Figure S5.

CD3, CD28, CD86, PD-1, CD80, and PD-L1 expression profiles and background ERK activation level. (A) CD3 and CD28 expression profiles following CRISPR/Cas9-mediated KO of CD28 and MACS-based purification. TCR55⁺ PD-1⁺ ERK-KTR-mScarlet or GFP-NFAT2 reporter Jurkat line is indicated. (B) PD-L1 versus CD86 expression profiles for CD86⁺ APC in the absence or presence of PD-L1 expression. (C) Jurkat reporter PD-1 expression profiles following lentiviral transduction of the LE-hPDCD1 vector and MACS-based purification. (D) PD-L1⁻, CD80⁺, PD-L1⁺, and CD80⁺ PD-L1⁺ APC expression profiles following lentiviral transduction and MACS-based purification. (E) PCA quantification of the impact of PD-L1:PD-1 engagement considering both MHCI and MHCII peptide ligands at various concentrations. Top: The loadings of signaling responses on PC1 (+0.64*ERK+0.66*NFAT2+0.40*p38). Middle: PCA of population response dynamics comparing the indicated peptide ligand quality versus quantity, in the absence versus the presence of PD-L1:PD-1 engagement, plotted along PC1. Bottom: PC1 variations between PD-L1⁻ and PD-L1⁺ conditions. ns: P value ≥0.05, Student’s t test. (F) The baseline is shown for antigen-specific pERK response in the absence of TCR55 expression or peptide ligand SQL. FACS gating strategy for human PBMC-derived PD-1⁺ CD8⁺ T cells is shown in Fig. 10 B. Population pERK response curves are shown for the top 20% PD-1⁺ CD8⁺ T cells comparing TCR55^−/+ versus SQL^−/+ versus MHCI-blocking antibody clone W6/32^−/+ versus PD-L1^−/+ control APC conditions. Dashed line indicates pERK background activation level in the presence of both HLA-B35 and TCR55 but in the absence of SQL peptide ligand.