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

Introduction/Rationale: 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.