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

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The effects of gemcitabine and capecitabine combination chemotherapy and of low-dose adjuvant GM-CSF on the levels of myeloid-derived suppressor cells in patients with advanced pancreatic cancer

doi: 10.1007/s00262-013-1502-y

Figure Lengend Snippet: No significant differences in the level of pro-inflammatory cytokines in patients with high MDSCs compared with those with low MDSCs. Peripheral blood mononuclear cells from 33 pancreatic cancer patients obtained pre-treatment (at baseline) were immunostained for HLA-DR-APC-Cy7, Lin1(CD3,14,16,19,20,56)-FITC and CD11b-PECy7 and with ViViD to discriminate live and dead cells. Following immunostaining, cells were analyzed using a MACSQuant flow cytometer with MACSQuantify software. In addition, serum cytokines TNFα, MCP-1, IL-1b, IL-6 and VEGF were determined using the Bio Rad Bio Plex 27 Pro Cytokine, Chemokine and Growth Factor assay using the Bio Rad Bio Plex Instrument. Baseline MDSCs dichotomized at median (1.85) plotted with baseline IL-6, IL-1b, VEGF, TNFα and MCP-1 (pg/ml). P value generated for Mann–Whitney test shows no significant differences

Article Snippet: Immunophenotypic analysis of cells Peripheral blood mononuclear cells were recovered and washed in 0.15 M phosphate-buffered saline, Dulbecco’s A (Oxoid, UK).

Techniques: Immunostaining, Flow Cytometry, Software, Generated, MANN-WHITNEY

Journal: Fly

Article Title: Characterizing a gene expression toolkit for eye- and photoreceptor-specific expression in Drosophila

doi: 10.1080/19336934.2021.1915683

Figure Lengend Snippet: Plasmids generated in this study

Article Snippet: The sRh1 or mRh1 promoter fragments were cloned into pBMPGal4LWL (Addgene #26270)[ 19 ] as NotI/BglII fragments to generate the pBMP-pRh1(−120/+67)-Gal4LWL and pBMP-pRh1(−450/+62)-Gal4LWL vectors and integrated in the attP2 and attP-9A sites, respectively, using ΦC31-mediated transformation by BestGene (CA).

Techniques: Generated

Journal: Cell

Article Title: Integrated analysis of multimodal single-cell data

doi: 10.1016/j.cell.2021.04.048

Figure Lengend Snippet: Applying WNN to additional multimodal technologies, related to Figure 2 (A) Analysis of a publicly available dataset of 11,351 PBMC processed with the 10x Genomics Multiome ATAC+RNA kit. UMAP visualizations of RNA and ATAC-seq data, as well as integrated WNN analysis. Cells are labeled by their WNN-annotated clusters. (B) Visualization of pseudobulk chromatin accessibility tracks of the CD8A locus for eight T cell subsets. Multiple peaks clearly separate CD8+ and CD8- T cells, exemplifying the information in ATAC-seq that can enhance parallel RNA measurements for defining cell states. (C) Enriched motifs within MAIT-specific open chromatin regions. Since multiple transcription factors (i.e., RORA, RORB, RORC) have very similar binding motifs, each exhibits strong evidence of enrichment. (D) Density plots, produced by the Nebulosa package, showing the RNA expression of RORC, RORA and RORB. (E) Visualization of RORC motif activity, as calculated by chromVAR, which mirrors the expression of the RORC as shown in (D). (F) Analysis of a published ASAP-seq dataset of 4,725 human PBMC where chromatin accessibility and surface expression of 227 surface proteins are simultaneously measured. UMAP visualizations of ATAC and protein data, as well as integrated WNN analysis. Cells are labeled by their WNN-annotated clusters. (G) Enriched motifs within MAIT-specific open chromatin regions in the ASAP-seq dataset are concordant with those identified in ATAC+RNA analysis. (H) Analysis of a publicly available dataset of 34,774 mouse skin cells from SHARE-seq, which generates paired single-cell profiles of gene expression and chromatin accessibility. UMAP visualizations of RNA and ATAC-seq profiles, as well as integrated WNN analysis. Cells are labeled by their annotations from (Ma et al., 2020b). (I) Four basal subpopulations were identified from WNN clustering, and cells from each subpopulation are highlighted in the UMAP visualizations from (H). Basal_4 and Basal_1 do not separate in transcriptomic analysis, but form distinct clusters in ATAC and WNN analysis. (J) Pseudobulk expression profiles of the Basal_4 and Basal_1 subpopulations demonstrate that the two groups exhibit similar transcriptomic profiles. (K) Top motifs exhibiting differential accessibility between Basal_4 and Basal_1, as identified by chromVar analysis. (L) chromVar motif activity scores for the p53 and CTCF motifs for all basal subpopulations. In each case, Basal_4 exhibits elevated accessibility at these motif sites. ∗∗∗ p value < 1e-5 based on Wilcoxon test. (M) Visualization of pseudobulk chromatin accessibility tracks of the Ct cf. locus for four basal subpopulations. In addition to exhibiting greater accessibility globally at CTCF motif sites, Basal_4 exhibits increased accessibility at the Ct cf. promoter.

Article Snippet: We applied WNN analysis to a dataset of 11,351 paired PBMC profiles generated by the 10x Genomics Multiome ATAC+RNA kit.

Techniques: Labeling, Binding Assay, Produced, RNA Expression, Activity Assay, Expressing, Gene Expression

Journal: Cell

Article Title: Integrated analysis of multimodal single-cell data

doi: 10.1016/j.cell.2021.04.048

Figure Lengend Snippet:

Article Snippet: We applied WNN analysis to a dataset of 11,351 paired PBMC profiles generated by the 10x Genomics Multiome ATAC+RNA kit.

Techniques: Expressing, Software

Journal: Cancer immunology research

Article Title: Human CD4 + T cells specific for Merkel cell polyomavirus localize to Merkel cell carcinomas and target a required oncogenic domain

doi: 10.1158/2326-6066.CIR-19-0103

Figure Lengend Snippet: TGEM was performed as outlined in the Methods testing seven allele types. PBMCs were cultured in the presence of pools of five MCPyV 20-mer peptides (WED, GNI, NSG, PNG; amino acid sequence indicated) for 10–14 days, followed by staining for pools of tetrameric complexes of 10 peptides (composed of two combined pools) bound to HLA class II molecules. After identification of CD4+tetramer+ T cells, the same cultures were stained with single tetramers containing each relevant peptide within the positive pool. Representative flow plots are shown for each peptide. For each epitope, four individuals were tested, and one to four persons were positive as detailed in column 7 of Table 1. The percentage of viable CD4+tetramer+ T cells (negative for CD8/CD14/CD19) in the lymphocyte forward/side scatter region are denoted.

Article Snippet: For tetramer-based sorting, PBMCs or TILs (5–20 X 10 6 ) were thawed, phosphate-buffered saline (PBS)-washed, and incubated with 100 nM dasatinib (SelleckChem, #S1021) for 10 minutes at 37°C in T-cell medium (TCM; RPMI, 8% human serum, 200 nM L-glutamine and 100 U/ml penicillin-streptomycin).

Techniques: Cell Culture, Sequencing, Staining

Journal: Cancer immunology research

Article Title: Human CD4 + T cells specific for Merkel cell polyomavirus localize to Merkel cell carcinomas and target a required oncogenic domain

doi: 10.1158/2326-6066.CIR-19-0103

Figure Lengend Snippet: A: Fresh tumor digest samples were acquired from three HLA-DRB1*0401-positive patients and stained with DRB1*0401-WEDLT209–228 dextramer. A WEDLT209–228 -specific CD4 clone generated against the WEDLT209–228 epitope was spiked into PBMCs as a positive control. TILs from an MCPyV-positive, HLA DB1*0401-negative patient were used as a negative control. Frequency of CD4+dextramer+ T cells shown for the indicated clones. Positive dextramer staining was consistent between 2 replicate experiments. B: Peripheral blood samples (n=13) and fresh tumor digest samples (n=3) from HLA-DRB1*0401-positive MCC patients were stained with DRB1*0401-WEDLT209–228 dextramer as described in Materials and Methods. The percent of viable CD4+dextramer+ T cells (negative for CD8/CD14/CD19) in the lymphocyte forward/side scatter region were divided by the total CD4+ T-cell population (in the lymphocyte forward/side scatter region, viable CD4+CD8−CD14−CD19−). The median number of WEDLT209–228 -specific CD4+ T cells of the total CD4 population was 0.0022% in the periphery compared to 0.56% within tumors (p = 0.0036, Mann-Whitney Test). C: TCR beta chain sequencing of the variable CDR3 region was performed on sorted cells from panel A. Pie slices depict the frequency of an individual clonotype within the dextramer-positive, WEDLT209–228 -specific CD4+ T-cell population. Exploded and starred slices indicate two TRB sequences with shared amino acid sequences between patients w876 and w1056.

Article Snippet: For tetramer-based sorting, PBMCs or TILs (5–20 X 10 6 ) were thawed, phosphate-buffered saline (PBS)-washed, and incubated with 100 nM dasatinib (SelleckChem, #S1021) for 10 minutes at 37°C in T-cell medium (TCM; RPMI, 8% human serum, 200 nM L-glutamine and 100 U/ml penicillin-streptomycin).

Techniques: Staining, Generated, Positive Control, Negative Control, Clone Assay, MANN-WHITNEY, Sequencing

Journal: Animal Models and Experimental Medicine

Article Title: Elimination of GGTA1 , CMAH , β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions

doi: 10.1002/ame2.12461

Figure Lengend Snippet: Construction and evaluation of 4KO piglets in xenotransplantation. (A) Schematic diagram of method for generating 4KO piglets. (B) Schematic of the 10 modified alleles. We generated the 4KO edits using CRISPR–Cas9 with gRNAs targeting the 2 copies of GGTA1 , 2 copies of CMAH , 4 copies of β4GalNT2 and 2 copies of the CIITA . (C) Deficiency of GGTA1 , CMAH , β4GalNT2 , and CIITA can impair the antigens of αGal, Neu5Gc, SDa, and SLA class II molecules, respectively. Flow cytometry was used to verify the deficiency of αGal, Neu5Gc, SDa, and SLA class II‐DR molecules at the cellular level in 4KO piglets. PBMCs of 4KO pigs had negative population cells (WT = 3, 4KO = 3). (D) Immunofluorescence results showed that αGal, Neu5Gc, SDa, and SLA class II molecules were not expressed in kidney tissue of 4KO piglets, and GGTA1 , CMAH , β4GalNT2 , and CIITA are genes were used to knockout the antigen of αGal, Neu5Gc, SDa and SLA class II molecules, respectively. Scale bar: 20 μm. (E) Verification by HE staining of the viability of 4KO tissue after gene editing; Scale bar: 100 μm.

Article Snippet: Stimulated pig cell samples were 2 × 10 5 WT or 4KO pig PBMCs treated with 50 μg/mL Mitomycin C (S8146, Selleck, Houston, TX, USA) for 20 min at 37°C to block their capacity for proliferation.

Techniques: Modification, Generated, CRISPR, Flow Cytometry, Immunofluorescence, Knock-Out, Staining

Journal: Animal Models and Experimental Medicine

Article Title: Elimination of GGTA1 , CMAH , β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions

doi: 10.1002/ame2.12461

Figure Lengend Snippet: Changes in the pig immune system induced by CIITA deficiency (WT = 3, 4KO = 3): (A) Percentage of the population of CD3 in WT and 4KO. (B) Percentage of the population of γδT in WT and 4KO. (C) 4KO CD3 T cells are not significantly different from WT. (D) 4KO γδT are not significantly different from WT. (E) Percentage of the population of CD4+ T and CD8+ T in WT and 4KO. (F) 4KO CD4+ T cells significantly decreased relative to WT; 4KO CD8+ T cells significantly increased relative to WT. (G) B cell population of WT and 4KO PBMCs.( n = 3). (H) No significant difference between WT and 4KO in the population of B cells. (I) Percentage of 4KO NK cell population significantly decreased relative to WT. (J) NK cell population of WT and 4KO PBMCs. ** p < 0.01; * p < 0.05.

Article Snippet: Stimulated pig cell samples were 2 × 10 5 WT or 4KO pig PBMCs treated with 50 μg/mL Mitomycin C (S8146, Selleck, Houston, TX, USA) for 20 min at 37°C to block their capacity for proliferation.

Techniques:

Journal: Animal Models and Experimental Medicine

Article Title: Elimination of GGTA1 , CMAH , β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions

doi: 10.1002/ame2.12461

Figure Lengend Snippet: (A) Verifying the function of CIITA deficiency, T cell proliferation was analyzed based on dilution of the proliferation dye CSFE with each cell division, Proliferation of human CD4+ T cells is depicted as percent of divided cell (WT = 3; 4KO = 3). (B) 4KO has lower immunogenicity to lead human CD4+ T proliferation than WT (WT = 3; 4KO = 3). (C) Verifying the Serum‐mediated antibody binding reaction of WT and 4KO, compared with WT, 4KO show significantly reduced binding to human IgG (a) and IgM (WT = 8; 4KO = 8). (D) Verifying the Human complement‐dependent cytotoxicity of WT and 4KO, 4KO show significantly lower antibody‐dependent complement cytotoxicity compared with WT (WT = 5; 4KO = 5). Unpaired two‐tailed t ‐test; ** p < 0.01; * p < 0.05).

Article Snippet: Stimulated pig cell samples were 2 × 10 5 WT or 4KO pig PBMCs treated with 50 μg/mL Mitomycin C (S8146, Selleck, Houston, TX, USA) for 20 min at 37°C to block their capacity for proliferation.

Techniques: Immunopeptidomics, Binding Assay, Two Tailed Test