Contributions
Abstract: EP604
Type: E-Poster Presentation
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
B-cell receptor (BCR) signalling plays a pathogenic role in Chronic Lymphocytic Leukemia (CLL); in CLL, there is constitutive activation of the BCR, and the CD40L-CD40 signalling is impaired. We have recently demonstrated that miR-181b is a downstream effector of B and activated T cells' interaction. Indeed, miR-181b is an effector of the CD40L-CD40 pathway, which induces death of CLL cells by increasing the cytotoxic activity of T cells versus the leukemic clone. We thus hypothesized that also miR-181a could stimulate this process. Considering the crosstalk between the CD40L-CD40 and the BCR pathway, we speculated the involvement of the miR-181a also in the BCR pathway.
Aims
Assess the involvement of miR-181a in BCR signalling pathway; evaluate the ability of miR-181a to induce the cytotoxic activity of T cells.
Methods
Since the BCR stimulation induces an heterogeneous response in the expression of miRNAs due to BCR stereotype, to assess the involvement of miR-181a in BCR signalling pathway, CLL cells were treated in vitro with Ibrutinib, a BCR inhibitor. The expression of the miR was then analysed in CLL samples from patients treated with Ibrutinib. To evaluate whether the transcription factor cFOS take part in BCR signalling, its expression was analysed after Ibrutinib treatment. To test whether the transcription factor cFOS regulates the miR-181a, the expression of the miR was analysed after overexpression or silencing of cFOS. To assess if the miR-181a could promote the cytotoxic activity of T cells, Cytotoxic T lymphocytes (CTLs) were generated by mixing CD40L-stimulated and miR-181a-overexpressed CLL cells with autologous T cells.
Results
We observed an increase in miR-181a expression after in vitro inhibition of BCR signalling; we observed the same effect also in vivo in most of CLL patients treated with Ibrutinib. The increased expression of miR-181a inversely correlated with the amount of White Blood Cells (WBC) and, specifically, it was higher in those patients for which the WBC decreased more than 50%. To explain a possible mechanism by which the expression of miR-181a increase, we focused on the transcription factor cFOS, which we already found involved in the transcriptional regulation of miR-181b in CLL; we found that cFOS expression decreased after Ibrutinib treatment in vitro and that overexpression of cFOS downregulated miR-181a, while its silencing increased the expression of the miR in CLL cell line.
Since the overexpression of miR-181a was associated with a reduction of the WBC in vivo, we sought to identify a mechanism by which the miR could affect cell survival. To this end, we focused on the IL10, whose downregulation through miR-181b increase the cytotoxic activity of CTL. We found that miR-181a overexpression downregulated the levels of IL10 after the CTL generation assay in half of the analysed patients, and this was associated with the increase in the percentage of CTL. Conversely, in those patients for which we did not observed a reduction of IL10, there were no effect on CTL amount. Previous studies demonstrated that the IL10 was upregulated by cFOS protein and that cFOS was a target of miR-181a. Considering that cFOS negatively regulates miR-181a, we suggest that the circuitry consisting of cFOS/miR-181a could play a role in BCR signalling and CLL cells death.
Conclusion
MiR-181a is upregulated by BCR signalling inhibition in vitro and in vivo. The overexpression of miR-181a promotes the generation of autologous cytotoxic T cell versus the leukemic clone through the downregulation of IL10.
Keyword(s): Chronic lymphocytic leukemia, CTL, Ibrutinib
Abstract: EP604
Type: E-Poster Presentation
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
B-cell receptor (BCR) signalling plays a pathogenic role in Chronic Lymphocytic Leukemia (CLL); in CLL, there is constitutive activation of the BCR, and the CD40L-CD40 signalling is impaired. We have recently demonstrated that miR-181b is a downstream effector of B and activated T cells' interaction. Indeed, miR-181b is an effector of the CD40L-CD40 pathway, which induces death of CLL cells by increasing the cytotoxic activity of T cells versus the leukemic clone. We thus hypothesized that also miR-181a could stimulate this process. Considering the crosstalk between the CD40L-CD40 and the BCR pathway, we speculated the involvement of the miR-181a also in the BCR pathway.
Aims
Assess the involvement of miR-181a in BCR signalling pathway; evaluate the ability of miR-181a to induce the cytotoxic activity of T cells.
Methods
Since the BCR stimulation induces an heterogeneous response in the expression of miRNAs due to BCR stereotype, to assess the involvement of miR-181a in BCR signalling pathway, CLL cells were treated in vitro with Ibrutinib, a BCR inhibitor. The expression of the miR was then analysed in CLL samples from patients treated with Ibrutinib. To evaluate whether the transcription factor cFOS take part in BCR signalling, its expression was analysed after Ibrutinib treatment. To test whether the transcription factor cFOS regulates the miR-181a, the expression of the miR was analysed after overexpression or silencing of cFOS. To assess if the miR-181a could promote the cytotoxic activity of T cells, Cytotoxic T lymphocytes (CTLs) were generated by mixing CD40L-stimulated and miR-181a-overexpressed CLL cells with autologous T cells.
Results
We observed an increase in miR-181a expression after in vitro inhibition of BCR signalling; we observed the same effect also in vivo in most of CLL patients treated with Ibrutinib. The increased expression of miR-181a inversely correlated with the amount of White Blood Cells (WBC) and, specifically, it was higher in those patients for which the WBC decreased more than 50%. To explain a possible mechanism by which the expression of miR-181a increase, we focused on the transcription factor cFOS, which we already found involved in the transcriptional regulation of miR-181b in CLL; we found that cFOS expression decreased after Ibrutinib treatment in vitro and that overexpression of cFOS downregulated miR-181a, while its silencing increased the expression of the miR in CLL cell line.
Since the overexpression of miR-181a was associated with a reduction of the WBC in vivo, we sought to identify a mechanism by which the miR could affect cell survival. To this end, we focused on the IL10, whose downregulation through miR-181b increase the cytotoxic activity of CTL. We found that miR-181a overexpression downregulated the levels of IL10 after the CTL generation assay in half of the analysed patients, and this was associated with the increase in the percentage of CTL. Conversely, in those patients for which we did not observed a reduction of IL10, there were no effect on CTL amount. Previous studies demonstrated that the IL10 was upregulated by cFOS protein and that cFOS was a target of miR-181a. Considering that cFOS negatively regulates miR-181a, we suggest that the circuitry consisting of cFOS/miR-181a could play a role in BCR signalling and CLL cells death.
Conclusion
MiR-181a is upregulated by BCR signalling inhibition in vitro and in vivo. The overexpression of miR-181a promotes the generation of autologous cytotoxic T cell versus the leukemic clone through the downregulation of IL10.
Keyword(s): Chronic lymphocytic leukemia, CTL, Ibrutinib