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.
Highlights
Peptides presented by human leukocyte antigen (HLA)1 molecules on the cell surface play a crucial role in immunol
The Peptides Constituting the HLA Ligandome—The pool of HLA peptides eluted from two Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (EBV-B-LCL), B-LCL-JYpp65 and B-LCLHHC, is quite complex
Three first dimension separations were chosen, reverse-phase C18 (RP-C18) chromatography, strong cation exchange chromatography (SCX), and peptide isoelectric focusing (IEF); these are based on a different separation mechanism and are the most commonly used
Summary
Sample Preparation—The Epstein-Barr virus (EBV)–transformed B lymphoblastic cell lines B-LCL-HHC (typing: HLA-A*0201, B*0702, B*4402, Cw*0501, and Cw*0702) and B-LCL-JY pp (typing: HLAA*0201, B*0702, and Cw*0702) were used as sources of HLA-class I molecules. The eluted peptides from B-LCL-HHC cell lines were divided into two equal portions, freeze dried, and dissolved in 95/3/ 0.1 water/ACN/FA, v/v/v. The eluted peptides from B-LCL-JYpp cell lines were divided into three equal portions, freeze dried, and dissolved in 95/3/0.1 water/ACN/FA, v/v/v. One portion of the eluted peptides from B-LCL-JY pp cell lines was fractionated on a homemade RP Reprosil-Pur C18-AQ column (200 m inner diameter, 3 m ϫ 15 cm) (Dr Maisch, GmbH, Ammerbuch, Germany). The samples were taken up in a make-up flow of 50/50/0.1 water/ACN/FA at 100 l/min supplied via a T-piece through the annular space between the separation capillary and an auxillary capillary In this way, 45 fractions, each half a minute wide, were collected; these were subsequently freeze dried and dissolved in solvent A for analysis via nano-LC-MS/MS. Couplings of these building blocks were identical to normal couplings, with one exception: a 3-fold excess of N-methylmorpholine was used instead of the routinely applied 2-fold excess
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