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H. El Hajj et al.
Figure 1. Arsenic and interferon-α 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 (IFNα) 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 (con- trol) and AS/IFNα-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 pri- mary 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/IFNα-treated primary mice into secondary SCID mice (n=3/condi- tion). Survival of untreated secondary SCID mice injected with CD25+ cells from control or AS/IFNα-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/IFNα-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/IFNα-treated primary mice are repre- sented 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.
Other materials and statistical analysis
Cell lines, their treatment and their transduction with short hair- pin RNA (shRNA), flow cytometry and cell sorting, apoptosis assays, quantitative PCR, immunoblot analysis, enzyme-linked immunosorbent assays (ELISA), b-glactosidase histochemical staining and the statistical analysis are described in the Online Supplementary Methods section.
Results
Arsenic and interferon-α target both Tax-driven leukemic cells and their microenvironment to abrogate adult T-cell leukemic-initiating cell activity
SCID mice were injected with tax transgenic malignant
reported21 and in agreement with the loss of LIC activity,21 treatment of primary mice with AS/IFNα reduced the tumor bulk (defined as spleen weight x tax DNA) in untreated secondary recipient SCID mice (Online Supplementary Figure S1J). Importantly, sorted CD25+ cells derived from AS/IFNα-treated primary mice exhibited preserved LIC activity, contrary to unsorted spleen cells (Figure 1A). This prompted us to investigate whether the effect of AS/IFNα on LIC activity was mediated by the Tax-negative non-ATL CD25– population. SCID mice
cells and subsequently left untreated or were treated with
either untreated or AS/IFNα-treated primary mice (Figure 1B). The survival of untreated secondary recipient mice was significantly increased only when they were injected with both CD25– and CD25+ cells derived from AS/IFNα- treated primary mice (Figure 1B). This result suggests that AS and IFNα exert a dual effect on both Tax-driven leukemic cells and non-leukemic cells to abrogate ATL LIC activity.
AS and IFNα for 3 days starting on day 18, then sacrificed.
As previously reported,21 this short-term treatment of pri-
mary mice did not affect tumor load (reflected by Tax
transgene DNA) or Tax expression in the leukemic cells
(Online Supplementary Figure S1A). One million unsorted
spleen-derived cells were then injected into untreated sec-
ondary recipient SCID mice. As previously reported,21 the
survival of secondary recipients transplanted with cells
from previously treated mice was longer than that of sec-
ondary recipients transplanted with cells from untreated
To directly assess whether AS/IFNα-induced loss of ATL LIC activity involves the innate immunity, we took advan- tage of the difference in the immune background of SCID and NOG SCID mice. While SCID mice are known to have natural killer (NK) cells and functional macrophages, NOG SCID mice lack NK cells and have impaired macrophage functions.26 Transplantation of spleen cells derived from AS/IFNα-treated primary SCID mice into secondary recipient SCID or NOG SCID mice revealed that the LIC activity was only abrogated in SCID mice (Figure 1C, compare red and blue histograms). All togeth- er, these results implicate the innate immune cells of both the donor and the recipient in clearance of LIC activity.
mice (Figure 1A), reproducing the formerly reported loss
of ATL LIC activity in mice treated with AS/IFNα.21 To
investigate the basis of this effect, we sorted spleen-
derived cells from untreated or AS/IFNα-treated primary
transplanted mice based on CD25 (CD25–, CD25+low or
CD25+high). CD25 is expressed on normal regulatory T cells
but is also highly expressed in ATL cells.8 Both CD25+high
and CD25+low fractions contained bona fide leukemic cells
A critical role for natural killer cells and macrophages in arsenic/interferon-α-mediated suppression of adult T-cell leukemic-initiating cell activity
The importance of the innate immunity in both donors and recipients in AS/IFNα-induced suppression of LIC activity prompted us to further investigate the role of macrophages and NK cells in the observed phenotype. SCID mice were injected with 106 CD25+ cells from untreated or AS/IFNα-treated primary mice along with 25,000 sorted macrophages (CD25–CD11b+F4/80+) or NK cells (CD25–CD335+) from AS/IFNα-treated primary mice (Figure 2A). Significantly increased survival was observed exclusively in untreated secondary recipients injected with CD25+ cells from AS/IFNα-treated primary mice together with either macrophages or NK cells also derived from AS/IFNα-treated primary mice (Figure 2A, Online Supplementary Figure S2A). These results demonstrate a
(Online Supplementary Figure S1B). Both CD25 – and CD25+low fractions displayed similar LIC activities, as all secondary recipients inoculated with 106 cells from either subpopulations developed leukemia (Online Supplementary Figure S1C-E) and succumbed at 3 weeks after injection (Figure 1A). Conversely, leukemia development was not observed in secondary recipients of CD25– cells (data not shown).
as they carried the tax transgene, unlike the CD25 cells +high
Treatment with AS/IFNα induced a slight decrease of the percentage of CD25+ cells, predominantly the CD25+high fraction (Online Supplementary Figure S1F). Importantly, fractions of CD25+ cells derived from AS/IFNα-treated primary recipients exhibiting features of apoptosis or senescence were similar to those of CD25+ cells from untreated primary recipients (Online Supplementary Figure S1G-I). Moreover, as previously
6–
were thus injected with 10 CD25 cells from AS/IFNα-
6+
treated primary mice along with 10 CD25 cells from
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haematologica | 2021; 106(5)