¹⁸F-FAC PET images brain-infiltrating leukocytes and peripheral immune activation in an experimental autoimmune encephalomyelitis model of multiple sclerosis

Introduction: Autoimmune diseases affect 17 million Americans¹. Multiple sclerosis (MS) is an autoimmune disease in which aberrant immune cells attack self-antigens in the central nervous system, leading to demyelination and neurodegeneration^2,3. A host of different therapies exist for treating MS patients, but the most effective therapies such as immune cell-depleting antibodies carry the greatest risk of significant side effects^2,3. Challenges remain in tailoring these therapeutic approaches so as to maximize efficacy while limiting side effects. The ability to non-invasively image a key cell type - brain-infiltrating leukocytes - during disease and in response to therapy would be an important advance. Experimental autoimmune encephalomyelitis (EAE) is the standard preclinical model for MS and is induced in C57Bl/6 mice by injecting an emulsion of MOG_35-55 peptide and Complete Freund's Adjuvant (CFA) followed by pertussis toxin⁴. ¹⁸F-FAC is a deoxycytidine analogue PET radiotracer that measures deoxyribonucleoside salvage activity, which is elevated in cells of the immune system and enhanced during immune cell activation⁵. Our primary goal in this work was to study whether ¹⁸F-FAC PET could be used to image brain-infiltrating leukocytes. A secondary goal was to use ¹⁸F-FAC PET to study immune cell activation and deoxyribonucleoside salvage in this model.

Methods: EAE was induced in male and female C57Bl/6 mice with a MOG_35-55 peptide/CFA emulsion and pertussis toxin. As a control, immunocompromised NOD scid gamma (NSG) mice were injected with the same peptide emulsion and pertussis toxin. The mice were imaged with ¹⁸F-FAC PET at different stages of disease: initiation (Day 7 post-EAE induction), peak disease (Day 14 post-EAE induction), maintenance (Day 21 post-EAE induction), and partial recovery (Day 28 post-EAE induction). Mice were treated with the immunomodulatory drug fingolimod and imaged with ¹⁸F-FAC PET. Tissues were analyzed by autoradiography, ex vivo gamma counting, and histology.

Results: ¹⁸F-FAC accumulation was higher in brains of EAE mice as early as Day 7 post-EAE induction and up through Day 21 post-EAE induction as quantified from the PET images and confirmed through ex vivo gamma counting. ¹⁸F-FAC accumulation in the brain localized to areas of high leukocyte infiltration and correlated with the number of brain-infiltrating leukocytes, suggesting that ¹⁸F-FAC can visualize brain-infiltrating leukocytes in this model. ¹⁸F-FAC accumulation was elevated in the spleen and lymph nodes of EAE mice from Day 7 to Day 28 post-EAE induction. No elevation in ¹⁸F-FAC accumulation was detected in the bone marrow or spinal cord of the EAE mice or in any tissues of the NSG mice treated with the EAE induction protocol. Similar results were obtained for male and female mice. These results suggest that ¹⁸F-FAC PET can visualize immune cell activation in this EAE mouse model. Fingolimod decreased ¹⁸F-FAC accumulation in the EAE mouse brain but had no effect on ¹⁸F-FAC accumulation in the spleen or lymph nodes of these mice, consistent with the proposed mechanism-of-action of Fingolimod to block lymphocyte egress from the secondary immune organs. These results suggest that ¹⁸F-FAC PET could be used to monitor the pharmacodynamics of immunomodulatory drugs.

Conclusions: ¹⁸F-FAC PET visualizes brain-infiltrating leukocytes and peripheral immune cell activation across different stages of disease in the EAE mouse model of MS and can further be used to monitor the effects of an immunomodulatory drug on immune cells in this model. These results suggest a possible role for ¹⁸F-FAC PET in improving the treatment of MS patients and patients suffering from other autoimmune diseases.