CXCL12-induced chemotaxis is impaired in T cells from patients with ZAP-70-negative chronic lymphocytic leukemia

Abstract

Background: T cells from patients with chronic lymphocytic leukemia may play an important role in contributing to the onset, sustenance, and exacerbation of the disease by providing survival and proliferative signals to the leukemic clone within lymph nodes and bone marrow.

Design and methods: By performing chemotaxis assays towards CXCL12, CCL21 and CCL19, we sought to evaluate the migratory potential of T cells from chronic lymphocytic leukemia patients. We next analyzed the chemokine-induced migration of T cells, dividing the chronic lymphocytic leukemia samples according to their expression of the poor prognostic factors CD38 and ZAP-70 in leukemic cells determined by flow cytometry.

Results: We found that T cells from patients with chronic lymphocytic leukemia are less responsive to CXCL12, CCL21 and CCL19 than T cells from healthy adults despite similar CXCR4 and CCR7 expression. Following separation of the patients into two groups according to ZAP-70 expression, we found that T cells from ZAP-70-negative samples showed significantly less migration towards CXCL12 compared to T cells from ZAP-70-positive samples and that this was not due to defective CXCR4 down-regulation, F-actin polymerization or to a lesser expression of ZAP-70, CD3, CD45, CD38 or CXCR7 on these cells. Interestingly, we found that leukemic cells from ZAP-70-negative samples seem to be responsible for the defective CXCR4 migratory response observed in their T cells.

Conclusions: Impaired migration towards CXCL12 may reduce the access of T cells from ZAP-70-negative patients to lymphoid organs, creating a less favorable microenvironment for leukemic cell survival and proliferation.

Figure 1.

T-cell migration towards CXCL12, CCL21 and CCL19 and the expression of CXCR4 and CCR7 in T cells from healthy donors and CLL patients. (A) PBMC from CLL patients or healthy adults were placed in the upper chamber of a 24-transwell plate; medium alone (control wells) or with the optimal dose of CXCL12 (1 μg/mL), CCL21 (1 μg/mL) or CCL19 (5 μg/mL) as chemoattractant was placed in the lower chamber. After 2 h of culture the migrating cells were suspended and divided into aliquots for counting with a FACSCalibur or for immunophenotyping. The migration index for each sample was calculated by determining the ratio of migrated T cells in chemokine-treated wells versus control wells, taking the spontaneous migration in control wells as 100%. NS: not significant; *P<0.01 Mann-Whitney test. (B) Freshly collected whole blood from CLL patients or healthy donors was incubated with saturating concentrations of PerCP-conjugated anti-CD3 antibodies and PE-conjugated anti-CXCR4 (clone 12G5) or anti-CCR7 (clone 3D12), or the appropriate isotype control antibody for 30 min at 4ºC. After selective red blood cell lysis the white cell pellet was evaluated by flow cytometry. Results are expressed as the mean fluorescence intensity (MFI) of CXCR4 and CCR7 expression on CD3⁺ cells for each sample analyzed. NS: not significant, Mann Whitney test.

Figure 2.

T-cell migration towards CXCL12, CCL21 and CCL19 in CLL patients separated according to CD38 and ZAP-70 expression. The figure shows the migration indices of T cells from CLL patients divided according to CD38 expression using cut-off values of 7% (A) and 30% (B) and according to ZAP-70 expression (C). NS: not significant, *P<0.01 Mann Whitney test. (D) Migration towards CXCL12 of T cells from ZAP-70⁻ and ZAP-70⁺ CLL patients and healthy donors. Black boxes correspond to CD38⁺ CLL patients and white boxes represent CD38⁻ CLL patients using the 7% cut-off value. T cells from ZAP-70⁻ CLL patients had lower migration indices towards CXCL12 than T cells from ZAP- 70⁺ CLL patients and healthy donors. **P<0.001; *P<0.05; NS: not significant; one-way ANOVA, Bonferroni’s multiple comparison test.

Figure 3.

The impaired migratory capacity of T cells from ZAP-70⁻CLL patients was not related to defective CXCR4 down-regulation or a particular actin polymerization defect. (A) PBMC from six ZAP-70⁻(dotted line), six ZAP-70⁺ CLL patients (dashed line) and six healthy donors (solid line) were incubated with medium alone (control) or with CXCL12 (1 μg/mL) at 37ºC for 0, 2, 5, 15 and 60 min and the remaining CXCR4 on CD3⁺ cells was assessed by flow cytometry. The graph shows the percentage of CXCR4 on the surface of CD3⁺ cells treated with CXCL12 relative to the control. (B) PBMC from ZAP-70⁻ and ZAP-70⁺ CLL patients and healthy donors were incubated for 60 min with different concentrations of CXCL12 (0, 10, 100, 1000 ng/mL) at 37ºC. The expression of remaining CXCR4 on the surface of T cells was then analyzed by flow cytometry. The data shown represent the mean±SEM (n=6) for the percentage of CXCR4 expression on the surface of CXCL12-treated T cells relative to control T cells (medium alone). (C) Intracellular F-actin was measured using FITC-labeled phalloidin in purified T cells from three ZAP-70⁻CLL patients, three ZAP-70⁺ CLL patients and three healthy donors after the addition of CXCL12 (1000 ng/mL) for 15, 30 and 300 sec. Results are shown as the mean±SEM of the percent of intracellular F-actin relative to the value before the addition of CXCL12. *P<0.05 Mann-Whitney test (CLL versus healthy donors).

Figure 4.

CLL cells from ZAP-70⁻ patients reduce T-cell migration towards CXCL12. Purified T cells from ZAP-70⁻ (n= 10) and ZAP-70⁺ CLL patients (n=9) were cultured in complete medium alone (pT cultures) or at a 1:4 ratio with autologous purified CLL cells (pT+pCLL cultures). Chemotaxis assays towards CXCL12 (1 μg/mL) were performed with freshly purified cells (A) and 48 h cultured cells (B). The migration of CD3⁺ cells was calculated by taking the migration index of T cells in pT+pCLL cultures as 100%. Bars represent the mean values±SEM for the CD3⁺ migration. NS: not significant, *P=0.001, Wilcoxon’s signed rank test. The lower panels show the migration indices of pT+pCLL and pT from 48 h cultured cells of each patient, *P<0.01, Wilcoxon’s signed rank test.