Fig. 4

WT/Tie-2-eGFP-CLN-5 chimeras highlight endothelial origin of leukocyte CLN-5. Bone marrow cells from WT, non-transgenic donor mice (CD45.1/CD45.2) were transplanted into lethally irradiated about 6-week-old Tie-2-eGFP-CLN-5 host mice (CD45.2) via retro-orbital injection. a At 10 weeks post-transplant, tail bleeds were performed to assess the efficacy of leukocyte substitution. FACS shows PBLs from non-irradiated, host eGFP-CLN-5 mice are all CD45.2⁺ (left), while those in chimeras are approx. 98% CD45.1⁺ CD45.2⁺ (right), indicating host PBLs were nearly entirely replaced. b Two days following confirmation of leukocyte substitution, EAE was induced in chimeras and age-matched non-irradiated, host eGFP-CLN-5 mice (at approx. 16 weeks of age; n = 4). At D8 EAE, PBLs were isolated from both groups of mice and percentages of eGFP-CLN-5⁺ CD45⁺ determined. Control PBLs from naïve, WT mice and PBLs labeled with isotype control antibodies were used to set the gates (data not shown). c z-stack confocal image from the spinal cord section of chimeric mouse at D9 EAE showing eGFP-CLN-5 (green) distribution along the intercellular boundaries of a CNS microvessel (arrows) and associated with an aggregate of perivascular leukocytes (arrowhead). Insert shows high-power field of aggregated eGFP-CLN-5⁺ leukocytes