Targeting the Idiotype: Nanobody-Based PET Imaging of Antigen-Specific T Cells in Experimental Autoimmune Encephalomyelitis 2260726

Abstract Introduction Multiple sclerosis (MS) is an autoimmune disease of the central nervous system characterized by demyelination and neuroinflammation. Autoreactive CD4+ T cells that recognize myelin-derived antigens, such as myelin oligodendrocyte glycoprotein (MOG), drive infiltration of immune cells into the brain and spinal cord. Experimental autoimmune encephalomyelitis (EAE), a well-established murine model of MS, recapitulates these features, including activation of MOG-specific T cells (2D2 T cells). Yet, the direct in vivo visualization of these antigen-specific T cells remains challenging. Methods An alpaca was immunized with recombinant 2D2 TCR, and phage display subsequently yielded nanobodies recognizing distinct 2D2 epitopes. We performed adoptive transfers (CD45.1 with CD45.2 2D2 CD4+ T cells) for binding and functional readouts. For imaging, D3 was site-specifically sortase-conjugated to DFO-PEG and labeled with 89Zr. Immune-PET was performed in 2D2-TCR transgenics, Rag⁻/⁻ recipients of 2D2 T cells, and C57BL/6 mice given 2D2 T cells and induced with MOG35—55 EAE. Results Six nanobodies (2A7, 2C1, 2C3, D3, E10, H4) bound distinct 2D2 epitopes with nanomolar affinity. In flow cytometry, D3 enabled single-reagent detection of 2D2 T cells with low background, obviating αTCR-Vα3.2/β11 panels and congenic gating. Functionally, 2C3 and D3 blocked antigen-dependent activation in vitro. 89Zr-DFO-PEG-D3 produced high-contrast PET: it specifically detected 2D2 T cells in 2D2-TCR mice; visualized homing to secondary lymphoid organs in Rag⁻/⁻ recipients; and revealed a distinct spinal-cord signal in EAE mice consistent with infiltrating 2D2 T cells. Conclusion Idiotype-specific nanobodies enable non-invasive, longitudinal imaging of defined antigen-specific T cells in vivo. The approach enables precise, longitudinal monitoring and functional modulation of defined T-cell subsets and can be generalized to additional TCR—pMHC pairs, providing immunologists a practical alternative to conventional antibody panels. Funding Source National Multiple Sclerosis Foundation Topic Categories Technological Innovations in Immunology (TECH)