PMID- 34321274 OWN - NLM STAT- MEDLINE DCOM- 20220111 LR - 20220111 IS - 2051-1426 (Electronic) IS - 2051-1426 (Linking) VI - 9 IP - 7 DP - 2021 Jul TI - Homocitrullination of lysine residues mediated by myeloid-derived suppressor cells in the tumor environment is a target for cancer immunotherapy. LID - 10.1136/jitc-2020-001910 [doi] LID - e001910 AB - BACKGROUND: Homocitrullination is the post-translational modification of lysine that is recognized by T cells. METHODS: This 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 gamma (IFNgamma)-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. RESULTS: Homocitrulline (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. CONCLUSION: We 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 IFNgamma, 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. CI - (c) Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. FAU - Cook, Katherine W AU - Cook KW AUID- ORCID: 0000-0002-7277-6700 AD - Scancell Ltd, Nottingham, UK. FAU - Xue, Wei AU - Xue W AD - Scancell Ltd, Nottingham, UK. FAU - Symonds, Peter AU - Symonds P AD - Scancell Ltd, Nottingham, UK. FAU - Daniels, Ian AU - Daniels I AD - Scancell Ltd, Nottingham, UK. FAU - Gijon, Mohamed AU - Gijon M AD - Scancell Ltd, Nottingham, UK. FAU - Boocock, David AU - Boocock D AD - School of Science and Technology, Nottingham Trent University, Nottingham, UK. FAU - Coveney, Clare AU - Coveney C AD - School of Science and Technology, Nottingham Trent University, Nottingham, UK. FAU - Miles, Amanda K AU - Miles AK AD - School of Science and Technology, Nottingham Trent University, Nottingham, UK. FAU - Shah, Sabaria AU - Shah S AD - Scancell Ltd, Nottingham, UK. FAU - Atabani, Suha AU - Atabani S AD - Biodiscovery Institute, University of Nottingham Faculty of Medicine and Health Sciences, Nottingham, UK. FAU - Choudhury, Ruhul H AU - Choudhury RH AUID- ORCID: 0000-0003-1472-9743 AD - Scancell Ltd, Nottingham, UK. FAU - Vaghela, Poonam AU - Vaghela P AD - Biodiscovery Institute, University of Nottingham Faculty of Medicine and Health Sciences, Nottingham, UK. FAU - Weston, Daisy AU - Weston D AD - Scancell Ltd, Nottingham, UK. FAU - Metheringham, Rachael L AU - Metheringham RL AD - Scancell Ltd, Nottingham, UK. FAU - Brentville, Victoria A AU - Brentville VA AUID- ORCID: 0000-0003-2000-5629 AD - Scancell Ltd, Nottingham, UK. FAU - Durrant, Lindy G AU - Durrant LG AD - Scancell Ltd, Nottingham, UK lindy.durrant@nottingham.ac.uk. AD - Biodiscovery Institute, University of Nottingham Faculty of Medicine and Health Sciences, Nottingham, UK. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PL - England TA - J Immunother Cancer JT - Journal for immunotherapy of cancer JID - 101620585 RN - 1190-49-4 (homocitrulline) RN - 29VT07BGDA (Citrulline) RN - K3Z4F929H6 (Lysine) SB - IM MH - Animals MH - Cell Line, Tumor MH - Citrulline/*analogs & derivatives/pharmacology/therapeutic use MH - Humans MH - Immunotherapy/*methods MH - Lysine/*metabolism MH - Mice MH - Myeloid-Derived Suppressor Cells/*immunology MH - Tumor Microenvironment PMC - PMC8320257 OTO - NOTNLM OT - CD4-positive T-lymphocytes OT - cellular OT - immunity OT - immunization OT - immunotherapy OT - vaccination COIS- Competing interests: KWC, WX, VAB and LGD have ownership interest in the patent. LGD is a director and shareholder in Scancell Ltd. All authors are employees of Scancell Ltd. except DB, CC and AKM. EDAT- 2021/07/30 06:00 MHDA- 2022/01/12 06:00 PMCR- 2021/07/28 CRDT- 2021/07/29 05:56 PHST- 2021/07/02 00:00 [accepted] PHST- 2021/07/29 05:56 [entrez] PHST- 2021/07/30 06:00 [pubmed] PHST- 2022/01/12 06:00 [medline] PHST- 2021/07/28 00:00 [pmc-release] AID - jitc-2020-001910 [pii] AID - 10.1136/jitc-2020-001910 [doi] PST - ppublish SO - J Immunother Cancer. 2021 Jul;9(7):e001910. doi: 10.1136/jitc-2020-001910.