il 12p40  (R&D Systems)

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: bioRxiv

Article Title: Sorting nexin 5 mediates antigen presentation and immunity against Mycobacterium tuberculosis

doi: 10.64898/2026.01.30.702915

Figure Lengend Snippet: (A) IFN-γ, (B) TNF-α, (C) IL-2, and (D) IL-12p40 were assessed in supernatants of homogenized lungs by ELISA. **p < 0.01 (for indicated comparison; Mann-Whitney test). (E-G) Lung explanted CD4 + T cells were characterized for cytokine expression, including (E) IFN-γ, (F) TNF-α, and (G) IL-2 using surface and intracellular cytokine staining for flow cytometry. Each circle corresponds to one animal. Bars are the mean ± S.D. for 3 to 5 animals per group per time point. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 (for indicated comparison; one-way ANOVA)

Article Snippet: MCP-1 (Biorad, 171G5019M), IL-6 (R&D Systems, M6000b), TNF-α (R&D Systems, MTA00B), and IL-12p40 (R&D Systems, MP400) were measured by ELISA according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, MANN-WHITNEY, Expressing, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Sorting nexin 5 mediates antigen presentation and immunity against Mycobacterium tuberculosis

doi: 10.64898/2026.01.30.702915

Figure Lengend Snippet: (A) TNF-α, (B) IL-12p40, (C) MCP-1, and (D) IL-6 were assessed in cell supernatants of infected macrophages, either primed or not with IFN-γ (100 UI/mL). Bars are the mean ± S.D. from one experiment performed in quadruplicate. The circles shown correspond to each replica. *p < 0.05 and ****p < 0.0001 (for indicated comparison; one-way ANOVA).

Article Snippet: MCP-1 (Biorad, 171G5019M), IL-6 (R&D Systems, M6000b), TNF-α (R&D Systems, MTA00B), and IL-12p40 (R&D Systems, MP400) were measured by ELISA according to the manufacturer’s instructions.

Techniques: Infection, Comparison

Journal: bioRxiv

Article Title: The active secretion of a subunit of IL-12 by tissue cells is regulated by Valosin-Containing Protein and intracellular calcium redistribution

doi: 10.64898/2026.01.28.702376

Figure Lengend Snippet: The IL-12p35 subunit is not secreted in the absence of the IL-12p40 binding partner: Development of two novel reporter systems to track the release of p35 from cells and subcellular location throughout p40-independent release. (A) Luciferase assay to detect p35NL release in the cell culture supernatant when co-expressed with p40. HEK cells were transfected with p35NL + empty vector (light red), p35NL + p40 (red), NanoLuc-only (NLO) + empty vector (light blue), or NLO + p40 (blue). Supernatants were collected 48 hours after transfection for luciferase assay (n=3). Luminescence data is represented in Raw Luminescence Units (RLUs). Functionality of p35NL as an IL-12 subunit when co-expressed with p40. SMARTA transgenic T cells were activated in vitro and re-stimulated with either recombinant p40 protein, recombinant IL-12, or supernatants from HEK cells co-transfected with p35NL and p40. After 2 hours of re-stimulation, IFN-γ production by T cells was measured using intracellular cytokine staining. (B) MFI of live CD4 + CD44 + IFN-γ + SMARTA transgenic T cells populations after restimulation (n = 3). (C) Percentages of MFI of live SMARTA CD4 + CD44 + IFN-γ + transgenic T cells populations after restimulation (n = 3). (D) Representative flow plots compare the percentage of live CD4+CD44+IFNγ+ cells among groups (n = 3). Characterization of fluorescent reporter system for tracking the intracellular location of p35 through ER and Golgi compartments. (E) Murine fibroblasts (L cells) were retrovirally transduced with p35-Scarlet, Golgi-eGFP (Beta-1,4-galactosyl transferase 1), and ER-Halotag (KDEL tagged with Halotag and developed with JFX650). Images are representative of steady-state cells during live cell fluorescent imaging at 100X. White scale bars correspond to 10µm. Characterization of fluorescent reporter system for tracking the association of p35 with ER chaperone, BiP. (F) L cells were retrovirally transduced with p35-Scarlet, Golgi-eGFP, and BiP-BFP. Images are representative of steady-state cells during live cell fluorescent imaging at 100X. White scale bars correspond to 10µm. (G) Pixel intensity of each marker in p35-Scarlet/Golgi-GFP/ER-Halotag L cells along the ROI line draw in ‘MERGED (F)’. ROI line is designated by an arrow. (H) Pixel intensity of each marker in p35-Scar/Golgi-GFP/BiP-BFP L cells along the ROI line draw in ‘MERGED (G)’. ROI line is designated by an arrow. Data is presented as the mean with SEM error bars. Significant changes in luminescence in (A) were determined using an unpaired t-test. Significant changes in percentage and MFI in (B) and (C) were determined using an unpaired t-test. p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant.

Article Snippet: IL-12p40-KO mice (B6.129S1- Il12b tm1Jm /J) were obtained from Jackson Laboratory and used to generate primary muscle cell lines.

Techniques: Binding Assay, Luciferase, Cell Culture, Transfection, Plasmid Preparation, Transgenic Assay, In Vitro, Recombinant, Staining, Transduction, Imaging, Marker

Journal: The Journal of Immunology Author Choice

Article Title: NKG2A-mediated immune modulation of natural killer cells by Staphylococcus aureus

doi: 10.1093/jimmun/vkaf174

Figure Lengend Snippet: TSST-1–mediated polarization toward a CD57 − NKG2A + NK cell phenotype involves an IL-12–independent mechanism. (A) Representative flow cytometry plots depicting expression levels of NKG2A vs CD57 on CD56 dim NK cells from human PBMCs collected from healthy controls that were cultured in medium alone (Unstim) or pretreated with anti-human IL-12p70 or an isotype control antibody (IgG 1 ) before being stimulated with TSST-1 for 48 h. (B) Scatter dot plot showing the frequency of NKG2C − NKG2A + (red filled circles) or CD57 − NKG2A + (green filled circles) subsets among CD56 dim NK cells from human PBMCs collected from healthy controls that were cultured in medium alone (Unstim) or pretreated with anti-human IL-12p70 or an isotype control antibody (IgG 1 ) before being stimulated with TSST-1 for 48 h. Each dot represents 1 donor. Line indicates mean value. (C) Scatter dot plots depicting geometric mean fluorescence intensity (gMFI) of NKG2A (green filled circles), CD16 (yellow filled circles), or CD56 (blue filled circles) expression on CD56 dim NK cells from human PBMCs collected from healthy controls that were cultured in medium alone (Unstim) or pretreated with anti-human IL-12p70 or an isotype control antibody (IgG 1 ) before being stimulated with TSST-1 for 48 h. Each dot represents 1 donor. Line indicates mean value. * P < 0.05, ** P < 0.01; 1-way ANOVA with Tukey post hoc test.

Article Snippet: Nunc MaxiSorp 96-well plates (BioLegend) were coated with an anti-IL-12p40 capture antibody (2 μg/ml; Mabtech; clone MT86/221) and incubated overnight at 4 °C.

Techniques: Flow Cytometry, Expressing, Cell Culture, Control, Fluorescence