10 only to target cells. Specific lysis was calculated using the following formula: ((experimental release - spontaneous release)/(maximum release - spontaneous release)) x 100.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Assays for direct translocation of phosphopeptides by TAP were
conducted as described (24, 25) by adding radiolabeled
peptides and phosphopeptides, which contain an N-linked
glycosylation sequon to streptolysin O-permeabilized cells. Fig. 1
shows the result of an assay for
translocation of the peptides 417, the serine substituted 417-S, and
the phosphorylated 417-P using the human LCL cells in the presence or
absence of ATP for each peptide. All peptides are transported across
the ER membrane in a strictly ATP-dependent manner by both the human
(Fig. 1) and murine (results not shown) TAP, as determined by the
resulting N-glycosylation and recovery of the iodinated
peptides with Con A-Sepharose. Peptide 417 as well as the
serine-substituted 417-S were very efficiently translocated with
recoveries of 4–5% for LCL, consistent with previously published data
(28). Phosphorylation of 417-S (giving 417-P) resulted in
translocation of similar high efficiency in LCL. These data show that
phosphorylated peptides can be efficiently transported from the cytosol
into the ER by TAP.
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Based on an extensive binding study4 with 73 potential MHC-binding peptides all containing a natural phosphorylation site from viral phosphoproteins as well as from phosphorylated oncogene products that display altered phosphorylation upon malignant transformation, nine peptides that showed strong binding were selected for phosphorylation. Four peptides were derived from human oncogene phosphoproteins and four peptides were from viral phosphoproteins. In addition, the model peptide K3 (FAPSNYPAL) (7) was included in the study. K3 is an analogue of the H-2Kb-restricted immunodominant Sendai virus NP324–332 epitope FAPGNYPAL.
The tyrosine-phosphorylated version of the CRKL-derived peptide OP42-P
(YAQPQTTTPL) bound strongly to HLA-A*0201 (Fig. 2A), illustrating that
phosphorylation did not prevent the efficient binding of this peptide
to class I MHC. However, we also observed several instances in which
the phosphorylation of nonanchor tyrosine, serine, or threonine
residues had either an intermediate or a severe negative effect on
peptide binding to class I .MHC alleles. Thus, the
phosphorylated c-Jun peptide OP27-P (GLLKLASPEL) only bound
weakly to HLA-A*0201 compared with the nonphosphorylated peptide (Fig. 2B), and the phosphorylated version of the
p53378–386 peptide OP39-P did not show any
binding to HLA-B*2705, whereas OP39 (SRHKKLMFK) bound better than the
positive control epitope (KRWIIMGLNK) (29).
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), and a
binding score was assigned to the different peptides.
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). Of the peptides
whose binding is blocked by phosphorylation, the influenza
M287–94 peptide P49
(DDSHFVSI) is the only one that is
phosphorylated on two sites in vivo (41). However, neither
the diphosphorylated version nor any of the two monophosphorylated
versions of the peptide showed any measurable binding to
H-2Kk. The fact that phosphorylation completely
abrogates class I MHC binding of some peptides examined is a strong
indication that dephosphorylation does not occur during the
peptide-binding assay.
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-amino terminus of the peptide
and the phosphate group may prevent peptide binding. Phosphopeptide-specific CTL responses
Both the nonphosphorylated and the phosphorylated version of the
synthetic peptide K3 (FAPSNYPAL) bound with high affinity to both
H-2Db and H-2Kb. Spleen
cells from C57BL/6 mice immunized with phosphopeptide K3-P-pulsed RMA-S
cells were harvested after 3 wk later and tested for lytic activity
against peptide-pulsed T2-Kb and
T2-Db cells. As seen in Fig. 3A, the spleen cell-derived
CTL showed a high degree of specificity against the phosphopeptide.
However, some CTL activity against the nonphosphorylated peptide was
also observed, either due to the coexistence of nonphosphorylated
peptide-specific CTL or due to cross-reacting CTL. Furthermore, the CTL
response against the phosphopeptide was H-2Kb
restricted, because only T2-Kb but not
T2-Db cells pulsed with K3-P were lysed.
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A), the CRKL-derived phosphopeptide OP42-P peptide was also
used to generate human CTL in vitro primary responses. Autologous DC
were generated from HLA-A2-positive PBMC in the presence of IL-4 and
GM-CSF. Next CTL were stimulated in vitro using irradiated
OP42-P-pulsed dendritic cells as APC in the presence of IL-2 and IL-7.
After five in vitro restimulations, the peptide specificity was tested
in standard 51Cr release assays using
peptide-pulsed autologous EBV-transformed KIG-BCL as target cells.
Target cells pulsed with OP42-P were lysed (Fig. 4A), whereas target cells
pulsed with OP42 or without peptide were not recognized. Thus, the
resulting CTL line was specific for the phosphopeptide OP42-P.
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B it is seen that a CTL clone raised against OP42-lysed
target cells pulsed with OP42, and that it did not recognize target
cells pulsed with phosphorylated OP42-P or without peptide. Thus, a
peptide-specific CTL clone was established that did not cross-react
with the phosphorylated peptide. Furthermore, these data indicate that
no dephosphorylation of peptide occurred during the CTL assay. These results clearly demonstrate the ability to generate phosphopeptide-specific class I MHC-restricted CTL responses, and suggest that the phosphate group is involved in a specific contact with TCR.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have investigated CTL responses against phosphopeptides in two model systems. We observed a HLA-A2-restricted phosphopeptide-specific primary response against OP42-P in vitro. We also immunized mice with the phosphopeptide K3-P analogue of the dominant Kb-restricted epitope from Sendai virus NP. Even though this phosphopeptide binds with high affinity to H-2Kb or H-2Db, we only observed a Kb-restricted CTL response against the peptide, thus resembling the natural H-2Kb-restricted response in C57BL/6 mice against Sendai virus. Our group has previously described that glycosylation of K3 peptide on the same serine residue at position 4 with N-acetylglucosamine (GlcNAc) does not effect peptide binding to H-2Kb or H-2Db, and that it is possible to elicit both H-2Kb- and H-2Db-restricted glycopeptide-specific CTL response (7). However, in our previous study, CTL were restimulated several times in vitro, which may have induced the Db-restricted response. Recently, we have solved the crystal structure of a glycosylated version of K3 (FAPS(O-ß-GlcNAc)NYPAL) in complex with H-2Db, and it was revealed that the carbohydrate residue is pointing out of the groove (42).
Four of the phosphopeptides examined did not show any binding to MHC when phosphorylated. The molecular basis for this inhibition is unclear because all modifications were made at positions outside the primary anchor residues. Two of the four affected peptides were phosphorylated at position 1, which could possibly affect the formation of the conserved bonding in the A pocket of the class I MHC-binding groove. The other two affected peptides were H-2Kk restricted, and binding to this allele, which requires a negatively charged position 2 anchor residue, may be adversely affected by the addition of a further highly negatively charged group. The negative influence of phosphorylation on class I MHC binding is of immunological interest, because it provides a novel potential immune escape mechanism. For example, the influenza NP473–481-derived phosphopeptide P26 did not show any appreciable binding to HLA-B*0702 and HLA-B*3501, whereas the nonphosphorylated version of the same peptide bound with high affinity to both alleles. Thus, if the source protein is phosphorylated in an infected cell, resulting in the generation of the phosphorylated peptide by the Ag-processing apparatus, it cannot be presented at the cell surface by the MHC molecule, thus allowing the virus to avoid immune elimination.
To date, only a few examples of posttranslationally modified natural class I MHC-restricted epitopes have been described. Skipper et al. identified a HLA-A*0201-restricted tyrosinase-derived melanoma peptide, in which the asparagine in position 3 was posttranslationally modified to aspartic acid (9). Meadows et al. identified a modified HLA-A*0201-restricted peptide epitope from the human minor histocompatibility Ag H-Y, describing T cells that only recognize posttranslationally altered forms of this peptide (10). One of these modifications involves attachment to the peptide of a second cysteine residue via a disulfide bond.
Our group has previously shown that class I MHC molecules can bind
synthetic glycopeptides and that the glycopeptides elicit a strong CTL
response that is glycopeptide specific (7). Furthermore,
we have recently shown that glycopeptides serve as natural substrates
for the TAP molecule, and that class I MHC molecules present a small
amount of glycosylated peptides in vivo (43). Van Stipdonk
et al. have recently described that murine class II MHC-restricted T
cells raised against differentially phosphorylated forms of
B-crystallin discriminate between the phosphorylated and the
nonphosphorylated peptides (44).
In this study, we have investigated the effect of phosphorylation on peptide binding to class I MHC molecules and the recognition of CTL. We have examined three different kinds of phosphoproteins as sources for the cellular production of phosphopeptides. First, because known phosphorylation sites in some proteins become constitutively phosphorylated during oncogene activation, the ensuing phosphopeptide fragments offer themselves as candidates for T cell Ags in line with overexpressed, nonmutated tumor Ags. Examples of such proteins include c-Jun and the tumor suppressor gene product p53. c-Jun becomes activated by phosphorylation of the two Ser residues 63 and 73, and has been reported to be phosphorylated in both leukemia and lung cancer cells (37). Phosphorylation of the p53 tumor suppressor gene product has been described for a number of serine residues, among these Ser9, Ser15, Ser37, Ser315, Ser392, and Ser378 (40, 45, 46, 47).
Second, upon the critical event leading to activation of the transforming phenotype, some protooncogenes characteristically acquire novel kinase activities leading to the appearance of novel phosphorylation sites. Thus, as a consequence of the (9, 22) translocation (the Philadelphia chromosome) that is associated with all chronic myeloid leukemia cases and 20% of adult acute lymphoblastic leukemia, the c-abl protooncogene becomes activated by its translocation to chromosome 22 giving rise to a BCR-ABL fusion protein (48, 49). A crucial difference between the fusion protein and the normal c-abl is that only the chimeric protein exhibits tyrosine kinase activity (14), resulting in autophosphorylation of a number of tyrosine residues (including Tyr177) of the BCR-ABL fusion protein (50, 51), as well as causing a constitutive increase in tyrosine phosphorylation of other cellular proteins. One of the best-described proteins is the CRKL, which is overexpressed and hyperphosphorylated on Tyr207 in Philadelphia chromosome-positive chronic myeloid leukemia cells (36, 52). Each of these new sites of phosphorylation could give rise to tumor-specific phosphopeptide neo-Ags. Furthermore, it is conceivable that transformation-associated posttranslational modification of a cellular protein could alter the pattern of peptide fragments that are generated from it.
Finally, viral phosphoproteins with known phosphorylation sites have also been included in the study. For example, the EBV protein EBNA-2 is phosphorylated at Ser469 and Ser470 (53), EBV LMP-1 at Ser313 and Thr324 (54), and EBV BZLF1 at Ser173 (55). Other viruses with multiple known phosphorylation sites include adenovirus, HIV, and influenza virus.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John S. Haurum, Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, 2100 Copenhagen OE, Denmark. E-mail address:
3 Abbreviations used in this paper: BCL, B lymphoblastoid cell line; BCR, breakpoint cluster region; ER, endoplasmic reticulum; GlcNAc, N-acetylglucosamine; LCL, lymphoblastoid cell line; NP, nucleoprotein.
4 M. H. Andersen, L. Tan, I. Søndergaard, J. Zeuthen, T. Elliott, and J. S. Haurum. Poor correspondence between predicted and experimental binding of peptides to class I MHC molecules. Submitted for publication
Received for publication May 19, 1999. Accepted for publication July 16, 1999.
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) was compared with
the nonphosphorylated OP42 (YAQPQTTTPL) (
) and the positive control
peptide C1 (
) (A). The binding of the phosphorylated
c-Jun67–76 peptide OP27-P
(GLLKLAS(-PO32-)PEL) (
) was compared with
the nonphosphorylated OP27 (GLLKLASPEL) (
) and the positive control
peptide C1 (
) was compared with
the nonphosphorylated OP39 (SRHKKLMFK) (
) and the positive control
peptide C4 (
) (C).
