Implication of STAT-5 Recruitment in the Expression of IRF-8 and in the Impairment of Human Plasmacytoid Dendritic Cells Differentiation Induced by BCR-ABL and GM-CSF

It is currently established that the immune system plays a critical role in the control of chronic myelogenous leukemia (CML). Several cell populations including T cells, dendritic cells (Dc) and NK cells have been shown to exhibit potential anti-leukemic activities. We have previously shown that plasmacytoid dendritic cells (pDc) differentiation and IFN-a production was impaired along the chronic phase (CP) of CML and that the Tyrosine- Kinase (TK) activity of BCR-ABL was responsible for this impairment. Moreover, patients in complete cytogenetic remission (CCyR) after imatinib mesylate (IM) or IFN-a therapy present a partial restoration of the pDc compartment and IFN-a production. Some recent results (Esashi et al., Immunity, 2008) indicate that, in mice, STAT-5 but not STAT-3, block the expression of IRF-8, a transcription factor required for the differentiation of pDc and that the GM-CSF-induced STAT5 activation interferes with pDc differentiation. As STAT3 and STAT5 proteins are important targets of BCR-ABL TK activity, we postulate that their recruitment could be responsible for the CML pDc impairment and could be involved in the persistent pDc dysfunction in CR patients.

We decided to investigate the effect of BCR-ABL or GM-CSF on STAT-3, STAT-5 and IRF8 status ex vivo, on CMN of patients who have achieved sustained CCyR and major molecular response (MMR) after IFN-a therapy and in whom treatment has been discontinued (n=8) compared to CMN of healthy subjects (HS) (n=5). The same investigations were also performed in vitro with the model of pDc differentiation of CD34⁺ haematopoietic progenitors from CP patients and HSs, using the combination of Stem Cell Factor, Flt3-Ligand and Thrombopoietin,.

Flow cytometry analysis showed that the homeostasis of the IFN-a production was altered in CCyR patients : pDc (determined by the coexpression of CD303, CD123 and CD4), represent 34.6 ± 14.9 % of the IFN-a producing cells after stimulation by Influenza virus and intracellular detection of IFN-a, whereas in healthy donors (HD) IFN-a producing cells were principally pDc (72.1 ± 20.5 %of IFN-a⁺ cells, p<0.05 in comparison with the CCyR patients group). The IRF-8 expression analysed by flow cytometry was also deregulated as low levels of IRF8 were detected in these cells when compared with pDc of HD (based on mean fluorescence analysis of the IRF-8 staining). Moreover, higher levels of P-STAT5 were observed in CMN and pDc from CCyR patients (65.1 ± 18 % of pDc was P-STAT5 ⁺) than from HD (0.75 ± 0.66 % of pDc are P-STAT5 ⁺, p<0.01 in comparison with the CR patients group). The involvement of Human STAT5 in the repression of pDc differentiation was confirmed in our in vitro model. We showed that GM-CSF blocked the pDc differentiation of normal CD34⁺ cells and enhanced the P-STAT5 levels. We showed higher levels of P-STAT5 along differentiation of CD34⁺ cells of CML patients in CP, in correlation with the lack of differentiation and maturation of pDc.

Altogether, our results suggest that in Human STAT5 is a target for BCR-ABL and GM-CSF and is responsible for the defect of pDc differentiation and IFN-a production. After CCyR, the pDc defect is only partially restored, principally because a STAT5 recruitment persists, possibly induced by soluble factors produced by the residual disease.