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. 2016 Sep 22;1(15):e84095. doi: 10.1172/jci.insight.84095

Figure 7. CD4+ cell depletion prevents lymphatic vessel sclerosis and improves lymphatic function.

Figure 7

(A) Representative images of hind limbs 18 weeks after DT administration and treatment with isotype control or CD4 mAb (beginning 9 weeks after DT injection). (B) Quantification of increase in foot diameter from baseline in isotype and CD4 mAb–treated mice over time. Red arrow indicates the time at which weekly isotype or CD4 mAb administration was initiated (n = 5/ group; *P < 0.001). (C) Representative H&E image of distal hind limb cross sections harvested 18 weeks after DT administration in isotype and CD4 mAb–treated mice. Fibroadipose tissue of the hypodermis is outlined with dotted black lines and marked with an asterisk (scale bar: 1 mm). (D) Quantification of fibroadipose tissue area in the distal hind limb of control and CD4 mAb–treated animals 18 weeks after DT treatment (*P < 0.001). (E) Representative high-power photomicrographs of collecting lymphatics (podoplanin, green; α-SMA, red) from the distal hind limb of control and CD4 mAb–treated mice 18 weeks following DT administration. Note the luminal narrowing and increase in perilymphatic smooth muscle cell deposition in isotype control–treated mice (scale bar: 20 μm). (F) Quantification of smooth muscle wall thickness surrounding collecting lymphatic vessels in the distal hind limbs of isotype control– and CD4 mAb–treated mice harvested 18 weeks after DT administration (*P = 0.004). (G) Quantification of collecting lymphatic vessel area in distal hind limbs of isotype and CD4 mAb–treated mice (*P = 0.005) 18 weeks after DT administration. (H) Representative NIR lymphangiography of control and CD4 mAb–treated mice demonstrating ICG-containing lymphatic vessels only in CD4-depleted mice (white arrows) (n = 5/group). (I) Representative colocalization of LYVE-1 and CD4+ cells in distal hind limb tissues of isotype and CD4 mAb–treated mice 18 weeks after DT administration (scale bar: 50 μm). (J) Quantification of lymphatic vessel density (LVD) in distal hind limbs of isotype or CD4 mAb–treated animals 18 weeks after DT administration (*P < 0.001). (K) Quantification of CD4+ cells in the tissues of the distal hind limbs of mice treated with isotype or CD4 mAbs 18 weeks after DT administration (*P < 0.001). (L) Representative low- and high-power photomicrographs of Prussian blue–stained popliteal lymph nodes harvested 18 weeks after DT administration from isotype- and CD4 mAb–treated mice (scale bar: 200 μm). (M) Quantification of Prussian blue (ferritin) staining area of popliteal lymph nodes in isotype- and CD4 mAb–treated mice (*P = 0.007). 2-tailed Student’s t test.