Figure 4.
HIV-Captured DCs Display Robust Migration and Lymphatic Vessel Entry
(A) Cell surface expression of CCR7 and S1PR1 on control or HIV-captured DCs.
(B) Transwell migration analysis of control or HIV-captured DCs in response to media, CCL19/21, or S1P. In some conditions, DCs were pre-treated with anti-CCR7 blocking antibody or FTY720-P before subjecting them to the migration assay. Migration index was calculated as fold increase in the number of DCs that responded to chemokine compared with media alone. Mean chemotactic responses +/− SEM toward CCL19/21 and S1P from three and two independent experiments, respectively, are presented. ∗p < 0.05, Mann-Whitney U test.
(C) Experimental design of the ex vivo skin crawl-in assay. A mixture of control and HIV-captured DCs (1:1) were pre-treated either with vehicle or with both anti-CCR7 antibody and FTY720-P, washed, and overlaid on dermal tissue. Cells that did not enter the tissue were washed and prepared for live-cell imaging. Right panel: representative micrograph of control and HIV-captured DC distributions in the presence or absence of chemokine receptor blockade.
(D) Relative distribution of control or HIV-captured DCs within the visualized dermal lymphatics or interstitium compartment. Mean values from four independent experiments +/− SEM are shown. n.s., not significant, chi-square test.
(E) Relative distribution of HIV-captured DCs before and after chemokine receptor blockade with the visualized dermal lymphatics or interstitium. ∗p < 0.05, chi-square test.
(F) Confinement ratio within the dermal skin layer before or after receptor blockade. Unpaired Student's t test was performed. n.s., not significant. ∗∗∗∗p < 0.001. Confinement ratio is defined as the ratio of the displacement of a cell to the total length of the path the cell has traveled. Data from four independent experiments are shown. Mean ± SEM are shown.
(G) Total displacement of DCs within the dermal skin layer before or after receptor blockade. Mann-Whitney U test was performed. n.s., not significant. ∗∗∗∗p < 0.001. Data from four independent experiments are shown. Mean ± SEM are shown.
(H) Time-lapse micrograph of an HIV-captured DC undergoing intraluminal crawling. Scale bar, 20μm.