American Society of Hematology

Figure 1

$Figure 1. Entrance of B cells through LN HEVs. (A) Firm adhesion of transferred B cells. Two images acquired by TP-LSM at 17 and 37 minutes after adoptive B-cell transfer. HEVs outlined by fluorescent nanodots. White arrows indicate transmigrating cells and red arrow direction of blood flow. (B) B cells adhere to the endothelium. The percentage of rolling B cells observed to adhere in 4 separate experiments (left panel) over a 10-minute imaging interval. The number of newly adherent B cells that persisted for at least a minute noted over the indicated time intervals in a single HEV outlined by fluorescent nanodots (similar results in 3 other experiments, right panel). (C) Transmigration of a B cell. Individual images from TP-LSM that illustrate the transmigration of fluorescently labeled B cell (white arrows). Direction of blood indicated with a red arrow. Time is from the initial adhesion. (D) Prolonged association of B cells with HEVs. Individual images from TP-LSM of HEVs revealed by fluorescent nanodots showing transferred B cells at 30 and 80 minutes after transfer in the same HEV, and at 130 and 160 minutes after transfer in another HEV. White arrows show direction of blood flow. (E) Differential usage of HEVs by wild-type and Ccr7−/− B cells. Different HEVs, 1 in the LN cortex (left image) and 1 located near the LN medulla (right image). HEVs revealed by fluorescent nanodots before adoptive transfer of wild-type (red) and 3 times as many Ccr7−/− (green) B cells. Images acquired by TP-LSM. (F) Differential localization of wild-type and Ccr7−/− B cells in the LN. Equal numbers of wild-type and Ccr7−/− B cells were transferred 24 hours before TP-LSM. The location of the medullary region was identified by injection of an LYVE-1 antibody to outline its strong lymphatic staining. Four LN follicles (Foll) are present in the image. Arrowheads indicate Ccr7−/− B cells. Scale bars are 30 μm. (G) Ratio of adherent wild-type B cells to Ccr7−/− B cell on HEVs fractionated by vessel diameter. Venule order 1 through 5 are: > 50 μm, 40-50 μm, 30-40 μm, 20- 30 μm, and < 20 μm, respectively. (H) Transmigration of adherent wild-type and Ccr7−/− B cells. Time for TEM of individual cells measured between images acquired 5 to 35 minutes after transfer. No significant difference in the TEM times was found. (I) Velocity, straightness, and displacement of wild-type and Ccr7−/− B cells were analyzed using images 3 to 4 hours after transfer (*P < .05).$

Entrance of B cells through LN HEVs. (A) Firm adhesion of transferred B cells. Two images acquired by TP-LSM at 17 and 37 minutes after adoptive B-cell transfer. HEVs outlined by fluorescent nanodots. White arrows indicate transmigrating cells and red arrow direction of blood flow. (B) B cells adhere to the endothelium. The percentage of rolling B cells observed to adhere in 4 separate experiments (left panel) over a 10-minute imaging interval. The number of newly adherent B cells that persisted for at least a minute noted over the indicated time intervals in a single HEV outlined by fluorescent nanodots (similar results in 3 other experiments, right panel). (C) Transmigration of a B cell. Individual images from TP-LSM that illustrate the transmigration of fluorescently labeled B cell (white arrows). Direction of blood indicated with a red arrow. Time is from the initial adhesion. (D) Prolonged association of B cells with HEVs. Individual images from TP-LSM of HEVs revealed by fluorescent nanodots showing transferred B cells at 30 and 80 minutes after transfer in the same HEV, and at 130 and 160 minutes after transfer in another HEV. White arrows show direction of blood flow. (E) Differential usage of HEVs by wild-type and Ccr7^−/− B cells. Different HEVs, 1 in the LN cortex (left image) and 1 located near the LN medulla (right image). HEVs revealed by fluorescent nanodots before adoptive transfer of wild-type (red) and 3 times as many Ccr7^−/− (green) B cells. Images acquired by TP-LSM. (F) Differential localization of wild-type and Ccr7^−/− B cells in the LN. Equal numbers of wild-type and Ccr7^−/− B cells were transferred 24 hours before TP-LSM. The location of the medullary region was identified by injection of an LYVE-1 antibody to outline its strong lymphatic staining. Four LN follicles (Foll) are present in the image. Arrowheads indicate Ccr7^−/− B cells. Scale bars are 30 μm. (G) Ratio of adherent wild-type B cells to Ccr7^−/− B cell on HEVs fractionated by vessel diameter. Venule order 1 through 5 are: > 50 μm, 40-50 μm, 30-40 μm, 20- 30 μm, and < 20 μm, respectively. (H) Transmigration of adherent wild-type and Ccr7^−/− B cells. Time for TEM of individual cells measured between images acquired 5 to 35 minutes after transfer. No significant difference in the TEM times was found. (I) Velocity, straightness, and displacement of wild-type and Ccr7^−/− B cells were analyzed using images 3 to 4 hours after transfer (*P < .05).

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