Journal: eLife

Article Title: Tumor-derived extracellular vesicles regulate tumor-infiltrating regulatory T cells via the inhibitory immunoreceptor CD300a

doi: 10.7554/eLife.61999

Figure Lengend Snippet: ( A ) Representative microscopy images of wild-type (WT) and Cd300a −/− bone marrow-derived dendritic cells (BMDCs) treated with pHrodo-labeled extracellular vesicles (EVs) to assess the localization of EVs (red) and early endosome antigen (EEA)-1 (green). Scale bar, 10 μm. Data are representative of two independent experiments. ( B ) Uptake of PKH-labeled tumor-derived EVs (TEVs) in WT ( n = 5) and Cd300a −/− BMDCs ( n = 5). ( C ) Representative microscopy images of WT and Cd300a −/− BMDCs treated with pHrodo-labeled exosomes to assess the localization of exosomes (green), TLR3 (red), and CD300a (blue). Scale bar, 10 μm. Data are representative of two independent experiments. ( D ) Quantitative RT-PCR analysis of Ifnb in WT and Cd300a −/− BMDCs treated with B16-derived exosomes in the presence of dimethyl sulfoxide (DMSO) (WT, n = 9; Cd300a −/− , n = 10), 100 nM TLR4 inhibitor ( n = 7 in each group), and 50 μM TLR3 inhibitor ( n = 6 in each group). ( E ) Quantitative RT-PCR analysis of Ifnb in WT, Cd300a −/− , ticam-1 −/− , and ticam-1 −/− ;Cd300a −/− mice-derived BMDCs treated with B16-derived EVs ( n = 5 in all group). ( F ) Representative immunoassay of WT and Cd300a −/− BMDCs left unstimulated (0 min) or stimulated for the indicated times with B16-derived exosomes, followed by immunoblot analysis of phosphorylated (p-) interferon regulatory factor 3 (IRF3) or total IRF3. Data are representative of two independent experiments. ( G and H ) Comparison of tumor growth and survival curves of B16 melanoma cells between ticam-1 −/− ( n = 6) and ticam-1 −/− ;Cd300a −/− ice ( n = 9) after inoculation of B16 melanoma. ( I and J ) Comparison of tumor growth and survival curves of B16 melanoma between MyD8 −/− ( n = 9) and MyD88 −/− ;Cd300a −/− mice ( n = 10) after inoculation of B16 melanoma. Data are given as means ± standard error of the means (SEMs). N.S.: not significant. *p < 0.05, **p < 0.01, and ***p < 0.001. p values were obtained by using the Student’s t -test ( B ), a two-way analysis of variance (ANOVA) followed by Bonferroni’s post-test ( D, E, G, and I ), and the log-rank test ( H and J ). Data were pooled from two ( B, E, and H ) or three ( D, I, and J ) independent experiments. Figure 5—source data 1. Source data for .

Article Snippet: For BMDC stimulation by EVs, 2 × 10 5 BMDCs were incubated in the presence of 3–5 μg/ml EVs for 2.5 h. To inhibit TLR3 and TLR4 signaling, a TLR3/dsRNA complex inhibitor (Merck) and a TLR4 inhibitor (TAK-242; Merck) were added to the cultures of BMDCs for 15 min before exosome stimulation.

Techniques: Microscopy, Derivative Assay, Labeling, Quantitative RT-PCR, Western Blot

Journal: eLife

Article Title: Tumor-derived extracellular vesicles regulate tumor-infiltrating regulatory T cells via the inhibitory immunoreceptor CD300a

doi: 10.7554/eLife.61999

Figure Lengend Snippet: Representative confocal microscopy images of bone marrow-derived dendritic cells (BMDCs) stained with anti-TLR3 and anti-CD300a mAbs. Data were pooled from two independent experiments.

Article Snippet: For BMDC stimulation by EVs, 2 × 10 5 BMDCs were incubated in the presence of 3–5 μg/ml EVs for 2.5 h. To inhibit TLR3 and TLR4 signaling, a TLR3/dsRNA complex inhibitor (Merck) and a TLR4 inhibitor (TAK-242; Merck) were added to the cultures of BMDCs for 15 min before exosome stimulation.

Techniques: Confocal Microscopy, Derivative Assay, Staining

Journal: eLife

Article Title: Tumor-derived extracellular vesicles regulate tumor-infiltrating regulatory T cells via the inhibitory immunoreceptor CD300a

doi: 10.7554/eLife.61999

Figure Lengend Snippet: RNA-containing TEV is incorporated into the endosomes in dendritic cells (DCs), where phosphatidylserine (PS) on TEV binds to CD300a and inhibits the TLR3–TRIF–IRF3 signaling pathway initiated by TEV-derived RNA binding to TLR3, resulting in the decrease in interferon-β (IFN-β) production by DCs and the number of tumor-infiltrating Treg cells. The Treg cells regulate tumor development.

Article Snippet: For BMDC stimulation by EVs, 2 × 10 5 BMDCs were incubated in the presence of 3–5 μg/ml EVs for 2.5 h. To inhibit TLR3 and TLR4 signaling, a TLR3/dsRNA complex inhibitor (Merck) and a TLR4 inhibitor (TAK-242; Merck) were added to the cultures of BMDCs for 15 min before exosome stimulation.

Techniques: Derivative Assay, RNA Binding Assay

Journal: eLife

Article Title: Tumor-derived extracellular vesicles regulate tumor-infiltrating regulatory T cells via the inhibitory immunoreceptor CD300a

doi: 10.7554/eLife.61999

Figure Lengend Snippet:

Article Snippet: For BMDC stimulation by EVs, 2 × 10 5 BMDCs were incubated in the presence of 3–5 μg/ml EVs for 2.5 h. To inhibit TLR3 and TLR4 signaling, a TLR3/dsRNA complex inhibitor (Merck) and a TLR4 inhibitor (TAK-242; Merck) were added to the cultures of BMDCs for 15 min before exosome stimulation.

Techniques: Purification, Sequencing, Recombinant, Isolation, Software