Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• RNA splicing

• neojunctions

•  neoantigens

• T cell receptors

Main Findings

Neoantigens, peptides uniquely expressed by cancer cells, are attractive targets for endogenous antitumor T cell responses and immunotherapeutic interventions. However, a lack of shared (public) neoantigens across individuals, low antigen expression, and intratumoral heterogeneity of neoantigen-encoding mutations makes it difficult to design neoantigen-directed immunotherapeutic strategies. Kwok et al. identify cancer-specific RNA splicing events (neojunctions) encoding highly clonal, shared neoantigens by analyzing RNA sequencing data from The Cancer Genome Atlas, their own repository of spatially mapped, multi-site brain cancer specimens, and normal tissue from the Genotype-Tissue Expression (GTEx) project. Transcript and peptide expression were further validated by bulk RNA sequencing and total mass spectrometry analysis of glioma cell lines. Major histocompatibility (MHC) class I restriction of neojunction-derived peptides was inferred using peptide processing and presentation prediction algorithms (MHCflurry and HLAthena). Finally, immunopeptidomic analysis of MHC-I ligands from HLA-null cells transfected with mRNAs encoding neojuntion transcripts and HLA-A*02:01, confirmed endogenous neojunction-derived peptide processing and presentation.

Multiple HLA-A*02:01-restricted peptides were synthesized for in vitrosensitization of naïve CD8 T cells by pulsing monocyte-derived dendritic cells prior to measuring interferon gamma secretion by enzyme linked immunosorbent assay to confirm peptide immunogenicity. These sensitization cultures also enabled the identification of neojunction-reactive T cell receptors (TCRs) by selecting clonotypes expressing T cell activation gene signatures (IFNG, GZMB, andTNFA) from single-cell RNA/TCR sequencing. TCR specificity was confirmed measuring triple-reporter activation of TCR-transduced Jurkat76 cells as well as expression of CD107a/CD137 on the surface or IFN-gamma/TNF-alpha by ELISA in peripheral blood mononuclear cell(PBMC)-derived CD8 T cells upon co-culture with peptide-pulsed T2 cells. Tumor cell killing was also assessed by co-culture of TCR transduced PBMCs with glioma cell lines. Finally, differential gene expression analysis combined with gene set enrichment revealed existence of disease subtype-specific RNA-splicing regulatory pathways in tumors causing discrepancies in neojunction expression (validated by small-interfering RNA knockdown experiments).

Limitations

• The breadth and potency of T cell responses targeting neojunction-derived peptides presented on MHC class II

• Confirmation that neojunction-specific T cell receptors/clones are able to infiltrate patient tumors

• In vivo validation of tumor control by neojunction-specific T cells

• Experimental validation of tumor specificity (e.g. custom PCR probes for neoantigen targets in normal tissue)

• Using whole-exome and bulk RNA sequencing from the same tumor specimens, in order to compare clonality, expression, and sharedness of DNA mutation-derived to neojunction-derived neoantigens

Significance/Novelty

Though this is not the first study to identify RNA splicing events encoding neoantigen peptides in cancer, this study is the first to extensively characterize the sharedness and clonality of neojunction-derived peptides across tumors. This study also comprehensively validates the immunogenicity of MHC-I-restricted neojunction-derived peptides and the ability for T cells transfected with neojunction-specific TCRs to recognize and kill tumor cells in vitro. Therefore, the findings in this study provide the framework for future investigation of the immunotherapeutic targeting of public neojunctions in cancers using “off-the-shelf” vaccines and adoptive cell transfer therapy.

Credit

Reviewed by Matthew Brown as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.

The author declares no conflict of interests in relation to their involvement in the review.