Figure 2.

Selection of 93 pHLA‐I complexes for heterotetramer combinatorial coding (HTCC). (a) Distribution of 133 predicted epitopes across HLA‐I and their binding avidity. Peptides with ≥ 50% binding to their respective HLA‐I allotype or previous confirmed as CD8⁺ T cell epitope were selected for CD8⁺ T cell analysis. (b) Distribution of selected peptides across viral proteins per HLA‐I allotype. (c) Overview of the HTCC approach. UV exposure cleaves the UV‐cleavable peptide in the HLA molecules and is exchanged for SARS‐CoV‐2‐specific peptides (red, green and blue). Next, pHLA‐I complexes were conjugated to two different fluorophores to generate the dual‐coded tetramers for each pHLA combination. Peripheral blood mononuclear cells were stained with the combinatorial encoded tetramers and analysed using flow cytometry. (d) Representative flow cytometry plots (donor D04) created by Boolean gating CD8⁺ T cell populations expressing tetramer fluorophores; associated gating strategy is provided in Supplementary figure 2. Each plot in the matrix represents a unique tetramer dual‐coding combination; cells that are double‐positive for both fluorophores are indicated in red, single‐positive cells are indicated in green, and cells negative for all tetramer fluorophores are in blue.