nk cell isolation kit  (Miltenyi Biotec)

Journal: Transplant International

Article Title: Blockade of CD155 and CD276 by Monoclonal Antibodies Fosters Immune Tolerance and Promotes Stable Engraftment of iPSC-Derived Islets in Allogeneic Humanized Mice

doi: 10.3389/ti.2025.15433

Figure Lengend Snippet: Blockade of CD276 and CD155 reduces missing-self recognition and NK-mediated killing of iPSC-derived EPs. (A) Experimental workflow. Both iPSCs and iPSC-derived EP clusters were co-cultured with KIR-genotyped NK cells that were isolated and expanded from healthy donor PBMCs in the presence of IL-2 and IL-15. NK-mediated cytotoxicity was evaluated by live-cell imaging over 6 h, in the presence or absence of blocking mAbs targeting the NKp30/CD276 and CD226/CD155 axes. (B) Quantification of NK-mediated killing (% of dead target cells) under KIR-HLA-matched (white bars) or mismatched (colored bars) conditions, with the latter in the presence of α-NKp30, α-CD226, α-CD276, α-CD155, or both α-CD276 and α-CD155 mAbs. N = 6. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. (C) Representative time-lapse images of NK cell infiltration and EP cluster lysis under matched and mismatched conditions (green = dead cells, red = eFluor670-labeled human NK cells). (D) Kinetic analysis of dead cell area expressed as Green Calibrated Unit (GCU) per square micrometer per field of view, under matched versus mismatched conditions. The line represents the mean. N = 5. *p < 0.05, ****p < 0.0001 by two-way ANOVA followed by Šídák’s post hoc multiple comparison test. (E) Kinetic analysis of NK cell-mediated infiltration and killing measured as the co-localization of GCU and Red Calibrated Unit (RCU) per field of view, under matched versus mismatched conditions. The line represents the mean N = 5. *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA followed by Šídák’s post hoc multiple comparison test. (F) Representative time-lapse images of NK cell infiltration and killing in untreated versus α-CD276 + α-CD155-treated EPs under mismatched conditions. (G) Kinetic analysis of dead cell area under untreated versus α-CD276 + α-CD155-treated EPs. The line represents the mean. N = 5. **p < 0.01, ****p < 0.0001 by two-way ANOVA followed by Šídák’s post hoc multiple comparison test. (H) Flow cytometry plots showing NK cell activation (HLA-DR + ) and degranulation (LAMP-1 + ) under untreated versus α-CD276 + α-CD155 treated conditions. (I) Violin plots representing the quantification of HLA-DR + and HLA-DR + /LAMP-1 + NK cells. N = 5. ****p < 0.0001 by two-tailed unpaired Student’s t-test.;

Article Snippet: NK cells were isolated from freshly obtained donor PBMCs using the CD56 + CD16 + NK Cell Isolation Kit (Miltenyi Biotec) and expanded for 12 days in NK MACS Medium (Miltenyi Biotec) supplemented with 5% human AB serum (Corning), 70 ng/mL IL-15, and 500 U/mL IL-2 (Peprotech).

Techniques: Derivative Assay, Cell Culture, Isolation, Live Cell Imaging, Blocking Assay, Lysis, Labeling, Comparison, Flow Cytometry, Activation Assay, Two Tailed Test

Journal: Transplant International

Article Title: Blockade of CD155 and CD276 by Monoclonal Antibodies Fosters Immune Tolerance and Promotes Stable Engraftment of iPSC-Derived Islets in Allogeneic Humanized Mice

doi: 10.3389/ti.2025.15433

Figure Lengend Snippet: Blockade of CD276 and CD155 modulates the activation of human immune cells. (A) Frequency of activated NK cells (CD56 + CD16 + CD69 + ) in the peripheral blood before and 7 days after EP TX in untreated or antibody-treated mice (α-CD276, α-CD155, or combination therapy). Data represent mean ± SEM. N = 4. ****p < 0.0001 by one-way ANOVA followed by Tukey’s post hoc multiple comparison test. (B) Frequencies of activated monocytes, evaluated by expression of HLA-DR and CD40L, before and 7 days after TX. Data represent mean ± SEM. N = 4. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post hoc multiple comparison test. (C) Frequencies of activated CD8 + T cells (CD8 + CD38 + HLA-DR + ) and PD-1 + CD8 + T cells before, 7 days, and 12 days after TX. Data represent mean ± SEM. N = 4. *p < 0.05, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post hoc multiple comparison test. (D) Frequency of CD4 + T cells expressing LAG-3 before and 7 and 12 days after TX. Data represent mean ± SEM. N = 4. *p < 0.05, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post hoc multiple comparison test. (E) Representative flow cytometry plots of CD4 + T cells showing Tconv (Foxp3 − Helios - ) and Treg (Foxp3 + Helios + ) subsets under untreated and α-CD155-treated conditions. (F) CD25 expression on Foxp3 + Helios + Tregs under untreated and α-CD155-treated groups. (G) Violin plots showing the quantification of the CD25 Mean Fluorescent Intensity (MFI) on Foxp3 + Helios + Tregs. N = 5. ****p < 0.0001 by two-tailed unpaired Student’s t-test.

Article Snippet: NK cells were isolated from freshly obtained donor PBMCs using the CD56 + CD16 + NK Cell Isolation Kit (Miltenyi Biotec) and expanded for 12 days in NK MACS Medium (Miltenyi Biotec) supplemented with 5% human AB serum (Corning), 70 ng/mL IL-15, and 500 U/mL IL-2 (Peprotech).

Techniques: Activation Assay, Comparison, Expressing, Flow Cytometry, Two Tailed Test