Phosphoantigen-Induced Inside-Out Stabilization of Butyrophilin Receptor Complexes Drives Dimerization-Dependent γδ TCR Activation

Rheumatology and Immunology Cell Biology Biochemistry Life Sciences Medicine Immunology Biophysics
phosphoantigens Butyrophilin receptor complexes γδ TCR dimerization cell activation

Academic Background

γδ T cells are a unique subset of the immune system, characterized by T cell receptors (TCRs) composed of γ and δ chains that recognize non-peptide antigens, such as phosphoantigens (PAgs) produced by microbial or tumor cells. Among them, Vγ9Vδ2 T cells are the most abundant γδ T cell subset in human circulation and play a critical role in anti-infection and anti-tumor immunity. However, the molecular mechanism by which PAgs activate γδ T cells through membrane receptors has long remained unclear.

Butyrophilin (BTN) family proteins (e.g., BTN3A1 and BTN2A1) have been identified as sensors of PAgs, but the assembly of BTN receptor complexes, PAg-induced conformational changes, and their interaction mechanisms with γδ TCRs were poorly understood. This study aims to elucidate the “inside-out” stabilization mechanism of PAg-induced BTN complexes and how they drive Vγ9Vδ2 TCR dimerization and T cell activation.

Paper Source

This paper was co-authored by Yuwei Zhu, Wenbo Gao, et al., with corresponding author Zhiwei Huang (School of Life Science and Technology, Harbin Institute of Technology). The research team resolved the high-resolution structure of the PAg-BTN-TCR complex using cryo-electron microscopy (cryo-EM). The findings were published in Immunity on July 8, 2025 (DOI: 10.1016/j.immuni.2025.04.012).

Research Process and Results

1. PAg Induces Formation of a 2:2 Tetrameric BTN2A1-BTN3A1 Complex

Experimental Design:
- Full-length BTN2A1 and BTN3A1 were co-expressed in 293F cells, and complex formation was analyzed by gel filtration after adding microbial PAg (HMBPP).
- The complex structure was resolved by cryo-EM (3.70 Å resolution), with local refinement improving the B30.2 domain resolution to 3.46 Å.

Key Findings:
- Structural Features: Both BTN2A1 and BTN3A1 form homodimers, which assemble into a 2:2 heterotetramer via intracellular B30.2 domains bound to HMBPP (Figure 1c).
- Transmembrane Interactions: BTN2A1 transmembrane helices stabilize homodimerization through hydrophobic interactions and disulfide bonds (C219-C219, C237-C237), while BTN3A1 helical domains (HD) form a “domain-swapped” dimer via hydrogen bonds (e.g., E282-R365) with B30.2.
- Conformational Changes: HMBPP binding induces BTN3A1 B30.2 conformational changes, enabling its interaction with BTN2A1 B30.2 and stabilizing the complex (Figure 1d).

Significance: First demonstration of PAg-triggered BTN complex assembly via intracellular binding, providing a structural basis for “inside-out” signaling.

2. BTN3A1-BTN3A2/BTN3A3 Heterodimers Enhance Complex Stability

Experimental Design:
- BTN2A1, BTN3A1, and BTN3A2 or BTN3A3 were co-expressed, and complexes were analyzed by gel filtration and cryo-EM (4.0 Å resolution).
- Limited proteolysis compared the stability of different complexes.

Key Findings:
- Heterodimer Advantage: BTN3A2 (lacking B30.2) or BTN3A3 (PAg-binding-deficient mutant) forms tighter HD heterodimers with BTN3A1 (Figures 2d-e).
- Enhanced Stability: BTN3A1-BTN3A2 heterodimers showed significantly higher resistance to elastase degradation than BTN3A1 homodimers (Figure 3b), correlating with stronger γδ T cell activation.

Significance: BTN3A2/BTN3A3 act as regulatory molecules, stabilizing BTN3A1 conformation to amplify PAg signaling.

3. BTN Complexes Drive Vγ9Vδ2 TCR Dimerization for Activation

Experimental Design:
- Cryo-EM resolved the BTN2A1-BTN3A1-BTN3A2 complex bound to Vγ9Vδ2 TCR ectodomains (4.05 Å resolution).
- Bioluminescence resonance energy transfer (BRET) validated TCR dimerization.

Key Findings:
- Dual-Binding Mode: One TCR is “sandwiched” between BTN2A1 and BTN3A2 IgV domains (binding site A, BSA=1070.2 Ų), while another binds free BTN2A1 IgV (binding site B, BSA=489.3 Ų) (Figure 4a).
- Conformational Rearrangement: TCR binding rotates the BTN2A1 Ig dimer by 25° and the BTN3A1-BTN3A2 heterodimer by 15° in the opposite direction (Figures 4c-e).
- Functional Validation: BRET confirmed HMBPP-treated BTN complexes significantly promote TCR dimerization (Figure 5i).

Significance: Proposes a “dimerization-dependent γδ TCR activation” model, explaining how BTN complexes optimize TCR signaling via spatial organization (Figure 6).

Conclusions and Impact

  1. Scientific Value:

    • Unveils the complete molecular mechanism of PAg-BTN-TCR signaling, filling a gap in γδ T cell immunology.
    • Clarifies the regulatory roles of BTN3A2/BTN3A3, advancing understanding of BTN family diversity.

  2. Applications:

    • Targeting BTN-TCR interfaces may inspire novel γδ T cell immunotherapies for cancer or infections.
    • Provides a structural basis for designing small-molecule PAg mimetics or BTN antagonists.

Highlights

  • Methodological Innovation: First cryo-EM structure of full-length BTN-TCR complexes, overcoming membrane protein challenges.
  • Theoretical Breakthrough: Proposes “inside-out stabilization” and “dimerization activation” models, redefining γδ TCR recognition paradigms.
  • Interdisciplinary Impact: Integrates structural biology, immunology, and computational modeling (e.g., molecular dynamics), fostering cross-disciplinary research.

Additional Information

  • Data Availability: Structural coordinates deposited in PDB (e.g., BTN2A1-BTN3A1 complex, accession number pending).
  • Limitations: Cryo-EM’s detergent environment may not fully mimic membranes; future studies using nanodiscs are warranted.