Super-resolution imaging reveals altered actin architecture at CAR-T cell immune synapses

Area of Science:

• Immunology
• Cell Biology
• Biophysics

Background:

• The actin cytoskeleton is crucial for T cell activation and immunological synapse (IS) formation.
• Understanding actin dynamics in chimeric antigen receptor (CAR)-T cells is vital for optimizing their therapeutic potential.

Purpose of the Study:

• To investigate and compare actin remodeling at the IS in CAR-T cells versus normal T cells using super-resolution microscopy.
• To elucidate how CAR engagement influences cytoskeletal organization during T cell activation.

Main Methods:

• Utilized stimulated emission depletion (STED) microscopy for super-resolution imaging of actin.
• Analyzed actin dynamics in single- and dual-target CAR-T cells activated on supported lipid bilayers.
• Quantitatively assessed structural features of the IS, including actin-depleted regions, actin foci, and microvilli.

Main Results:

• CAR-T cells formed distinct IS with reduced actin-depleted regions and fewer actin foci compared to untransduced T cells.
• Persistent microvilli-like protrusions were observed in CAR-T cell synapses, indicating incomplete cytoskeletal contraction.
• CAR-T cell synapses exhibited impaired actin network reorganization and partial maturation.

Conclusions:

• CAR-T cell-mediated signaling involves fundamentally different actin dynamics than T cell receptor (TCR)-mediated signaling.
• Defective actin remodeling in CAR-T cells may lead to unstable synapses, altered signaling, and potential off-target effects.
• These findings offer insights for engineering safer and more effective CAR-T cell therapies.