Fig. 4: Postvaccination expanded TCR clones identified in the tumor are reactive to PTCV-encoded antigens.

a, Most frequent TCRs identified by TCRseq and RNAseq in a patient (before vaccination versus week 9 after vaccination, pairwise scatterplots). Different superscript letters show selected high-frequency new T cell clones detected in PBMCs after vaccination and their abundance in the tumor. Orange, green, and gray circles represent expanded, contracted and not significantly changed T cell clones, respectively. b, CDR3 sequences of the three TCRs (from patient 8; TCR 1, TCR 2 and TCR 3) selected for cloning and their frequency (freq.) in the tumor before (pre-Vax) and after (post-Vax) vaccination. Selected cloned TCRs were present in high frequency only in the peripheral blood and tracked into the tumor after treatment. c, UMAP (Uniform Manifold Approximation and Projection) and stacked barplot indicating the single-cell cluster identities and number of cells for each of the three TCRs selected for cloning. d, Patient-specific clonal TCR sequences were gene optimized and inserted into the pMXs-IRES-GFP retroviral plasmid vector containing the viral packaging signal, transcriptional and processing elements, and the GFP reporter gene. MuLV, murine leukemia virus; Mo-MuLV, Moloney MuLV; LTR, long terminal repeat; Amp^R, ampicillin resistance. e, TCR-engineered T cells (GFP⁺) from unvaccinated patient-derived PBMCs were stimulated for 6 h with epitope pools or the nonspecific epitope CTA1 (10 µg ml⁻¹), and CD69 expression was evaluated by flow cytometry. Peptide pool 1 included the most reactive epitopes measured by ELISpot, whereas pool 2 (consisting of peptides corresponding to epitopes 21–40) served as an internal negative control.