Figure 2.

ZAP70 regulates CCR7 and CXCR4 expression, and CCR7 inhibition is relevant for homing processes. A: CCR7 and CXCR4 expression in 697-shZAP70 in comparison to 697-shGFP cells as measured by qRT-PCR (left) and by extracellular FACS staining (right), black: isotype, red: 697-shGFP and blue: 697-shZAP70. Unpaired t-test, two-sided P-value. B: ZAP70, CCR7 and CXCR4 expression in 697 cells transduced with either pMIG or pMIG-ZAP70, unpaired t-test, two-sided P-value. C: 697-shGFP or -shZAP70 cells were subjected to a migration assay toward CCL19 or CXCL12. Cells in the lower chamber were counted by flow cytometry or trypan blue. The migration index is defined as the number of transmigrating cells in the presence of the chemokine divided by the number of transmigrating cells to control medium. Results are shown as the mean ± SEM of 4 independent experiments (unpaired t-test, two-sided P-value). D: Migration assay of 697-pMIG and 697-pMIG-ZAP70 toward CCL19 or CXCL12 (unpaired t-test, two-sided P-value). E: 697 or JURKAT cells were injected into NSG mice, 5 mice were treated with 1 mg/kg anti-CCR7 antibody on day +1, +3 and +7, 5 mice were treated with vehicle alone. Spleen volume as assessed by the formula longest length × highest height × broadest width (unpaired t-test, two-sided P-value). F: Spleen and BM infiltration by human leukemic blasts in control and treated animals (unpaired t-test, two-sided P-value). G: Semi-quantitative CNS scoring of control and treated animals (Fisher´s exact test, one-sided P-value. *P is significant). *P<0.05; **P<0.01, ***P<0.001, n.s. = not significant. BM: bone marrow; CNS: central nervous system; Sp: spleen; shGFP: shRNA against GFP(green fluorescent protein); shZAP70: shRNA targeting ZAP70 (zeta-chain-associated protein kinase 70).