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, CD4T^TMD or CD4T^Δbind, 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-E^k. Data are representative of four independent experiments with independently generated cell lines. (D) IL-2 secretion from four independently generated CD4T^TMD 58α^-β^- T cell hybridomas after 16 hours of co-culture with MCC:I-E^k+ 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-E^k (CHO E^k) 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).