Fig. 8

Th1 cells required for retinal endothelial VCAM-1 upregulation in glaucoma. A Flow cytometry was performed to quantify peripheral blood β1⁺ CXCR3⁺ CD4⁺ T cells (Ctr, n = 10; GL, n = 8) (n refers to the number of enrolled mice, and shown are representative results of one of three independent experiments). B Flow cytometry was performed to quantify retina-infiltrating β1⁺ CXCR3⁺ CD4⁺ T cells (Ctr, n = 10; GL, n = 10; shown are pooled data from multiple experiments; n refers to the number of flow cytometry tests; 4 retinas from 4 individual mice (one retina from each mouse) were combined for each flow cytometry test; the total number of enrolled mice were: Ctr = 40; GL = 40). C, D Shown are representative results of one of three independent experiment. C Anti-CXCR3 or D anti-IFN-γ treatment was performed as described in Fig. 5H or Fig. 4E. Retina flat-mounts were prepared and stained for VCAM-1 and fluorescence intensity (FI) of VCAM-1 was quantified. n = 4, n refers to the number of retinas used for staining and one random retina was analyzed per mouse. E Shown is a schematic of the cell co-culture design: Th1_GL (cells pooled from 8 mice) and Th1_Ctrl (cells pooled from 8 mice) cells sorted by FACS were co-cultured in vitro with mRMECs (a mouse retinal microvessel cell line), respectively. After 3 days of co-culture, each well was washed to remove non-adhesive T cells. F The mRNA expression of VCAM-1 in remaining cells was determined by qRT-PCR. Each RNA test for qRT-PCR was pooled from 3 wells of cells. n = 4 tests. G Immunofluorescence staining for VCAM-1 was performed after non-adhesive T cells were removed. Representative images of VCAM-1-expressing mRMECs (scale bar: left, 20 μm; right, 20 μm). VCAM-1 fluorescence intensity (FI) was evaluated. In each culture well, at least 3 microscopic fields (size: 159.73 mm²) were analyzed. n = 5. H 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. I Shown are results representing one of three independent experiment. Retina flat-mounts of Th1_GL and Th1_Ctr recipients were stained for VCAM-1 (scale bar: 500 μm; inset, 20 μm). For determination of VCAM-1 expression, fluorescence intensity (FI) was quantified. n = 4, n refers to the number of retinas used for staining for VCAM-1 and one random retina of each recipient mouse was analyzed. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired Student’s t test was performed