Fig 2: Identification of the optimal EP concentration for the read-out of viral-specific CD8+ and CD4+ cells after in vitro expansion. PBMCs were cultured with INF- or EBV-derived short peptides or matched EPs (20 mers, N- and/or -C-terminal elongations are indicated) for 12 days. On day 1, cells were stimulated with 1 µg/mL (HLA class I) or 5 µg/mL (HLA class II) short peptide (standard concentrations) or with 1, 10, or 50 µg/mL of the EPs plus 20 µg/mL Poly-ICLC (x-axis). On day 12, cells were re-stimulated in the ICS with 10 µg/mL short peptide or with 10 µg/mL or 50 µg/mL of the respective EP (graph legends). Single live CD8+ or CD4+ lymphocytes were analyzed for their specific IFN-? and TNF expression (percentage of IFN-?+TNF+ cells; background of the negative control was subtracted). Shown are the percentages of double-positive cytokine CD8+ cells ((A–C), HD9) or CD4+ cells ((D), HD7) relative to the cytokine response detected with the standard protocol. Absolute frequencies with short peptide stimulation: 11.9% IFN-?+TNF+ CD8+ (INF); 16.3% IFN-?+TNF+ CD4+ (EBV EBNA2). Full gating strategy is depicted in Figure S1A,B.

Fig 3: 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 4: Ex vivo detection of virus-specific T cell responses using EPs can be suboptimal. PBMCs from healthy donors (n = 7 HDs in total, n = 3 per specificity) were stimulated with 10 µg/mL HLA class I and class II viral-derived epitopes from CMV, EBV (BRFL1/EBNA1/EBNA2), and Influenza virus (INF) of short length, or with their respective EP version (20 mers, N-peptide source-C: N- and C-terminal elongated; N-peptide source: N-terminal elongated; peptide source-C: C-terminal elongated) for 12 h. Two EP purities (>50% and >90%) were used. The percentages of peptide-specific (background cytokine production in the negative control was subtracted) CD8+ (A) and CD4+ (B) cells that produced both cytokines (IFN-?+TNF+) were calculated. The anti-EP cytokine response is depicted related to the response to the matched short peptide (range 0.012–2.22% of IFN+TNF+ cells within the CD4+ or CD8+ subsets), which is normalized to 100%. The full gating strategy is depicted in Figure S1A,B and frequencies of specific cells are given in Table S2.

Fig 5: The single addition of Poly-ICLC is superior to the combination of Poly-ICLC and GM-CSF for the detection of viral-specific CD4+ and CD8+ T cells using EPs. PBMCs were stimulated with virus-derived HLA class I and HLA class II EPs (10 µg/mL of 20 mers; N- and/or -C-terminal elongations are indicated) on day 1. In addition to the peptide stimulation on day 1, Poly-ICLC (20 µg/mL, P), GM-CSF (0.8 µg/mL, G), or the combination of P+G (same concentration as for the single addition) were added. Control stimulation was performed with peptide only (w/o). (A,B) IFN-? ELISpot analysis at day 12 of the background spot counts ((A), negative control; 300,000 cells/well seeded, normalized to 50,000 cells) or spot counts after restimulation with 50 µg/mL EPs ((B), 50,000 cells/well seeded). EPs were CMV (N-CMV-C) and EBV EBNA2 (N-EBNA2-C) (HLA class II peptides), or CMV (N-CMV) and Influenza virus (N-INF) (HLA class I peptides). n = 2 HDs. Tests were performed in duplicate. Box and whisker plots of the mean IFN-? spot counts per 50,000 cells are shown (min to max and median values are indicated). Statistical analysis: Friedman test with post-test Dunn’s multiple comparison. * p = 0.05. Representative ELISpot wells and spot counts (technical replicates) of PBMCs from HD5 for the negative control or the EP N-INF stimulation are shown with spot counts. (C–F) ICS results. Cells were re-stimulated on day 12 with 10 µg/mL short peptide or 50 µg/mL of the respective EP. Specific frequencies (background subtracted) of cytokine+ (IFN?+TNF+) CD8+ (HD1; (C–E)) or CD4+ (HD4, (F)) cells are shown relative to the responses against the matched short peptides. Absolute frequencies with short peptide stimulation: 27.7% IFN?+TNF+CD8+ (HLA class I, INF); 4.70% IFN?+TNF+CD4+ (HLA class II, CMV). Full gating strategy is depicted in Figure S1A,B.