Nanoscale CAR Organization at the Immune Synapse Correlates with CAR-T Effector Functions

Area of Science:

• Immunology
• Cell Biology
• Biophysics

Background:

• Chimeric antigen receptor (CAR)-T cell therapy is a promising cancer treatment.
• The nanoscale organization of CARs at the CAR-T cell interface is critical for T cell activation but poorly understood.

Purpose of the Study:

• To investigate the nanoscale organization of CARs targeting CD138 proteoglycans at the CAR-T cell interface.
• To correlate CAR nanoscale organization with CAR-T cell effector functions.

Main Methods:

• Utilized single-molecule localization microscopy (SMLM) on fixed and live CAR-T cell interfaces.
• Studied CARs targeting CD138 in interactions with on-target (SKOV-3, CAG, FaDu) and negative (OVCAR-3) cancer cells.

Main Results:

• Observed significant CAR self-association into nanoclusters, enhanced with on-target cells.
• Demonstrated efficient CAR segregation from CD45 at the interface with on-target cells.
• Found correlations between CAR clustering/CD45 segregation and calcium influx/target cell killing.

Conclusions:

• Elucidated the nanoscale organization of CARs during CAR-T cell interactions with target cells.
• Highlighted the functional significance of CAR nanoclustering and CD45 segregation for CAR-T cell efficacy and safety.