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(Figure 5E) in the ATL-derived cell line, which expresses high levels of Tax (HuT-102), and in the ATL-derived cell line, which does not express detectable Tax protein (MT- 1) (Figure 5E, Online Supplementary Figure S5C), but not in HTLV-1-negative Jurkat cells (Figure 5E). Although Tax protein was undetectable in the MT-1 cell line (Online Supplementary Figure S5C), these cells have been shown to sporadically express Tax and such expression is critical for the survival of the whole cell population.11 These results highlight the role of Tax in IL-10 production and confirm in human primary ATL cells and ATL-derived cell lines that AS/IFNα treatment decreases IL-10 production.
The triple combination of arsenic, interferon-α and anti-interleukin-10 monoclonal antibodies cures murine adult T-cell leukemia/lymphoma
The critical role of IL-10 shutdown in suppression of ATL LIC activity incited us to combine IL-10 suppressive therapy with AS and IFNα. SCID mice were injected with 106 spleen cells and treated as indicated in Figure 6A. Treatment with AS/IFNα or with anti-IL-10 monoclonal antibodies significantly prolonged the survival of primary ATL SCID mice, whereas treatment with the triple combi- nation yielded a 6-month survival of around 80% of mice (Figure 6A).
To assess the effects of these combinations on LIC activ- ity, primary ATL SCID mice received short-term treat- ment (for 3 days) with AS/IFNα, anti-IL-10 antibodies, or their triple combination (see experimental design in Figure 6B). No significant changes in spleen weight, in tax DNA or in the percentage of CD25+ cells were observed follow- ing short-term treatment of these primary mice, as com- pared to the untreated group (Online Supplementary Figure S6A). Importantly, significant increases of the production of the pro-inflammatory cytokines IFN-γ and IL-15 as well as MCP-1 and MIP-1α were observed exclusively in the CD25– spleen cell subpopulation sorted from the treated groups. Furthermore, an additive effect of AS/IFNα and anti-IL-10 antibodies was noted (Online Supplementary Figure S6B). Conversely, IL-10 transcripts (Online Supplementary Figure S6B) and protein (Online Supplementary Figure S6C) were produced by the CD25+ population and were downregulated after treatment with AS/IFN, but only IL-10 protein levels were downregulated by anti-IL-10 antibodies (Online Supplementary Figure S6B and C).
As previously reported,21 short-term treatment of pri- mary ATL mice with AS/IFNα significantly decreased the ability of Tax-driven leukemic cells to initiate leukemia in untreated secondary recipients. This was demonstrated by the increased survival of untreated secondary SCID mice from a median of 23 days (range, 20-24) to a median of 70 days (range, 67-76) (Figure 6B). Similar results were observed upon short-term treatment of primary mice with anti-IL10 antibodies, which significantly increased sur- vival of untreated secondary mice to a median of 55 days (range, 45-62) (Figure 6B). Strikingly, treatment of primary mice with the triple combination of AS/IFNα/anti-IL10 antibodies almost totally abrogated ATL LIC activity in untreated secondary mice (Figure 6B).
Finally, ATL LIC activity was rescued completely when secondary SCID mice, injected with spleen cells derived from primary SCID mice treated with the triple combina- tion of AS/IFNα/anti-IL10 antibodies, were further treated with clodronate or anti-NK1.1 antibodies, confirming the
critical role of macrophages and NK cells in the therapeu- tic efficacy of this triple combination (Figure 6B). These results provide a strong rationale for testing the potential therapeutic effect of the combination of AS/IFNα and anti-IL10 in patients with ATL.
Discussion
In this study, we unraveled the mechanisms of the cur- ative therapeutic effect of AS/IFNα in murine ATL. We demonstrated that this combination targets both malig- nant cells and innate immune cells to suppress ATL LIC activity. Indeed, malignant growth was impaired only when malignant cells were exposed to AS/IFNα in the presence of intact innate immunity in both primary donor mice and secondary recipient mice (Figure 6C). Yet, innate immunity alone was not able to prevent growth of untreated malignant cells. At the molecular level, we showed that ATL long-term self-renewal is dependent on IL-10 signaling and AS/IFNα therapy abrogated IL-10 pro- duction by the malignant cells. This triggers activation of innate immunity, which subsequently eliminates ATL LIC activity (Figure 6C).
LIC rely on a tight crosstalk between signals from leukemic cells and their neighboring immune microenvi- ronment cells, through their respective cytokine produc- tion, for their activity and self-renewal. Indeed, malignant cells release suppressive cytokines and chemokines to evade immune detection and hamper the activity of immune cells.29,30 In that sense, patients with ATL exhibit an immunosuppressive cytokine profile characterized by high levels of IL-10 at diagnosis.23,24,29 IL-10 can be pro- duced by ATL cells, HTLV-1-infected non-ATL cells or even by HTLV-1-negative cells.23 Furthermore, besides its potential immunosuppressive properties,31 IL-10 promotes proliferation of HTLV-1-infected cells through the STAT3 and IRF4 pathways.25 Interestingly, it was demonstrated that IRF4 and nuclear factor-κB drive ATL maintenance.32 Similarly, a recent study showed that the HTLV-1 anti- sense bZIP factor HBZ upregulates expression of IL-10, interacts with STAT1 and STAT3 and modulates the IL- 10/JAK/STAT signaling pathway.33 In this study, we demonstrate that IL-10 is produced by malignant cells in Tax-driven murine ATL and, importantly, AS/IFNα, known to induce Tax degradation, decreased IL-10 levels. Similarly, ex-vivo treatment of ATL-derived cell lines or pri- mary cells from patients with ATL with AS/IFNα decreased IL-10 expression. These results demonstrate a great similarity with the high IL-10 levels in patients with ATL and the decrease of IL-10 following AS/IFNα/AZT therapy.24
One prominent observation in our study is the crucial role of innate immunity, particularly NK cells and macrophages, in therapy-induced suppression of ATL LIC and disease cure. This innate immunity is activated by the decrease in IL-10 levels, known to suppress macrophage activity.31 Loss of IL-10, following treatment with AS/IFNα or monoclonal antibodies, activates innate immunity. This triggers the production of pro-inflammatory cytokines by NK cells, macrophages, and potentially other components of innate immunity such as dendritic cells and neutrophils, eventually leading to abrogation of ATL LIC activity (Figure 6C). Our results presumably indicate that ATL cells evade the innate immune system through the production
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