In vivo B-cell–primed T cells (TofB) preferentially migrate to lymph nodes. T cells primed by peptide-loaded DC and B cells were differentially labeled with live cell dyes. Equal numbers of TofDCs and TofBs were adoptively transferred intravenously, and distribution of the cells was determined 24 hours after transfer by organ removal and FACS analysis. (A) T cells for adoptive transfer were labeled with CFSE; TofDCs were additionally labeled with CTO and TofBs with DiD. (B) Upon organ analysis after transfer, equal numbers of TofDCs and TofBs as transferred can be found in the spleen, whereas in lymph nodes TofBs clearly dominate. (C) Distribution of the transferred T-cell populations in lymphatic organs 24 hours after transfer. When analysis was performed at day 7 after transfer, a similar pattern was observed (data not shown). The data shown are representative of at least 3 independent experiments. Within the lymph nodes, TofBs migrate to T-cell areas and are excluded from B-cell follicles. A simplified drawing illustrates lymph node architecture showing B-cell areas (blue), T-cell dominated areas [green], and the imaged area shown above (red rectangle) at the transition of the 2 zones. (D) An image stitched together of 6 individual extended focus projections of each 200 × 200 × 144 μm shows the transition from a B-cell follicle dominated by B cells (blue) to a mixed T-B zone containing mostly naive T cells (green). Note that TofBs (red) are found only in the T-cell area. Scale bar: 100 μm. (E) A quantification of TofB distribution analyzing 14 image stacks from within and outside B-cell follicles. TofBs clearly show T-cell homing behavior and localize within T-cell–dominated areas but are excluded from B-cell follicles. Bars represent mean plus or minus SD.