Fig. 5

Th1 cells aggregate microglia and deteriorate glaucomatous RGC loss. A Pooled data from 3 independent experiments are shown. Flow cytometry was performed to quantify retina-infiltrating CXCR3⁺ CD4⁺ T cells of Ctr (n = 10) and GL (day 20, n = 10). n refers to the number of flow cytometry tests. Four retinas from 4 individual mice (one retina from each mouse) were combined for each flow cytometry test. B Representative confocal image of a whole-mount retina of a MB-injected mouse (from 8 mice) with CD4 specific expression of Tomota (red), stained for CXCR3 (green) (scale bar: 20 μm). C, D Shown are results representing one of three independent experiments. C qRT-PCR was performed to measure the retinal expression levels of Cxcl10 mRNA. Ctr, n = 8; GL, n = 6. n refers to the number of RNA tests. Two retinas from 2 mice (one retina from each mouse) were combined for each RNA test. D Representative confocal image of a whole-mount retina of GL and Ctr mouse, stained for Iba1, CXCL10, and DAPI (scale bar: 20 μm). The number of Iba1⁺ CXCL10⁺ cells were quantified. n = 5, n refers to the number of retinas used for Iba1 staining and one retina was analyzed per mouse. E Shown is a schematic of the cell co-culture design: Splenic CXCR3⁺ Th1 cells sorted by FACS from GL (cells pooled from 8 mice, day 20, referred to as Th1_GL) and Ctr mice (cells pooled from 8 mice, referred to as Th1_Ctrl) were co-cultured in vitro with BV2 cells (a mouse microglial cell line), respectively. After 48 h of co-culture, each well was washed to remove non-adhesive T cells. RNA-seq was performed to analyze the gene expression profile in remaining BV2 cells (two BV2 samples per group). F Immune response-associated pathway analysis of genes enriched in BV2 cells. G Heatmap of DEG involved in immune responses. H Experimental design: glaucoma was induced and mice were intraperitoneally injected with anti-CXCR3 (αCXCR3) or isotype antibody every 7 days from day 15 after MB injection to the end of indicated experiments. I–K Shown are results representing one of three independent experiments. In each experiment, one random retina from each recipient mouse was used for RGC density and the other was for Iba1 and GFAP staining (isotype, n = 5; αCXCR3, n = 4; n refers to the number of retinas for analysis). RGC number, J the percentage of GFAP⁺ area per microscopic field (size: 319.45 μm²), and K the number of microglia (magnification: morphological changes) were calculated. I scale bar: 500 μm; inset, 20 μm. J, K scale bar: 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance. two-tailed unpaired Student’s t test was performed