Spleen vascular smooth muscle cells and lung increase CCL9 expression during cGVHD. (A) Immunofluorescence staining (×200 magnification) of spleen sections of BM only and cGVHD mice at day 58 post-HCT. White arrow indicates CCL9 was dominantly expressed by spleen vasculature, but not CD11b cells or cells in the GC areas. Quantification of CCL9 staining areas suggests cGVHD mice have increased CCL9 expression in spleen. (B) Costaining of αSMA with CCL9 in the spleen. Images were taken at ×400 magnification. αSMA-positive cells of cGVHD mice had increased CCL9 expression that was not altered by CCL9 blockade. Images were quantified by Fiji software “color segmentation” plugin. (C) CCL9 expression in lung. Quantification was done by Fiji software “measure” function. Data shown are representative of 2 to 3 independent experiments with 5 to 8 mice per group. An unpaired Student t test was used when comparing 2 groups. Significance: *P < .05; **P < .01; ***P < .001; ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole.