CCR7 signalling as an essential regulator of CNS infiltration in T-cell leukaemia

Abstract

T-cell acute lymphoblastic leukaemia (T-ALL) is a blood malignancy afflicting mainly children and adolescents. T-ALL patients present at diagnosis with increased white cell counts and hepatosplenomegaly, and are at an increased risk of central nervous system (CNS) relapse. For that reason, T-ALL patients usually receive cranial irradiation in addition to intensified intrathecal chemotherapy. The marked increase in survival is thought to be worth the considerable side-effects associated with this therapy. Such complications include secondary tumours, neurocognitive deficits, endocrine disorders and growth impairment. Little is known about the mechanism of leukaemic cell infiltration of the CNS, despite its clinical importance. Here we show, using T-ALL animal modelling and gene-expression profiling, that the chemokine receptor CCR7 (ref. 5) is the essential adhesion signal required for the targeting of leukaemic T-cells into the CNS. Ccr7 gene expression is controlled by the activity of the T-ALL oncogene Notch1 and is expressed in human tumours carrying Notch1-activating mutations. Silencing of either CCR7 or its chemokine ligand CCL19 (ref. 6) in an animal model of T-ALL specifically inhibits CNS infiltration. Furthermore, murine CNS-targeting by human T-ALL cells depends on their ability to express CCR7. These studies identify a single chemokine-receptor interaction as a CNS 'entry' signal, and open the way for future pharmacological targeting. Targeted inhibition of CNS involvement in T-ALL could potentially decrease the intensity of CNS-targeted therapy, thus reducing its associated short- and long-term complications.

Figure 1. Notch1 activation induces T-ALL and targets leukaemic cells into the CNS

a, Induction of T-ALL in a transplantation model (WT/WT^Notch1-IC). Peripheral blood smears (left), and fluorescence-activated cell sorting (FACS, right) analysis using CD4 and CD8 antibodies are shown. ‘WT^MIG’ denotes wild-type bone marrow infected with a control MIG retrovirus. b, Notch1-IC⁺ EGFP ⁺ cells in the brain meningeal spaces of transplanted mice. c, Infiltrating lymphocytes surrounding a brain vessel in leukaemic (bottom panel) but not in healthy (control, top panel) recipients. Co-staining with CD31 antibodies (blue) indicates endothelial cells within the infiltrating lymphocytes.

Figure 2. CCR7 expression and response to CCL19/CCL21 is induced by Notch1 activation

a, Heat diagram of selected adhesion/migration regulators that are controlled by Notch1-IC. A few classical Notch targets (Dtx1, Gata3, Hey1 and Nrarp) are also included. For all genes, P < 0.001. Yellow and blue denote increased and decreased mRNA abundance, respectively. b, c, Real-time PCR (b) and FACS (c) analysis showing the induction of CCR7 gene and protein expression in haematopoietic progenitors in response to Notch1-IC expression; n = 4. d, Notch1-IC expression induces the chemotaxis of wild-type (WT^Notch1-IC), but not CCR7(KO)^Notch1-IC progenitors towards both CCL19 and CCL21; n = 3. Error bars define s.d. for all experiments.

Figure 3. CCR7 expression is sufficient for CNS infiltration of human T-ALL cells

a, Kaplan–Meyer analysis of recipients that received identical numbers of CEM, DND41 or DND41/mCCR7⁺ cells; n =5. b, Bioluminescent imaging of mice 2 weeks after transplantation with the indicated cell lines. c–d, Infiltration of T-ALL cells into the CNS of recipient mice as shown using bioluminescence and histochemistry.

Figure 4. CCR7–CCL19 interactions are essential for CNS infiltration in an animal model of T-ALL

a, Leukaemic cells in the brain meningeal spaces of the indicated transplanted recipients. A positive control (WT/WT^Notch1-IC) is also included. b–g, Immunofluorescent staining of brain sections: magnification of a brain microvessel in plt/WT^Notch1-IC (b) and WT/WT^Notch1-IC (c) mice. Endothelial, CD31⁺ cells are shown in blue. d–g, CCL19 (red) expression in WT/WT^Notch1-IC (e, f, g) but not plt/WT^Notch1-IC (d) microvessels. f, g, Magnification of a WT/WT^Notch1-IC microvessel and costaining with CCL19 (red) and CD31 (blue).