Loss of interleukin-10 activates innate immunity to eradicate adult T-cell leukemia-initiating cells

Abstract

Adult T cell leukemia/lymphoma (ATL) is associated to chronic human T cell leukemia virus type 1 (HTLV-1) infection and carries a poor prognosis. Arsenic trioxide (AS) and interferon-alpha (IFNα) together selectively trigger Tax viral oncoprotein degradation and cure Tax-driven murine ATL. AS/IFNα/zidovudine treatment achieves a high response rate in patients with chronic ATL. Interleukin 10 (IL-10) is an immuno-suppressive cytokine whose expression is activated by Tax. Here we show that, in ATL, AS/IFNα-induced abrogation of leukemia initiating cell activity requires IL-10 expression shutoff. Loss of IL-10 secretion drives production of inflammatory cytokines by the microenvironment, followed by innate immunity-mediated clearance of Taxdriven leukemic cells. Accordingly, anti-IL-10 monoclonal antibodies significantly increased the efficiency of AS/IFNα therapy. These results emphasize the sequential targeting of malignant ATL cells and their immune microenvironment in leukemia initiating cell (LIC) eradication and provide a strong rational to test AS/IFNα/anti-IL10 combination in ATL.

Figure 1.

Arsenic and interferon-target adult T-cell leukemia/lymphoma cells and their innate immunity to abrogate adult T-cell leukemic-initiating cell activity. (A) SCID mice were injected with 106 cells, derived from the tumoral spleen of tax transgenic mice that developed adult T-cell leukemia/lymphoma (ATL), treated with arsenic trioxide (AS) and interferon-alpha (IFN) from day 18, for 3 days, then sacrificed. One million unsorted or sorted CD25+high or CD25+low spleen-derived cells from primary recipients were injected into secondary SCID mice. Survival of untreated secondary recipients injected with unsorted spleen cells from untreated (control) and AS/IFN-treated primary mice are shown in black and red, respectively (n=10 per condition). Survival of untreated secondary recipients injected with CD25+high or CD25+low cells from untreated primary mice (control) are shown in black crossed and dashed histograms, respectively; those from AS/IFNα-treated primary mice are shown in red crossed and dashed histograms, respectively (n=4/condition). (B) One million CD25+ cells from control or AS/IFNα-treated primary mice were injected alone (n=5 per condition) or together with 106 spleen-derived CD25- cells from AS/IFN-treated primary mice into secondary SCID mice (n=3/condition). Survival of untreated secondary SCID mice injected with CD25+ cells from control or AS/IFN-treated primary mice are represented in black and red histograms, respectively. Survival of secondary SCID mice injected with CD25+ cells from control mice and CD25- cells from AS/IFN-treated primary mice are represented by a dashed red histogram (black contour line). Survival of secondary SCID mice injected with CD25+ cells and CD25- cells from AS/IFN-treated primary mice are represented by a dashed red histogram (red contour line). (C) Survival of untreated secondary SCID (red histograms) and NOG SCID (blue histograms) mice injected with 106 unsorted spleen cells from control or AS/IFNα-treated primary SCID mice (n=7/condition). The t-test was performed to validate significance. *P≤0.05, **P≤0.01, and ***P≤0.001. P-values ≤0.05 were considered statistically significant.

Figure 2.

A critical role for natural killer cells and macro-phages in arsenic/interferon-mediated suppression of adult T-cell leukemic-initiating cell activity. (A) The same experimental design as shown in Figure 1A was followed. Histograms represent survival of untreated secondary SCID mice injected with 106 CD25+ cells from untreated (control; n=5 black histograms) or arsenic trioxide (AS)/interferon alpha (IFN)-treated primary mice (n=5 red histograms); survival of untreated secondary SCID mice injected with 106 CD25+ cells from untreated primary mice and 25,000 CD25– CD335+ NK cells from AS/IFN- treated primary mice (n=3 vertically dashed red histogram with black contour line); survival of untreated secondary SCID mice injected with 106 CD25+ cells from AS/IFN-treated primary mice and 25,000 CD25– CD335+ cells from AS/IFN- treated primary mice (n=3 vertically dashed red histogram with red contour line); survival of untreated secondary SCID mice injected with 106 CD25+ cells from untreated primary mice and 25,000 CD25–CD11b+ F4/80+ cells (macrophages) from AS/IFN-treated primary mice (n=4 oblique dashed red histogram with black contour line); and survival of untreated secondary SCID mice injected with 106 CD25+ cells from AS/IFN-treated primary mice and 25,000 CD25–CD11b+F4/ 80+ cells from AS/IFN-treated primary mice (n=4 oblique dashed red histogram with red contour line). (B) Secondary SCID mice were injected with 106 unsorted spleen cells from untreated (black histograms) or AS/IFN-treated (red histograms) primary SCID mice wth adult T-cell leukemia/lymphoma (ATL). The secondary mice were treated with clodronate or their corresponding empty liposomes, or anti-NK1.1 antibody or mouse IgG isotype control starting on day 3 after injection of cells (n=5/condition). (C) Enzymelinked immunosorbent assay on supernatant of homogenized CD25+ or CD25– sorted cells derived from the spleen of untreated (control) or AS/IFN-treated primary mice for pro-inflammatory cytokines (IL-12 and IFN), as well as the pro-inflammatory monocyte chemoattractant protein-1 (MCP- 1/CCL2) and the macrophage inflammatory protein 1-alpha (MIP-1) chemokines. The t-test was performed to validate significance. *P≤0.05, **P≤0.01, and ***P≤0.001. P-values ≤0.05 were considered statistically significant.

Figure 3.

A critical role for the loss of interleukin-10 production in arsenic/interferon-mediated eradication of adult T-cell leukemic-initiating cell activity. (A) Enzyme-linked immunosorbent assay (ELISA) on supernatant of homogenized CD25+ or CD25– sorted cells derived from the spleen of untreated (control) or arsenic trioxide (AS)/interferon alpha (IFN)-treated primary mice. (B) ELISA on plasma of normal SCID mice (n=3; gray histogram) or SCID mice with adult T-cell leukemia/lymphoma (ATL) injected with 106 spleen cells derived from tax transgenic mice, and left untreated (n=5; black histogram) or treated with AS and IFNfrom day 18 until day 21 (n=5; red histogram). (C) Survival of untreated secondary SCID mice injected with 106 or ten unsorted spleen cells from untreated (control) primary ATL mice (n=3 and n=4, respectively; black), 106 cells from primary mice treated with AS/IFNfor 3 days (n=8; red), or ten cells from untreated primary mice together with 106 cells from primary mice treated with AS/IFNfor 3 days (n=8; red-black dashed). (D) Survival and spleen weight of secondary recipient SCID mice injected with 106 unsorted spleen cells derived from primary mice treated with AS/IFN. These secondary recipients were left untreated (control; black histograms) or treated with recombinant murine (Rec-m) interleukin 10 (IL-10) (red; n=7/condition). (E) Spleen weight and survival of primary ATL SCID mice (n=7/condition) treated with anti-IL-10 antibodies (green histogram) or control isotype (black histogram). (F) Quantitative reverse transcriptase polymerase chain reaction analysis of homogenized CD25+ or CD25- sorted cells derived from the spleens of untreated primary mice or primary mice treated with anti-IL-10 antibodies. The t-test was performed to validate significance. *P≤0.05, **P≤0.01, and ***P≤0.001. P-values ≤0.05 were considered statistically significant.

Figure 4.

Loss of leukemia-initiating cell activity requires innate immunity. (A) Primary mice injected with cells, derived from the tumoral spleen of tax transgenic mice that developed adult T-cell leukemia/lymphoma (ATL), were treated with arsenic trioxide (AS) and interferon alpha (IFN) for 3 days then sacrificed. One million unsorted spleen-derived cells were injected into 50 secondary SCID mice that were left untreated and sacrificed on a weekly basis (5 per week). (B, C) One million unsorted spleen cells derived from weekly sacrificed untreated secondary SCID mice were injected into tertiary SCID (green histograms) (B) or NOG SCID (dark blue histograms) (n=10/condition) (C) mice that were left untreated. (D) Enzyme-linked immunosorbent assay on supernatant of homogenized spleen-derived CD25+ and CD25– sorted cells from weekly sacrificed untreated secondary mice injected with 106 unsorted spleen cells from AS/IFNα-treated primary mice. Control indicates secondary mice injected with 106 unsorted spleen cells from untreated primary mice. The t-test was performed to validate significance. *P≤0.05, **P≤0.01, and ***P≤0.001. P-values ≤0.05 were considered statistically significant.

Figure 5.

Tax knock down in adult T-cell leukemia/lymphoma-derived cells decreased interleukin-10 production. (A) The same experimental design as that illustrated in Figure 1A was followed and secondary SCID mice were left untreated and sacrificed on a weekly basis (left). Interleukin 10 (IL-10) levels were determined by enzyme-linked immunosorbent assay (ELISA) in the supernatant of CD25+ sorted spleen cells from untreated secondary mice injected with 106 unsorted spleen cells from arsenic trioxide (AS)/interferon alpha (IFNα)-treated primary mice sacrificed at week 2 to 4 (each condition represents pooled cells from 3 mice, middle). IL-10 determined by ELISA in the supernatant of homogenized spleen-derived CD25+ and CD25– sorted cells from untreated secondary mice injected with 106 unsorted spleen cells from AS/IFN-treated primary mice sacrificed at week 4 to 8 (right). (B) Western blot for p-STAT3 and STAT3 in untreated secondary mice injected with 106 unsorted spleen cells derived from primary mice with adult T-cell leukemia/lymphoma (ATL) treated with AS/IFN. These secondary mice were sacrificed at weeks 4, 6 and 8. (C) Transcript levels of human IL-10 in untreated ATL-derived (HuT-102 and MT-1) or HTLV-1-negative (Jurkat) cell lines (black histograms) or after 24 h of ex-vivo treatment with AS/IFNalone (red histograms) or combined with the proteasome inhibitor PS-341 (green histograms). (D) Transcript levels of human IL-10 in CD25– (blue histograms) or CD25+ (red histograms) sorted cells from freshly isolated peripheral blood mononuclear cells from six patients with ATL, after exvivo treatment with AS/IFNα. (E) IL-10 levels (ELISA) of green fluorescent protein-positive (GFP+) sorted cells from ATL-derived (HuT-102 and MT-1) or HTLV-1-negative (Jurkat) cell lines following transduction with GFP-lentiviral vectors encoding scrambled shRNA (shScr) or shRNA against Tax (shTax). Gray and red histograms correspond to un-transduced, shScr, or shTax transduced GFP+ sorted cells as indicated. The t-test was performed to validate significance. *P≤0.05, **P≤0.01, and ***P≤0.001. P-values ≤0.05 were considered statistically significant.

Figure 6.

Combination of an anti-interleukin-10 monoclonal antibody, arsenic and interferon abrogates adult T-cell leukemic-initiating cell activity. (A) Kaplan-Meier analysis of overall survival of primary SCID mice injected with 106 unsorted spleen cells and treated with an anti-interleukin (IL)-10 antibody (blue line; n=5), arsenic trioxide (AS)/ interferon alpha (IFN) (red line; n=7) or the triple combination AS/IFN/anti-IL-10 antibody (green line; n=5) or left untreated (control; black line; n=5). (B) Survival of secondary recipient SCID mice injected with 106 unsorted spleen cells derived from primary SCID mice with adult T-cell leukemia/lymphoma (ATL) treated with an anti-IL-10 antibody (blue line; n=5), AS/IFNα (red line; n=6) or AS/IFN/anti-IL-10 antibody (green line; n=10) or untreated controls (black line; n=10). Secondary SCID mice injected with 106 spleen cells derived from primary ATL SCID treated with AS/IFN/anti-IL-10 were treated with clodronate (light blue line; n=5) or anti-NK1.1 (purple line; n=5) starting on day 3 after injection of leukemic cells. (C) Proposed model for therapy-induced ATL cure: ATL cells depend on IL-10 signaling to escape from the innate immune system. Treatment with AS/IFNα induces Tax degradation, resulting in decreased IL-10 production, activation of NK cells and macrophages and innate immunity-mediated abrogation of ATL LIC activity.