PO-408 New targets for the immunotherapy of adult B-cell acute lymphocytic leukaemia

Abstract

Results and discussions Between 6373 and 2243 peptides were identified for each cell line, but only peptides found in all replicates for each cell line analysed further. 1806 peptides were identified in all three replicates of the fibroblast cell line, while 455 and 379 peptides were common to all three replicates for the DFT1-IFNg and DFT2 cell lines respectively. Analysis of peptide length shows a preference for 8mers and 9mers in devil fibroblasts and DFT2 cells and a preference for 9mers in DFT1-IFNg. We next searched for binding motifs for the 8mers and 9mers across all cell lines and found potential anchor residues at position 3 and position 8/9, where there was a preference for hydrophobic amino acids (in particular Leucine). We then identified 61 and 55 peptides unique to DFT1-IFNg and DFT2 respectively. Conclusion This is the first study to characterise the repertoire of peptides bound to MHC molecules in contagious cancers and represents a pivotal step for understanding the immunological features of transmissible cancers. Introduction Cancer stem cells (CSCs) are more resistant to chemo-and radiation therapy than the bulk of the tumour. CSCs, surviving traditional therapies, possess self-renewal capacity and contribute to tumour re-growth. We are investigating the susceptibility of CSCs to antigen-specific killing by T cells. We generated a dataset incorporating all HLA Class I epitopes of the DU145 prostate cancer cell line. We aim to identify novel CSC epitopes which may serve as T cell targets. Material and methods We isolated HLA Class I:peptide complexes from DU145 cells by immunoaffinity precipitation and analysed the epitope sequences by liquid chromatography tandem-mass spectrometry (LC-MS/MS). We selected epitopes in the dataset which showed low gene expression in healthy tissues (www.GTexportal.org) and high predicted HLA-binding affinity (http://tools.immuneepitope.org/mhci/). DU145 CSCs were isolated based on aldehyde dehydrogenase (ALDH) activity using FACS. ALDH high and ALDH low DU145 cells were determined to have CSC or non-CSC characteristics, respectively, by in vivo tumour initiation. Gene expression of the epitopes was measured by qPCR in ALDH high and ALDH low DU145 cells. Protein expression was determined by immunofluorescence. We performed homology modelling of the HLA: peptide interfaces and quantified the binding interactions using PISA interface analysis software. We measured T cell activation by selected peptides analysing cytokine release by flow cytometry. Results and discussions We selected 42 epitopes from the DU145 dataset with low global tissue expression and predicted HLA binding percentile rank <1%. We identified 11 CSC epitopes by qPCR; 6 upregulated in ALDH high DU145 cells (e.g. TACSTD2) and 5 abundant in both ALDH high and ALDH low DU145 cells (e.g. XPO1). We identified 3 HLA:epitope complexes for which the PISA P-value indicated high affinity interface interactions. CD8 + T cells that release cytokines in response to one of these epitopes have been identified. Th...