Increased diversity of the HLA-B40 ligandome by the presentation of peptides phosphorylated at their main anchor residue

Abstract

Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I-associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with high-resolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy.

Fig. 1.

Analysis of the HLA-B*40:02-associated ligandome. A, length distribution of the ligands identified from the peptide pool bound to B*40:02. B, molecular weight distribution of the same set of peptides. The mean (± S.D.) molecular weight of the identified peptides is shown. C, Venn diagram showing the number of sequences identified in the flow-through (continuous line), the IMAC (dashed line), and the TiO₂ (dotted line) fractions.

Fig. 2.

Residue usage at P2 among B*40:02 ligands. The observed frequency (f_obs) of each amino acid residue at P2 is compared with the frequency of the same residue in the database (f_exp). Statistically significant differences (p < 0.05 after multiple testing correction) are indicated with gray bars.

Fig. 3.

Residue usage at the peptide C terminus among B*40:02 ligands. The observed frequency (f_obs) of each amino acid residue at PΩ is compared with the frequency of the same residue in the database (f_exp). Conventions are the same as in Fig. 2.

Fig. 4.

Fine mapping of the B*40:02 binding motif. Residues statistically overrepresented (p < 0.05 after multiple testing correction) at any peptide position are shown ordered downward according to their f_obs/f_exp ratio. Binding motifs were arbitrary classified as strong (f_obs/f_exp > 5) or weak (f_obs/f_exp < 5).

Fig. 5.

Sequence confirmation of phosphorylated ligands. The identity of each reported phosphopeptide was confirmed by fragmentation of the corresponding synthetic peptide. A, experimental MS2 spectrum of a molecular species at m/z = 523.74 fragmented during LC-MS analysis of the B*40:02-bound peptidome. B, MS2 spectrum of the synthetic peptide S[pS]YGNIRAV. C, the retention time of each phosphorylated peptide in the IMAC fraction (Rt - IMAC) is plotted against the retention time of the corresponding synthetic peptide (Rt - Syn.) in an independent LC-MS run. The data fit a linear function (R² = 0.9991).

Fig. 6.

Competitive binding assay of peptides with phosphoserine at P2 to HLA-B*40:02. C1R-B*40:02 cells were acid stripped to denature surface HLA class I complexes. After extensive washing, β2m, the reference peptide GEFGGXGSV (X = fluoresceine-labeled cysteine), and different concentrations of the indicated test peptides were added. HLA class I complexes were left to reassociate overnight at 4 °C. A, binding of the reference peptide to C1R (blue line) and C1R-B40 (red line). The percentage of the maximum fluorescence is plotted against the peptide concentration. The mean ± S.D. of three independent experiments is shown. B, the inhibition of the binding of the reference peptide is plotted against the concentration of the test peptides (colored dots). The mean of three independent experiments is shown. Experimental data were fitted to sigmoid curves (colored lines) to allow the estimation of the IC₅₀ values. C, estimated IC₅₀ values of the test peptides.