Understanding the constitutive presentation of MHC class I immunopeptidomes in primary tissues

CD8+ T cells can eradicate abnormal cells through the recognition of small peptides presented by major histocompatibility complex (MHC, human leukocyte antigen (HLA) in humans) class I molecules. The large collection of peptides associated with the MHC is referred to as the immunopeptidome. The complexity of the immunopeptidome is due to the interindividual and intraindividual heterogeneity of the MHC-I immunopeptidome. In humans, up to six different HLA-I allotypes are expressed at the individual level, and at least thousands of different HLA-I allotypes are expressed across human populations. Moreover, recent studies have shown that the MHC-I immunopeptidome is tissue-dependent. However, to date immunopeptidome biogenesis studies have been almost exclusively conducted in transformed cells.

In this study, a system-level and cross-species analysis of the MHC-I immunopeptidome from 29 primary human tissues from 21 different subjects and 19 primary mouse tissues was conducted to understand how the MHC-I immunopeptidome is being shaped in different primary tissues in vivo. The results validated that HLA-I allotypes express unevenly across different tissues and individuals, considerably contributing to the overall heterogeneity of the human MHC-I immunopeptidome. Expectedly, immune-related organs (e.g. spleen) yielded high numbers of MHC-I numbers, while nonimmune related organs such as brain and testis yielded low numbers of MCH-I peptides, in both mouse and human. In addition, a significant proportion of MHC-I peptides were found to be  tissue-specific whereas a small proportion of peptides are shared across various immune and nonimmune organs, with highly abundant and strong MHC-I binding peptides being widely shared across tissues. Further correlative analysis between proteins and MHC-I peptides allowed for the identification of novel players of the antigen processing network. For instance, four poorly characterised carboxypeptidases showed significant correlations between protein abundance and number of MHC-I peptides across tissues. Collectively, this study provides insights into the biogenesis of the mammalian MHC-I immunopeptidome and further exploration of new proteolytic enzymes in antigen processing in vivo.

How was PEAKS used?

Raw data from the mouse and human immunopeptidome datasets were downloaded and re-analysed using PEAKS. For the mouse immunopeptidome data, a PEAKS project was generated for each pulldown experiment. A precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.01 Da were applied. Oxidation (M) and deamidation (NQ) were included as variable post translational modifications (PTM). Peptides identified with a false discovery rate (FDR) of less than 5% were exported and further assessed for binding to the MHC-I alleles H2Kb and H2Db. For the human immunopeptidome data, a precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.015 Da were applied. The following PTMs were used: oxidation (M), pyro-Glu from Q (N-term Q), and carbamidomethylation (C). Matched peptides were filtered to 10% FDR and peptides were predicted as a binder to the corresponding HLA alleles.

Kubiniok, Peter, et al. “Understanding the constitutive presentation of MHC class I immunopeptidomes in primary tissues.” Iscience 25.2 (2022): 103768. doi:10.1016/j.isci.2022.103768


Understanding the molecular principles that govern the composition of the MHC-I immunopeptidome across different primary tissues is fundamentally important to predict how T cells respond in different contexts in vivo. Here, we performed a global analysis of the MHC-I immunopeptidome from 29 to 19 primary human and mouse tissues, respectively. First, we observed that different HLA-A, HLA-B, and HLA-C allotypes do not contribute evenly to the global composition of the MHC-I immunopeptidome across multiple human tissues. Second, we found that tissue-specific and housekeeping MHC-I peptides share very distinct properties. Third, we discovered that proteins that are evolutionarily hyperconserved represent the primary source of the MHC-I immunopeptidome at the organism-wide scale. Fourth, we uncovered new components of the antigen processing and presentation network, including the carboxypeptidases CPE, CNDP1/2, and CPVL. Together, this study opens up new avenues toward a system-wide understanding of antigen presentation in vivo across mammalian species.