Fig. 3: The cholesterol molecule has an inhibitory role in TCR signalling.
a, Two molecules (cholesterol-like and unassigned densities) were embedded into the TMD of the Vγ9Vδ2 TCR–CD3 complex. The electrostatic surface potential map of the Vγ9Vδ2 TCR–CD3 complex (left) and a magnified view of the interactions between the cholesterol-like molecules and the complex (right) are shown. The cryo-EM densities are contoured at 9σ. b, Flow cytometry analysis of CD69 expression on Jurkat-76 cells transduced with WT (n = 3 per group) and mutant variants of Vγ9Vδ2 TCR (n = 6 per group) after co-culture for 15 h with K562 cells expressing CD1d or ZIM3–dCas9 (ref. 52) (parental). c, Quantitative analysis of cholesterol content in purified WT or mutant Vγ9Vδ2 TCR–CD3 complex using liquid chromatography coupled with tandem MS (LC–MS/MS; n = 6 per group). d, Magnified views of the TMD of the TMα and AAA Vγ9Vδ2 TCR–CD3 complex. The cryo-EM maps are shown as a black mesh and contoured at 8σ. The position of the cholesterol binding site in the Vγ9Vδ2 TCR–CD3 complex is indicated by a dashed circle. e, Structural comparison of the TMDs of the WT, AAA and TMα Vγ9Vδ2 TCR–CD3 complex (left). Right, structural comparison of the TMDs of Vγ9Vδ2, WT αβ (PDB: 7FJD)40 and gain-of-function (GOF) αβ TCR–CD3 complexes (PDB: 7FJE)40. f, Flow cytometry analysis of CD69 expression on Jurkat-76 cells that were transduced with Vγ9Vδ2 TCR and Vγ5Vδ1 TCR, with or without treatment with 0.5 μM ALOD4 and 0.5 μM ALOD4 non-binding mutant (ALOD4-mut) for 12 h. n = 4 per group. Results are representative of three (b and f) and two (c) independent experiments. Each symbol corresponds to a biologically independent experiment. Data are mean ± s.d. P values were calculated using one-way ANOVA with Dunnett’s multiple-comparison test. For c, mutant complexes were compared with the WT complex.
