Direct visualization of chimeric antigen receptors on primary human T cells using dSTORM super-resolution microscopy

Area of Science:

• Immunology
• Biotechnology
• Microscopy

Background:

• Chimeric antigen receptor (CAR) T-cell therapy shows promise for hematological malignancies and is being explored for solid tumors and autoimmune diseases.
• Accurate assessment of CAR organization and spatiotemporal expression is vital for understanding CAR T-cell mechanisms and evaluating product potency and safety.

Purpose of the Study:

• To develop and apply a novel, tag-free method for direct CAR labeling and visualization using super-resolution microscopy.
• To determine CAR surface expression on human primary T cells with single-molecule resolution.
• To investigate factors influencing CAR surface expression and its impact on CAR T-cell activation.

Main Methods:

• Application of an IgG4-targeted F(ab)2 fragment for direct CAR labeling.
• Utilizing direct stochastic optical reconstruction microscopy (dSTORM) for super-resolution imaging.
• Combining direct CAR detection with phenotypic assessment of CAR T cells.

Main Results:

• Successfully detected surface expression of CARs targeting SLAMF7, BCMA, and CD19 with minimal background.
• Identified T cell subtype, donor material, and CAR construct as factors influencing CAR surface expression.
• Observed a potential link between CAR surface expression levels and CAR T-cell activation state.

Conclusions:

• A novel, tag-free super-resolution microscopy approach provides insights into CAR surface expression.
• This method can inform the application of synthetic immune receptors in CAR T-cell therapy.
• Potential to improve CAR T-cell therapy efficacy, predict outcomes, and enhance patient care.