FIGURE 3.
Antibody-mediated cross-linking of LYVE-1 triggers HA binding in native untransfected lymphatic endothelial cells. The effects of LYVE-1 cross-linking on bHA binding were assessed by incubating suspensions of live unfixed and untransfected HDLEC with bHA together with bivalent hLYVE-1 mAbs under various different conditions as indicated in the figure. A and B show representative FACS histograms for HDLEC after initial staining for LYVE-1 (blue, control IgG; red, LYVE-1/Alexa 488 conjugate; A) or following incubation in the presence of bHA either alone or together with the hLYVE-1 mAb 8C or isotype-matched control IgG or a combination of mAb 8C and the hLYVE-1 HA-blocking mAb 893, prior to fixation and detection with streptavidin Alexa 647- and Alexa 488-conjugated Abs as appropriate. C and D, the effects on HA binding of incubating HDLEC with either bHA and IgG (control); bHA alone, followed by washing and reincubation with mAb 8C; bHA and mAb 8C together; mAb 8C alone, followed by washing and reincubation with bHA; or bHA + mAb 8C + HA-blocking mAb 891 added together. Data are shown as two-color FACS analyses for bHA binding as a function of LYVE-1 surface level (C) and as a bar chart in which the binding data are analyzed quantitatively (D; data are mean ± S.D. (error bars), n = 3). The graph in E shows the levels of bHA binding in relation to LYVE-1 surface expression in HDLEC co-incubated with bHA and the individual hLYVE-1 mAbs indicated or control IgG prior to analysis by flow cytometry. Values are mean fluorescence intensity (MFI) of bHA binding (streptavidin Alexa 647) within set gated intervals of LYVE-1 surface expression (mean fluorescence intensity; Alexa 488-conjugated Ab). Data shown are in each case from single representative experiments that were repeated at least twice.