Discovery of T Cell Epitopes Implementing HLA-Peptidomics into a Reverse Immunology Approach

Abstract

T cell recognition of minor histocompatibility Ags (MiHA) plays an important role in the graft-versus-tumor effect of allogeneic stem cell transplantation. Selective infusion of T cells reactive for hematopoiesis-restricted MiHA presented in the context of HLA class I or II molecules may help to separate the graft-versus-tumor effects from graft-versus-host disease effects after allogeneic stem cell transplantation. Over the years, increasing numbers of MiHA have been identified by forward immunology approaches, and the relevance of these MiHA has been illustrated by correlation with clinical outcome. As the tissue distribution of MiHA affects the clinical outcome of T cell responses against these Ags, it would be beneficial to identify additional predefined MiHA that are exclusively expressed on hematopoietic cells. Therefore, several reverse immunology approaches have been explored for the prediction of MiHA. Thus far, these approaches frequently resulted in the identification of T cells directed against epitopes that are not naturally processed and presented. In this study we established a method for the identification of biologically relevant MiHA, implementing mass spectrometry–based HLA-peptidomics into a reverse immunology approach. For this purpose, HLA class I binding peptides were eluted from transformed B cells, analyzed by mass spectrometry, and matched with a database dedicated to identifying polymorphic peptides. This process resulted in a set of 40 MiHA candidates that were evaluated in multiple selection steps. The identification of LB-NISCH-1A demonstrated the technical feasibility of our approach. On the basis of these results, we present an approach that can be of value for the efficient identification of MiHA or other T cell epitopes.

Analysis of Major Histocompatibility Complex (MHC) Immunopeptidomes Using Mass Spectrometry*

“…By integrating exome sequencing, MHC peptide ligand prediction algorithms and targeted MS, Gubin et al recently identified tumor-specific mutant MHC class I peptides as targets of this form of immunotherapy (88). Proteogenomics approaches (139, 140) using DDA MS were also recently used for the identification of mutant as well as polymorphic MHC class I peptides (5,51,(141)(142)(143)(144). Notably, the detection of tumor-specific mutant MHC peptides has not been achieved yet using DIA MS.…”

Therapeutic targeting of the BCR-associated protein CD79b in a TCR-based approach is hampered by aberrant expression of CD79b

“…45 PBMCs were incubated with PE-labeled pMHC tetramers for 1 hour at 4°C. Cells were washed twice and incubated with anti-PE magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany).…”

Identification of minor histocompatibility antigens based on the 1000 Genomes Project

“…Ultimately, this improved knowledge of mHags may lead to superior identification strategies through the design of algorithms directed towards the computational prediction of true immunotherapeutic mHags. 11,32,33 In conclusion, the new approach integrating all variations of the 1000 Genomes Project currently represents the most attractive method for highly efficient mHag identification. This methodology will conceivably facilitate the identification of the broad set of mHags that are not only necessary for the optimal setup of mHag-based immunotherapy trials, but also for a better understanding of mHag immunobiology.…”

“…The genetic bases of these polymorphisms are either single nucleotide polymorphisms (SNPs), base-pair insertions or deletions (indels) or copy number variations (CNVs). 11 In the mid-1990s, the discovery that a specific set of mHags is solely expressed on malignant and non-malignant hematopoietic cells, shifted the paradigm on mHags and suggested that hematopoietic-restricted mHags could be ideal targets for separating GvT from GvHD. 1,12,13 Nonetheless, a thorough clinical evaluation of this attractive concept has not yet been realized, because the small number of genuinely hematopoietic system-specific mHags identified so far permits the inclusion of not even 20% of the transplant patients in the slow-running clinical trials.…”