Citrullinated Epitopes Identified on Tumour MHC Class II by Peptide Elution Stimulate Both Regulatory and Th1 Responses and Require Careful Selection for Optimal Anti-Tumour Responses.

ABSTRACT

Background: Somatic mutations or post-translational modifications of proteins result in changes that enable immune recognition. One such post-translational modification is citrullination, the conversion of arginine residues to citrulline. Citrullinated peptides are presented on MHC class II (MHCII) via autophagy which is upregulated by cellular stresses such as tumourigenesis. Methods: Peptides were eluted from B16 melanoma expressing HLA-DP4 and analysed by mass spectrometry to profile the presented citrullinated repertoire. Initially, seven of the identified citrullinated peptides were used in combination to vaccinate HLA-DP4 transgenic mice. Immune responses were characterised from the combination and individual vaccines by ex vivo cytokine ELISpot assay and assessed for tumour therapy. Results: The combination vaccine induced only weak anti-tumour therapy in the B16cDP4 melanoma model. Immune phenotyping revealed a dominant IFNγ response to citrullinated matrix metalloproteinase-21 peptide (citMMP21) and an IL-10 response to cytochrome p450 peptide (citCp450). Exclusion of the IL-10 inducing citCp450 peptide from the combined vaccine failed to recover a strong anti-tumour response. Single peptide immunisation confirmed the IFNγ response from citMMP21 and the IL-10 response from citCp450 but also showed that citrullinated Glutamate receptor ionotropic (citGRI) peptide stimulated a low avidity IFNγ response. Interestingly, both citMMP21 and citGRI peptides individually, stimulated strong anti-tumour responses that were significantly better than the combined vaccine. In line with the citGRI T cell avidity, it required high dose immunisation to induce an anti-tumour response. This suggests that as the peptides within the combined vaccine had similar binding affinities to MHC-II the combination vaccine may have resulted in lower presentation of each epitope and weak anti-tumour immunity. Conclusion: We demonstrate that tumours present citrullinated peptides that can stimulate Th1 and regulatory responses and that competition likely exists between similar affinity peptides. Characterisation of responses from epitopes identified by peptide elution are necessary to optimise selection for tumour therapy.