Vaghela, Poonam
(2022)
T cell recognition of stress-induced post-translational modifications.
PhD thesis, University of Nottingham.
Abstract
Background: Citrullination is the post-translational modification of arginine to citrulline mediated by peptidylarginine deiminases (PADs). We have previously shown that vaccination with citrullinated peptides from vimentin and α-enolase which are presented on HLA-DR4 or -DP4 in tumour cells mediate strong anti-tumour responses [1, 2]. This response is dependent upon autophagy and presentation of citrullinated peptides on MHC-II. Cytotoxic CD4 T cells can directly kill tumours expressing MHC-II and cognate peptide. As most tumours do not express MHC-II unless induced to do so by IFNγ we propose that citrullination is a normal stress response but that citrullinated epitopes are only presented on viable cells in the presence of inflammation.
Results: Exposure to nutrient starvation induced citrullinated vimentin expression in the murine melanoma cell line B16F1 grown in vitro, measured using the anti-Citrullinated vimentin rabbit polyclonal anti-serum GB17002 (ECV-1), but was also associated with apoptosis. Interestingly, up to 70% of low density plated cells expressed citrullinated vimentin which was mostly cytoplasmic with some cases showing punctate staining, increased phagosome associated LC3-II and increased proliferation marker Ki67 compared to high density cultures, suggesting actively growing cells undergo more autophagy and have greater potential to express citrullinated epitopes. The role the HIPPO pathway and Epithelial-to-Mesenchymal transition (EMT) plays in citrullination was investigated however results were inconclusive and require further work. Approximately 27-55% of human cancers were positive for MHC-II. The expression of MHC-II was associated with survival advantage in breast cancer patients (P=0.004), and positively correlated with citrullinated vimentin (P<0.001), PAD2 (P<0.001), IFNγ receptor (P=0.003) and Ambra1 (P=0.016) in ovarian cancer. In a panel of triple negative breast cancer (TNBC) and ovarian cancer cell lines, 4/8 lines showed expression of MHC-II. This was enhanced following exposure to IFNγ which was dependent on activation of the Class II transactivator (CIITA) and is controlled by the pIV promoter. In addition, a strong correlation between IRF-1 and CIITA mRNA expression was found in 10 cancer types analysed from the TCGA database and in 4 cancer types CIITA expression alone correlated with good prognosis. Interestingly, IFNγ also increased citrullinated vimentin expression in the B16F1, suggesting this cytokine upregulates citrullination and MHC-II. Analysis of the human CD4 T cell repertoire responding to the citrullinated peptides, Vim28-49cit, Vim415-433cit and Eno241-260cit, showed oligoclonal proliferation of mainly T effector memory (Tem) cells but also an increase in the cytotoxic CD45RA expressing Tem population (TEMRA). In total, 28 high frequency TCRs were identified by RNA sequencing of proliferating CD4 T cells in response to Vim28cit (16), Eno241cit (4) and Vim415cit (8). These were transduced using lentiviral technology into Jurkat 76 and/or T cells and tested in antigen presentation assays to confirm their reactivity to citrullinated peptides by upregulation of the activation marker CD69 and the potent T cell stimulating cytokine, IFNγ. Despite numerous improvements to the lentiviral constructs, transfection/transduction methods and recognition assays, no positive responses were detected with any citrullinated peptide transduced TCRs. This suggests that the in vitro assays to detect citrulline specific T cell responses did not sufficiently expand the antigen specific T cells or that the TCR that did expand were of too low affinity for in vitro peptide recognition. Repeat stimulation assays to select for higher affinity more specific TCRs are required.
Summary: Lack of cell-to-cell contact induced the greatest levels of citrullination in viable cells which were also highly proliferative and had upregulated autophagy. For stress induced post-translational modifications (siPTMs) to be recognised by T cells, MHC-II expression is also required. This data shows MHC-II is present on human tumour cells at low levels but can be further induced by the inflammatory cytokine IFNγ and is dependent on CIITA expression. Citrullinated peptides presented by MHC-II stimulated oligoclonal proliferation of CD4 T cells that expressed an effector phenotype. IFNγ released from these cells can induce MHC-II expression on tumours thus allowing presentation of siPTM epitopes. Citrullinated peptide vaccines have shown that siPTMs are good targets for immunotherapy but further results are required to determine if specific TCRs can be identified and used for adoptive T cell therapy.
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