Fig. 6. Acidification destabilizes binding of CD80 to CD28, but not to CTLA4.

a, b Superimposition of acidification-induced changes in electrostatic potential (ΔE) on monomeric human CD28 or CTLA4 or respective dimers interacting with CD80. Ligand-interaction and homodimer regions of CD28 and CTLA4 are marked in pink and cyan, respectively. Histidines are marked in red. c, d Molecular dynamic simulations of monomeric CD28 (C) or CTLA4 (D) interacting with CD80 receptor-binding domain. Ligand interaction sites of CD28 and CTLA4 are highlighted in pink. Note that acidification induces a reorientation of the ligand-interaction site of CD28, but not of CTLA4. e Cartesian coordinates of normal vector tips of CD28 and CTLA4 interacting with CD80. f Umbrella sampling simulations of monomeric receptor-ligand interactions. Shaded areas depict error estimation. g Molecular dynamic simulations of CD28 homodimers at pH 7 vs. 5. Ligand-interaction and homodimer regions are marked in pink and cyan, respectively. Histidines are marked in red. h Inhibition of acidification stabilizes endosomal conformation of CD28. CTLA4-TagGFP or CD28/CTLA4(IC)-TagGFP expressing 58αβ A2 T cells were cultured with or without Bafilomycin. CD80-Fc staining was performed on fixed, permeabilized cells. Plot depicts increase of αIgG-AF647-signal normalized to TagGFP gMFI upon Bafilomycin treatment. i, j Analysis of CD28-CD80- (i) and CTLA4-CD80- (j) stability upon acidification. 58αβ A2 T cells expressing CD28 or CTLA4 were incubated on ice with titrated amounts of CD80-Fc and subsequently treated with PBS (pH 7.2) or citric buffer (pH 5.5). After washing bound CD80-Fc was detected using αIgG-AF647 antibody by flow cytometry. See also Figures S5–8. Statistics: 6 h: Pooled data from 4 independent experiments. Two-sided paired t-test (n = 4 biologically independent samples per group). i: Representative data from 1 of 2 independent experiments. Extra sum-of-squares F test. p-value: 3 × 10⁻¹⁵ (n = 3 biologically independent samples per group). j: Representative data from 1 of 2 independent experiments. Extra sum-of-squares F test (n = 3 biologically independent samples per group). Source data are provided as a Source data file.