Supplementary Figure 5: Substitution of G135D in LAT enables the activation of T cells by low-affinity antigen in a gain-of-function manner.

a. Naive OT-I⁺ CD8 T cells (left panels) were isolated and transduced with retrovirus expressing wild-type LAT-P2A-BFP or G135D LAT-P2A-BFP. Cells were rested for one day before they were subjected to stimulation with various peptides-pulsed TCR Cα-deficient splenocytes over a range of peptide concentrations (10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0 μM for OVA or G4 peptide; 10 μM, 1 μM, 0 μM for VSV peptide). Or, naive OT-II⁺ CD4 T cells were used for experiments. OT-II⁺ CD4 T cells were stimulated with agonist OVA- or partial agonist E336Q-pulsed splenocytes overnight (10 μM, 3 μM, 1 μM, 0.1 μM, 0 μM for OVA or E336Q peptide; 10 μM, 1 μM, 0 μM for CLIP peptide). Representative histograms are shown. Peptides used for stimulation are indicated at the left. The expression of IRF4 or CD69 was analyzed. Data are representative of three independent experiments. b. Statistical analysis of p-ERK activation for OT-I⁺ CD8 T cells (left) or OT-II⁺ CD4 T cells (right) as experiments done in (a). TCR Cα-deficient splenocytes were pulsed with 1 μM of OVA, T4, or G4 peptide, 10 μM of Catnb peptide or 10 μM of VSV peptide. Each symbol represents an independent replicate (mean ± s.d). **P = 0.0043 (OVA); **P = 0.0022 (T4, G4, Catnb); ns: not significant P = 0.3095. Mann-Whitney test. Statistical analysis of p-ERK induction of OT-II⁺ T cells was shown on right. Each symbol represents an independent replicate (n=6 samples from two independent experiments). **P = 0.0022; ns: not significant P > 0.9999. Two-tailed Mann-Whitney test. c. Cells were prepared as in (a) to retrovirally express wild-type LAT-P2A-BFP or G135D LAT-P2A-BFP, loaded with the calcium-sensitive dye Indo-I, and incubated with 1:100 or 1:200 biotinylated OVA/H-2K^b, or 1:100 T4/H-2K^b, G4/H-2K^b, or VSV/H-2K^b monomers. Cells were then moved to 37^oC and subjected to flow cytometry-based calcium assays. Cells were first recorded for 30 sec to obtain a baseline calcium level. Streptavidin (SA) was added at the 30th sec. Ionomycin (Iono) was added at the 240th sec. Representative calcium traces are shown. Monomers used for stimulation are indicated above the calcium plots. Data are representative of two independent experiments.