Probes for CD3

Murine astrovirus 2 (MuAstV2) is a novel murine astrovirus recently identified in laboratory and wild mice. MuAstV2 readily transmits between immunocompetent mice yet fails to transmit to highly immunocompromised mouse strains-a unique characteristic when contrasted with other murine viruses including other astroviruses. We characterized the viral shedding kinetics and tissue tropism of MuAstV2 in immunocompetent C57BL/6NCrl mice and evaluated the apparent resistance of highly immunocompromised NOD- Prkdcem26Cd52Il2rgem26Cd22 /NjuCrl mice to MuAstV2 after oral inoculation. Temporal patterns of viral shedding were determined by serially measuring fecal viral RNA. Tissue tropism and viral load were characterized and quantified by using in-situ hybridization (ISH) targeting viral RNA. Cellular tropism was characterized by evaluating fluorescent colocalization of viral ISH with various immunohistochemical markers. We found a rapid increase of fecal viral RNA in B6 mice, which peaked at 5 d after inoculation (dpi) followed by cessation of shedding by 168 dpi. The small intestine had the highest percentage of hybridization (3.09% of tissue area) of all tissues in which hybridization occurred at 5 dpi. The thymus displayed the next highest degree of hybridization (2.3%) at 7 dpi, indicating extraintestinal viral spread. MuAstV2 RNA hybridization was found to colocalize with only 3 of the markers evaluated: CD3 (T cells), Iba1 (macrophages), and cytokeratin (enterocytes). A higher percentage of CD3 cells and Iba1 cells hybridized with MuAstV2 as compared with cytokeratin at 2 dpi (CD3, 59%; Iba1, 46%; cytokeratin, 6%) and 35 dpi (CD3, 14%; Iba1, 55%; cytokeratin, 3%). Neither fecal viral RNA nor viral hybridization was noted in NCG mice at the time points examined. In addition, mice of mixed genetic background were inoculated, and only those with a functioning Il2rg gene shed MuAstV2. Results from this study suggest that infection of, or interaction with, the immune system is required for infection by or replication of MuAstV2.

Cancer cells have an imbalance in oxidation-reduction (redox) homeostasis. Understanding the precise mechanisms and the impact of the altered redox microenvironment on the immunologic reaction to tumors is limited.We isolated exosomes from ovarian cancer cells through ultracentrifuge and characterized by Western-blots and Nanoparticle Tracking Analysis. 2D, 3D-coculture tumor model, and 3D live cell imaging were used to study the interactions between tumor cells, macrophages and CD3 T cells in vitro. The role of exosomal miR-155-5p in tumor growth was evaluated in xenograft nude mice models and immune-competent mice models. Flow cytometry and flow sorting were used to determine the expression levels of miR-155-5p and PD-L1 in ascites and splenic macrophages, and the percentages of CD3 T cells subpopulations.The elevation of reactive oxygen species (ROS) greatly downregulated exosomal miR-155-5p expression in tumor cells. Neutralization of ROS with N-acetyl-L-cysteine (NAC) increased the levels of miR-155-5p in tumor exosomes that were taken up by macrophages, leading to reduction of macrophage migration and tumor spheroid infiltration. We further found that programmed death ligand 1 (PD-L1) is a functional target of miR-155-5p. Co-culture of macrophages pre-treated with NAC-derived tumor exosomes or exosomal miR-155-5p with T-lymphocytes leading to an increased percentage of CD8+ T-lymphocyte and a decreased CD3+ T cell apoptosis through PD-L1 downregulation. Tumor growth in nude mice was delayed by treatment with NAC-derived tumor exosomes. Delivery of tumor exo-miR-155-5p in immune-intact mice suppressed ovarian cancer progression and macrophage infiltration, and activated CD8+ T cell function. It is of note that exo-miR-155-5p inhibited tumor growth more potently than the PD-L1 antibody, suggesting that in addition to PD-L1, other pathways may also be targeted by this approach.Our findings demonstrate a novel mechanism, ROS-induced down-regulation of miR-155-5p, by which tumors modulate the microenvironment that favors tumor growth. Understanding of the negative impact of ROS on the tumor immune response will improve current therapeutic strategies. Targeting miR-155-5p can be an alternative approach to prevent formation of an immunosuppressive TME through downregulation of PD-L1 and other immunosuppressive factors.

Abstract BACKGROUND: IL10 receptor (IL10R) deficiency causes severe infantile-onset inflammatory bowel disease. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell functions in mice. We have previously reported a key role of IL10 in the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4 T-cell function. METHODS: Peripheral blood mononuclear cells and intestinal biopsies cells were collected from IL10/IL10R-deficient patients and controls. Frequencies of CD4 T-cell subsets, naive T-cell proliferation, regulatory T cell (Treg)-mediated suppression, and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling was performed by NanoString and quantitative real-time polymerase chain reaction. RNA in situ hybridization was used to determine the quantities of various transcripts in intestinal mucosa. RESULTS: Analysis of 16 IL10- and IL10R-deficient patients demonstrated similar frequencies of peripheral blood and intestinal Tregs, compared with control subjects. In addition, in vitro Treg suppression of CD4 T-cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naive cells exhibited higher proliferative capacity, a strong TH17 signature, and an increase in polarization toward TH17 cells, compared with controls. Moreover, the frequency of TH17 cells was increased in the colon and ileum of IL10R-deficient patients. Finally, we show that stimulation of IL10R-deficient Tregs in the presence of IL1β leads to enhanced production of IL17A. CONCLUSIONS: IL10R signaling regulates TH17 polarization and T-cell proliferation in humans but is not required for the generation and in vitro suppression of Tregs. Therapies targeting the TH17 axis might be beneficial for IL10- and IL10R-deficient patients as a bridge to allogeneic hematopoietic stem cell transplantation.