Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A to G ) PBMCs from the patients with irAE were cultured in the plate coated with anti-CD3 and anti-CD28 (10 μg/ml) in the presence of IgG1 isotype control or anti–human IL-6R (50 μg/ml), anti–human IL-12p40 (50 μg/ml), and anti–human IFNAR1 (50 μg/ml) for 3 days; n = 9. (A) Expression of CD38 and CD127 on CD8 + T cells. Right: Percentage of CD38 + CD127 − CD8 + T cells. (B and C) CD8 + T cells activated for 5 days were restimulated with PMA, ionomycin, and monensin for 5 hours. (B) Expression of granzyme B and IFN-γ. Right: Percentage of granzyme B + IFN-γ + CD8 + T cells. (C) Expression of perforin and IFN-γ. Right: Percentage of perforin + IFN-γ + CD8 + T cells. [(D) and (E)] MFIs of GluCy5 (D), TMRM, and MTDR (E) in CD8 + T cells were presented. [(F) and (G)] CD4 + T cells activated for 5 days were restimulated with PMA, ionomycin, and monensin for 5 hours. (F) Expression of IL-21 and IL-2. Right: Percentage of IL-21 + IL-2 + CD4 + T cells. (G) Expression of CD38 and CXCR5 on CD4 + T cells. Right: Percentage of CD38 + CXCR5 − CD4 + T cells. Data in graphs represent mean ± SEM. Significance was tested paired Student’s t test [(A) to (G)].

Article Snippet: PBMCs from patients with irAE were thawed and rested in a complete medium for 1 hour and then activated with plate-coated anti–human CD3 and anti–human CD28 (10 μg/ml) with IgG1 isotype control (Bio X Cell, catalog no. CP174) or a combination of anti–human IL-6R 50 μg/ml; Bio X Cell, catalog no. SIM0014), anti–human IL-12p40 (50 μg/ml; Bio X Cell, catalog no. SIM0020), and anti–human IFNAR1 (50 μg/ml; Bio X Cell, catalog no. SIM0022) for 3 days.

Techniques: Cell Culture, Control, Expressing

Journal:

Article Title: Human IL-23-producing type 1 macrophages promote but IL-10-producing type 2 macrophages subvert immunity to (myco)bacteria

doi: 10.1073/pnas.0400983101

Figure Lengend Snippet: Differentiation of human blood monocytes into functionally distinct Mϕ subsets, Mϕ-1 and Mϕ-2. (A) In contrast to mo.DCs, Mϕ-1 and Mϕ-2 highly expressed CD14 but showed no or only weak expression of CD1a or CD83 (the latter after activation by LPS) as determined by flow cytometry. Cytokine secretion was measured up to 72 h after stimulation with M. tuberculosis sonicate (myc) (▪), LPS (•), or medium control (♦). (B) Whereas activated Mϕ-1, similar to DCs, secreted high levels of IL-12p40, Mϕ-2 failed to secrete IL-12p40. (C) IL-10, in contrast, was most predominant in Mϕ-2. Similar cytokine profiles were obtained with cells from at least 10 independent donors.

Article Snippet: IL-23 was measured by ELISA using anti-IL-12p40 monoclonal antibody BP40 (Diaclone) for coating and rat-anti-hp19 monoclonal antibody 12F12 for detection (with a sensitivity of 60 pg/ml).

Techniques: Expressing, Activation Assay, Flow Cytometry

Journal:

Article Title: Human IL-23-producing type 1 macrophages promote but IL-10-producing type 2 macrophages subvert immunity to (myco)bacteria

doi: 10.1073/pnas.0400983101

Figure Lengend Snippet: Type 1 cytokine secretion by monocyte-derived Mϕ and mo.DCs. The capacity of mo.DCs, Mϕ-1, and Mϕ-2 to secrete IL-12 (p40/p35) and IL-23 (p40/p19) proteins was determined 24 h after microbial stimulation in the absence (black bars) or presence (gray bars) of 500 units/ml IFN-γ. Mϕ-1 secreted IL-23 but failed to produce IL-12 after activation with mycobacterial sonicate (10 μg/ml) or LPS (10 ng/ml) unless IFN-γ was added. Also, DCs showed IFN-γ-enhanced IL-12 secretion, but unlike Mϕ-1 they yielded decreased rather than elevated IL-23 levels with IFN-γ. Mϕ-2 failed to produce IL-12 or IL-23. Depicted are average protein levels plus standard deviation (n = 3; n = 5 for IL-12 in DCs and Mϕ-1).

Article Snippet: IL-23 was measured by ELISA using anti-IL-12p40 monoclonal antibody BP40 (Diaclone) for coating and rat-anti-hp19 monoclonal antibody 12F12 for detection (with a sensitivity of 60 pg/ml).

Techniques: Derivative Assay, Activation Assay, Standard Deviation

Journal:

Article Title: Human IL-23-producing type 1 macrophages promote but IL-10-producing type 2 macrophages subvert immunity to (myco)bacteria

doi: 10.1073/pnas.0400983101

Figure Lengend Snippet: Type 1 cytokine mRNA levels in monocyte-derived Mϕ and mo.DCs. (A) Quantitative reverse transcription–PCR analyses 8 h after LPS stimulation of Mϕ-1 revealed that expression of IL-12-specific p35, but not p40 or p19 mRNA, required costimulation with IFN-γ (500 units/ml). DCs showed low but significant transcription of p35 in response to LPS and profound levels of p40 and p19 mRNA. Mϕ-2 failed to induce any of these cytokine transcripts. (B) Whereas stimulation with 10 μg/ml mycobacterial sonicate strongly induced the production of p40 and p19 mRNA, high-level transcription of p35 required costimulation with IFN-γ. (Note that the y axes are logarithmic in B!) Stimulation indexes were calculated from the average level of mRNA from two (DCs) to four (Mϕ-1) experiments. Activation-induced p19 transcription was inhibited by IFN-γ in both mo.DCs and Mϕ-1.

Article Snippet: IL-23 was measured by ELISA using anti-IL-12p40 monoclonal antibody BP40 (Diaclone) for coating and rat-anti-hp19 monoclonal antibody 12F12 for detection (with a sensitivity of 60 pg/ml).

Techniques: Derivative Assay, Expressing, Activation Assay

Journal: Cell Reports Medicine

Article Title: MAIT cell activation is associated with disease severity markers in acute hantavirus infection

doi: 10.1016/j.xcrm.2021.100220

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-IL-12 (p40/p70) , Milteny Biotec , Cat#130-095-755, clone C8.6.

Techniques: Control, Virus, Clinical Proteomics, Recombinant, Staining, Luminex, Enzyme-linked Immunosorbent Assay, Labeling, Software

Journal: The Journal of Experimental Medicine

Article Title: Interferon-α and Interleukin-12 Are Induced Differentially by Toll-like Receptor 7 Ligands in Human Blood Dendritic Cell Subsets

doi: 10.1084/jem.20020207

Figure Lengend Snippet: TLR-7 ligands induce the production of IL-12 and IFN-α from MDCs and PDCs, respectively. (A and B) After 24-h culture with various stimuli, concentration of IL-12 p40+p70 (A) and IFN-α (B) in the culture supernatants of MDCs, PDCs, and PBMCs were measured by ELISA. The data are shown by means ± SEM of five independent experiments. (C) After 5-h culture with R-848 (10 −6 M), intracellular staining of IL-12 and IFN-α in MDCs and PDCs, together with the staining of surface expression of CD40, was performed. Percentages of the respective cytokine producing DCs are indicated. This figure represents the results from one of three experiments.

Article Snippet: 10 μg/ml brefeldin A (Sigma-Aldrich) was added during the last 1 h. After the stimulation, MDCs and PDCs were stained with Cy-Chrome-labeled CD40 (5C3; BD PharMingen), and then fixed, permeabilized (FIX and PERM kit; Caltag Laboratories), and stained with FITC-labeled anti–IL-12 p40+p70 mAb (B-P24; DIACLONE Research) or unconjugated mouse anti–human IFN-α mAb (MC-16; Genzyme).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Expressing