Journal: iScience

Article Title: TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

doi: 10.1016/j.isci.2023.108401

Figure Lengend Snippet: NK cells are activated by T cell-derived soluble mediators to conduct a TRAIL-dependent cytotoxicity against HPMC (A) TRAIL expression on CD335+ ascites-derived NK cells was measured after treatment with CM from unstimulated and α-CD3 Ab stimulated T cells by flow cytometry (n = 13 different patients). The geometric MFI was calculated after subtracting the isotype control. (B) Corresponding to the TRAIL expression on NK cells induced by the CM of T cells (+/− α-CD3 Ab stimulation), HPMC apoptosis induced by these activated NK cells was also analyzed by annexin V/PI staining (n = 13 different patients). (C) CD3 activation of ascites-derived T cells induce the secretion of cytokines fitting the terms “NK cell activation” and “TRAIL” in the Genecards database. Protein signals were determined by PEA-based affinity proteomics. Results are expressed as the fold change of T cells activated with α-CD3 Ab relative to untreated T cells (n = 5 matched pairs of different patients). Boxplots show the median (line), upper and lower quartiles (box), range (whiskers) and outliers (circles). (D) Upregulation of TRAIL expression on NK cells upon stimulation with rh-TNFα and rh-IL-21 together with rh-IL-2 (n = 5 experiments) and combinations of all 3 cytokines (n = 3 experiments) was analyzed by flow cytometry. (E) Co-culture of HPMC with NK cells previously activated with rh-TNFα (n = 6 patients) and rh-IL-21 (n = 4 patients) combined with rh-IL-2 were conducted and HPMC apoptosis induction was measured by flow cytometry. (F) A TRAIL-dependent apoptosis induction in HPMC by NK cells activated with rh-IL-2/IL-21/TNFα was confirmed by adding α-TRAIL blocking Ab (n = 6 patients). (G) To evaluate the contribution of TNFα and IL-21 on NK cell activation resulting in HPMC apoptosis, CM of T cells (α-CD3 Ab stimulated) were incubated with blocking Ab against IL-21 and TNFα (Infliximab) prior to NK cell treatment. The effect on HPMC apoptosis induction was analyzed in co-culture experiments (n = 5 experiments). Horizontal bars or boxes indicate the mean and vertical error bars represent the standard deviation in panels A, B, and D–G. p values were determined by two-sided, paired t test and Benjamini-Hochberg adjustment: ∗ FDR < 0.05; ∗∗ FDR < 0.01; ∗∗∗ FDR < 0.001; ∗∗∗∗ FDR <0.0001.

Article Snippet: In individual experiments, NK cells were treated with the following cytokines – alone or in combination – for two days: 10 ng/mL rh-TNFα (PeproTech, Hamburg, Germany), 10 ng/mL rh-IFNγ (Biomol), 20 ng/mL rh-IL-2 (ImmunoTools, Friesoythe, Germany), and 10 ng/mL rh-IL-21 (PeproTech).

Techniques: Derivative Assay, Expressing, Flow Cytometry, Staining, Activation Assay, Co-Culture Assay, Blocking Assay, Incubation, Standard Deviation

Journal:

Article Title: A hypermorphic I?B? mutation is associated with autosomal dominant anhidrotic ectodermal dysplasia and T cell immunodeficiency

doi: 10.1172/JCI18714

Figure Lengend Snippet: PBMC and T cell analysis. (a) Peripheral blood cells from patient P, a healthy control, and patient X-EDA-ID with OL-EDA-ID were stimulated by PHA, PMA-ionomycin, IL-12, IL-12 + IL-1β, or PHA, and IFN-γ secretion was measured by ELISA. Results from one representative experiment are shown. (b) Peripheral blood cells were stimulated by PMA-ionomycin or LPS + IFN-γ, and TNF-α secretion was measured by ELISA. Results from one representative experiment are shown. (c) CD45RA and CD45RO expression on control (C) and patient (P) T cells at day 0 and day 10 of PHA + IL-2 stimulation in vitro, as detected by flow cytometry. (d) T cell proliferation (left) after 64 hours of stimulation with anti-CD3 alone, or anti-CD3 in combination with anti-CD28. Production of IFN-γ (right) in culture supernatants after 48 hours of stimulation with anti-CD3 alone or in combination with anti-CD28. Results from one representative experiment are shown. Data are normalized for 106 cells.

Article Snippet: T cell blasts were generated by stimulating frozen PBMC with 5 μg/ml PHA (Sigma-Aldrich), 50 U/ml rh-IL-2 (PeproTech, Inc.), and allogeneic irradiated PBMCs (5 × 10 5 allogeneic irradiated PBMCs per 1 × 10 6 PBMCs).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, In Vitro, Flow Cytometry