The human leukocyte antigen-presented ligandome of B lymphocytes

Abstract

Peptides presented by human leukocyte antigen (HLA) molecules on the cell surface play a crucial role in adaptive immunology, mediating the communication between T cells and antigen presenting cells. Knowledge of these peptides is of pivotal importance in fundamental studies of T cell action and in cellular immunotherapy and transplantation. In this paper we present the in-depth identification and relative quantification of 14,500 peptide ligands constituting the HLA ligandome of B cells. This large number of identified ligands provides general insight into the presented peptide repertoire and antigen presentation. Our uniquely large set of HLA ligands allowed us to characterize in detail the peptides constituting the ligandome in terms of relative abundance, peptide length distribution, physicochemical properties, binding affinity to the HLA molecule, and presence of post-translational modifications. The presented B-lymphocyte ligandome is shown to be a rich source of information by the presence of minor histocompatibility antigens, virus-derived epitopes, and post-translationally modified HLA ligands, and it can be a good starting point for solving a wealth of specific immunological questions. These HLA ligands can form the basis for reversed immunology approaches to identify T cell epitopes based not on in silico predictions but on the bona fide eluted HLA ligandome.

Fig. 1.

Venn diagrams of identified HLA ligands per separation. The number of identified unique peptides per mass spectrometric replicate for IEF (A), SCX (C) and RP-C18 (E) for cell line B-LCL JYpp65, and for IEF (B) and SCX (D) for cell line B-LCL HHC. The area of the circles corresponds with the number of peptides. Total, fractional, and overlapping numbers of unique peptides are indicated.

Fig. 2.

Venn diagrams of cumulative number of identified HLA ligands. Cumulative number of identified unique peptides per cell line for B-LCL HHC (A) and cell line B-LCL JYpp65 (B). C, cumulative number of identified unique peptides for B-LCLs JYpp65 and HHC. The area of the circles corresponds with the number of peptides. Total, fractional, and overlapping numbers of unique peptides are indicated.

Fig. 3.

Shotgun nature of the experiment. The weighted intensity distribution of the peptides found in the various regions in the Venn diagrams of Fig. 2B. Three regions were defined (x-axis): (1) peptides found with only a unique first dimension separation technique (i.e. the “outer” regions), (2) peptides found in either of two overlapping regions, and (3) peptides always found irrespective of the first dimension separation technique. Average peptide intensity increases from region 1 to region 3, as expected in a shotgun-type of proteomics experiment. The median peptide intensity is indicated.

Fig. 4.

Peptide length distribution of the HLA ligandome. Of note are the longer peptides and the relatively high number of HLA-B7 binding peptides of 8-mer length, and the relatively wide spread of HLA-B44 binding peptides over 9 to 11 amino acids length. Number of 13-mers ∼ 80 per allele; number of 14-mers ∼ 20 per allele.

Fig. 5.

Binding motif of various HLA-A2 length variants. Icelogo plots of HLA-A2 binding peptides of 9-mer (n = 3302), 12-mer (n = 137), and 14-mer (n = 17) length. Peptides of different lengths clearly display similar binding motifs.

Fig. 6.

Occupation of the ligandome. The intensity distribution of peptides constituting the ligandome is shown for B-LCL JYpp65. A, the outer rim of the pie chart displays the percentage occupation of the HLA ligandome, and the number within each pie segment is the number of unique peptides in that segment. Of the total number of 6500 identified peptides, the top five abundant proteins occupy 6% of all HLA molecules, the top 50 abundant peptides occupy 27% of all HLA molecules, etc. Note that the lowest abundant 5500 peptides occupy only 17% of all HLA molecules. B, the numbers in panel A have been converted to copy numbers (i.e. the number of ligands per cell). The outer rim of the pie chart displays the number of unique peptides, and the number within each pie segment is the average copy number for the peptides in that segment. The top five abundant peptides are on average present with 16,500 copies on the cell surface, peptides 5–50 are on average present with 5700 copies, etc. The average copy number of the lower abundant peptides is 43; the very low abundant peptides are present at copy numbers close to 1 or even less.

Fig. 7.

Representation of proteins in the HLA ligandome. A, this figure depicts the number of proteins as a function of the number of peptides identified per protein. HLA ligands derived from 7059 unique proteins are in our dataset. In our dataset, about 50% of the proteins are represented by one peptide, 20% are represented by two peptides, 10% are represented by three peptides, and so on. As can be seen, there is a gradual decrease in the number of peptides by which a protein is represented. Up to 41 peptides/protein are present in our dataset. B, the protein length is depicted as a function of the number of peptides by which the protein is represented in HLA. The number of peptides in HLA of a protein correlates well with its protein mass.

Fig. 8.

HLA ligandome physicochemical properties. A, distribution of HLA peptides over the pI range as a function of the first dimension separation technique used. As can be seen, IEF, SCX, and RP-C18 do not discriminate among peptides based on their theoretical pI. The only exception is that peptides with low pI values are clearly underrepresented in the IEF process. This is caused by the actual pI in the pI 3–10 strips, which in fact appeared to be pI 4–10, as also independently checked with a pH indicator after IEF. B, distribution of HLA peptides over the hydrophobicity range depending on the first dimension separation technique used. As can be seen, IEF, SCX, and RP-C18 do not discriminate among peptides based on their hydrophobicity. The plots illustrate that our HLA ligandome is a good representation of the real HLA ligandome.

Fig. 9.

Allele-specific distribution of HLA peptides over the hydrophobicity range. These plots show a clear average hydrophobicity shift going from HLA-B44 (most hydrophilic, median GRAVY index of −0.84) to HLA-B7 (median GRAVY index of −0.08) to HLA-A2 (median GRAVY index of 0.64). The positions of the three distributions are as expected and illustrate the overall quality of our dataset.