Peptide mutagenesis and superagonist peptide screen.

A peptide library was generated by single mutation of each Qa-1 anchoring position (p 2, 3, 6, 7, and 9) of the FL9 peptide (FYAEATPML) with 20 aa, which is composed of 96 FL9 variant peptides. FL9 TCR⁺ 58C hybridomas were incubated with EL4 cells that were pulsed with each FL9 variant. After 12 hours, CD69 expression and levels of TCR expression were measured by flow cytometry. For analysis of the binding strength of FL9 TCR with Qa-1-FL9 variants, trogocytosis was measured directly by the detection of FL9 TCR (Vα3.2⁺Vβ5⁺) on EL4 cells. FL9 TCR⁺ 58C hybridomas were cocultured with EL4 cells that were pulsed with FL9 variant peptides from the library. After 2 hours, the percentage of Vα3.2⁺Vβ5⁺ EL4 cells was assessed by flow cytometry as a measurement of trogocytosis.