Structural characterization of two γδ TCR/CD3 complexes

Abstract

The T-cell receptor (TCR)/CD3 complex plays an essential role in the immune response and is a key player in cancer immunotherapies. There are two classes of TCR/CD3 complexes, defined by their TCR chain usage (αβ or γδ). Recently reported structures have revealed the organization of the αβ TCR/CD3 complex, but similar studies regarding the γδ TCR/CD3 complex have lagged behind. Here, we report cryoelectron microscopy (cryoEM) structural analysis of two γδ TCRs, G115 (Vγ9 Vδ2) and 9C2 (Vγ5 Vδ1), in complex with CD3 subunits. Our results show that the overall subunit organization of the γδ TCR/CD3 complexes is similar to αβ TCRs. However, both γδ TCRs display highly mobile extracellular domains (ECDs), unlike αβ TCRs, which have TCR ECDs that are rigidly coupled to its transmembrane (TM) domains. We corroborate this finding in cells by demonstrating that a γδ T-cell specific antibody can bind a site that would be inaccessible in the more rigid αβ TCR/CD3 complex. Furthermore, we observed that the Vγ5 Vδ1 complex forms a TCR γ5 chain-mediated dimeric species whereby two TCR/CD3 complexes are assembled. Collectively, these data shed light on γδ TCR/CD3 complex formation and may aid the design of γδ TCR-based therapies.

Fig. 1. CryoEM reconstruction and modeling of the G115 (Vγ9 Vδ2) TCR/CD3 complex.

a Left and middle: two views of a 3.27 Å resolution map of the G115 TCR/CD3 complex bound by OKT3 fab. Right: cryoEM map was Gaussian filtered to 2.5 σ and the contour level was reduced to allow visualization of a weak γδ TCR ECD density. b Structure of the G115 TCR/CD3 complex. Interfaces between TCRγ/TCRδ (c), TCRγ/CD3εγ (d), TCRγ/CD3ζ (e), TCRδ/CD3εδ (f), and TCRδ/CD3ζζ (g). Chains are shown in cartoon representation and color-coded as indicated and interfacial residues are shown as sticks. Underlined residues highlight conserved electrostatic interactions between αβ and γδ TCR-CD3 interaction networks.

Fig. 2. Structural comparison of the G115 TCR/CD3 complex to an αβ TCR/CD3 complex.

a The G115 TCR/CD3 complex was aligned to an αβ TCR/CD3 complex (PDB:7FJD) via the TCR γ and β chains using matchmaker command in ChimeraX. G115 TCR chains are colored as indicated, while the entire αβ TCR/CD3 complex is colored white. b, c Cross sectional and top-down views are depicted from regions indicated in a. The rotation angles shown in b were estimated between the centroids of each domain (CD3εδ or CD3εγ ECDs, comparing γδ and αβ TCRs) calculated in ChimeraX.

Fig. 3. CryoEM structures of γδ TCR ECDs bound by anti-TCRVδ Fabs.

a CryoEM map of G115 TCR bound by Fab 1. b CryoEM map of 9C2 TCR bound by Fab 2. c CryoEM map of 9C2 TCR bound by Fab 3. Densities are color coded based on the built atomic model. Each left panel shows Gaussian filtered maps (2.5 σ) at low threshold with fitted atomic models to enable visualization of the TMD and micelle densities. Locally refined (a, b) or higher threshold sharpened (c) maps shown on the right display higher resolution features for the TCR ECD and bound Fab V domains.

Fig. 4. Vγ5 Vδ1 TCR dimer interface and its sequence conservation.

a Structure of the 9C2 TCR ECD (Fab 2 complex used, Fab models hidden for clarity) with each TCRγ protomer colored a different shade of blue. Analysis of the TCRVγ5-mediated dimer interface with interacting residues shown as sticks and dashed lines representing putative hydrogen bonds. Note: TCRγ Y72 of protomer 1 forms a π-π interaction with Y72 of protomer 2. b Sequence alignment of various germline encoded TCR γ-chain variable regions. Residues that have been identified in the dimerization interface are labeled. CDRs are indicated with a faint yellow tint.

Fig. 5. Fab 3 binding region is masked in the rigid αβ TCR/CD3 complex.

a Depiction of the Fab 3 binding site in a Fab 3 bound 9C2 γδTCR ECD structure. The Fab variable region is shown as cartoon, whereas the TCR ECD is shown as surface. The Fab 3 epitope is highlighted in yellow. b Bound Fab 3 was modeled onto the αβ TCR from PDB ID 8ES7 through alignment of the TCR β-chain to TCRγ. The Fab variable region is shown as a cartoon, whereas the TCR ECD is shown as surface view. Apparent steric clash between Fab 3 and CD3δ is highlighted in red. Expansion of Vδ1 γδ T-cells from human donor PBMCs following culture with Ab 3. T-cell purity (c, gated on live, CD45+ cells), and counts (d) were assessed on day 13. N = 1 for flow cytometry experiments.

Fig. 6. γδ TCR ECD flexibility correlates with the diversity of antigen recognition by the γδ TCR.

Immune receptor models are organized by their ligand binding geometries. αβ TCR/CD3/pMHC complex (rightmost) is characterized as having a rigid ECD and conserved pMHC-docking mode. BCR (left most, IgM receptor shown) has highly mobile antigen binding (Fab) regions and unlimited ways of engaging antigens. γδTCRs (hypothetical composite models of TM/CD3 and ECD/ligand complexes shown, with flexible linkers depicted as dotted lines) have a αβTCR-like architecture but use their mobile ECDs to engage ligands in diverse ways. Arrows represent flexibility in ligand/antigen recognition domains. PDB ID 7XQ8 was used for the IgM BCR. PDB ID 8DFW was used for γδ TCR ECD/BTN2A1 complex. PDB ID 6MWR was used for the γδ TCR ECD/MR1 complex (cleft distal). PDB ID 7LLI was used for γδ TCR ECD/MR1 complex (cleft proximal). PDB ID 4LHU was used for γδ TCR ECD/CD1d complex. PDB ID 8ES8 was used for αβ TCR/CD3/pMHC complex.