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Targeting IL-10 abrogates ATL LIC
Conversely, treatment of secondary recipients with an anti-IL-10 monoclonal antibody significantly decreased their splenomegaly and prolonged their survival (Figure 3E, Online Supplementary Figure S3B). Importantly, untreat- ed secondary (see Figure 6B) or tertiary (Online Supplementary Figure S3C) SCID mice serially transplanted with 106 spleen cells derived from anti-IL-10-treated pri- mary recipients survived significantly longer than those transplanted with spleen cells derived from untreated pri- mary recipients. These results demonstrate a critical role for IL-10 in ATL LIC activity. Further asserting this pivotal role of IL-10, treatment of ATL SCID mice with an anti-IL- 10 monoclonal antibody significantly increased the pro- duction of the pro-inflammatory cytokines MCP-1, MIP- 1α, IFNγ, IL-12 and IL-15 (Figure 3F), exclusively in the CD25– spleen cell population. These results provide direct evidence implicating the loss of IL-10 production in the observed activation of innate immunity.
and IL-15 (Figure 4D, Online Supplementary Figure S4B) was observed exclusively in the CD25– non-leukemic fraction, starting in week 6 or 7. This delayed production of pro- inflammatory cytokines by non-malignant cells in the microenvironment is concomitant with the delayed loss of ATL LIC activity.
Loss of interleukin-10 production by Tax-driven leukemic cells is an early event preceding the loss of leukemic-initiating cell activity
Contrary to pro-inflammatory cytokines, IL-10 was selectively produced by the Tax-driven CD25+ leukemic subpopulation. IL-10 protein and transcript levels signifi- cantly and rapidly decreased in untreated secondary recip- ients injected with spleen cells from AS/IFNα-treated pri- maries (Figure 5A, Online Supplementary Figure 5A). The decrease of IL-10 levels was accompanied by the reduc- tion of its downstream STAT3 signaling (Figure 5B). Similarly, treatment of primary ATL SCID mice with anti- IL-10 monoclonal antibody resulted in a decrease of pSTAT3 (Online Supplementary Figure 5B). Collectively, these results demonstrate that treatment of Tax-driven leukemic cells with AS and IFN abrogates their produc- tion of IL-10 followed by increased production of pro- inflammatory cytokines by non-leukemic cells and innate immunity-mediated loss of ATL LIC activity.
Tax knock-down in human adult T-cell leukemia/lymphoma cells decreased interleukin-10 production
To translate the findings from our murine model to human cells, we treated ATL-derived cell lines HuT-102 or MT-1 with AS/IFNα for 24 h. Tax protein was detectable in HuT-102 cells but not in MT-1 cells or HTLV-1-negative Jurkat cells (Online Supplementary Figure S5C). As previous- ly reported,28 AS/IFNα induced proteasomal-mediated Tax degradation (Online Supplementary Figure S5C). Treatment with AS/IFNα significantly decreased IL-10 expression, exclusively in ATL-derived cell lines (Figure 5C). Importantly, this AS/IFNα-induced loss of IL-10 produc- tion was rescued upon co-treatment with PS-341 (Figure 5C), suggesting that the effect of AS/IFNα on IL-10 pro- duction involves Tax degradation by the proteasome. Importantly, no effect was observed on the HTLV-1-nega- tive Jurkat T cell line (Figure 5C). Similar results were observed upon ex-vivo treatment of PBMC from six patients with ATL (3 with acute ATL and 3 with chronic ATL). These PBMC were treated ex-vivo with AS/IFNα for 24 h, before sorting the CD25+ and CD25– subpopulations of cells. Of note, and as previously reported, baseline IL- 10 transcripts levels were significantly higher in the PBMC of patients with ATL than in those of HTLV-1-negative controls24 (Online Supplementary Figure S5D). In patients with ATL, IL-10 was predominantly produced by the CD25+ cells and significantly decreased upon treatment with AS/IFNα (Figure 5D). These results are consistent with the previously reported high IL-10 plasma levels in patients with ATL as compared to levels in healthy donors24 and the decrease of IL-10 production following AS/IFNα/AZT therapy.24
To directly demonstrate the role of Tax in IL-10 produc- tion, we knocked down Tax in HuT-102 or MT-1 cell lines using shRNA. Tax knock-down was verified by real-time PCR (Online Supplementary Figure S5E). Extinction of Tax resulted in a significant decrease of IL-10 protein levels
Finally, AS/IFNα-induced abrogation of IL-10 produc- tion was rescued upon co-treatment with the proteasome inhibitor PS-341 (Online Supplementary Figure S3D), in agreement with our previous finding that the curative effect of AS/IFNα requires intact proteasome activity, like- ly involving Tax degradation by the proteasome.21
Arsenic and interferon-α induced loss of adult T-cell leukemic-initiating cell activity is associated with delayed activation of innate immunity
Our results indicate that both AS/IFNα-induced preven- tion of IL-10 production by Tax-driven leukemic cells and AS/IFNα-induced activation of innate immunity are mandatory for the suppression of ATL LIC activity. To dis- sect the sequence of events, 106 unsorted spleen-derived cells from AS/IFNα-treated primary mice were injected into 50 SCID mice that were left untreated. Subsequently, five of these secondary recipients were sacrificed each week until week 9 (Figure 4A). The loss of ATL LIC activ- ity was investigated by serial transplantation of spleen cells from secondary recipients into tertiary SCID or NOG SCID mice that were left untreated. Transplantation of unsorted spleen-derived cells from untreated secondary SCID mice injected with spleen cells from AS/IFNα-treat- ed primary mice into tertiary SCID mice revealed a delayed and progressive loss of LIC activity starting at week 7 and reaching complete exhaustion by week 9 (Figure 4B). Interestingly, spleen cells derived from untreated secondary mice sacrificed at weeks 4, 5 and 6 maintained their long-term LIC activity in serial transplan- tation experiments in tertiary and quaternary recipient SCID mice (Figure 4B green histograms, Online Supplementary Figure S4A). Hence, early isolation of spleen- derived ATL cells from their environment in untreated sec- ondary SCID mice allowed the cells to maintain their leukemia-initiating activity, which would have been lost if the cells had been kept in their original environment. LIC activity was preserved when unsorted spleen-derived cells from untreated secondary SCID mice were transplanted into tertiary NOG SCID mice (Figure 4C), pointing to a critical role of recipient innate immunity in this delayed eradication of LIC activity. To confirm this finding, we measured levels of pro-inflammatory cytokines in sorted CD25+ or CD25– cells from spleens of untreated secondary SCID mice sacrificed at weeks 4 to 8 (see experimental design in Figure 4D). A gradual and significant increase of the production of IL-12, IFNγ, MCP-1, MIP-1α, RANTES
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