A single amino acid substitution converts T10
d into a strong G8 γδ TCR agonist.
a) Graphs depict stimulation of the G8 hybridoma, as measured by activity of a NFAT-dependent alkaline phosphatase reporter, by the indicated wild-type or mutant versions of T22 (left; ◆=R35L, □=D58G, ▲=M89V, ■=P124H,
=α3, ●=T22 and
=negative control, m157) and T10
d (right; ◆=L35R, □=G58D, ▲=V89M, ■=H124P,
=α3, ●=T10
d and
=negative control, m157). Graphs depicting P124H and H124P mutants are drawn in orange and blue, respectively. All stimulation assays were performed in triplicate.
b) Location of position 124 in the interface between the G8 γδ TCR and T22. The proline is colored in orange, and is located on the edge of the platform domain of T22, in direct contact with the G8 γδ TCR.
c) Melting temperatures (T
m) of wild-type and mutant T22 and T10
d. At least two independent measurements were made for each sample; error bars represent the standard error of these measurements.