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

BORGE M; NANINNI P; MORANDE P; ZANETI S; GALLETTI J; BEZARES RF; SANCHEZ AVALOS JC; GIORDANO M; GAMBERALE R

Workshop; XIII International Workshop on CLL; 2009

T-cells from chronic lymphocytic leukemia (CLL) patients may actually 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. By performing chemotaxis assays towards CXCL12 (1g/ml), CCL21 (1g/ml) and CCL19 (5g/ml), we sought to evaluate the migratory potential of T cells from CLL patients. We found that T cells from CLL patients are less responsive to these chemokines than T cells from healthy adults (p<0.01, n=28 CLL samples and n=14 healthy donors), despite similar CXCR4 and CCR7 expression. We next analyzed the data from chemokine-induced migration of T cells by discriminating CLL samples in high or low risk groups. To this aim, we evaluated the expression of the poor prognostic factors CD38 and ZAP-70 in CLL cells by flow cytometry. We observed that, although not statistically significant, T cells from CD38- patients were less responsive to CXCL12 and CCL19, while a comparable response was found for CCL21 compared to CD38+ samples. Of note, when we analyzed T cell migration in ZAP-70+ and ZAP-70- subsets, a lower migratory capacity towards CXCL12 in ZAP-70- samples (n=16) compared to ZAP-70+ samples (n=12) was found. It should be mentioned that this different migratory capacity was highly significant (p=0,009) despite T cells from both groups of CLL patients expressed similar CXCR4 surface levels.     Once we compared the migration indexes of T cells towards CXCL12 from healthy donors and CLL groups segregated by ZAP-70, we surprisingly found that only T cells from ZAP-70- samples showed a significant defective migratory capacity compared to healthy donors (p<0.001). By contrast, no statistically significant differences were found between T cells from ZAP-70+ CLL patients and healthy donors. Given that CXCL12-induced migration seems to be regulated by intricated mechanisms besides CXCR4 expression, we further investigated other molecules and mechanisms capable to regulate CXCL12 responsiveness of T cells from CLL patients. We found that CXCL12 induces similar time- and dose-dependent CXCR4 endocytosis in T cells from healthy donors, ZAP-70+ and ZAP-70- CLL patients. Moreover, since it was previously reported that ZAP-70, CD38 and CD45 expression was associated with an enhanced response through CXCR4, we evaluated their expression in T cells from CLL patients without finding any significant difference between ZAP-70+ and ZAP-70- groups.    Finally, given that previous work has demonstrated that the presence of leukemic cells can induce specific changes in T cells from CLL patients, we decided to evaluate whether CLL cells from ZAP-70- and ZAP-70+ samples can modulate autologous T cell responses towards CXCL12. To this aim, purified T cells were cultured alone or with autologous CLL cells (ratio T cells: CLL cells = 1:4). T cell chemotaxis towards CXCL12 was evaluated with freshly purified cells and 48hs cultured cells. A comparable migratory capacity towards CXCL12 were found between freshly purified T cells alone (pT) or with autologous CLL cells (pT+pCLL) in both ZAP-70- and ZAP-70+ samples. Similar results were obtained with T cells from ZAP-70+ patients after 48hs of culture (n=9).  By contrast, in all of the ZAP-70- patients evaluated (n=10), pT cells cultured for 48hs alone displayed a higher migratory capacity towards CXCL12 (p=0.001) compared to T cells cultured with autologous CLL cells (pT+pCLL), although they both showed similar CXCR4 surface expression.     In summary, the results presented in this study demonstrate that T cells from ZAP-70- patients have a defective migratory response to CXCL12 which might be caused by inhibitory signals provided by the leukemic clone itself. We propose that leukemic cells from ZAP-70- patients would impair T cell migratory responses towards CXCL12, restricting the entry of circulating T cells to lymphoid organs. Since T cells in proliferation centers may help CLL cells to survive and proliferate, the low migratory response towards CXCL12 in T cells from ZAP-70- CLL patients might favor the indolent clinical course of the diseases in these patients.