Discriminating Post-Transplant Rejection from Infection by Detecting TCR-CD3 Oligomerization on Extracellular Vesicles Using a Ratiometric Caliper Probe

Post-transplant rejection and infection remain significant obstacles to long-term patient survival. However, there is currently no standardized assay to simultaneously assess an individual patient’s risk for both complications. Here, we demonstrate that T cell receptor (TCR)-CD3 oligomers on extracellular vesicles (EVs) represent a promising biomarker for acute cellular rejection. To leverage this, we developed a caliper-shaped aptamer probe to quantify the ratio of TCR-CD3 oligomeric to monomeric EVs derived from CD8+ cytotoxic T cells. This method inherently normalizes the differences in overall EV abundance and minimizes variability arising from plasma input volume (5–15 μL) and storage conditions, thereby ensuring highly robust results. In murine models, the assay yielded markedly higher fluorescence ratios in mice experiencing allograft rejection than in those with infection (mean 0.26 vs 0.14). Crucially, in a clinical cohort of 34 transplant recipients, the assay reliably distinguished rejection and infection with overlapping clinical manifestation and also accurately identified rejection even in patients with concurrent rejection and infection. At a diagnostic cutoff value of 0.69, the assay demonstrated robust performance, with an area under the curve of 0.85, corresponding to a sensitivity of 71% and a specificity of 90%. Ultimately, this approach offers a minimally invasive tool for routine immune monitoring, with the potential to guide immunosuppressive therapy and reduce the reliance on biopsies.