Complex cellular interactions between the immune system and cancer can impact tumour development, growth, and progression. T cells play a key role in these interactions, however, the challenge for T cells is to recognise tumour antigens whilst minimising cross reactivity with antigens associated with healthy tissue. Some tumour cells including those associated with viral infections, have clear, tumour-specific antigens that can be targeted by T cells. High mutational burden can lead to increased numbers of mutational neoantigens that allow very specific immune responses to be generated but also allow escape variants to develop. Other cancer indications and those with low mutational burden are less easily distinguished from normal tissue. Recent studies have suggested that cancer-associated alterations in tumour cell biology including changes in post translational modification (PTM) patterns may also lead to novel antigens that can be directly recognised by T cells. The PTM derived antigens provide tumour specific T cell responses that both escape central tolerance and avoid the necessity for individualised therapies. PTM specific CD4 T cell responses have shown tumour therapy in murine models and highlight the importance of CD4 T cells as well as CD8 T cells in reversing the immunosuppressive tumour microenvironment. Understanding which cancer-specific antigens can be recognised by T cells and the way that immune tolerance and the tumour microenvironment shapes immune responses to cancer is vital for future development of cancer therapies.

Citrullination and homocitrullination are stress induced post‐translational modifications (siPTMs) which can be recognized by T cells. Peripheral blood mononuclear cells isolated from healthy donors and rheumatoid arthritis (RA) patients were stimulated with nine siPTM‐peptides. CD45RA/CD45RO depletion was employed to determine if peptide‐specific responses are naïve or memory. Human leucocyte antigen (HLA)‐DP4 and HLA‐DR4 transgenic mice were immunized with siPTM‐peptides and immune responses were determined with ex vivo ELISpot assays. The majority (24 out of 25) of healthy donors showed CD4 T cell‐specific proliferation to at least 1 siPTM‐peptide, 19 to 2 siPTM‐peptides, 14 to 3 siPTM‐peptides, 9 to 4 siPTM‐peptides, 6 to 5 siPTM‐peptides and 4 to 6 siPTM‐peptides. More donors responded to Vim28‐49cit (68%) and Bip189‐208cit (75%) compared with Vim415‐433cit (33%). In RA patients, the presentation of citrullinated epitopes is associated with HLA‐SE alleles; however, we witnessed responses in healthy donors who did not express the SE allele. The majority of responding T cells were effector memory cells with a Th1/cytotoxic phenotype. Responses to Vim28‐49cit and Eno241‐260cit originated in the memory pool, while the response to Vim415‐433cit was naïve. In the HLA‐DP4 and HLA‐DR4 transgenic models, Vim28cit generated a memory response. Peptide‐specific T cells were capable of Epstein–Barr virus transformed lymphoblastoid cell line recognition suggesting a link with stress due to infection. These results suggest siPTM‐peptides are presented under conditions of cellular stress and inflammation and drive cytotoxic CD4 T cell responses that aid in the removal of stressed cells. The presentation of such siPTM‐peptides is not restricted to HLA‐SE in both humans and animal models.

IntroductionPost translational modification of proteins plays a significant role in immune recognition. In particular the modification of arginine to citrulline which is mediated by PAD enzymes is increased during cellular stress (autophagy) which permits the presentation of modified epitopes upon MHC class II molecules for recognition by CD4 T cells. Citrullination also occurs in tumour cells as a result of continuous environmental stresses and increased autophagy. We have shown in animal models the efficient stimulation of citrullinated epitope specific CD4 T cells resulting in dramatic elimination/regression of tumours. The ER chaperone glucose-regulated protein 78 (GRP78) is known to also be required for stress-induced autophagy and is directly linked to autophagosome formation. GRP78 is known to be highly expressed by many tumour types. In this study we investigate the potential of targeting citrullinated GRP78 for cancer therapy.MethodsA citrullinated GRP78 specific antibody was used to assess citrullinated GRP78 expression in murine and human tumour cells by flow cytometry. Five peptides were selected and used to vaccinate HLA transgenic mice and immune responses were characterised by ex vivo cytokine ELISpot assay. T cell repertoire in humans was assessed through proliferation assays and cytokine ELISpot assay. Citrullinated peptide was identified in murine B16 melanoma by mass spectrometry and the peptide vaccine was assessed for tumour therapy in a mouse melanoma model.ResultsWe show the identification CD4 T cell responses to one citrullinated GRP78 epitope that are restricted through HLA DP*0401 and HLA-DR*0101 alleles. This peptide is detected by mass spectrometry in B16 melanoma grown in vivo and citrulline specific CD4 responses to two peptides spanning this epitope mediate efficient therapy of established B16 melanoma tumours in HHDII/DP4 (p<0.0001) transgenic mouse model. Finally, we demonstrate the existence of a repertoire of responses to the citrullinated GRP78 peptide in healthy individuals (p=0.0023) with 13/17 (76%) individuals showing a response to this peptide.ConclusionWe propose that citrullinated GRP78 is a candidate tumour antigen and vaccination against citrullinated GRP78 may provide a promising tumour therapy approach.