(A) On the left is a diagram showing the region of the inguinal lymph node that was imaged. The right panel shows the xz projection view of an image stack collected immediately prior to time-lapse imaging, demonstrating the location of a B cell follicle containing transferred B cells (green and red). The collagen-rich lymph node capsule is visualized by second harmonic emission (blue). The dashed white rectangle shows the region used in the time-lapse image analysis.
(B) Time-lapse images of HEL-engaged Ig-tg B cells (green) clustering at the follicle–T zone boundary and naive non-tg B cells (red) in the follicle. The 0-min image is approximately 1 h after antigen exposure (see Video S1). The pathways of an Ig-tg B cell (light blue circle and dotted line) and a non-tg B cell (pink circle and dotted line) are traced as examples. The traced non-tg B cell moved out of the imaging stack at the 57.5-min timepoint. Scale as in (C).
(C and D) Tracks of antigen-engaged Ig-tg (C) and naive non-tg (D) B cells in the xy-, xz-, and yz-planes. The boundary as defined by the area of Ig-tg B cell accumulation at 120 min is shown in lighter gray. Tracks cover 30 to 112 min (C) and 30 to 57 min (D). Circles indicate the end point of tracking.
(E) Velocity distribution for naive (upper histogram) and antigen-engaged (lower histogram) B cells. Data are shown for 44 Ig-tg cells and 31 non-tg cells. Medians are indicated by arrows.
(F) Velocity distributions for non-tg B cells at 1–20 h (red, n = 25) or Ig-tg B cells at 1–3 h (green, n = 29), 6–8 h (light blue, n = 21), or 18–20 h (dark blue, n = 39) after antigen injection. The data are for cells imaged in the boundary regions. Medians are indicated by arrows.
(G) Ratios of log (displacement) to log (path length) of naive B cells (20 cells), antigen-engaged B cells that moved to the boundary (22 cells), or antigen-engaged B cells that did not move to the boundary (22 cells). Points indicate data for individual cells collected at cumulative 30-sec time intervals. The formula for each regression line is indicated. The correlation coefficients, R², were 0.63, 0.80, and 0.67, respectively. Data are from three experiments and are representative of 12 to ∼18 cells from each recording.

(A and B) Lymph node sections of wild-type mice that received CCR7^+/+ Ig-tg (A) or CCR7^−/− Ig-tg B cells (B) and 1 mg of HEL intravenously for 6 h, stained to detect all B cells (brown) and HEL-binding B cells (blue).
(C) B cell (upper three panels) or T cell (lower panel) staining of lymph node sections from wild-type and plt/plt mice as indicated. The wild-type lymph nodes shown in the upper panels were from Igh^a mice that had received CCR7^−/− Igh^b B cells 1 d before. Staining was to detect all B cells (B220, brown) and transferred CCR7^−/− B cells of the Igh^b allotype (dark blue) or T cells (CD3, red) as indicated.
(D and E) Detection of CCL21 concentration gradients in follicle. In (D), adjacent sections were stained for CCL21. The dashed lines indicate the B/T boundary determined by the distribution of B220 staining. The pixel intensity at each position along the filled line extending from the follicle into the deep T zone (line 1) or the interfollicular T zone (line 2) was averaged across 50 μm perpendicular to the line (the width shown by the bar at the T zone end of each line) and plotted against distance from the follicular end of the line (E) in red for line 1 and blue for line 2. As a negative control, the pixel intensity was determined along a line located in the equivalent location in a CD3-stained section (bottom panel of [C] and shown as a green line in [E]). Pixel intensity was measured using Metamorph software after converting colored pixels to gray scale. A decrease in transmitted light intensity indicates an increase in CCL21. Data are representative of more than five lymph nodes from two mice of each type.

(A) Numbers of Ig-tg B cells and TCR7 CD4⁺ T cells in draining (two inguinal) lymph nodes plotted against time after cell transfer. Shown are means ± standard errors of more than three experiments. Immunization of recipient mice with HEL in adjuvant was done 8 h before cell transfer.
(B) Time course of B and T cell division in draining lymph nodes, determined by CFSE dilution.
(C–F) Immunohistochemical analysis of draining lymph nodes at days 1–5 following immunization, stained as indicated. Note redistribution of B cells into interfollicular clusters in the presence of helper T cells. An enlarged version of the boxed region in (C) is shown in Figure S2.
(G) Distribution of Ig-tg B cells at day 1 in the absence of helper T cells, stained as indicated.
Objective magnification in (C), (D), and (G), 5×, and in (E) and (F), 10×.

(A) The histogram shows contact time distribution for Ig-tg B cells and TCR7 CD4⁺ T cells 30 to 50 h after immunization with antigen with adjuvant. Open bars show conjugates that were tracked for the duration of contact and shaded bars show conjugates that could not be tracked for the entire period of contact because the cells entered the field as a conjugate, left the field as a conjugate, or both.
(B) Contact time distribution for Ig-tg B cells and OT-II CD4⁺ T cells 1 to 2 d after antigen priming. Open and shaded bars as in (A).

(A) The xz projection view of an image stack collected immediately prior to time-lapse imaging, demonstrating the location of the B cell follicle containing transferred B cells (green and red). The collagen-rich lymph node capsule is visualized by second harmonic emission (blue). The dashed white rectangle shows the region used in the time-lapse image analysis.
(B) Time-lapse images of HEL-engaged Ig-tg B cells (green) clustering at the follicle–T zone boundary and naive non-tg B cells (red) in the follicle. The 0-min image is approximately 1 h after antigen exposure (see Video S2). Square boxes indicate regions used for directionality analysis, shown in (C). Scale bar is as shown in (A).
(C) Tracks of antigen-engaged B cells originating from 30-μm follicular cubes. The centers of the boxes are placed proportionally to the actual positions of the boxes shown in (A), with each box corresponding to a 30 μm × 30 μm × 30 μm cube. Tracks cover 8 to 95 min. The histograms show the percentage of cells that moved across the sides of the square (solid lines) that face the boundary (left histograms) and the percentage of cells that moved across the opposite sides (dashed lines) of the squares (right histograms). The bottom, middle, and top histograms show the results with cubes that are 20–80, 80–140, and 140–200 μm away, respectively, from the boundary. Data shown in the histograms are pooled from three experiments. *, p < 0.05; **, p < 0.01.
(D) Tracks of naive B cells originating from inside of the middle blue box shown in (A). Tracks cover 5 to 25 min. The histogram shows the percentage of cells that moved across the solid or dashed sides of cubes that are 20–80 μm away from the boundary. Data shown in the histogram are from three experiments.
(E) The dot plots show ratios of the displacement to the path length of 8-min tracks of antigen-engaged B cells (left three graphs) or naive B cells (far right graph) originating from the cubes described in (B–D). The distances from the cubes to the boundary are indicated above the graphs. The left and right plots in each graph are the data of tracks that cross the solid and dashed sides of the cubes, respectively. All (100%) of Ig-tg B cells and 94% of non-tg B cells that crossed the sides of the cubes could be tracked for 8 min. The means of each data group are shown as red bars. *, p < 0.05; **, p < 0.01.

(A) Time-lapse images of HEL-engaged CCR7^−/− (green) and CCR7^+/+ (red) Ig-tg B cells in an inguinal lymph node. The 0-min image is ~3.5 h after antigen exposure (see Video S4). Square boxes indicate regions used for directionality analysis, shown in (B). A CCR7^−/− Ig-tg B cell (light blue circle and line) and a CCR7^+/+ Ig-tg B cell (pink circle and line) are traced as examples.
(B and C) Tracks of antigen-engaged CCR7^+/+ (B) and CCR7^−/− (C) Ig-tg B cells originating from 30-μm follicular cubes, analyzed as described in Figure 2. Tracks cover 3–17 min for CCR7^+/+ cells and 3–22 min for CCR7^−/− cells.
(D) Dot plot showing the percentage of cells that moved across the solid or dashed sides of the cubes. Filled symbols correspond to the data shown in (A–C) and Video S4, open symbols correspond to data obtained in a second time-lapse movie collected 2.5–3.5 h after antigen injection. The cubes were located approximately 20–80 μm from the site of accumulation of CCR7^+/+ Ig-tg B cells.
(E) The dot plots show ratios of the displacement to the path length of 8-min tracks of antigen-engaged CCR7^+/+ or CCR7^−/− Ig-tg B cells originating from the cubes described in (A–D). The left and right plots for each cell graph are the data of tracks that cross the solid and dashed sides of the cubes, respectively. A total of 74% of wild-type B cells and 81% of CCR7^−/− B cells that crossed the sides of the cubes could be tracked for 8 min. The means of each data group are shown as red bars. *, p < 0.01.
(F) Velocity distribution data for CCR7^−/− Ig-tg B cells (green, n = 27) and CCR7^+/+ Ig-tg B cells (red, n = 40) tracked 2.5–4.2 h after antigen injection. The data are pooled from two time-lapse movies. Medians are indicated by arrows.

(A) Time-lapse images of Ig-tg B cells interacting with TCR7 CD4⁺ T cells ~30 h after immunization with HEL in adjuvant, showing T cells moving along behind B cells. The pathways of a B cell (pink dotted line) and a T cell (blue dotted line) remaining bound to each other for more than 1 h are shown (see also Video S5).
(B) Time-lapse images showing the dynamics of a B-T conjugate.
(C) The t-x, t-y, t-z plots of the interacting B (red line) and T (green line) cells traced in (A). Note the B cell makes turns before the T cell (arrows).
(D and E) Velocity measurements of unpaired (D) and paired (E) B and T cells, showing that paired T cells slow to the velocity of the B cell. Velocity data are from 16 cells of each type.
(F) Time-lapse images of a B cell interacting with two T cells.
(G) Time-lapse images showing an encounter of a B cell and a T cell to form a conjugate.