Fig 2: siRNA targeting PRKACA transcript decreases expression of both transcript variants of LINC00473 in A9 clone.(a) siRNA directed at PRKACA effectively decreases both PRKACA and DP protein levels following 48 and 72 hours of treatment. Full-length blots are presented in S1 Fig. (b-c) Treatment with PRKACA-siRNA demonstrates statistically significant decrease in LINC00473 TV1 and TV2 transcript levels at 48 and 72 hours. (*-p<0.005; **-p<0.05).

Fig 3: Mass spectrometry-based identification of naturally presented DNAJB1-PRKACA-derived HLA class I and HLA class II ligands.a Schematic overview of Doxycycline (Dox)-inducible DNAJB1-PRKACA fusion gene expression. Hepatocellular carcinoma (HCC) cell lines transduced with DNAJB1-PRKACA Dox-inducible or control plasmid were treated with Dox followed by mass spectrometry (MS)-based immunopeptidome analysis of the HCC cell line, or of mature monocyte-derived dendritic cells (moDC) of a healthy volunteer (HV) incubated with HCC cell line lysate (created with BioRender.com). b Dox-induced DNAJB1-PRKACA expression by immunoblotting of HCC cell lines (HLE, SMMC-7721, and HepG2) carrying the DNAJB1-PRKACA Dox-inducible (+) or the control plasmid (-) with and without Dox treatment using an anti-Protein Kinase A (PKA) antibody (n = 3). The black arrow indicates the endogenous PKA band. GAPDH served as a loading control. c, f MS-identified peptides of c HCC cell lines (HLE n = 1, SMMC-7721 n = 3, and HepG2 n = 3) carrying the DNAJB1-PRKACA Dox-inducible or the control plasmid after Dox-treatment (data are presented as mean values ± SD) and f of mature moDCs of HVs incubated with HCC cell line lysate with or without expression of DNAJB1-PRKACA protein, respectively. d, g Distribution of MS-identified HLA ligands over the DNAJB1-PRKACA fusion protein sequence plotted with the frequency of amino acids (AA) per sample for, d HLA class I ligands of the HCC cell lines (n = 3) expressing DNAJB1-PRKACA and g HLA class II peptides of mature moDCs of HV3, HV4, and HV5 (n = 3) incubated with HCC cell line lysate expressing DNAJB1-PRKACA. e, h Fragment spectra (m/z on the x-axis) of the experimentally eluted peptides (e) of experimentally eluted HLA class I-presented ligands EIFDRYGEEV (PA*68/A*02, left) and IFDRYGEEV (PC*04/C*05, right) extracted from the DNAJB1-PRKACA expressing cell lines SMMC-7721 and HepG2, respectively (identification) by comparison to the respective synthetic peptide (validation, mirrored on the x-axis) with the calculated spectral correlation coefficient (R2). h Validation of the experimentally eluted HLA class II-presented peptide EVKEFLAKAKEDFLKK (PII-2) extracted from mature moDCs of a HV (identification) by comparison to the respective synthetic peptide isotope labeled on AA position two (validation, mirrored on the x-axis) with the calculated spectral correlation coefficient (R2). Identified b- and y-ions are marked in red and blue, respectively. Isotope-labeled ions are marked with asterisks. Source data are provided as a Source Data file.

Fig 4: Single-cell RNA sequencing of vaccine-induced PII-1 specific CD4+ T cells.a–f Single-cell RNA-sequencing analysis of CD4+ T cells sorted from PII-1-stimulated PBMCs of the FL-HCC patient 31 weeks after the second vaccination with a personalized DNAJB1-PRKACA-derived peptide vaccine. a Uniform Manifold Approximation and Projection (UMAP) plot showing distinct T cell clusters. b Heat map of cluster defining log normalized gene expression for activated, exhausted/late effector, and naïve resting T cells. c UMAP plots depicting log normalized IFNG, GZMB (encoding granzyme B), TNF, and CCL3 gene expression defining the activated T cell cluster. d Functional enrichment of the log normalized gene expression for the hallmark cancer gene sets “TNF signaling via NF-?B” and “inflammatory response”. e UMAP plot showing T cell receptor (TCR) clonality of sequenced CD4+ T cells. The color code indicates the number of cells belonging to an expanded clonotype. f Distribution of the 10 largest TCR clonotypes across the cell type clusters identified in PII-1-specific CD4+ T cells. g Physiochemical properties and amino acid (AA) sequences of the CDR3-a/-ß region of the most frequent TCR clones in comparison to their target peptide PII-1. The y-axes indicate the hydrophilicity according to the Hopp-Woods scale82. AAs are grouped by their physiochemical properties with color code. h Clustering of variable sequences of the CDR3-a and CDR3-ß region of the ten largest TCR clonotypes across the cell type clusters identified in PII-1-specific CD4+ T cells (upper panel), compared to all identified naïve single cell clones (lower panel). Position-wise Pearson correlation coefficient (PCC) calculation between positive and negative dataset (middle panel), with the significance of correlation calculated using a two-sided Pearson correlation test. The clustering was conducted using GibbsCluster 2.080. Source data are provided as a Source Data file.

Fig 5: Expression of LINC00473 is upregulated in HEK-DP clones.(a) The two known transcript variants (TV1 and TV2) of LINC00473 are significantly upregulated in A9 and A11 clones. The expression of the DNAJB1-PRKACA fusion transcript (DP) is demonstrated in A9 and A11.