The bidirectional tumor - mesenchymal stromal cell interaction promotes the progression of head and neck cancer

Introduction: Mesenchymal stromal cells (MSC) are an integral cellular component of the tumor microenvironment. Nevertheless, very little is known about MSC originating from human malignant tissue and modulation of these cells by tumor-derived factors. The aim of this study was to isolate and characterize MSC from head and neck squamous cell carcinoma (HNSCC) and to investigate their interaction with tumor cells. Methods: MSC were isolated from tumor tissues of HNSCC patients during routine oncological surgery. Immunophenotyping, immunofluorescence and in vitro differentiation were performed to determine whether the isolated cells met the consensus criteria for MSC. The cytokine profile of tumor-derived MSC was determined by enzyme-linked immunosorbent assay (ELISA). Activation of MSC by tumor-conditioned media was assessed by measuring cytokine release and expression of CD54. The impact of MSC on tumor growth in vivo was analyzed in a HNSCC xenograft model. Results: Cells isolated from HNSCC tissue met the consensus criteria for MSC. Tumor-derived MSC constitutively produced high amounts of interleukin (IL)-6, IL-8 and stromal cell-derived factor (SDF)-1α. HNSCC-derived factors activated MSC and enhanced secretion of IL-8 and expression of CD54. Furthermore, MSC provided stromal support for human HNSCC cell lines in vivo and enhanced their growth in a murine xenograft model. Conclusions: This is the first study to isolate and characterize MSC from malignant tissues of patients with HNSCC. We observed cross-talk of stromal cells and tumor cells resulting in enhanced growth of HNSCC in vivo

Differential expansion of circulating human MDSC subsets in patients with cancer, infection and inflammation

Background Myeloid-derived suppressor cells (MDSC) are a functional myeloid cell subset that includes myeloid cells with immune suppressive properties. The presence of MDSC has been reported in the peripheral blood of patients with several malignant and non-malignant diseases. So far, direct comparison of MDSC across different diseases and Centers is hindered by technical pitfalls and a lack of standardized methodology. To overcome this issue, we formed a network through the COST Action Mye-EUNITER (www.mye-euniter.eu) with the goal to standardize and facilitate the comparative analysis of human circulating MDSC in cancer, inflammation and infection. In this manuscript, we present the results of the multicenter study Mye-EUNITER MDSC Monitoring Initiative, that involved 13 laboratories and compared circulating MDSC subsets across multiple diseases, using a common protocol for the isolation, identification and characterization of these cells. Methods We developed, tested, executed and optimized a standard operating procedure for the isolation and immunophenotyping of MDSC using blood from healthy donors. We applied this procedure to the blood of almost 400 patients and controls with different solid tumors and non-malignant diseases. The latter included viral infections such as HIV and hepatitis B virus, but also psoriasis and cardiovascular disorders. Results We observed that the frequency of MDSC in healthy donors varied substantially between centers and was influenced by technical aspects such as the anticoagulant and separation method used. Expansion of polymorphonuclear (PMN)-MDSC exceeded the expansion of monocytic MDSC (M-MDSC) in five out of six solid tumors. PMN-MDSC expansion was more pronounced in cancer compared with infection and inflammation. Programmed death-ligand 1 was primarily expressed in M-MDSC and e-MDSC and was not upregulated as a consequence of disease. LOX-1 expression was confined to PMN-MDSC. Conclusions This study provides improved technical protocols and workflows for the multi-center analysis of circulating human MDSC subsets. Application of these workflows revealed a predominant expansion of PMN-MDSC in solid tumors that exceeds expansion in chronic infection and inflammation

Chemoirradiated neutrophils and T cells differentially affect immune functions of APCs

Extracorporeal photopheresis (ECP) is known as an immunomodulatory therapy with few side effects, which is mainly used in the treatment of cutaneous T cell lymphoma, graft-versus-host disease, and allograft rejection. During ECP, leukocytes are separated from whole blood by leukapheresis, subsequently chemoirradiated with 8-methoxypsoralen and UVA light, and re-infused into the patient. Although clinically effective, its mode of action has not been fully elucidated. In the present study, we analyzed the interaction of chemoirradiated neutrophils and CD3+ lymphocytes with APC in an in vitro model. We report that chemoirradiated CD3+ T cells induced increased expression of activation markers on dendritic cells (DC), macrophages, and monocytes. Coculture of chemoirradiated CD3+ T cells with these APC also led to significantly increased secretion of TNF-alpha. Although less pronounced, additional activation of APC took place when APC were stimulated with LPS or IFN-gamma. In contrast, chemoirradiated neutrophils did not show activating effects on APC. The presence of chemoirradiated neutrophils during LPS and IFN-gamma stimulation of DC rather diminished DC and macrophage activation. In line with these findings DC cocultured with chemoirradiated CD3+ T cells, but not neutrophils, showed significantly increased activation of CD3+ responder lymphocytes in a mixed lymphocyte reaction. With this study, we demonstrate that chemoirradiated leukocytes have differential indirect immunomodulatory effects. Whereas chemoirradiated CD3+ T cells activate APC, chemoirradiated neutrophils suppress activation of APC in the presence of other activating factors, suggesting that the composition of the ECP-treated buffy coat might be of importance for its immunomodulatory effects

Activated Tissue-Resident Mesenchymal Stromal Cells Regulate Natural Killer Cell Immune and Tissue-Regenerative Function

The interaction of mesenchymal stromal cells (MSCs) with natural killer (NK) cells is traditionally thought of as a static inhibitory model, whereby resting MSCs inhibit NK cell effector function. Here, we use a dynamic in vitro system of poly(I:C) stimulation to model the interaction of NK cells and tissue-resident MSCs in the context of infection or tissue injury. The experiments suggest a time-dependent system of regulation and feedback, where, at early time points, activated MSCs secrete type I interferon to enhance NK cell effector function, while at later time points TGF-β and IL-6 limit NK cell effector function and terminate inflammatory responses by induction of a regulatory senescent-like NK cell phenotype. Importantly, feedback of these regulatory NK cells to MSCs promotes survival, proliferation, and pro-angiogenic properties. Our data provide additional insight into the interaction of stromal cells and innate immune cells and suggest a model of time-dependent MSC polarization and licensing

Cutting Edge: An Inactive Chromatin Configuration at the IL-10 Locus in Human Neutrophils

To identify the molecular basis of IL-10 expression in human phagocytes, we evaluated the chromatin modifica-tion status at their genomic locus. We analyzedposttranslational modifications of histones associatedwith genes that are active, repressed, or poised for tran-scriptional activation, including H3K4me3, H4Ac,H3K27Ac, and H3K4me1 marks. Differently from au-tologous IL-10–producing monocytes, none of the marksunder evaluation was detected at the locus of rest-ing or activated neutrophils from healthy subjects ormelanoma patients. By contrast, increased H3K4me3,H4Ac, H3K4me1, and H3K27Ac levels were detectedat syntenic regions of the locus in mouse neutro-phils. Altogether, data demonstrate that human neutro-phils, differently from either monocytes or mouseneutrophils, cannot switch on the gene becauseits locus is in an inactive state, likely reflecting a neutro-phil-specific developmental outcome. Implicitly, dataalso definitively settle a currently unsolved issue on thecapacity of human neutrophils to produce IL-10

Mesenchymal stem cells augment the anti-bacterial activity of neutrophil granulocytes.

BackgroundMesenchymal stem cells (MSCs) participate in the regulation of inflammation and innate immunity, for example by responding to pathogen-derived signals and by regulating the function of innate immune cells. MSCs from the bone-marrow and peripheral tissues share common basic cell-biological functions. However, it is unknown whether these MSCs exhibit different responses to microbial challenge and whether this response subsequently modulates the regulation of inflammatory cells by MSCs.Methodology/principal findingsWe isolated MSCs from human bone-marrow (bmMSCs) and human salivary gland (pgMSCs). Expression levels of TLR4 and LPS-responsive molecules were determined by flow cytometry and quantitative PCR. Cytokine release was determined by ELISA. The effect of supernatants from unstimulated and LPS-stimulated MSCs on recruitment, cytokine secretion, bacterial clearance and oxidative burst of polymorphonuclear neutrophil granulocytes (PMN) was tested in vitro. Despite minor quantitative differences, bmMSCs and pgMSCs showed a similar cell biological response to bacterial endotoxin. Both types of MSCs augmented anti-microbial functions of PMNs. LPS stimulation, particularly of bmMSCs, further augmented MSC-mediated activation of PMN [corrected].Conclusions/significanceThis study suggests that MSCs may contribute to the resolution of infection and inflammation by promoting the anti-microbial activity of PMNs. This property is exerted by MSCs derived from both the bone-marrow and peripheral glandular tissue

Additional file 2: Figure S2. of Human mesenchymal stromal/stem cells acquire immunostimulatory capacity upon cross-talk with natural killer cells and might improve the NK cell function of immunocompromised patients

CD56dim NK cells do not respond to CCR2 ligands. NK cells were incubated in the absence (–) or presence of 0.5 ng/ml of recombinant human CCL2, CCL8, CCL7, and CCL12, or with conditioned medium (CM) from MSCs for 12 h. Thereafter, the cells were stimulated with IL-12 (1 ng/ml) and IL-18 (5 ng/ml). Dot plots of intracellular staining of IFN-γ in gated CD3–CD56dim NK cells (gating strategy as shown in Fig. 1d). Data are representative for one out of five experiments. The threshold of positive staining for IFN-γ was set according to the isotype control (iso). Numbers indicate the percentage of IFN-γ-positive NK cells. MSC mesenchymal stromal/stem cell, CCL C-C ligand, IFN interferon. (PDF 36 kb

Activation of the NF-κB, JAK-STAT and TRAF pathways.

<p>(A/B) mRNA expression of bmMSCs and pgMSCs was determined in unstimulated and stimulated (LPS, 10 ng/mL for 4 h) MSCs by qPCR. mRNA was pooled from 4 different donors respectively for bmMSCs and pgMSCs. Gene expression of unstimulated MSCs was set as 1 (reference value). Values ≥ 2.0 are set as a significant increase of gene expression and values≤0.5 are set as a significant decrease of gene expression after LPS stimulation. 2^−ddCT = 2^{−(dCT Target – dCT Reference) LPS stimulated – (dCT Target – dCT Reference)}^unstimulated. (C/D) Classical (canonical) NF-κB pathway: immunofluorescence analysis of translocation of p65 (C) and c-REL (D) was determined in unstimulated and stimulated (LPS, 10 ng/mL for 0, 30, 60, 90, 120 min) bmMSCs and pgMSCs. One representative experiment of two independent experiments is shown. (E) Analysis of the phosphorylation status of Acetyl-NF-κB p65 and phospho-NF-κB (p-NF-κB) detected by western blot (unstimulated and stimulated with LPS for 0, 30, 60, 90, and 120 min).</p

Cytokine secretion profile of bmMSCs and pgMSCs.

<p>Cytokines were quantified by ELISA. MSCs were (A) left untreated or (B/C) were stimulated with LPS [10 ng/ml] (bmMSCs, n = 7; pgMSCs, n = 5; passage 3). (C) Cytokine secretion of untreated MSCs was set as 1 (reference value). Mann-Whitney test was used for statistical analysis; significance was set at the level of <i>P</i>≤0.05.</p