Fig. 2: Detection of neoepitope-reactive T cell responses in mUC cancer patients.

a Overview of workflow for detection of neoantigen specific T cells in mUC cancer patients. Experimental steps include building of DNA-barcoded pMHC multimer libraries from predicted neopeptides, collection of multimer panels, staining of patient PBMCs with multimers, sorting of multimer-binding CD8⁺ T cells, amplification and sequencing of DNA barcodes, and data analysis. Created with BioRender.com. b Representative output from patient #2389, screening of detected NART responses. Log₂ fold change (fc) of sequenced pMHC associated barcodes enriched by T cell sorting over the input library at each timepoint: pre-treatment, three weeks, nine weeks, 63 weeks, 159 weeks and at 273 weeks post-treatment. Labelled points represent significantly enriched pMHC co-attached barcodes with Log₂ fc >2, count fraction >0.1% and p < 0.001, determined as T cell responses, among the sorted multimer positive CD8⁺ T cell populations. T cell responses are colored based on peptide-presenting HLA-type, text labelled with peptide sequence, and sized according the estimated frequency of the peptide-recognizing T cell population. If the peptide is derived from a virus, the virus is annotated in brackets, otherwise it is a mutation-derived neopeptide. Grey points represent non-enriched barcodes. Horizontal line at Log₂ fc = 2. Vertical line separating peptide-presenting HLA-types. c Tetramer validations of three NART responses in patient #2389 at 9-weeks post-treatment. CD8+ cells shown, gated for tetramer⁺ populations. Frequency of tetramer pMHC population given in percentage, together with estimated frequency derived from multimer screening given in brackets. d Library sizes of predicted HLA-I-feasible neopeptides per patient, colored bars according to predicted HLA-I binding strength, compared to total no. of unique NART responses across screened samples for each patient (stars). Source data are provided as a Source Data file.