⁸⁹Zr-labeled PEGylated anti-CD8 VHH detects CD8 T cells. (A) Structure of the biorthogonal sortase substrate. The azide functionality allows installation of PEG groups, and the DFO chelator is used to install ⁸⁹Zr for PET imaging. (B) Schematic representation of preparing PEGylated ⁸⁹Zr-labeled VHHs for PET imaging. (C–G and C-II–G-II) PET-CT images of anti-CD8 ⁸⁹Zr-labeled X118-VHH with and without different-size PEG functionalities in wild-type C57BL/6 and RAG-KO mice (n = 3 for each experiment). Images were acquired 24 h p.i. of radiolabeled VHHs. (C-II–G-II, top) Whole-body maximum intensity projections. (C-II–G-II, bottom) Transverse PET-CT images of cross sections through the spleen, showing specific staining and a reduction in accumulation of label in the kidney with increasing PEG size. (H) Characterization of functionalized VHHs. LC-MS analysis confirms formation of X118-DFO and X118-DFO-azide. (I) Biodistribution of anti-CD8 X118-VHH with and without different-size PEGs 24 h p.i. (n = 3 for each cohort). Error bars represent standard deviation.