Integration of affinity and abundance parameters for the identification of novel CD8+ T cell specificities in the CT26 tumor model

Abstract

The CT26 model is one of the most widely tested systems for cancer immunotherapy. Over 70% of the CD8+ CTL responses in CT26 tumors are directed against two peptides: SPSYVYHQF (AH1) presented by H-2Ld, or GGPESFYCASW (GSW11) presented by H-2Dd. Both peptides derive from the ecotropic murine leukaemia virus gp70 envelope glycoprotein, which is the highest expressed gene in CT26, and have relative low affinity values for their respective MHC-I. However, there is a lack of knowledge about other CTL specificities in CT26, the relationship between abundance and affinity of such peptides, and their roles in tumor rejection. We identified 96 potential epitopes restricted by MHC-I molecules in BALB/c mice based on previously published immuno-transcriptomic data. We used a mathematical algorithm integrating the peptides relative affinity to MHC-I measured by NetMHC4.0, and their abundance in the proteome, which resulted in a prediction of the likelihood of peptide presentation. Screening was performed in splenocytes from Treg-depleted CT26-challenged mice via IFNγ intracellular cytokine staining and T cell proliferation assays. Our approach led to the identification of a novel CTL specificity restricted by H-2Kd (Kd34), which shows moderate IFNγ responses in splenocytes from mice responding to the therapy. Moreover, using Kd34-specific dextramers, we were able to detect a small population of tumor-infiltrating lymphocytes (TILs) specific for this peptide, with similar phenotypic traits to AH1- and GSW1-specific TILs. The identification of such novel CD8+ T cell specificities will improve the understanding about the relationship between immunodominance and epitope abundance/affinity to better tailor vaccine strategies in cancer.