Tumor‐associated mesenchymal stem cells inhibit naïve T cell expansion by blocking cysteine export from dendritic cells

Abstract

Mesenchymal stem cells (MSCs) represent an important cellular constituent of the tumor microenvironment, which along with tumor cells themselves, serve to regulate protective immune responses in support of progressive disease. We report that tumor MSCs prevent the ability of dendritic cells (DC) to promote naïve CD4(+) and CD8(+) T cell expansion, interferon gamma secretion and cytotoxicity against tumor cells, which are critical to immune-mediated tumor eradication. Notably, tumor MSCs fail to prevent DC-mediated early T cell activation events or the ability of responder T cells to produce IL-2. The immunoregulatory activity of tumor MSCs is IL-10- and STAT3-dependent, with STAT3 repressing DC expression of cystathionase, a critical enzyme that converts methionine-to-cysteine. Under cysteine-deficient priming conditions, naïve T cells exhibit defective cellular metabolism and proliferation. Bioinformatics analyses as well as in vitro observations suggest that STAT3 may directly bind to a GAS-like motif within the cystathionase promoter (-269 to -261) leading to IL-10-STAT3 mediated repression of cystathionase gene transcription. Our collective results provide evidence for a novel mechanism of tumor MSC-mediated T cell inhibition within tumor microenvironment.

Neem Leaf Glycoprotein Restrains VEGF Production by Direct Modulation of HIF1α-Linked Upstream and Downstream Cascades

“…B16Mel cells were subjected to hypoxia w/wo NLGP for 4 h, and Co-IP of Sp1/3, CBP, p300 with HIF1α was performed as described (23).…”

“…STAT3 specific si-RNA (Santacruz Biotechnology, Dallas, TX) was added in 70% confluent B16Mel culture as described (23). Finally, expression of stat3 and hif1α was checked both in untreated, NLGP treated and siRNA transfected B16Mel cells by RT-PCR.…”

NLGP Attenuates Murine Melanoma and Carcinoma Metastasis by Modulating Cytotoxic CD8+ T Cells

“…Anti-CD25 (PE), anti-MHC-I [clone 28-8-6; clone 25-D1. 16; PE, FITC, and antigen-presenting cell (APC)], anti-MHC-II (PE), anti-CD80 (PE), anti-CD86 (PE), anti-CCR7 (PE), anti-CD44 (PE), and anti-CD69 (FITC) were purchased from Biolegends (San Diego, CA, USA). Purified anti-mouse transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and interferon γ (IFN-γ) mAbs, secondary antibodies either fluorescence or peroxidase labeled, were obtained from e-Biosciences (San Diego, CA, USA).…”

“…Flow-cytometric analysis was done as described (16). The data were generated by fluorometric analysis of 5,000, 10,000, 50,000, or 100,000 events as per experimental requirement.…”

“…Ki67 staining was done as described (16). In brief, cells were harvested and fixed in chilled 75% ethanol with vortexing, followed by incubation at −20 • C overnight.…”