OR55: DEEP LIGAND SEQUENCING REVEALS OVER 200 HLA-A*02:01 TOXOPLASMA GONDII LIGANDS

Abstract

Aim Advancements in mass spectrometry and nano-scale high pressure liquid chromatography have allowed for a more sensitive, rapid, and through characterization of the HLA ligandome. Coupled with an abundant and pure source of HLA, we set out to detect and identify ligands that are at or below the sensitivity of T-cell detection. The overall objective of this study was to apply cutting-edge proteomics techniques to dramatically improve our understanding of the peptide ligands that are presented by HLA of infected cells. Methods To identify HLA presented peptide ligands at high sensitivity, we utilized Deep Ligand Sequencing (DLS), an approach that funnels milligrams of HLA into a lingand pool that are analyzed in a two-dimensional LCMS system. Here, the characterized HLA was sHLA-A*02:01 harvested from T. gondii infected THP-1 cells. Peptide ligands were released from A*02:01 with an acid boil and separated into 40 fractions by RP-HLPC at pH10. Peptides in each of these 40 fractions were separated again using nano RP-HPLC at pH2, and the peptides were analyzed with an AB Sciex 5600 triple TOF mass spectrometer as they eluted from the nano column. Peptide sequences are determined using PEAKS and MASCOT at a 1% False Discovery Rate. All T. gondii sequences were validated with fragmentation of corresponding synthetic peptides. Results The DLS technique found that 201 novel T. gondii ligands from 97 proteins are presented by A*02:01. Of these ligands, 15% (31/201) came from the known antigenic dense granular proteins and 36 of the ligands were from 21 different hypothetical T. gondii proteins, confirming the expression of these hypothetical proteins. Strikingly, an additional 113 ligands share sequence between T. gondii and H. sapiens. Conclusions Cutting-edge mass spectrometers coupled to nano HPLC systems and high throughput bioinfromatic software has allowed us to survey the ligandome with an unprecedented depth. Here we applied these techniques to an intracellular pathogen and identified over 200 novel T. gondii ligands from a single HLA. These data will revolutionize T cell therapies for targeting infected cells. C. McMurtrey: Consultant; Company/Organization; Pure Protein LLC . W. Hildebrand: Scientific/Medical Advisor; Company/Organization; Pure Protein LLC.