Fig. 6

Circulating Th1 cells undergo phenotypic transformation before their infiltration into the retina in glaucoma. A–C Splenic Th1_GL (day 20 days after MB injection, cells pooled from 8 mice) and Th1_Ctr (cells pooled from 8 mice) were sorted by FACS and subjected them to transcriptomic analysis using RNA-seq. A The main category terms associated with cell migration in the biological process class of GO (Gene Ontology) analysis. B Heatmap of DEG involved in cell migration. C The main category terms of KEGG pathway analysis. D Experimental design: Th1_GL and Th1_Ctr were labeled with a CellTrace CFSE Cell Proliferation Kit, and then transferred into naïve WT recipient mice via tail vein (2 × 10⁶ cells per recipient mice), respectively. After 4 days of reconstitution, all recipients were sacrificed and their retinas were collected. E–G Shown are representative results of one of three independent experiments. In each experiment, one random retina from each recipient mouse was used to show Isolectin⁺ vessels and CFSE⁺ cells, and the other was for Iba1 and GFAP staining. n = 4. n refers to the number of retinas for analysis. E Representative confocal images of CFSE-labeled cells (green) in retina flat-mounts, stained for Isolectin (red) and DAPI (blue) (scale bar: 20 μm; inset, 5 μm). Quantification of extravascular CFSE-labeled cells (per microscopic field, size: 266.21 μm²) in retina flat-mounts is presented. In each flat-mount, at least 3 microscopic fields were analyzed in each quadrant and 4 quadrants were examined. F, G Retina flat-mounts of Th1_GL and Th1_Ctr recipients were also stained for F Iba1 and G GFAP. For determination of F Iba1 and G GFAP, the percentage of antibody-labeled areas per microscopic field (size: 319.45 μm²) was quantified. F, G scale bar: 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired Student’s t test was performed