FIGURE 6.

TE-B cells do not promote trophoblast invasion or migration by indirect effect on T cells. A, Timeline of T-cell stimulation, resting period, and collection of media is shown. B, For invasion assays, trophoblast spheroids were incubated on matrigel combined with medium from TCR-stimulated T cells alone (Tstim) or in the presence of conditioned medium from TE-B cells (TE-B_CM) or normal B cells (B-CM). Medium alone or stromal cell medium was used as negative and positive controls, respectively. No effect on invasion by any of the conditioned media was observed. Representative photographs at 48- and 96-h timepoints of the trophoblast spheroids in the matrigel invasion assay are shown. C, Independent invasion (left panel) and migration (right panel) experiments were assessed and given an activity score. Combined data from at least three biologically independent experiments are shown. D, For migration assays, trophoblast spheroids were individually monitored for attachment and migration of trophoblast cells for 48 h. No migration was induced by stimulated T-cell culture media with or without conditioned media from TE-B or normal B cells. Representative photographs of trophoblast spheroids at 0-, 12-, 24-, 36-, and 48-h timepoints in the migration assay are shown. All photographs were taken using the Revolve Pro microscope or the IncuCyte ZOOM System at 200× magnification and are representative of three other independent experiments performed in duplicate