Background: Haematopoietic stem cell transplantation (HSCT) is one of the therapeutic options for high-risk paediatric leukaemia. One of the adverse effects of HSCT is graft-versus-host disease (GvHD). Ruxolitinib is one of the drugs used as GvHD treatment. In this context, alloreactive NK cells have been correlated with a lower relapse rate, decreasing GvHD with maintenance of beneficial graft-versus-leukaemia (GvL) effect.
Aims: We aimed to evaluate the role of Ruxolitinib on the NK cell mediated GvL effect.
Methods: Purified NK cells were obtained from healthy donors. NK cells were incubated for 16h with LPS and CpG in presence or absence of Ruxolitinib (0.1, 1, 10 µM). Their gene expression and production of pro-inflammatory cytokines IL-1β, TNFα, IFNγ and IL-6 were analysed by RT-qPCR and ELISA, respectively. Functional capacity was also assessed using DELFIA Cell Cytotoxicity Assay and CD107a degranulation assay against leukaemic cell lines by flow cytometry. To evaluate proliferative capacity, activated and expanded NK cells (NKAEs) were monitored in presence or absence of Ruxolitinib. Phosphorylation of STAT3 and STAT5 of JAK/STAT pathway was studied by western blot.
Results: Stimulated NK cells treated with increasing doses of Ruxolitinib show decreased mRNA expression of TNFα, IFNγ and IL-6 in a dose-dependent manner. In terms of proinflammatory cytokine release, activated NK cells produce lower levels of pro-inflammatory cytokines (TNFα, IFNγ and IL-6) in the presence Ruxolitinib in a dose-dependent manner. The cytotoxic and degranulation capacity of stimulated NK cells was reduced in the presence of increasing doses of Ruxolitinib but not completely abolished, versus K562, SEM and MV-4-11 cell lines. Treatment with 1 and 10 uM Ruxolitinib impairs NKAES cell proliferation, while at low doses of Ruxolitinib NKAES cells proliferate until day 14. Ruxolitinib inhibits phosphorylation of the JAK/STAT pathway with no effect on Toll-Ligand Receptors signalling pathway (activated by LPS and CpG ligands).
Summary/Conclusion: The JAK1/JAK2 inhibitor Ruxolitinib decreases functional and proliferative capacities of NK cells from healthy donor, inhibiting their GvL effect in vitro.