Isolation of Structurally Heterogeneous TCR-CD3 Extracellular Vesicle Subpopulations Using Caliper Strategy

Cells in different states can release diverse types of extracellular vesicles (EVs) that participate in intracellular communication or pathological processes. The identification and isolation of EV subpopulations are significant to explore their physiological functions and clinical value. In this study, structurally heterogeneous T-cell receptor (TCR)-CD3 EVs were proposed and verified for the first time using a caliper strategy. Two CD3-targeting aptamers were designed in the shape of a caliper with an optimized probe distance and were assembled on gold nanoparticles (Au-Caliper) to distinguish TCR-CD3 monomeric and dimeric EVs (m/dCD3 EVs) in skin-transplanted mouse plasma. Phenotyping and sequencing analysis revealed clear heterogeneity in the isolated m/dCD3 EVs, providing the potential for mCD3 EVs as a candidate biomarker of acute cellular rejection (ACR) and holding great prospects for distinguishing EV subpopulations based on protein oligomerization states.

Automated summary

Cells in different states release diverse extracellular vesicles (EVs) that play roles in cell communication and pathological processes. This study introduces a caliper strategy to identify and isolate structurally heterogeneous T-cell receptor (TCR)-CD3 EVs. The use of CD3-targeting aptamers assembled on gold nanoparticles allows distinguishing TCR-CD3 monomeric and dimeric EVs (m/dCD3 EVs) in skin-transplanted mouse plasma. Phenotyping and sequencing analysis reveal heterogeneity in the isolated m/dCD3 EVs, showing potential for mCD3 EVs as a biomarker for acute cellular rejection (ACR) and the ability to distinguish EV subpopulations based on protein oligomerization states.