An autonomous CDR3delta is sufficient for recognition of the nonclassical MHC class I molecules T10 and T22 by gammadelta T cells

Abstract

It remains unclear whether gammadelta T cell antigen receptors (TCRs) detect antigens in a way similar to antibodies or alphabeta TCRs. Here we show that reactivity between the G8 and KN6 gammadelta TCRs and the major histocompatibility complex class Ib molecule T22 could be recapitulated, with retention of wild-type ligand affinity, in an alphabeta TCR after grafting of a G8 or KN6 complementarity-determining region 3-delta (CDR3delta) loop in place of the CDR3alpha loop of an alphabeta TCR. We also found that a shared sequence motif in CDR3delta loops of all T22-reactive gammadelta TCRs bound T22 in energetically distinct ways, and that T10(d), which bound G8 with weak affinity, was converted into a high-affinity ligand by a single point mutation. Our results demonstrate unprecedented autonomy of a single CDR3 loop in antigen recognition.

Figure 1

Three dimensional structure of the G8 γδ TCR in complex with the MHC class Ib molecule T22. The G8 δ-chain is shown in blue, and the G8 γ-chain in green; T22 is shown in yellow and the G8 CDR3_δ loop in red. Inset, right, shows magnified view of the G8 CDR3_δ loop (red) in contact with T22 (yellow). The residues of the W…GYEL motif found in all T22-binding γδ TCRs are labeled.

Figure 2

Transfer of CDR3_δ onto CDR3_α. a. Top, ribbon representation of the full-length G8 γδ TCR (grey, with the CDR3_δ loop shown in sticks and colored red), the 172 αβ TCR scFc (blue), and the fusion in which the CDR3_δ loop was grafted onto the CDR3_α loop. Bottom, sequences of CDR3δ loops grafted from the G8 and KN6 γδ TCRs (red) onto the CDR3_α (replaced residues are in blue).

Figure 3

CDR3_δ grafted into αβ TCR retains wild-type affinity for T10 and T22 ligands. SPR measurements of binding between T22 and wild-type or fusion G8 and KN6 γδ TCRs. Columns (G8, left; KN6, right) show sensograms corresponding to the wild-type full-length γδ TCR (top), fusions of the CDR3_δ loop with the single chain 172 αβ TCR (172sv) expressed in E. Coli or insect cells (middle), and the wild-type full-length 172 αβ TCR expressed in insect cells (bottom).

Figure 4

Heterogeneous energetic landscapes of G8 and KN6 CDR3_δ interactions with T10 and T22. a) Alanine scanning mutagenesis of the G8 (top) and KN6 (bottom) CDR3_δ loops. +, mutations that resulted in immeasurable binding. Bars are colored spectrally red through blue based on the affect of the mutation on binding, with red indicating a large ΔΔG penalty on binding versus blue for little or no ΔΔG penalty. b) The interface between the G8 γδ TCR and T22 showing the residues of the CDR3_δ loop color-coded according to their energetic contribution to binding as in a.

Figure 5

The G8 γδ TCR binds to strong and weak stimulatory ligands with different affinity. SPR measurements of T22, T10^b and T10^d binding to the G8 γδ TCR. K_D and association and dissociation rates are listed in Table X.

Figure 6

Mutations responsible for differential G8 γδ TCR binding and stimulation by T22 and T10^b versus T10^d. SPR measurements of G8 γδ TCR binding to T22 mutants (left sensograms) and T10^d mutants (right sensograms). T22 and T10^d wild-type residues are indicated in blue and orange, respectively. The position of each point mutation and the corresponding calculated affinity (K_D) are indicated in the upper right region of each sensogram. Structures in left column depict position of each point mutation in the structure of T22.

Figure 7

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.