Fig 1.
The CD4 TMD contains highly conserved glycine residues.
(A) CD4 transmembrane domain (TMD) sequence alignment showing highly conserved glycine residues shaded red. (B) Ribbon diagram model or (C) space-filled model of the CD4 TMD with alanine and glycine residues of interest highlighted in magenta (generated with PyMol).
Fig 2.
CD4T contributes to T cell activation.
(A) 58α-β- T cell hybridomas were retrovirally transduced with the 5c.c7 TCR and either CD4WT, CD4T or no CD4. IL-2 secretion from CD4T 58α-β- T cell hybridomas after 16 hours of co-culture with MCC:I-Ek+ M12 cells was normalized to the matched no CD4 controls within the same experiment to determine a relative IL-2 concentration. Bars represent mean values +/- SEM from four independently generated matched sets of cell lines. (B) IL-2 secretion after 16 hours of co-culture with MCC:I-Ek+ M12 cells normalized to matched CD4WT controls within the same experiment to determine relative IL-2 concentration. Bars represent mean values +/- SEM from four independently generated matched sets of cell lines. (*p<0.05; Mann-Whitney).
Fig 3.
Mutating the CD4 transmembrane domain GGxxG motif impairs T cell activation.
(A) Alignment of the wild-type CD4 TMD with the CD4 TMD mutant (G403V/G406L). Mutated residues are highlighted in red. Bulky side chains were introduced to disrupt the glycine patch. (B) 58α-β- T cell hybridomas were retrovirally transduced with the 5c.c7 TCR and either CD4T, CD4TTMD or CD4TΔbind, which is mutated in the region known to bind MHC class II. Surface expression of TCR and CD4 were assessed by flow cytometry as labeled. (C) IL-2 secretion from 58α-β- T cell hybridomas after 16 hours of co-culture with M12 cells expressing agonist (MCC), weak agonist (T102S), or null (HB) peptide tethered to I-Ek. Data are representative of four independent experiments with independently generated cell lines. (D) IL-2 secretion from four independently generated CD4TTMD 58α-β- T cell hybridomas after 16 hours of co-culture with MCC:I-Ek+ M12 cells normalized to matched CD4T controls within the same experiment to determine relative IL-2 concentration. (E) IL-2 secretion from 58α-β- T cell hybridomas after 16 hours of co-culture with Chinese hamster ovary (CHO) cells ectopically expressing I-Ek (CHO Ek) pulsed with MCC peptide at the indicated concentrations. Data are representative of three independent experiments with independently generated cell lines. (*p<0.05; Mann-Whitney).
Fig 4.
Mutating the CD4 TMD GGxxG motif does not impair dimerization at steady-state.
(A) Grey-scale images of mCherry (top) and GFP (bottom) intensities pre and post mCherry ablation for representative cells. (B) Plot of relative mCherry values pre- and post-ablation for all cells analyzed. The average ablation of all populations was below 10% and did not differ significantly from each other. (C) FRETE values for CD28GFP/Ch, PD1GFP/Ch and CD4TGFP/Ch cells. Representative of two experiments with independently generated cell lines. (D) FRETE values for CD4WTGFP/Ch vs. CD4TGFP/Ch cells. Concatenated data from two independently generated cells lines is shown because the CD4WTGFP/Ch cells had a broader range of donor and acceptor levels that limited the number of cells available for subset analysis (methods). (E) FRETE values for CD4TGFP/Ch vs. CD4TTMD-GFP/Ch cells. Representative of two experiments with independently generated cell lines. Matched expression for analysis was based on median pre-bleach intensity. Dots represent single cells and green bars represent median values (*p<0.05, **p<0.001; ***p<0.0001; Mann-Whitney).
Fig 5.
Mutating the CD4 TMD GGxxG motif does not impair dimerization upon TCR engagement.
(A) Mobility of lipid bilayers was assessed by measuring recovery of streptavidin-PE molecules into a bleached region of the lipid bilayer and normalized to a reference region that was not bleached. (B) FRETE values for CD28GFP/Ch, PD1GFP/Ch and CD4TGFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-Ek). Data are representative of those obtained with two independently generated cell lines. (C) FRETE values for CD4WTGFP/Ch vs. CD4TGFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-Ek). Data is concatenated from two independently generated cell lines as in Fig 4D. (D) FRETE values for CD4TGFP/Ch vs. CD4TTMD-GFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-Ek). Data are representative of those obtained with two independently generated cell lines. Analysis was performed as for Fig 4. Dots represent single cells and green bars represent median values (*p<0.05, **p<0.001; ***p<0.0001; Mann-Whitney).