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H. El Hajj et al.
Zidovudine (AZT) and interferon-alpha (IFNα) have demonstrated clinical efficacy in patients with leukemic subtypes of ATL.13-17 However, the disease in several patients is primary or secondary resistant to the therapy and progresses or relapses even after a long period of dis- ease control. This highlights the need for more effective therapies to increase the response rate, improve long-term disease control and, most importantly, eradicate the dis- ease. One approach to improve therapy is targeting ATL leukemia-initiating cells (LIC). Arsenic trioxide (AS) and IFNα, in combination, degrade Tax and selectively induce cell cycle arrest and apoptosis in ATL cells.18,19 Patients with ATL treated with a combination of AS, IFNα and AZT achieved complete remissions20 and some of them had a long-lasting responses, even after treatment with- drawal,21 suggesting an effect of the treatment on ATL LIC. Similarly, a recent study showed unprecedented pro- longed remissions of ATL in patients who received AS/IFNα/AZT as a consolidation therapy, with molecular studies demonstrating disappearance of a predominant malignant clone in one patient.22 Although relapse occurred later, it was due to a different clone.22 Likewise, in Tax-driven murine ATL, AS and IFNα cooperate to cure leukemia through an immediate loss of LIC, and a delayed exhaustion of tumor cells, proving the feasibility of selec- tive targeting of ATL LIC.21 Of note, LIC activity was measured and defined as the ability of these cells to initi- ate leukemia in serial transplantation assays.
At diagnosis, the immunosuppressive profile of patients with ATL is mostly characterized by high levels of inter- leukin-10 (IL-10),23,24 which stimulates the proliferation of HTLV-1-infected cells through activation of STAT3 signal- ing.25 In patients with ATL responding to AS/IFNα/AZT treatment, we documented a sharp decrease of IL-10 and a significant increase of IL-2 and IFN-gamma (IFNγ), hence restoring an immunocompetent profile.24
In the present report, we demonstrate a critical role for innate immunity, particularly macrophages and natural killer (NK) cells in AS/IFNα-induced abrogation of ATL LIC activity. Mechanistically, this combination induces loss of IL-10 production by Tax-driven leukemic cells, triggering a subsequent production of pro-inflammatory cytokines by the microenvironment, and activation of innate immunity that eventually mediates clearance of ATL LIC activity. Treatment with the triple combination of AS, IFNα and anti-IL-10 monoclonal antibodies yielded high cure rates in a mouse ATL model. This provides the rationale for clinical trials combining AS/IFNα/AZT with IL-10 suppressive therapy to treat ATL. Beyond ATL, our data reinforce the concept of dual targeting of malignant cells and their immune microenvironment to eradicate LIC activity.
Methods
Treatments
AS was obtained from Sigma-Aldrich (St Louis, USA) and recombinant human IFNα (Roferon®) from Hoffman-La Roche (Basel, Switzerland). The proteasome inhibitor PS341 or borte- zomib (Velcade®) was purchased from Millenium Pharmaceuticals. For in vivo experiments, mice received AS (5 μg/g/day) intraperitoneally, IFNα (106 IU/day) subcutaneously, and bortezomib (0.5 mg/Kg/day) through mini-osmotic pumps (Alzet, Charles River). Treatment protocols were initiated at day 15 (bortezomib) or day 18 (AS and IFNα) following intraperitoneal
injection of spleen cells from tax transgenic mice (spleen cells), and given for the duration of 6 days (bortezomib) or 3 days (AS and IFNα ).
Anti-IL-10 antibody (JES5-2A5, BioXCell, EUROMEDEX) or its isotype control (InVivoMAb rat IgG1, BioXCell), were adminis- tered by intraperitoneal injection, starting on day 7 after injection of spleen cells. These antibodies were given at the dose of 500 mg on a biweekly basis for 2 weeks, followed by a continuous biweekly dose of 200 mg in surviving mice until the animals’ death or sacrifice.
Recombinant mouse IL-10 (Biolegend, Ozyme) was given once weekly by intraperitoneal injection at the dose of 200 ng to sec- ondary ATL mice starting on day 7 following injection of spleen cells from AS/IFNα -treated primary mice.
Clodronate or empty liposomes were obtained from Encapsula NanoSciences LLC and administered intraperitoneally at the dose of 50 mg/kg, starting on day 3 following intraperitoneal injection of spleen cells twice weekly over 2 consecutive weeks, followed by one injection in the third week.
Anti-NK1.1 antibody (clone PK136) or its mouse IgG isotype control was obtained from BioXCell. Starting on day 3 after injec- tion of spleen cells in SCID mice, both antibodies were adminis- tered intraperitoneally, at the dose of 250 mg, on a weekly basis, until the death of the mice.
Mice
A murine ATL transplantation model in which 106 unsorted spleen cells from two independent tax transgenic mice that devel- oped murine ATL8,21 was used throughout the study. Spleen cells were inoculated intraperitoneally into SCID mice (Charles River, France). All recipient mice rapidly developed massive hyperleuko- cytosis, splenomegaly, hypercalcemia, multiple organ invasion, and constitutive activation of nuclear factor-κB. Tax expression was detectable only at very low levels by quantitative polymerase chain reaction (PCR) (Online Supplementary Figure S1A). These fea- tures are reminiscent of those in patients with ATL or tax trans- genic mice. All untreated mice died within 3 to 4 weeks and these murine ATL could be serially passaged for years, with very con- stant time-to-death in all recipients, pointing to the remarkable stability of this murine ATL model.
To study the role of the innate immune microenvironment in recipient mice, primary ATL SCID mice were treated with AS/IFNα for 3 days. Spleen-derived cells were transplanted into secondary SCID or NOG SCID mice (Jackson, USA) that were left untreated and sacrificed weekly. In serial transplantation assays, spleen-derived cells from weekly sacrificed secondary ATL SCID mice were injected into tertiary SCID or NOG SCID mice and subsequently quaternary SCID mice.
All mice protocols were approved by the Institutional Animal Care and Utilization Committee of the American University of Beirut. All animals were housed in specific pathogen-free housing. Animals were sacrificed by cervical dislocation following deep anesthesia with isoflurane.
Patients’cells
Peripheral blood mononuclear cells (PBMC) from two normal donors, three patients with chronic ATL and three patients with acute ATL were separated using Ficoll and cultured in RPMI sup- plemented with 10% fetal bovine serum and antibiotics. Blood was collected after informed consent in accordance with the Declaration of Helsinki. PBMC were treated, ex-vivo, with arsenic (1 mM) and IFNα (103 IU) for 24 h, and sorted based on CD25 expression. Transcript levels of IL-10 were quantified by real-time PCR in both CD25+ and CD25– subpopulations using appropriate primers (Table 1).
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haematologica | 2021; 106(5)