(A) A library composed of 96 FL9 peptide variants (crude peptides) was generated by amino acid mutagenesis at the Qa-1 anchoring positions (p2, p3, p6, p7, and p9). FL9 TCR⁺ hybridomas were incubated with EL4 cells (Qa-1⁺) loaded with each FL9 peptide variant for 12 hours and CD69 expression and TCR downregulation were measured as an indication of TCR stimulation. (B) Activation of FL9 TCR⁺ 58C hybridoma after stimulation with FL9 peptide variants. CD69 expression by FL9 TCR⁺ 58C hybridoma after stimulation with each FL9 peptide variant (left). Downregulation of TCR is shown as ΔTCR MFI based on calculation 100–(Testing TCR MFI/Control TCR MFI) × 100 (%) (middle). Expression of Vα3.2 and Vβ5 on EL4 cells (trogocytosis) was measured (right). (C) Activation of FL9.2 T cells after stimulation with FL9 variants selected from library screen above. Dose-dependent activation of FL9 T cells was measured by culturing FL9.2 T cells with EL4 (Qa-1⁺) at various concentrations of indicated peptides (0, 1, 3, and 10 μg/mL). (D) CD45.1⁺ B6 hosts were adoptively transferred with FL9.2 T cells and immunized i.p. with PBS, FL9, or FL9-68 in CFA on day 0. After 6 days, proliferation of FL9.2 T cells (CD45.2⁺Vα3.2⁺Vβ5⁺) was measured by CFSE dilution (left). (E) CD45.1⁺ B6 mice that were vaccinated with FL9-68 in CFA or CFA alone on day 0 were immunized with Ova_323-339 peptide in CFA on day 6. The frequency of I-A^b/Ova_323-339 Tet⁺ CD4 cells in activated (CD44⁺) CD4 cells was analyzed on day 14. (F) Comparison of high-affinity antibody and auto-antibody responses in WT and Qa-1.D227K mice. WT B6 and Qa-1.D227K.KI mice were immunized with NP₁₉-KLH/CFA and boosted with NP₁₉-KLH/IFA on day 10. High affinity anti-NP responses were measured on day 15. Levels of anti-dsDNA antibody were measured on day 21. Mean ± SEM is indicated. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.