The CD28-Transmembrane Domain Mediates Chimeric Antigen Receptor Heterodimerization With CD28

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Anti-CD19 chimeric antigen receptor (CD19-CAR) T-cells are established therapies for cancers.
• The roles of hinge (HD) and transmembrane domains (TMD) in CAR structure and function are not fully understood.

Purpose of the Study:

• To investigate the impact of different hinge and transmembrane domains on CD19-CAR T-cell function.
• To elucidate the mechanism by which TMD influences CAR T-cell activity.

Main Methods:

• Generated a series of CD19-CARs with varying HD (CD8, CD28, IgG4) and TMD (CD8, CD28).
• Utilized co-immunoprecipitation and anti-CD28 stimulation assays to assess heterodimerization and T-cell proliferation.
• Analyzed CD28 cell-surface expression and response to CD80/CD86 stimulation.

Main Results:

• CARs with CD28-TMD, unlike CD8-TMD, formed heterodimers with endogenous CD28 in T-cells.
• Heterodimerization efficiency depended on HD and polar amino acids within the CD28-TMD.
• CD28-CAR heterodimers showed reduced CD28 cell-surface expression and did not respond to CD80/CD86.

Conclusions:

• A significant difference exists between CD28-TMD and CD8-TMD in CAR T-cells.
• The CD28-TMD can modulate CAR T-cell activity through interactions with endogenous CD28.
• Understanding TMD function is crucial for optimizing CAR T-cell therapy design.