Journal: Frontiers in Immunology

Article Title: Signaling Through gp130 Compromises Suppressive Function in Human FOXP3 + Regulatory T Cells

doi: 10.3389/fimmu.2019.01532

Figure Lengend Snippet: Loss of suppressive function in FOXP3 + Treg cells is driven by APC-derived factors. Primary FOXP3 + clones were generated from healthy donors as described above, and were analyzed for their ability to suppress allogeneic CD4 + CD25 − Teff cells in a 1:1 Treg:Teff suppression assay in the presence or absence of irradiated PBMCs (APCs). (A) Representative FACS plots showing CD25 and FOXP3 expression in representative FPSP, FPSN, and FNSN clones and their suppressive function in the presence or absence of APCs. (B) FOXP3 expression in FPSP, FPSN, and FNSN clones before, or 48 h after re-stimulation with anti-CD3 and irradiated APCs. (C) Suppressive potency of FOXP3 + and FOXP3 − clones in the presence or absence of APCs, or APC-derived supernatants. Shown are the suppression values for 61 FPSP, 31 FPSN, and 9 FNSN clones from one representative experiment of three different experiments where clones were generated from three different healthy individuals. Statistical analysis was done with the one-way ANOVA followed by the Tukey post-test. ** p < 0.01, **** p < 0.0001.

Article Snippet: Peripheral blood mononuclear cells (PBMC) were purified from buffy coats of healthy donors (Sanguine Biosciences) using Ficoll-Paque PLUS density gradient (GE Healthcare), and were cryopreserved.

Techniques: Derivative Assay, Clone Assay, Generated, Suppression Assay, Irradiation, Expressing

Journal: Frontiers in Immunology

Article Title: Signaling Through gp130 Compromises Suppressive Function in Human FOXP3 + Regulatory T Cells

doi: 10.3389/fimmu.2019.01532

Figure Lengend Snippet: Gp130 is preferentially expressed on naïve CD4 + T cells. PBMCs were isolated from healthy individuals and analyzed ex vivo by flow cytometry for the expression of the indicated markers. (A) The expression of gp130 on naïve vs. memory CD4 + T cells. Statistical analysis was performed using the student t- test. (B) The expression of gp130 on naïve vs. memory Treg cells and its correlation with the expression of FOXP3, Helios, TIGIT, and FCRL3. Shown are the combined results from seven different healthy individuals. Statistical analysis was done with the one-way ANOVA followed by the Tukey post-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Peripheral blood mononuclear cells (PBMC) were purified from buffy coats of healthy donors (Sanguine Biosciences) using Ficoll-Paque PLUS density gradient (GE Healthcare), and were cryopreserved.

Techniques: Isolation, Ex Vivo, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Signaling Through gp130 Compromises Suppressive Function in Human FOXP3 + Regulatory T Cells

doi: 10.3389/fimmu.2019.01532

Figure Lengend Snippet: High gp130 expression identifies Treg cells with reduced suppressive capacity ex vivo . Naïve and memory Treg cells (CD4 + CD25 High CD127 Low ) cells were FACS-sorted according to their gp130 expression levels and co-cultured with CFSE-labeled, FACS-sorted CD4 + CD25 − Teff cells in the presence of anti-CD3 and irradiated PBMCs for 96 h. Shown are representative FACS plots (A) and the percentage of suppression (B) of Teff cells at multiple Treg:Teff ratios in two different experiments on two different healthy donors. Statistical analysis was done with the one-way ANOVA followed by the Dunnett post-test. * p < 0.05, *** p < 0.001.

Article Snippet: Peripheral blood mononuclear cells (PBMC) were purified from buffy coats of healthy donors (Sanguine Biosciences) using Ficoll-Paque PLUS density gradient (GE Healthcare), and were cryopreserved.

Techniques: Expressing, Ex Vivo, Cell Culture, Labeling, Irradiation

Journal: Frontiers in Immunology

Article Title: Signaling Through gp130 Compromises Suppressive Function in Human FOXP3 + Regulatory T Cells

doi: 10.3389/fimmu.2019.01532

Figure Lengend Snippet: Functional blockade of gp130 augments Treg cell function. (A) Healthy human PBMCs were incubated with the gp130 inhibitor LMT-28 (10 μM) for 1 h, followed by stimulation with rhIL-6 (10 ng/mL) for 10 min. STAT3p expression was evaluated using multi-parametric flow cytometry. Shown is STAT3 expression in gated CD4 + T cells. (B) Naive Treg (CD4 + CD45RA + CD25 + CD127 Low ) cells were sorted from healthy human PBMCs and co-cultured with CD25 − Teff cells in the presence of irradiated PBMCs and soluble anti-CD3 (30 ng/mL) for 96 h. Cells were plated at a 0:1 or 1:8 Treg: Teff cell ratio along with rhIL-6 (10 ng/mL) with or without LMT-28 (10 μM). Shown data is representative of 3 independent experiments. Statistical analysis was done with the one-way ANOVA followed by the Dunnett post-test. * p < 0.05, **** p < 0.0001.

Article Snippet: Peripheral blood mononuclear cells (PBMC) were purified from buffy coats of healthy donors (Sanguine Biosciences) using Ficoll-Paque PLUS density gradient (GE Healthcare), and were cryopreserved.

Techniques: Functional Assay, Cell Function Assay, Incubation, Expressing, Flow Cytometry, Cell Culture, Irradiation

Journal: Future Science OA

Article Title: Promising approach for targeting ROBO1 with CAR NK cells to combat ovarian cancer primary tumor cells and organoids

doi: 10.2144/fsoa-2023-0135

Figure Lengend Snippet: Strategy and materials in this study. (A) Diagram of the procedure in the study. The primary tumor cells and tumor organoids were generated from patients' tumor tissues and ROBO1-NK were constructed from patients' PBMCs. (B) IHC of ovarian tumor tissues. Negative control was used irrelevant IgG replacing primary antibody for incubating. Strong positive of ROBO1 expression on cell membrane was marked by red circles. (C) ROBO1 positively expressed in tumor tissues of the clinical ovarian cancer patient #01, #02, #03 and #04.

Article Snippet: In brief, PBMCs were isolated from the patient's peripheral blood using the MACSprepTM PBMC Isolation Kit (130-115-169, Miltenyi Biotec, Bergisch Gladbach, GER) employing the density gradient centrifugation method.

Techniques: Generated, Construct, Negative Control, Expressing, Membrane

Journal: Future Science OA

Article Title: Promising approach for targeting ROBO1 with CAR NK cells to combat ovarian cancer primary tumor cells and organoids

doi: 10.2144/fsoa-2023-0135

Figure Lengend Snippet: Characters of ROBO1-NK cells. (A) Morphology comparison of PBMCs (upper panel), PBMC-NK cells (mid panel) and ROBO1-NK cells (bottom panel) under bright field. (B) Biomarkers determination of ROBO1-NK cells by flow cytometry. For the biomarkers CD34, CD45, CD3, CD56 and CD16, negative controls were used irrelevant IgG replacing primary antibody for incubating, presented as red peaks. For the ROBO1-CAR detection, negative control was used PBMC-NK incubating with anti-CAR antibody, presented as red peak. Samples in detection were presented as blue peaks.

Article Snippet: In brief, PBMCs were isolated from the patient's peripheral blood using the MACSprepTM PBMC Isolation Kit (130-115-169, Miltenyi Biotec, Bergisch Gladbach, GER) employing the density gradient centrifugation method.

Techniques: Comparison, Flow Cytometry, Negative Control

Journal: Future Science OA

Article Title: Promising approach for targeting ROBO1 with CAR NK cells to combat ovarian cancer primary tumor cells and organoids

doi: 10.2144/fsoa-2023-0135

Figure Lengend Snippet: ROBO1-NK lyse ovarian cancer cell line and primary cancer cells. (A) Positive expression of ROBO1 on SKOV-3 cells. (B) Rate of SKOV-3 cell lysis with different E-T ratio. E: number of effective cells; T: number of target cells. (C) Time curve of different NK cells, including Mock-CAR-NK (blue), PBMC-NK (green), ROBO1-NK (red) and control (medium, black), lysing SKOV-3 cells. (D) Lysis rate of primary ovarian tumor cells from patient #01, #02, #03 and #04 by PBMC-NK and ROBO1-NK after 24 h, detected with CCK-8 assay. (E) Photograph of PBMC-NK and ROBO1-NK lysing efficacy on primary ovarian tumor cells after 48 h co-culturing. The primary cells were presented as spindle-like form while NK cells were spherical form with clusters.

Article Snippet: In brief, PBMCs were isolated from the patient's peripheral blood using the MACSprepTM PBMC Isolation Kit (130-115-169, Miltenyi Biotec, Bergisch Gladbach, GER) employing the density gradient centrifugation method.

Techniques: Expressing, Lysis, Control, CCK-8 Assay