Journal: PLOS Pathogens

Article Title: Regulatory T cell-like response to SARS-CoV-2 in Jamaican fruit bats ( Artibeus jamaicensis ) transduced with human ACE2

doi: 10.1371/journal.ppat.1011728

Figure Lengend Snippet: Four bats were transduced with human ACE2 and infected 5 days later. After 12 days, splenocytes were recovered for AIM testing and gene expression analsysis. A. Splenocytes from hACE2-transduced bats 12 days post infection with SARS-CoV were cultured with or without SARS-CoV-2 nucleocapsid peptide library for 6 hours in the presence of anti-CD40 blocking antibody (AIM testing) or 24 hours without anti-CD40 blocking antibody (cellular RNA). B. Cells were stained with anti-CD154 and anti-CD4 and analyzed by flow cytometry. Violin plots for the individual bats, plus the pooled sample and rank data. In the presence of peptide, CD154 expression was significantly increased on splenocytes (red) compared to no peptide controls (blue) (Wilcoxon rank sum test). C. Heat map of splenocyte qPCR (ΔΔCq) gene expression in response to peptide stimulation determined 3 of the 4 bats expressed a profile consistent with a regulatory T cell response. D. Peptide-induced gene expression changes in splenocyte cultures of bats 1, 2 and 4 from panel C; IL-10, TGFβ and CD4 gene expression were significantly elevated, whereas CXCR4 was nearly significant (two-tailed t-test).

Article Snippet: Test samples were then resuspended in a 1:20 dilution of anti-mouse CD40 PE (Tonbo, clone FGK45), or anti-human CD154 PE (Tonbo, clone 5C8) in FACS buffer and incubated for 30 minutes at 4°C in the dark.

Techniques: Transduction, Infection, Gene Expression, Cell Culture, Blocking Assay, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Regulatory B Cells Dysregulated T Cell Function in an IL-35-Dependent Way in Patients With Chronic Hepatitis B

doi: 10.3389/fimmu.2021.653198

Figure Lengend Snippet: Suppression effect on effector cells (ECs). B cells were isolated from PBMC using CD19+ microbead and stimulated with CD40L/CpG/LPS with or without HBVcore peptide. Stimulated B cells were then co-cultured with autologous CD19-depleted PBMCs at the ratio of 2:1 in anti-CD3/CD28 coated plate for 48hours. As a control, CD19-depleted PBMCs were also cultured alone without B cells (CD19-depleted PBMCs non-activated and CD19-depleted PBMCs activated). (A) Suppression effect on IFN-ɤ-producing CD4+T cells; (B) Suppression on IL-4-producing CD4+T cells; (C) Suppression effect on IL-17A-producing CD4+T cells; (D) Suppression on IFN-ɤ-producing CD8+T cells; (E) Suppression on IL-4-producing CD8+T cells. Non-parametric Kruskal-Wallis ANOVA test and Tukey’s Multiple Comparison test was used to analysis the data. P value<;0.05 was considered statistically significant.

Article Snippet: For Breg subset and B10 or IL-35+B cell detection, PBMCs (2 × 10 6 ) were stimulated with CD40L (5 μg/mL, Purified Anti-Human CD154 (CD40L), Tonbo Biosciences, USA) plus CpG-ODN (1.5 μM, TLR9 Agonist-Stimulatory Class B tlrl-2006, InvivoGen) and lipopolysaccharide (LPS, 1 μg/mL, eBioscience) for 48 h. Furthermore, cells were activated for another 4 h at 37°C in 5% CO 2 with 50 ng/mL phorbol myristate acetate, 1 mmol/L ionomycin (both from Sigma, St. Louis, MO, USA), and 10 mg/mL brefeldin A (Tocris Cookson, Bristol, UK) in complete RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, USA).

Techniques: Isolation, Cell Culture, Control, Comparison

Journal: Pain

Article Title: Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation

doi: 10.1097/j.pain.0000000000000341

Figure Lengend Snippet: PI3K-δ and -γ antagonists block macrophage infiltration. (A) Intraplantar carrageenan elicits macrophage infiltration into the tissue 4 hours after injection. This is reduced by pretreatment with antagonists to PI3K-δ and -γ, but not PI3K-α and -β. ** P ≤ 0.001, * P ≤ 0.01, compared to vehicle + carrageenan. N = 4 to 6 rats per group. (B) Intraplantar carrageenan elicits macrophages to associate with the vascular endothelium. This is reduced by pretreatment with antagonists to PI3K-δ and -γ, but not PI3K-α and PI3K-β in the same pattern as seen for tissue infiltration. * P ≤ 0.01, compared to vehicle + carrageenan. (C-I) Representative tissue sections stained for laminin (green) and CD11b (red) harvested 1 or 4 hours after injection. (C) Naive tissue; (D) Tissue parallel to and near needle entry harvested 1 hour after carrageenan + vehicle injection; (E) Tissue 2-mm away from needle path, harvested 1 hour after injection; (F) Carrageenan injection with vehicle pretreatment harvested 4 hours after injection; (G) Carrageenan-injected animals with PI3Kα antagonist pretreatment; (H) Carrageenan-injected animals with PI3K-β antagonist pretreatment; (I) Carrageenan-injected animals with PI3K-δ antagonist pretreatment; (J) Carrageenan-injected animals with PI3K-γ antagonist pretreatment. Arrow indicates macrophage on blood vessel.

Article Snippet: In other tissue, infiltrating proinflammatory macrophages were labeled using a mouse anti-CD11b monoclonal antibody (1:5000; Serotec, Raleigh, NC, lot #MCA2756A).

Techniques: Blocking Assay, Injection, Staining