Homocitrullination of lysine residues mediated by myeloid-derived suppressor cells in the tumor environment is a target for cancer immunotherapy

Abstract

BackgroundHomocitrullination is the post-translational modification of lysine that is recognized by T cells.MethodsThis study identified homocitrullinated peptides from aldolase, enolase, cytokeratin and binding immunoglobulin protein and used human leukocyte antigen (HLA) transgenic mice to assess immunogenicity by enzyme-linked immunosorbent spot assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma expressing constitutive or interferon γ (IFNγ)-inducible major histocompatibility complex class II (MHC-II) as represented by most human tumors. To determine the mechanism behind the therapy, immune cell infiltrates were analyzed using flow cytometry and therapy studies in the presence of myeloperoxidase (MPO) inhibitor and T-cell depletion performed. We assessed the T-cell repertoire to homocitrullinated peptides in patients with cancer and healthy donors using flow cytometry.ResultsHomocitrulline (Hcit) peptide vaccination stimulated strong CD4 T-cell responses and induced significant antitumor therapy in an established tumor model. The antitumor response was dependent on CD4 T cells and the effect was driven mainly via direct tumor recognition, as responses were only observed if the tumors were induced to express MHC-II. In vitro proliferation assays show that healthy donors and patients with cancer have an oligoclonal CD4 T-cell repertoire recognizing homocitrullinated peptides. Inhibition of cyanate generation, which mediates homocitrullination, by MPO inhibition reduced tumor therapy by the vaccine induced T cells (p=0.0018). Analysis of the tumor microenvironment (TME) suggested that myeloid-derived suppressor cells (MDSCs) were a potential source of MPO. The selected B16 melanoma model showed MDSC infiltration and was appropriate to see if the Hcit vaccine could overcome the immunosuppression associated with MDSCs. The vaccine was very effective (90% survival) as the induced CD4 T cells directly targeted the homocitrullinated tumor and likely reversed the immunosuppressive environment.ConclusionWe propose that MPO, potentially produced by MDSCs, catalyzes the buildup of cyanate in the TME which diffuses into tumor cells causing homocitrullination of cytoplasmic proteins which are degraded and, in the presence of IFNγ, presented by MHC-II for direct CD4 T-cell recognition. Homocitrullinated proteins are a new target for cancer vaccines and may be particularly effective against tumors containing high levels of MPO expressing MDSCs.

Vaccine Can Induce CD4-Mediated Responses to Homocitrullinated Peptides via Multiple HLA-Types and Confer Anti-Tumor Immunity

“…Mouse MHC II from Balb/c and C57Bl/6 strains can mediate homocitrulline specific responses (19,27), while for human HLA alleles, responses to the synthetic JED homocitrullinated peptide can be induced in transgenic HLA-DR4 mice (27). Furthermore, we have previously shown responses to several homocitrullinated peptides in transgenic HLA-HHDII/DP4 mice (22).…”

“…We have previously shown that homocitrullinated peptides can stimulate responses restricted through the HLA-DP4 allele (22). In this study we first aimed to determine whether peptide immunization can induce homocitrulline-specific responses via HLA types other than the HLA-DP4 allele.…”

“…In inflammatory conditions cyanate accumulation is driven by the myeloperoxidase (MPO) enzyme which is released by neutrophils (21). In addition to neutrophils, we have previously demonstrated a role for MPOproducing tumor-associated myeloid derived suppressor cells (MDSCs) in MPO production and ultimately in causing homocitrullination within the tumor microenvironment (22).…”

A Novel Pyroptosis-Related Signature for Predicting Prognosis and Indicating Immune Microenvironment Features in Osteosarcoma

“…The enrichment analysis results showed that the DEGs between high and low PRS-score subgroups were mainly enriched in interferon-gamma mediated signaling pathways, antigen processing, and peptide antigens presented via MHC class II, peptide binding. The MHC-II is the critical component of adaptive anti-tumor immunity, and its upregulation is closely associated with increased levels of interferon-gamma in tumors (Dubrot et al, 2014;Cook et al, 2021). During inflammation, epithelial cells could act as accessory antigen-presenting cells along with the expression of MHC-II (Ghasemi et al, 2020).…”

The analysis of the pyroptosis-related genes and hub gene TP63 ceRNA axis in osteosarcoma

“…These cells can inhibit tumor progression by recognizing and killing tumor cells. Thus, immunotherapy has emerged as an effective therapeutic approach to killing tumor cells by activating immune responses ( 12 , 13 ). However, as compared to other cancers, there have been fewer investigations into immunotherapy for OS.…”