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 5

Quantitative PCR validates differences with ENC1 expression increases in MDC-MSCs and MAB21L2 expression decreases in monocytes exposed to MDS-MSCs, both of which lead to suppression mediated by TGF-β.

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Quantitative PCR validates differences with ENC1 expression increases in...
Total RNA (n = 3–5) was isolated from monocytes alone, cytokine -derived MDSCs, monocytes precultured with HD-MSCs and MDS-MSCs, or HD-MSCs and MDS-MSCs, and the total mRNA was reverse transcribed into cDNA and analyzed by real-time quantitative PCR for (B) MAB21L2 and INHBA or (A) ENC1 gene expression. (C) Alternatively, RNA was isolated from MDS BM monocytes to evaluate MAB21L2 expression. Gene expression was normalized to the housekeeping gene GAPDH and calculated relative to control. (D) HD-MSCs and MDS-MSCs (n = 4) were analyzed by flow cytometry for total ROS following treatment with or without H2O2 (250 μM). (E) Monocytes cultured with HD-MSCs and MDS-MSCs were evaluated for intracellular TGF-β (sTGFβ). Representative and cumulative data (n = 5) are shown as mean ± SEM. (F) Monocytes cultured with HD-MSCs and MDS-MSCs were evaluated for membrane-bound TGF-β (mTGFβ). Representative and cumulative data (n = 3) are shown as mean ± SEM. (G) Cryopreserved BM mononuclear cells were cultured in medium overnight for recovery from freezing prior to CD14 cell isolation. Cells were stimulated with LPS for 6 hours prior to staining for mTGFβ expression in monocytes. Representative and cumulative data (n = 5) are shown as mean ± SEM. Statistical analysis were performed using the Mann-Whitney U test.