Fig. 6.

Competitive interactions between LPS mOVA B cells and pre-established CpG mOVA B-cell–OTII T-cell pairs. (A) A typical partner-swapping event initiated by an LPS mOVA B cell contacting an established CpG mOVA B-cell–OTII T-cell pair. A short-lived three-way interaction (*) is followed by displacement of the responding T cell and establishment of a new long-lived pair. (Imaging time in min:s; scale bar, 10 μm.) (B) Reciprocal partner swap by an LPS mOVA B cell with an established CpG mOVA B–OTII pair forming a three-way contact between the cell types (*) followed by a partner swap and then a reciprocal swap also preceded by a three-way contact (*). (Imaging time in min:s; scale bar, 10 μm.) (C) Percentage of contacts initiated by an approaching B cell with either the B cell (79%) or T cell (21%) in three-way contacts (n = 94, five separate experiments). (D) Duration of contacts between OTII T cells paired with LPS B cells (8.6 ± 1.9 min, n = 58) or with CpG B cells (11.3 ± 1.4 min, n = 113) or in a triplet with both B-cell types (4.7 ± 0.59 min, n = 57). Data are from seven separate experiments. Filled circles indicate contacts in which the start and/or finish was not visualized. (E) Bars show the percentages of different outcomes of three-way contacts initiated by either LPS (Left) or CpG mOVA B cells (Right); n = 94 in five separate experiments. Swarms were not used to determine partner-swapping outcomes. No significant differences were apparent in partner retention (69%), partner exchange (25% for both LPS and CpG B cells), or dissolution of all contacts (5% for LPS and 7% for CPG B cells). (F) The outcome of three-way contacts after renormalizing to account for the sixfold-higher rate of three-way contact initiation in favor of LPS over CpG mOVA B cells. (G) An LPS mOVA B-cell swarm attracted to a newly formed CpG mOVA B-cell–OTII T-cell pair [formed at 5:45 (min:s), red arrow], at the peak of the swarm [22:31 (min:s), blue arrow], and after a partner swap in favor of an LPS mOVA B cell followed by the swarm dissipating [30:06; (min:s)] (Movie S9). (H) The mean instantaneous velocity of the swarming B cells (shown in G) that were in-frame at the time of the formation of a CpG mOVA B-cell–OTII T-cell pair is plotted against time. The red bar indicates a mean overall velocity 8.0 μm/min. A fourth B cell that entered from out of frame was not included in the analysis. The red arrow indicates the initiation of CpG mOVA B-cell–OTII T-cell contact; the blue arrow indicates the time when the LPS mOVA B-cell swarm contained the greatest number of cells.