American Society of Hematology

Figure 3

$Figure 3. CCR7 mediates prosurvival signals in vivo. (A) Proliferation rate of transplanted Wt (n = 3) and CCR7-deficient lymphoma cells (n = 4) in congenic recipient mice was assessed 16 hours after intraperitoneal injection of 1 mg/animal BrdU. As a control, B cells (B220+) from untreated mice (n = 2), without lymphoma cell and BrdU application, are shown in the top panel. Viable lymphoma cells (gated on B220+/CD45.2+) were stained for BrdU and 7-AAD; gated boxes represent the fraction of BrdU+ proliferating cells (bottom panel). (B) Congenic mice were transferred with 1 × 105 Wt or CCR7−/− Eμ-Myc lymphoma cells. Six (n = 4 mice per group) and 15 days (n = 6 or 7 mice per group) after tumor challenge, gated tumor cells (CD45.2+) were stained with annexin V and 7-AAD and analyzed by flow cytometry. Graph represents fraction of late apoptotic cells (annexin V+/7-AAD+). *P ≤ .05; **P ≤ .01. (C) Frozen spleen sections were stained for CD45.2+ (red; top panel). Tumor cell load for Wt (day 11; n = 3) and CCR7−/− lymphomas (day 28; n = 3) in splenic red pulp was comparable. Scale bars represent 100 μm. TUNEL staining (brown; bottom panel) of paraformaldehyde-fixed spleen sections (Wt, n = 4; CCR7−/− Eμ-Myc splenic tumors, n = 6). Scale bars represent 50 μm. (D-F) Akt phosphorylation on stimulation with CCL19 or CCL21. Freshly isolated LN-derived Wt or CCR7−/− Eμ-Myc lymphoma cells were stimulated with CCL21 (D) and CCL19 (E). In addition, splenic Wt or CCR7−/− B cells were stimulated with CCL21 (F). Cell lysates were analyzed by immunoblotting (top panels) for activation-induced Akt phosphorylation (p-Akt), and quantitation of phosphorylation relative to total Akt is shown in the bottom panels (D-F). One representative experiment of 13 (Wt Eμ-Myc lymphoma cells) and 4 (CCR7−/− Eμ-Myc lymphoma cells) independent experiments (D). One representative experiment of 4 (Wt lymphoma cells) and 1 (CCR7−/− lymphoma cells) independent experiments (E). One representative experiment of 4 (Wt B cells) and 2 (CCR7−/− B cells) independent experiments (F). (G) Chemotaxis of tumor cells derived from Wt Eμ-Myc transgenic mice toward CCL21 (100nM) in the presence or absence of Akt inhibitor (20μM). Error bars represent SD for 4 independent experiments with triplicates per each group. *P ≤ .05.$

CCR7 mediates prosurvival signals in vivo. (A) Proliferation rate of transplanted Wt (n = 3) and CCR7-deficient lymphoma cells (n = 4) in congenic recipient mice was assessed 16 hours after intraperitoneal injection of 1 mg/animal BrdU. As a control, B cells (B220⁺) from untreated mice (n = 2), without lymphoma cell and BrdU application, are shown in the top panel. Viable lymphoma cells (gated on B220⁺/CD45.2⁺) were stained for BrdU and 7-AAD; gated boxes represent the fraction of BrdU⁺ proliferating cells (bottom panel). (B) Congenic mice were transferred with 1 × 10⁵ Wt or CCR7^−/− Eμ-Myc lymphoma cells. Six (n = 4 mice per group) and 15 days (n = 6 or 7 mice per group) after tumor challenge, gated tumor cells (CD45.2⁺) were stained with annexin V and 7-AAD and analyzed by flow cytometry. Graph represents fraction of late apoptotic cells (annexin V⁺/7-AAD⁺). *P ≤ .05; **P ≤ .01. (C) Frozen spleen sections were stained for CD45.2⁺ (red; top panel). Tumor cell load for Wt (day 11; n = 3) and CCR7^−/− lymphomas (day 28; n = 3) in splenic red pulp was comparable. Scale bars represent 100 μm. TUNEL staining (brown; bottom panel) of paraformaldehyde-fixed spleen sections (Wt, n = 4; CCR7^−/− Eμ-Myc splenic tumors, n = 6). Scale bars represent 50 μm. (D-F) Akt phosphorylation on stimulation with CCL19 or CCL21. Freshly isolated LN-derived Wt or CCR7^−/− Eμ-Myc lymphoma cells were stimulated with CCL21 (D) and CCL19 (E). In addition, splenic Wt or CCR7^−/− B cells were stimulated with CCL21 (F). Cell lysates were analyzed by immunoblotting (top panels) for activation-induced Akt phosphorylation (p-Akt), and quantitation of phosphorylation relative to total Akt is shown in the bottom panels (D-F). One representative experiment of 13 (Wt Eμ-Myc lymphoma cells) and 4 (CCR7^−/− Eμ-Myc lymphoma cells) independent experiments (D). One representative experiment of 4 (Wt lymphoma cells) and 1 (CCR7^−/− lymphoma cells) independent experiments (E). One representative experiment of 4 (Wt B cells) and 2 (CCR7^−/− B cells) independent experiments (F). (G) Chemotaxis of tumor cells derived from Wt Eμ-Myc transgenic mice toward CCL21 (100nM) in the presence or absence of Akt inhibitor (20μM). Error bars represent SD for 4 independent experiments with triplicates per each group. *P ≤ .05.

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