Mesenchymal stromal cells shape the MDS microenvironment by inducing suppressive monocytes that dampen NK cell function
Dhifaf Sarhan, Jinhua Wang, Upasana Sunil Arvindam, Caroline Hallstrom, Michael R. Verneris, Bartosz Grzywacz, Erica Warlick, Bruce R. Blazar, Jeffrey S. Miller
Dhifaf Sarhan, Jinhua Wang, Upasana Sunil Arvindam, Caroline Hallstrom, Michael R. Verneris, Bartosz Grzywacz, Erica Warlick, Bruce R. Blazar, Jeffrey S. Miller
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Research Article Immunology

Mesenchymal stromal cells shape the MDS microenvironment by inducing suppressive monocytes that dampen NK cell function

Abstract

Altered BM hematopoiesis and immune suppression are hallmarks of myelodysplastic syndrome (MDS). While the BM microenvironment influences malignant hematopoiesis, the mechanism leading to MDS-associated immune suppression is unknown. We tested whether mesenchymal stromal cells (MSCs) contribute to this process. Here, we developed a model to study cultured MSCs from patients with MDS (MDS-MSCs) compared with those from aged-matched normal controls for regulation of immune function. MDS-MSCs and healthy donor MSCs (HD-MSCs) exhibited a similar in vitro phenotype, and neither had a direct effect on NK cell function. However, when MDS- and HD-MSCs were cultured with monocytes, only the MDS-MSCs acquired phenotypic and metabolic properties of myeloid-derived suppressor cells (MDSCs), with resulting suppression of NK cell function, along with T cell proliferation. A MSC transcriptome was observed in MDS-MSCs compared with HD-MSCs, including increased expression of the ROS regulator, ENC1. High ENC1 expression in MDS-MSCs induced suppressive monocytes with increased INHBA, a gene that encodes for a member of the TGF-β superfamily of proteins. These monocytes also had reduced expression of the TGF-β transcriptional repressor MAB21L2, further adding to their immune-suppressive function. Silencing ENC1 or inhibiting ROS production in MDS-MSCs abrogated the suppressive function of MDS-MSC–conditioned monocytes. In addition, silencing MAB21L2 in healthy MSC-conditioned monocytes mimicked the MDS-MSC–suppressive transformation of monocytes. Our data demonstrate that MDS-MSCs are responsible for inducing an immune-suppressive microenvironment in MDS through an indirect mechanism involving monocytes.

Authors

Dhifaf Sarhan, Jinhua Wang, Upasana Sunil Arvindam, Caroline Hallstrom, Michael R. Verneris, Bartosz Grzywacz, Erica Warlick, Bruce R. Blazar, Jeffrey S. Miller

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Figure 6

Knockdown of MDS-MSC ENC1 abrogates suppressive function and knockdown of MAB21L2 promotes recapitulates suppression function.

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Knockdown of MDS-MSC ENC1 abrogates suppressive function and knockdown o...
(A) NK cells were cultured for 6 days with monocytes and HD-MSCs or MDS-MSCs (n = 8–10) in direct contact or separated by (T) Transwell inserts in the presence of IL-15 (10 ng/ml) and stimulated with IL-12 and IL-18 and anti-CD16 6 hours prior staining. NK cell function was evaluated for IFN-γ production and proliferation (Ki67) by flow cytometry. (B) (Left) Monocytes were cultured with HD-MSCs and MDS-MSCs in direct contact or separated by Transwell inserts, and their ability to induce MDSC (Lineage−/HLA-DR−/CD33+/CD11b+) was evaluated by flow cytometry. (Right) NK cells were cultured for 6 days with monocytes from cultures in the images to the left in direct contact in the presence of IL-15 (10 ng/ml) and stimulated with IL-12 and IL-18 and anti-CD16 6 hours prior staining. NK cell function was evaluated for IFN-γ production by flow cytometry. Pooled data (n = 3) are shown as mean ± SEM. (C) Monocytes (n = 3) were transfected with nontargeting or MAB21L2-targeting siRNA and cultured with HD-MSCs and evaluated for the production of TGF-β. Alternatively, monocytes (n = 3) were cocultured with MDS-MSCs transfected with nontargeting or ENC1-targeting siRNA and evaluated for the production of TGF-β. (D) NK cells (n = 4) were cultured with monocytes, monocytes cultured with MDS-MSCs for 5–6 days, and MDS-MSCs transfected with nontargeting or ENC1-targeting siRNA and NK cells. IFN-γ production was evaluated by flow cytometry after 6 hours of stimulation with IL-12 and IL-18 and anti-CD16 prior to analysis. Additionally, NK cells were cultured with monocytes cultured with HD-MSCs and monocytes transfected with nontargeting or MAB21L2-targeting siRNA. (E) NK cells (n = 4) were cocultured with monocytes precultured with MDS-MSCs or HD-MSCs treated with catalase. Alternatively, NK cell monocyte cocultures were treated with anti–TGF-β and anti-PD1, and NK cell IFN-γ production was evaluated by flow cytometry after 6 hours of stimulation with anti-CD16 and IL-12 and IL-18. Pooled data are shown as mean ± SEM, and statistical analyses were performed using 1-way ANOVA.