Extracellular domain, hinge, and transmembrane determinants affecting surface CD4 expression of a novel anti-HIV...

Area of Science:

• Immunology
• Molecular Biology
• Virology

Background:

• Chimeric antigen receptor (CAR)-T cell therapy shows promise for HIV treatment.
• Current CAR designs require optimization for efficacy against chronic HIV infection.

Purpose of the Study:

• To identify critical structural elements in novel anti-HIV CARs for improved surface expression.
• To evaluate the impact of extracellular, hinge, and transmembrane (TM) domains on CAR function.

Main Methods:

• Constructing novel anti-HIV CAR variants with modifications in extracellular, hinge, and TM domains.
• Assessing CAR surface and intracellular expression using flow cytometry and confocal microscopy.
• Evaluating the cytotoxic activity of engineered CAR T cells against HIV-infected cells.

Main Results:

• A novel CAR incorporating truncated CD4 extracellular domain and CD8α hinge/TM initially showed poor surface expression.
• Shortening the CD8α hinge and adding the LYC motif to the CD8α TM domain fully restored both intracellular and extracellular CAR expression.
• Mutations in the LYC motif (TTA or TTC) severely abrogated CAR expression.
• Removing the FQKAS motif enhanced CD4-CAR surface expression and led to cytotoxic killing of HIV Env+ target cells.

Conclusions:

• Specific motifs within the hinge and TM domains, particularly the LYC motif, are crucial for optimal CAR surface expression.
• Structural modifications can significantly enhance the efficacy of CAR T cells for HIV therapy.
• Further structural analysis is needed to establish design guidelines for effective CAR development.