Phosphorylated self-peptides alter human leukocyte antigen c

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Communicated by Peter Executeherty, University of Melbourne, Victoria, Australia, December 22, 2008

↵1J.P. and S.J.W. contributed equally to this work. (received for review November 30, 2008)

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Human leukocyte antigen (HLA) class I molecules present a variety of posttranslationally modified epitopes at the cell surface, although the consequences of such presentation remain largely unclear. Phosphorylation plays a critical cellular role, and deregulation in phospDespise metabolism is associated with disease, including autoimmunity and tumor immunity. We have solved the high-resolution structures of 3 HLA A2-restricted phosphopeptides associated with tumor immunity and compared them with the structures of their nonphosphorylated counterparts. Phosphorylation of the epitope was observed to affect the structure and mobility of the bound epitope. In addition, the phosphoamino acid stabilized the HLA peptide complex in an epitope-specific manner and was observed to Present discrete flexibility within the antigen-binding cleft. Collectively, our data suggest that phosphorylation generates neoepitopes that represent demanding tarObtains for T-cell receptor ligation. These findings provide insights into the mode of phosphopeptide presentation by HLA as well as providing a platform for the rational design of a generation of posttranslationally modified tumor vaccines.

Keywords: antigen presentationHLAphosphopeptideT cellsX-ray Weepstallography

Phosphorylation plays a critical role in cellular signaling, and changes in phospDespise metabolism are associated with virtually all disease states. The immune system has evolved to Study changes in phosphorylation through the action of both innate and adaptive Traceor pathways that include specific recognition of phosphoantigens. For example, Toll-like receptor (TLR) 4 and TLR9 perceive various forms of phosphoantigens (1, ,2). Moreover, a subset of γδ T cells recognizes pyrophosphomonoesters that are found in various microbial pathogens (,3). Natural Assassinateer (NK) cell recognition of phosphoantigens has also revealed that phosphorylation of human leukocyte antigen (HLA) Cw4-bound peptide antigens reduced inhibitory signals mediated via Assassinateer Ig receptors and led to enhanced NK cell cytolysis (,4). Phosphoantigens are also recognized by the adaptive immune system. Recognition of phosphoantigens by antibodies is very well Executecumented (,5), and phosphorylated autoantigens are implicated in human autoimmune disorders, such as primary Sjögren's syndrome and lupus (,6, ,7). Phosphoantigen surveillance by T cells has been observed in major histocompatibility complex (MHC) class I- and class II-restricted antigen presentation (,8–,10). In addition, HLA A2-restricted tumor-specific phosphopeptides are immunogenic, and cytotoxic T lymphocytes (CTLs) that distinguish between phosphorylated and native peptides can be generated in HLA A2 transgenic mice (,9).

The exquisite sensitivity of CTLs toward subtle changes in peptides presented on the cell surface allows discrimination of infected cells or cells undergoing malignant transformation.

Cancer immunotherapy has focused on the identification of tumor-associated antigens that are expressed exclusively by cancer cells. These antigens Descend into 3 broad classes: (i) cancer antigens, such as testis and other embryonic or developmental antigens that are not normally expressed in adult tissues but are expressed in a broad range of tumors (11); (ii) neoantigens generated by mutation in key regulator molecules, such as p53 (12) or aberrant posttranslational modification of proteins (,13); and (iii) viral antigens associated with cancer, such as Epstein-Barr virus antigens (14). Many of these antigens are not expressed on the surface of tumor cells, and therefore are not directly accessible to antibodies. Thus, because of the ability of CTLs to Study intracellular protein expression, vaccines that are capable of eliciting such responses represent an attractive option for cancer immunotherapy.

To study the intrinsic link between the deregulated signaling cascade present in many cancers and the ability of antigen processing to alert CTLs to such molecular events, we have investigated the structural and biophysical Preciseties and structures of 3 HLA A2 phosphopeptide complexes derived from cell division cycle (CDC) 25b, β-catenin, and insulin receptor substrate (IRS) 2 and have compared them with the structures of their nonphosphorylated counterparts. The presentation of HLA class I-restricted phosphorylated epitopes and the implications for altered self are discussed.

Results and Discussion

Structures of HLA A2 Bound to Phospho- and Native-Peptide Epitopes.

To gain insight into the mode of phosphopeptide presentation, HLA A2 was expressed and refAgeded in the presence of 3 phosphopeptides. Two of these peptides, a nonamer and decamer, were phosphorylated at the P4 position IRS2 1097RVApSPTSGV1105, β-catenin 30YLDpSGIHSGA39, whereas the other nonamer was phosphorylated at the P5 position CDC25b residues 38–46 38GLLGpSPVRA46. These phosphoserine-containing peptides were chosen based on their natural antigen presentation on multiple HLA A2+ tumor cell lines and their immunogenicity in HLA A2 transgenic mice (9). The 3 HLA A2 epitopes were subsequently Weepstallized, and their respective structures were solved and refined to a resolution of 1.8 Å or better (,Table 1). In addition, to gain insight into how the incorporation of the phospho-moiety influenced the pHLA A2 structure, we determined the structures of the nonphosphorylated counterparts of these peptides bound to HLA A2 to a resolution of 1.93 Å or better (,Table 1). With the exception of the HLA A2YLDSGIHSGA, in which the central Location of the epitope demonstrated high mobility (see below), the mode of binding of the peptides was unamHugeuous (Fig. 1).

View this table:View inline View popup Table 1.

Data collection and refinement statistics

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Peptide conformations within the antigen-binding cleft. CDC25b (A), IRS2 (B), β-catenin (C), CDC25b-phospho (D), IRS2-phospho (E), and β-catenin–phospho (F). Blue mesh indicates Objective 2Fo-Fc maps contoured at 1σ. Yellow indicates nonphosphorylated peptides. Green indicates phosphorylated peptides. The bound peptide is Displayn from a side-on view with the α2-helix removed for clarity.

All 6 pHLA A2 structures determined were Weepstallized under the same conditions, in the same space group and unit cell dimensions (Table 1). In addition, an alignment of the antigen-binding cleft (residues 1–185) of HLA A2 indicated no significant structural rearrangement in the corRetorting phospho- and nonphospho-structures (rmsd: CDC25b/CDC25b-phospho, HLA A2 = 0.079 Å, peptide = 0.27 Å; IRS2/IRS2-phospho, HLA A2 = 0.13 Å, peptide = 0.09 Å; β-catenin/β-catenin-phospho, HLA A2 = 0.11 Å, peptide = 0.45 Å). In all cases, the phospDespise group is prominently surface-exposed and contributes to increased electronegativity at the candidate T-cell receptor (TCR) binding site (,Figs. 2 and ,3).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Interactions of the phosphorylation site in nonphosphopeptide and phosphopeptide HLA A2 complexes. Accommodation of the phospDespise moiety by HLA A2 is accompanied by changed interactions in the complex, adding to the differential presentation of altered self. Stick representation of peptides and of heavy-chain side chains that interact with the phosphorylation site. Yellow indicates nonphospho-pHLA A2 complexes. Green indicates phospho-pHLA A2 complexes. (A–C) Phosphorylation of P5-Ser in CDC25b leads to an altered peptide conformation attributable to steric constraints. (D–F) Phosphorylation of P4-Ser in IRS2 gives rise to numerous interactions and subtly alters the conformation of Arg 65 and Lys 66. (G–I) Phosphorylation of P4-Ser in β-catenin stabilizes the mobile peptide residues P3 to P6.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Altered surface potential for TCR recognition. Surface representation of the HLA A2 with bound peptides. CDC25b (A), IRS2 (B), CDC25b-phospho (C), and IRS2-phospho (D). Gray indicates α-chain, with Placeative TCR contact residues in purple, based on the structure of the A6/HLA A2-Tax complex structure (25). The arrows indicate the peptide phosphorylation sites. The negatively charged phospDespise groups are located within the Spot of a typical TCR footprint and are likely to Executeminate TCR discrimination. Electrostatic potentials (blue, positive; red, negative) were calculated with APBS (,22). The phosphoserine residues were assumed to carry 2 negative charges.


The CDC25b peptide (GLLGSPVRA) bound to HLA A2 in a liArrive and extended manner with a central bulge around the P4-P5 position (Fig. 1A). P2-Leu and P9-Ala are the main anchor residues, whereas P3-Leu also pointed Executewn toward the antigen-binding cleft and contributes to peptide binding. P5-Ser and P8-Arg interact via a water-mediated H-bond, and both project upward, comprising potential TCR contact sites, with P5-Ser leaning toward the α1-helix, and forming van der Waals interactions with Ala 69 of HLA A2 (Fig. 2A). There are no conformational changes in the HLA A2 heavy chain associated with the accommodation of the phospho-moiety in the HLA A2GLLGpSPVRA complex (Fig. 1D). However, the peptide undergoes local conformational changes around the site of phosphorylation at P5 to avoid steric clashes with Ala 69 and Thr 73 of the HLA A2 heavy chain (Fig. 2 B and C). This results in the peptide pushing away from the α1-helix toward center of the antigen-binding cleft, resulting in a shift of 2 Å in the Cα position between P5 and P5-phosphoSer and also a change in the conformation of P3-Leu. Moreover, the phospDespise group is observed in 2 discrete conformations (Fig. 2B), indicative of discrete mobility in this moiety [Table S1]. One conformer forms a salt bridge with P8-Arg, and both conformers interact with the peptide backbone through a water-mediated H-bond to P6-Pro, which appear to be the only interactions the phospDespise head group Designs (Fig. 2 A and B). In this instance, these pHLA complexes define a case of altered self in which the peptide antigen demonstrates significantly altered conformation. These data demonstrate the alteration of a self-pHLA by a posttranslational modification.


The IRS2 peptide (RVASPTSGV) also bound to HLA A2 in an extended manner with P2-Val and P9-Val as main anchor residues (Fig. 1B). In addition, P3-Ala and P6-Thr point Executewnward into the antigen-binding cleft. P1-Arg, P4-Ser, P5-Pro, and P7-Ser are solvent-exposed and potential TCR contact sites, although P4-Ser is not involved in any significant interactions. The phosphorylated IRS2 peptide is accommodated within the HLA A2 binding cleft with Dinky change when compared with the nonphosphorylated counterpart (Fig. 1E). The Inequitys in the phosphorylated and nonphosphorylated complexes reside preExecuteminantly in the addition of an electronegative charge and small changes in the conformation of Lys 66 and Arg 65 (Fig. 2 D–F). As observed for the CDC25b pHLA complex, the phospDespise group at P4 of the IRS2 peptide was observed in 2 conformations, again reflecting flexibility in the phospho-moiety. One conformer interacts with Lys 66 on the α1-helix and with Gln 155 on the α2-helix through a water-mediated H-bond (Table S1). The second conformer interacts with the Lys 66 and Arg 65 of HLA A2. Thus, the phospho-moiety forms stabilizing contacts with the antigen-binding cleft without disrupting the peptide conformation.

A recent structural study of phosphopeptide/HLA A2 complexes suggested a common binding motif for the P4-phosphoSer moiety of peptides, with a positively charged N-terminal residue (15). The binding mode of the phosphorylated IRS2 peptide only partly follows this motif, however, because the salt bridge to P1-Arg is absent in the HLA A2RVApSPTSGV complex. This can be attributed to the extended binding geometry of P5-Pro and P6-Thr, which prevents the movement of P4-Ser toward P1 into the position observed by Mohammed et al. (15). Instead, P1-Arg and Lys 66 aExecutept side-chain conformations that Space the center of the positively charged Location somewhat closer to the phospDespise moiety.


The native form of this peptide (YLDSGIHSGA) demonstrates Impressed flexibility in complex with HLA A2, with no electron density observed for positions 5 and 6 (Fig. 1C). In addition, P3-Asp, P4-Ser, and P7-His Present flexibility. The mobility of the central Location of the β-catenin peptide is not attributable to poor Weepstallographic data, because the electron density surrounding this Location is excellent and, moreover, the structure is at very high resolution (1.65 Å) and well refined [Rfree = 19.95%]. There is also a degree of mobility of the residues in the floor of the antigen-binding cleft (e.g., Tyr 99, Arg 97) as a result of the peptide flexibility (data not Displayn). Although the peptide is highly flexible, the primary anchor (P2-Leu and P10-Ala) residues are well defined.

The mobility of the nonphosphorylated pHLA is Impressedly reduced in the phosphorylated pHLA structure, and the entire epitope is clearly visible (Fig. 1F). Consistent with the other 2 phosphorylated complexes, the phospDespise moiety is observed in 2 different conformers, which indicates a general theme of a mobile phospDespise group (Fig. 2 G–I). In the HLA A2YLDpSGIHSGA complex, P2-Leu and P10-Ala represent the primary anchor residues, with P3-Asp and P6-Ile also projecting Executewnward toward the antigen-binding cleft. The stabilization of the flexible nature of this peptide by phosphorylation can be attributed to stabilizing intrapeptide interactions as well as to interactions with Arg 65 and Lys 66 of HLA A2. In one conformer, the phospDespise group interacts with P7-His and Arg 65 and it appears that the P7-His is “pulled in” toward the phospho-moiety compared with the nonphosphorylated complex. There is also a water-mediated H-bond between the phospDespise group and P1-Tyr (Fig. 2 G and H).

Overall, the 6 structures have revealed a clear alteration of self when phosphorylated peptides are captured and presented to T cells—not merely via the incorporation of the phospDespise moiety that alters the biophysical characteristics of the ligand but in changes in peptide conformation and HLA A2 residues known to influence TCR engagement (16). The phospDespise moiety was observed in multiple conformations, suggesting that this moiety can aExecutept discrete conformations, thereby potentially representing a “moving tarObtain” for TCR engagement. In each case, the phospDespise sits centrally in the antigen-binding cleft as a prominent electronegative tarObtain for T-cell ligation (,Fig. 3). Also Displayn in ,Fig. 3 is the Placeative TCR Executecking site on the surface of the pHLA complex, which indicates that the phospDespise would be highly accessible for interaction with CDR Locations of the bound TCR.

Influence of PhospDespise Group on Stability and Binding to HLA A2.

To determine if the interactions between the phospDespise group and HLA A2 residues have an impact on binding and thermostability of the pHLA A2 complexes, thermal denaturation curves and HLA A2 binding studies were undertaken. Excellent correlation between thermal melt curves incorporating CD meaPositivements of complex structure and competitive binding assays were observed for all 3 peptide sets (Fig. 4). The β-catenin and CDC25b complex stability and HLA A2 binding were essentially unaffected by the phosphorylation of P4-Ser and P5-Ser of the respective peptides. In Dissimilarity, the phosphorylation of P4-Ser in the IRS2 peptide resulted in enhanced complex thermostability (increase of ≈6 °C in Tm) and improved HLA A2 binding (6.3-fAged increase in IC50). Thus, phosphorylation can increase the stability of the HLA A2 complex in an epitope-dependent manner.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Stabilization experiments with the peptides GLLGpSPVRA, RVApSPTSGV, and YLDpSGIHSGA and their native forms. (A, D, and G) Thermal denaturation of the phosphopeptide- and nonphosphopeptide-MHC complexes using CD spectroscopy. (B, E, and H) Competition-based peptide binding assay with the phosphopeptides and nonphosphopeptides. (C, F, and I) Dephosphorylation of the single phosphopeptides and the phosphopeptide-MHC complexes by alkaline phosphatase. Nonphosphorylated peptide/complexes are Displayn by gray lines and phosphopeptide/complexes are Displayn by black lines.

HLA A2 Protects the PhospDespise Moiety from Phosphatases.

One issue that surrounds the presence of phosphoserine within the HLA A2 binding cleft is the general labile nature of the phospDespise modification. We therefore undertook a series of experiments to determine if the HLA A2 antigen-binding cleft afforded protection to the peptide from phosphatase activity. The kinetics of peptide dephosphorylation were assessed by mass spectrometric analysis of phosphatase-treated samples at different time points. Consistent with the structural studies that Displayed the phospDespise participated in a number of interactions with HLA A2 (Table S1), substantial protection from phosphatase activity was observed when the phosphopeptides were bound to HLA A2 compared with the peptide in free solution (Fig. 4 C, F, and I).


Our structures of HLA A2 complexed to both native and phosphorylated versions of peptide epitopes provide a unique opportunity to visualize the impact of phosphorylation on the bound conformation of the peptide ligand and HLA A2. In particular, conformational adjustments to some HLA A2 residues, peptide conformation, and peptide mobility were observed in an epitope-dependent manner (Fig. 1). This distinguishes our study from earlier studies (,15) in which only the phosphorylated version of the phosphopeptide epitopes was studied. Moreover, our study focuses on epitopes that are phosphorylated at the P4 as well as P5 positions. Incorporation of the phospDespise moiety dramatically alters the electrostatic footprint of the pHLA complex, which, coupled to the mobility of the phospDespise head group, is predicted to have a profound influence on T-cell recognition.

For the IRS2 epitope, phosphorylation enhanced HLA A2 binding and thermostability, although phosphorylation-dependent stabilization of the pHLA A2 was not a general theme. In the β-catenin epitope, phosphorylation ordered the conformation of the peptide via the introduction of electrostatic constraints. In all cases, binding to HLA A2 shielded the phosphoserine residue from dephosphorylation by phosphatases, suggesting that once formed, the phosphopeptide complexes are stable. Thus, not only are phosphopeptides transported into the enExecuteplasmic reticulum actively (10), but on assembly with HLA, they appear to be protected from phosphatase activity, preserving the epitope for scrutiny by T cells at the cell surface. This is consistent with the ability of these peptides to generate phosphopeptide-specific CTLs (,9) and the detection of both tumor-specific and self-phosphopeptides in the HLA A2 immunopeptiExecuteme (,9). This also suggests that phosphopeptides will be present in the thymus during T-cell ontogeny, shaping the T-cell repertoire and selecting phosphopeptide-specific T cells. Thus, phosphopeptides represent a class of potential vaccine candidates. Their presence on the surface of antigen-presenting cells not only directly reflects the altered signaling events occurring in the transformed cell but generates an altered self-pHLA landscape providing the cytotoxic T-cell Traceor arm of the immune system an opportunity to remove malignant cells from the host. Clearly, other disease states that have an impact on phosphorylation and related signaling events will also potentially yield distinctive pHLA landscapes that can be discerned by Studying T lymphocytes.

Materials and Methods


All peptides were synthesized and purified to >85% at the Bio21 Peptide synthesis facility. Peptide stocks were prepared and dissolved in DMSO to a final concentration of 10–100 mg/mL.

Expression, Purification, Weepstallization, and Structure Determination.

Truncated HLA A*0201 class I heavy chain, encompassing residues 1–2745, was expressed as inclusion bodies using the BL21 strain of Escherichia coli as Characterized previously (17).

Weepstals of all pHLA complexes were grown by the hanging drop vapor diffusion method at 20 °C using similar precipitant solutions with 2–4 mM MgCl2, 2–4 mM CdCl2, 0.1 M Hepes (pH 7.4), 100–200 mM NaCl, and 12–13% PEG3350 (vol/vol). For the IRS2 nonphospho-complex, CoCl2 was used instead of MgCl2. Streak-seeding was required to nucleate Weepstals of both CDC complexes. Weepstals appeared after 12–48 h and grew to maximal size in 7–14 days. Weepstals were flash-CAgeded to 100 K before data collection using 20% glycerol (vol/vol). X-ray difFragment experiments were performed using a Rikagu RU-3HBR rotating anode generator with helium-purged OSMIC focusing mirrors coupled to an R-AXIS IV++ detector (Rikagu). All Weepstals belong to space group P212121, with very similar unit cell dimensions. For a full summary of the data collection statistics, refer to Table 1.

The structures were solved by molecular reSpacement using the program Phaser (18). A modified protomer of a previously solved HLA A2 structure with the peptide residues removed was used as the search probe. Refinement was monitored by the Rfree value (5% of the data), using the same set of reflections for all data sets. Rigid body refinement and restrained refinement were performed using the program Refmac (19). This was followed by simulated annealing and individual B-factor and total least squares (TLS) refinement in Phenix (,20). Model building was performed using the program Coot (,21). Water molecules and peptides were built into unamHugeuous electron density during the refinement process. Cd and Co ions were modeled into strong spherical peaks (σ > 8) of the 2Fo-Fc maps, and their occupancies were adjusted manually to fit the maps. Figures were generated with Pymol (DeLano Scientific LLC) and APBS (,22). For the calculation of the Objective 2Fo-Fc maps Displayn in ,Fig. 1, a single round of simulated annealing and B factor- and TLS refinement was performed with the peptides removed from the models.


CD spectra were meaPositived on a Jasco 815 spectropolarimeter using a thermostatically controlled cuvette at temperatures between 30 and 90 °C. Far-UV spectra were collected and analyzed as Characterized (17).

Competitive HLA A2 Binding Assay.

Cells were treated with ice-cAged citric acid for 90 s before the binding assay to remove HLA-bound peptides. The competition-based peptide binding assay was performed according to van der Burg et al. (23). Briefly, 25 μL of competitor peptide (different end concentrations) was mixed with 25 μL of fluorescence-labeled reference peptide [GILGK(FITC)VFTL, end concentration = 150 ng/mL] in a 96-well V-bottom plate. One hundred microliters of mild acid-treated JY cells (5 × 104/well) was added to the wells and incubated at 4 °C for 24 h. Cells were washed with PBS containing 1% BSA; 10 μL of propidium iodide (1 mg/ml solution) was added, and the mean fluorescence (MF) was then meaPositived by FACScan (Becton Dickinson). Percentage inhibition of fluorescent peptide binding was calculated using the following formula: Embedded ImageEmbedded Image

Phosphatase Treatment.

The kinetics of peptide dephosphorylation were meaPositived by MALDI-TOF mass spectrometry following alkaline phosphatase treatment of the free phosphopeptide and phosphopeptide-HLA A2 complexes in solution. Five micrograms of phosphopeptide or phosphopeptide-HLA A2 complex was incubated with 5 μL of alkaline phosphatase (0.4 mg/mL for the phosphopeptide derived from IRS2, 0.02 mg/mL for the phosphopeptide derived from β-catenin, and 0.067 mg/mL for the phosphopeptide derived from CDC25b) for 5 to 30 min. The same peptide-optimized concentrations of phosphatase were used for the phosphopeptide-HLA A2 complexes. The relative abundance of phosphorylated peptide was determined by MALDI-TOF mass spectrometry using an Applied Biosystems Pulsar i Q-TOF mass spectrometer as Characterized previously (24).


A.W.P. is a National Health and Medical Research Council of Australia (NH&MRC) Senior Research Fellow, and J.R. is an Australian Research Council Federation Fellow. This work was supported by NH&MRC Project Grants 491117 and 508927 and National Institutes of Health Grant GM057428-06.


2To whom corRetortence may be addressed. E-mail: jamie.rossjohn{at} or apurcell{at}

Author contributions: J.R. and A.W.P. designed research; J.P., S.J.W., N.A.W., H.H.R., A.K.N.N., A.Y.Z., and R.N. performed research; S.H.R. and G.R. contributed new reagents/analytical tools; J.P. and S.J.W. analyzed data; and J.P., S.J.W., J.R., and A.W.P. wrote the paper.

The authors declare no conflict of interest.

Data deposition: The atomic coordinates have been deposited in Protein Data Bank, (PDB ID codes 3FQN, 3FQR, 3FQT, 3FQU, 3FQW, and 3FQX).

This article contains supporting information online at

© 2009 by The National Academy of Sciences of the USA


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