Correlation between cytokines and MDSCs as well as M2 macrophages in ECA patients.

<p>A: Correlation between the plasma concentration of Arg1 and the percentages of circulating MDSCs in ECA patients. Positive correlation was found between Arg1 and MDSCs (r = 0.493, <i>P</i> = 0.006). b: Correlation between the plasma concentration of Arg1 and the mRNA level of IL-4 from ECA patients. Positive correlation was found between Arg1 and IL-4 mRNA (r = 0.510, <i>P</i> = 0.009). c: Correlation between the plasma concentrations of Arg1 and IL-13 from ECA patients. Positive correlation was found between Arg1 and IL-13 (r = 0.455, <i>P</i> = 0.017). d: Correlation between the mRNA level of IL-4 and plasma level of IL-13 from ECA patients. Positive correlation was found between IL-4 and IL-13 (r = 0.484, <i>P</i> = 0.016). e: Correlation between the mRNA level of IFN-γ and plasma level of Arg1 from ECA patients. Negative correlation was found between IFN-γ and Arg1 in ECA patients (r = −0.381, <i>P</i> = 0.038). f: Correlation between the number of CD163⁺ macrophages in cancer tissues and the percentages of circulating MDSCs in PBMC from ECA patients (r = 0.410, <i>P</i> = 0.003). g: Correlation between the number of CD163⁺ macrophages in cancer tissues and the plasma concentration of IL-13 from ECA patients (r = 0.405, <i>P</i> = 0.036).</p

Immunohistochemical analysis of CD68 and CD163 expression in esophageal cancer and adjacent tissues.

<p>Representative immunohistochemical pictures showed the expression of CD68 in a cancer-adjacent tissue (a). and a cancer tissue (b). Representative immunohistochemical pictures showed the expression of CD163 in a cancer-adjacent tissue (c). and a cancer tissue (d). Normal esophageal squamous epithelium (haematoxylin-eosin/HE staining) (e). Squamous cell esophageal carcinoma (HE staining) (f). Analysis of the number of CD68⁺ macrophages (g). and CD163+ macrophages (h). in cancer and cancer-adjacent tissues, the results showed that most cancer tissues had larger number of CD68+ and CD163+ macrophages infiltration than that in the cancer-adjacent tissues.</p

Relation between CD163 expression in cancer tissues and MDSC% in PBMC from ECA patients.

<p>Relation between CD163 expression in cancer tissues and MDSC% in PBMC from ECA patients.</p

Expression of CD68/CD163 in esophageal cancer tissues and adjacent tissue.

<p>*Ten high magnification views were selected to count all cells, the cell color depth and the percentage of stained cells as the judgment result basis. According to the staining color (A): 0 = no color in cytoplasm; 1 = light yellow; 2 = pale brown; 3 = brown color. According to the percentage of stained cells (B): “0” indicates the percentage of positive cells <5%; “1″ indicates the percentage of positive cells 5%–25%; “2″ indicates the percentage of positive cells 26%–50%; “3” represents the percentage of positive cells >50%. The two scores of A plus B as a final judgment result: 0∼1: “−”; 2∼3: “+”; 4∼6: “++”.</p

The frequency of circulating MDSCs (HLA-DR⁻/CD14⁻/CD11b⁺/CD33⁺ cells) increased in ECA patients.

<p>The frequency of MDSCs in PBMC was analyzed by flow cytometry, the patients and healthy controls used in this experiment had been matched with sex, age and number. a: Representative diagram of flow cytometry analysis for circulating MDSCs from healthy controls. b: Representative diagram of flow cytometry analysis for circulating MDSCs from ECA patients. MDSCs, myeloid-derived suppressor cells; ECA, esophageal cancer; FSC, forward scatter; FITC, fluorescein isothiocyanate; PerCP-Cy5.5, peridinin chlorophyll protein-cyanin 5.5; PE, phycoerythrin; APC, allophycocyanin.</p

Plasma concentrations of cytokines in ECA patients and healthy controls.

<p>The patients and healthy controls used in this experiment had been matched with sex, age and number. Plasma concentrations of IFN-γ (a), IL-4 (b), IL-6 (c), Arg1 (d) and IL-13 (e) were determined by ELISA. Data were analyzed by the Student’s t-test. *<i>P</i><0.05, ***<i>P</i><0.001 vs. control group. NS: no significant difference, ECA: esophageal cancer, ELISA: enzyme linked immunosorbent assay, IFN: interferon, IL: interleukin, Arg1: arginase 1.</p

Primers used in real-time PCR.

<p>IFN, Interferon; IL, Interleukin; U, Upper; L, Lower.</p

The mRNA level of each gene in PBMC from ECA patients and healthy controls.

<p>The mRNA levels of IFN-γ (a), T-bet (b), IL-4 (c), GATA3 (d) and IL-12 (e) were determined by real-time PCR. Data were analyzed by the Student’s t-test. *<i>P</i><0.05, ***<i>P</i><0.001 vs. control group. NS, no significant difference. ECA, esophageal cancer; PCR, polymerase chain reaction; IFN, interferon; IL, interleukin. The patients and healthy controls used in this experiment had been matched with sex, age and number.</p

The Closely Related CD103⁺ Dendritic Cells (DCs) and Lymphoid-Resident CD8⁺ DCs Differ in Their Inflammatory Functions

<div><p>Migratory CD103⁺ and lymphoid-resident CD8⁺ dendritic cells (DCs) share many attributes, such as dependence on the same transcription factors, cross-presenting ability and expression of certain surface molecules, such that it has been proposed they belong to a common sub-lineage. The functional diversity of the two DC types is nevertheless incompletely understood. Here we reveal that upon skin infection with herpes simplex virus, migratory CD103⁺ DCs from draining lymph nodes were more potent at inducing Th17 cytokine production by CD4⁺ T cells than CD8⁺ DCs. This superior capacity to drive Th17 responses was also evident in CD103⁺ DCs from uninfected mice. Their differential potency to induce Th17 differentiation was reflected by higher production of IL-1β and IL-6 by CD103⁺ DCs compared with CD8⁺ DCs upon stimulation. The two types of DCs from isolated lymph nodes also differ in expression of certain pattern recognition receptors. Furthermore, elevated levels of GM-CSF, typical of those found in inflammation, substantially increased the pool size of CD103⁺ DCs in lymph nodes and skin. We argue that varied levels of GM-CSF may explain the contrasting reports regarding the positive role of GM-CSF in regulating development of CD103⁺ DCs. Together, we find that these two developmentally closely-related DC subsets display functional differences and that GM-CSF has differential effect on the two types of DCs.</p></div

CD103⁺ DCs and CD8⁺ DCs differ in expression of costimulatory molecules, inflammasomes and TLR.

<p>(<b>A</b>) Cells of pooled cutaneous LNs from Langerin-EGFP mice and Langerin-EGFP/<i>CD103−/−</i> mice were analyzed. CD103⁺ DCs were identified as CD326⁻CD205⁺langerin⁺ within migratory DCs (mDC, CD11c^intMHC II^high); CD8⁺ DCs were identified as CD205⁺ CD8⁺ within cDCs (CD11c^highMHC II^int). Histograms show the expression of CD103 and langerin-EGFP by CD8⁺ and CD103⁺ DCs. For CD103 expression, CD8⁺ DCs (grey dot line) and CD205⁺CD11b⁻ migratory DCs (equivalent of CD103⁺ DCs, black dot line) from CD103−/− mice were included. (B) CD8⁺ and CD103⁺ DCs from B6 mice were analyzed for the expression of costimulatory molecules. (C&D) CD8⁺ and CD103⁺ DCs from B6 mice were sorted. RT-qPCR was performed for the indicated transcripts with 3 reference genes as controls. One of three repeated experiments is shown.</p