Fig. 2: SCT optimization and characterization.

a Axial view of HLA-A*02:01 SCT crystal structure (RDB ID: 6APN). Highlighted regions of interest: H74 (blue), Y84 (green), A139 (cyan), and the first three amino acids of the L1 linker (black). b Table: L1 and HLA amino acid modifications for each SCT template. Heatmap: Relative expression of each SCT combination (n = 3), designated by template (row) and peptide (column), and exemplified by reduced SDS-PAGE of 18 SCTs constructed using design template D9. Previously expressed and a purified aliquot of WT1 SCT is used as a positive control (+) for band intensity quantification. c Thermal melting profiles of SCTs. The negative of the change in fluorescence over the change in temperature (−δF/δT) is measured for SCTs encoding WT1 peptide (c.i). Local minima representing Tm values (see the boxed region of WT1 plot) are plotted (c.ii) for each SCT template and peptide (n = 3). d WT1 SCTs constructed according to each of the six template designs were paired with a MART-1 SCT (D3 template) to identify cognate TCR-transduced cells. The number/color at the top right of each plot indicates the SCT template used for WT1 SCT tetramer in the flow assay. Percentages indicate the proportion of the total cell population captured in the WT1 SCT-positive quadrant. e Capture of CMV-specific T cells using SCT or refolded pMHC format. Unique paired TCR clonotypes identified by 10× single-cell sequencing of tetramer-positive cells. CDR3a and CDR3b sequences of the twelve most frequently captured clonotypes from SCT tetramer along with LD to publicly reported CMV-specific clonotypes from VDJdb are reported in the Table. L1 linker 1, WT1 RMFPNAPYL, MART-1 ELAGIGILTV, CMV NLVPMVATV, LD Levenshtein distance, VDJdb VDJ database, ACN allophycocyanin, PE phycoerythrin.