Regulatory T cells (Tregs) could maintain the stemness property and inhibit the differentiation of hematopoietic stem/progenitor cells (HSPCs) in normal hematopoietic system. Recent studies have shown that Tregs, as an important component of acute myeloid leukemia (AML) microenvironment, helped AML cells to evade immunoesurveillance. However their function in regulating the stemness property of AML cells remains elusive.

In this study, we analyzed the proportion of Tregs and leukemic stem cells (LSCs) in 68 patients' bone marrow, and found that the proportion of LSCs in Tregs high group (Tregs ≥ 9%) was higher than that in Tregs low group (Tregs < 9%) (Figure A). So, we hypothesized that Tregs up-regulated the stemness property of AML cells. Therefore, we treated Kasumi-1 and SKNO-1 with the supernatant of Tregs culture medium (condition medium, CM) which was isolated from donors, and verified that the proportion of side population (SP) cells was increased (Figure B) and the efficiency of sphere formation was enhanced (Figure C) by CM treatment. Further, Tregs promoted the initiation of AML and shortened the life span of mice in AML1-ETO mice model (Figure D).

As is well known that, Tregs could secrete IL10 cytokine to establish a suppressive microenvironment. Therefore, we analyzed the expression level of IL10 in 35 patients' bone marrow, and found that the expression of IL10 in Tregs high group was higher than that in Tregs low group (Figure E), meanwhile, the expression of IL10 in LSCs high group (LSCs ≥ 5%) was higher than that in LSCs low group (LSCs < 5%). Thus, we treated Kasumi-1 and SKNO-1 with human recombinant IL10 cytokine (rhIL10). rhIL10 treatment increased proportion of SP, enhanced the efficiency of sphere formation and induced both activating phosphorylation of PI3K and AKT at S473 with similar kinetics in a dose-dependent (with concentrations ranging from 1 to 100 nM) manner. Given the ability of AKT to increase the expression of OCT4 and nanog, we analyzed changes in the expression of OCT4 and nanog. As expected, expression levels of OCT4 and nanog increased after treatment with IL10.

To investigate whether IL10 is responsible for stemness property induction, we blocked PI3K/AKT signaling pathway by IL10R neutralizing antibody (IL10R Ab) and PI3K/AKT inhibitor- LY294002 following CM treatment. In contrast to the control (CM treatment only), LY294002 and IL10R Ab could greatly reduce the proportion of SP, lessen the efficiency of sphere formation, and decrease the expression of OCT4 and nanog.

Consequently, our study revealed that Tregs increased the proportion of LSCs in AML through IL10/PI3K/AKT/OCT4/nanog signaling pathway and indicated that targeting on the communication between Tregs and AML cells might be a promising strategy in AML therapy.

Figure
Figure.
View largeDownload slide
Disclosures

No relevant conflicts of interest to declare.

Topics:
culture media, cytokine, interleukin-10, leukemia, myelocytic, acute, regulatory t-lymphocytes, signal pathway, signal transduction pathways, 1-phosphatidylinositol 3-kinase, proto-oncogene proteins c-akt, leukemic hematopoietic stem cell

Author notes

*

Asterisk with author names denotes non-ASH members.

© 2017 by The American Society of Hematology
2017
Sign in via your Institution