Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Involvement of CD56brightCD11c+ cells in IL-18-mediated expansion of human γδ T cells.

doi: 10.4049/jimmunol.1001919

Figure Lengend Snippet: FIGURE 1. Effect of IL-18 on the growth of gd T cells and CD56brightCD11c+ cells. A, Effect of IL-18 (100 ng/ml) on expansion of gd T cells. PBMCs derived from a representative healthy adult individual were stimulated by ZOL (1 mM)/IL-2 (10 ng/ml) or 2M3BPP (100 nM–1 mM)/IL-2 (solid bar); ZOL/IL-2/IL-18 or 2M3BPP/IL-2/IL-18 (open bar); or ZOL/IL- 2/anti–IL-18Ra mAb (2 mg/ml; gray bar) or 2M3BPP/IL-2/anti–IL-18Ra mAb. The total number of gd T cells was quantified by trypan blue dye exclusion and flow cytometry. **p , 0.001. B, Massive cell aggregation induced by ZOL/IL-2/IL-18. Cell clusters were photo- graphed using a Nikon Digital Sight TE300- HM-2 (310) microscope (Nikon, Tokyo, Ja- pan) after 5 d of culture and analyzed by Lu- mina Vision Software (Mitani, Tokyo, Japan). C, Proportion of gd TCR-bearing cells in cul- ture on days 0 and 14. PBMCs were incubated in the presence of ZOL/IL-2/IL-18 for 14 d and analyzed for expression of CD3 and gd TCR. D, Phenotypic analysis of PBMCs in culture on days 0 and 14. PBMCs incubated with ZOL/ IL-2 or ZOL/IL-2/IL-18 were analyzed for expression of CD11c, CD3, and CD56 on days 0 and 14. CD56intCD11c2, CD56intCD11c+, CD56brightCD11c2, and CD56brightCD11c+ cells were gated, respectively. E, Time course of the expansion of gd T cells and CD56bright

Article Snippet: Depletion of CD3 +, CD56+, and CD14+ cells from PBMCs was conducted using LD columns and microbeads conjugated with mAbs to CD3, CD56, and CD14 (Miltenyi Biotec, Auburn, CA), respectively. gd T cells and CD14+ cells were purified by positive selection using MS columns (anti-TCR gd MicroBeads kit; Miltenyi Biotec).

Techniques: Derivative Assay, Cytometry, Microscopy, Software, Incubation, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Involvement of CD56brightCD11c+ cells in IL-18-mediated expansion of human γδ T cells.

doi: 10.4049/jimmunol.1001919

Figure Lengend Snippet: FIGURE 2. Effect of depletion of CD56+ or CD14+ cells on the expansion of gd T cells. Whole PBMCs and PMBCs depleted of CD56+ or CD14+

Article Snippet: Depletion of CD3 +, CD56+, and CD14+ cells from PBMCs was conducted using LD columns and microbeads conjugated with mAbs to CD3, CD56, and CD14 (Miltenyi Biotec, Auburn, CA), respectively. gd T cells and CD14+ cells were purified by positive selection using MS columns (anti-TCR gd MicroBeads kit; Miltenyi Biotec).

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Involvement of CD56brightCD11c+ cells in IL-18-mediated expansion of human γδ T cells.

doi: 10.4049/jimmunol.1001919

Figure Lengend Snippet: FIGURE 3. Expansion of CD56brightCD11+ cells by IL-2/IL-18 in CD3-depleted PBMCs. A, Expan- sion of CD56brightCD11c+ cells in CD3-depleted PBMCs. CD3-depleted PBMCs were incubated in the presence of ZOL/IL-2 or ZOL/IL-2/IL-18 and then analyzed for expression of CD3, CD11c, and CD56. B, Proliferation of PBMCs depleted of CD3+ cells. CD3-depleted PBMCs were incubated in the presence of various cyto- kines in combinations, as indicated, and the number of living cells was counted at indicated time points. C, Expansion of CD56brightCD11c+ cells in CD3- depleted PBMCs. The total number of CD56bright

Article Snippet: Depletion of CD3 +, CD56+, and CD14+ cells from PBMCs was conducted using LD columns and microbeads conjugated with mAbs to CD3, CD56, and CD14 (Miltenyi Biotec, Auburn, CA), respectively. gd T cells and CD14+ cells were purified by positive selection using MS columns (anti-TCR gd MicroBeads kit; Miltenyi Biotec).

Techniques: Incubation, Expressing

Journal: Cell Reports Medicine

Article Title: Merkel cell polyomavirus-specific and CD39 + CLA + CD8 T cells as blood-based predictive biomarkers for PD-1 blockade in Merkel cell carcinoma

doi: 10.1016/j.xcrm.2023.101390

Figure Lengend Snippet:

Article Snippet: 114 - CD56 (clone: REA196) , Miltenyi Biotec , Cat# 130-108-016, RRID: AB_2658728.

Techniques: Recombinant, Control, Sequencing, RNA Sequencing, Software

Journal: International Journal of Molecular Sciences

Article Title: Glycan Structures in Osteosarcoma as Targets for Lectin-Based Chimeric Antigen Receptor Immunotherapy

doi: 10.3390/ijms25105344

Figure Lengend Snippet: CD301-CARs display specific cytotoxicity against osteosarcoma cell lines. ( A ) Schematic representation of the CD301 LEC-CAR: ( B ) Expression of CD301 CAR at the cell surface of NK92 cells. NK92 cells were retrovirally transduced and sorted based on the GFP expression. The expression of the CD301 CAR construct was measured by flow cytometry using an antibody specific to the CD301 CRD. Color denotes areas of high and low population density. ( C ) Detection of CD301 ligands on target cells: osteosarcoma cell lines were stained with fluorescently labeled recombinant CD301 and analyzed by flow cytometry. ( D ) Cytotoxicity assay: NK92 cells expressing the CD301-CAR were cocultured for 3 h with calcein-labeled target cells with an effector-to-target ratio of 3:1 and 5:1, respectively, and measured as quadruplicates. Columns represent the median. Error bars show standard deviation. p < 0.01 is indicated by **, respectively.

Article Snippet: The degranulation of NK92 CAR and wild-type NK92 cells was induced upon interaction with target cells at an E:T ratio of 1:1 for 4 h at 37 °C and was assessed by measuring the expression of CD107a with a PE/Cy7-labeled anti-CD107a antibody (Biolegend, #328617) at the surface of FITC-labeled anti-CD56-stained NK92 cells (Miltenyi, #130-114-740).

Techniques: Expressing, Construct, Flow Cytometry, Staining, Labeling, Recombinant, Cytotoxicity Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Glycan Structures in Osteosarcoma as Targets for Lectin-Based Chimeric Antigen Receptor Immunotherapy

doi: 10.3390/ijms25105344

Figure Lengend Snippet: Lytic activity of CD301-CAR-expressing NK92 cells correlates with interferon-gamma secretion and increased degranulation. ( A ) CAR expression leads to enhanced interferon-gamma secretion of NK92 cells upon engagement with osteosarcoma cell lines. ( B ) CAR expression leads to enhanced degranulation of NK92 cells upon engagement with osteosarcoma cells. Columns represent the mean of three independent experiments measured in triplicates. Error bars show the standard deviation of the mean. p < 0.05, p < 0.01, and p < 0.001 are indicated by *, **, or *** respectively.

Article Snippet: The degranulation of NK92 CAR and wild-type NK92 cells was induced upon interaction with target cells at an E:T ratio of 1:1 for 4 h at 37 °C and was assessed by measuring the expression of CD107a with a PE/Cy7-labeled anti-CD107a antibody (Biolegend, #328617) at the surface of FITC-labeled anti-CD56-stained NK92 cells (Miltenyi, #130-114-740).

Techniques: Activity Assay, Expressing, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Glycan Structures in Osteosarcoma as Targets for Lectin-Based Chimeric Antigen Receptor Immunotherapy

doi: 10.3390/ijms25105344

Figure Lengend Snippet: Combination of immune checkpoint inhibition with CD301 CAR immunotherapy. ( A ) Flow cytometry analysis showing the surface expression of the TIGIT and PVRIG on NK92 cells. Respective antibody staining is shown as a red histogram, and the isotype control and unstained control are depicted as blue and gray histograms. ( B ) Target cell expression of PVR and PVRL2. Expression was analyzed by flow cytometry. The binding of anti-PVR and anti-PVRL2 antibodies are shown as orange histograms. The isotype and unstained control are shown as blue and gray histograms. ( C ) Cytotoxicity assay: NK92 cells expressing the CD301-CAR were cocultured for 3 h with calcein-labeled targets with an effector target ratio of 3:1, respectively, in the presence or absence of different amounts of the inhibitory anti-TIGIT antibody. Measurements were performed as quadruplicates. Columns represent the mean of three independent experiments. Error bars show the standard deviation of the mean. p < 0.05 and p < 0.01 are indicated by * and **, respectively.

Article Snippet: The degranulation of NK92 CAR and wild-type NK92 cells was induced upon interaction with target cells at an E:T ratio of 1:1 for 4 h at 37 °C and was assessed by measuring the expression of CD107a with a PE/Cy7-labeled anti-CD107a antibody (Biolegend, #328617) at the surface of FITC-labeled anti-CD56-stained NK92 cells (Miltenyi, #130-114-740).

Techniques: Inhibition, Flow Cytometry, Expressing, Staining, Control, Binding Assay, Cytotoxicity Assay, Labeling, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Ex vivo- generated lymphoid progenitors encompass both T cell and innate lymphoid cell fates

doi: 10.3389/fimmu.2025.1617707

Figure Lengend Snippet: ProTcell’s subsets express innate lymphoid cell markers delineating a second cell fate. ProTcells were produced from mPB and CB CD34 + cells through an ex vivo system and their transcriptome (scRNAseq) or protein expression (CyTOF) were assessed in a single cell fashion, and restricted to CD7 + progenitors, as described in Figures 1A, B . Violin plots showing the normalized expression level of (A) ILC- and (B) NK-related genes in the 7 clusters. Density plots illustrating the gene expression of (C) the main ILC markers KLRB1, ID2, NFIL3 and KIT and (D) NK-related markers GZMA, GZMB, PRF1 and CD56. (E) CyTOF data was visualized by UMAP dimension reduction technic. UMAP projections illustrating the protein expression of CD7, CD161, KIT and CD5. The green line delineates the CD161-expressing cells along with KIT enrichment, i.e. the ILC-primed cells; the red contour excludes the CD161-expressing cells and includes CD5 + progenitors. (F) UMAP projection showing the pseudotime lineages calculated by Slingshot that describes the progressive transition along CD7 + clusters.

Article Snippet: 176Yb , CD56 , Fluidigm , NCAM16.2 , 3176008C.

Techniques: Produced, Ex Vivo, Expressing, Gene Expression

Journal: Frontiers in Immunology

Article Title: Ex vivo- generated lymphoid progenitors encompass both T cell and innate lymphoid cell fates

doi: 10.3389/fimmu.2025.1617707

Figure Lengend Snippet: Both CD161 + and CD161 - ProTcell subsets retain T and NK cell potential as well as thymus seeding ability. (A–E, G, H) ProTcells were produced from CB CD34 + cells in 7 days as indicated in Figure 1A and sorted by FACS regarding their CD161 expression for further NK or T cell differentiation and functional in vivo experiment. To ensure the consistency, we used the same cell lot and the same sorted cell populations to perform the experiments shown in this figure, including NK cell differentiation, T cell differentiation, and the in vivo engraftment assay. (A) Experimental scheme for CD7 + CD161 - (red frame) and CD7 + CD161 + (green frame) cell subset sorting before NK or T cell differentiation. Differentiating cell phenotype was assessed by flow cytometry. (B, C) CD7 + CD161 +/- lymphoid progenitors were cultured for 14 days in the presence of IL15 without any feeder cell for NK differentiation. Bar plots represent the mean ± SD of (B) the proportion and (C) number of CD56 + NK-primed cells in the culture. (D, E) CD7 + CD161 +/- lymphoid progenitors were co-cultured for 4 weeks with OP9-DLL1 to advance T cell differentiation. Line plots depicting the mean ± SD of the (D) proportion and (E) number of CD3 + developing T cell throughout the culture. *p<0.05 values are for multiple paired Student’s t-test. (F) ProTcells were produced from mPB and CB CD34 + cells and their protein expression (CyTOF) was assessed in a single cell fashion, and restricted to CD7 + progenitors, as described in Figures 1A, B . CyTOF data was visualized by UMAP dimension reduction technic. UMAP projections illustrate the protein expression of several adhesion/homing molecules CCR9, CCR7, CXCR4, CD44, CD62L, SELPLG, ITGA4 and ITGB4. The black line separates the CD161-enriched from the CD161-devoid cells. (G) CD7 + CD161 - or CD7 + CD161 + cell subset has been injected separately into NSG neonates (<4-day-old). T cell reconstitution was analyzed by flow cytometry in the thymus at 6-week-post-transplantation. Bar plot representing the human chimerism (human CD45 + cells/human + murine CD45 + cells) and (H) the proportion of mature CD3 + TCRαβ + T cells in the thymus of mice injected either CD161 + or CD161 - ProTcells. ns, non significant for (B, C) Wilcoxon signed-rank test, (D) multiple paired Student's t-test, (G, H) Mann-Whitney U test.

Article Snippet: 176Yb , CD56 , Fluidigm , NCAM16.2 , 3176008C.

Techniques: Produced, Expressing, Cell Differentiation, Functional Assay, In Vivo, Flow Cytometry, Cell Culture, Injection, Transplantation Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Ex vivo- generated lymphoid progenitors encompass both T cell and innate lymphoid cell fates

doi: 10.3389/fimmu.2025.1617707

Figure Lengend Snippet: Activation of BCL11B regulatory elements does not restrict NK cell potential. (A) Experimental design for assessing BCL11B role in human T cell commitment. EGFP-BAC reporter was integrated at exon 1 of endogenous BCL11B gene in hPSC, resulting in a monoallelic disruption of this gene and the creation of BCL11B -EGFP reporter cell line. BCL11B -EGFP hPSCs were differentiated into hematopoietic progenitors (HP) for 9 days. The generated HP cells were isolated for early T cell differentiation on OP9-DLL4 for 14 more days. At this time, T9+T14, BCL11B -EGFP negative and positive progenitor cells had been sorted by FACS and secondary cultures were performed to assess their myeloid, NK and T cell potential. Phenotypes were assessed at each stage by flow cytometry for myeloid, lymphoid, NK or T membrane markers. (B) Representative dot plot of flow cytometry analysis of BCL11B -EGFP hPSC-derived progenitors upon sorting at day T9+T14 after culture on OP9-DLL4 for 14 days. At this stage, CD45 + CD56 - CD7 + cells are clearly distributed into two populations, according to BCL11B -EGFP expression. (C, D) BCL11B-EGFP neg/+ progenitors were co-cultured for 10 days with OP9-DLL4 with IL15 for NK differentiation. (C) Representative contour plots of flow cytometry analysis illustrating the expression of CD56, delineating NK differentiation, and CD16, translating NK maturation. (D) Bar plot representing the mean ± SD of CD56-expressing NK-primed cells in three independent experiments. *p<0.05 values are for paired Student’s t-test. (E, F) BCL11B -EGFP neg/+ progenitors were co-cultured 5 days with OP9 for myeloid differentiation. (E) Representative contour plots of flow cytometry analysis illustrating CD11b expression translating myeloid priming. (F) Bar plot representing the mean ± SD of CD11b-expressing myeloid progenitors in three independent experiments. *p<0.05 values are for paired Student’s t-test. (G, H) BCL11B -EGFP neg/+ progenitors were co-cultured 7 days with OP9-DLL4 devoid of IL15 for T cell differentiation. (G) Representative contour plots of flow cytometry analysis illustrating the expression of CD7 and CD5 translating T cell differentiation. (F) Bar plot representing the mean ± SD of CD7 and CD5-expressing T cell progenitor cells in four independent experiments.

Article Snippet: 176Yb , CD56 , Fluidigm , NCAM16.2 , 3176008C.

Techniques: Activation Assay, Disruption, Generated, Isolation, Cell Differentiation, Flow Cytometry, Membrane, Derivative Assay, Expressing, Cell Culture

Journal: iScience

Article Title: Defects in NK cell immunity of pediatric cancer patients revealed by deep immune profiling

doi: 10.1016/j.isci.2024.110837

Figure Lengend Snippet:

Article Snippet: Anti-CD56 (clone B159, conjugated to 155Gd) , Standard BioTools , Cat3155008B.

Techniques: Purification, Clinical Proteomics, Recombinant, Blocking Assay, Staining, Saline, Mass Cytometry, Software, Cytometry