Journal: Science Advances

Article Title: Targeted knockdown of Kv1.3 channels in T lymphocytes corrects the disease manifestations associated with systemic lupus erythematosus

doi: 10.1126/sciadv.abd1471

Figure Lengend Snippet: ( A ) IHC of CD8 (brown signal) and CD4 (pink signal) expression in representative kidney biopsies from LN, DN, and NK individuals. Scale bar, 100 μm. Magnified tissue from the highlighted area (yellow rectangles) is presented in the bottom panels. ( B to D ) Number of CD8 + T cells (B) and CD4 + T cells (C) in LN, DN, and NK were quantitated as described in Materials and Methods. (D) The ratio of CD8 + T cells to CD4 + T cells measured in LN kidneys, DN kidneys, and NKs. ( E ) Representative confocal images of kidney biopsy sections stained for CD8 (green) and CD45RO channels (magenta) in patients with LN and DN. Scale bar, 50 μm. ( F and G ) Number of CD45RO + cells (F) and CD8 + CD45RO + cells (G) were quantitated as described in Materials and Methods. In (B) to (D), data are presented as box and whisker plots. The data are reported as the median (horizontal line), first (top box), and third (bottom box) quartiles in biopsy samples from 10 LN kidneys, 10 DN kidneys, and 10 NKs. In (F) and (G), the data are presented as box and whisker plots in biopsy samples from four LN kidneys and four DN kidneys where at least five fields were imaged per patient. Data in (B) to (D) were analyzed by one-way analysis of variance (ANOVA) ( P < 0.001), and post hoc testing was performed by Tukey’s test, while data in (F) and (G) were analyzed by Student’s t test.

Article Snippet: For CD8 functionality, sections were stained with mouse monoclonal anti-human CD8 antibody (clone 4B1, Novus Biologicals), rabbit monoclonal anti-human Ki-67 (clone EPR3612, Abcam), and guinea pig polyclonal anti-human Kv1.3 (Alomone Labs).

Techniques: Expressing, Staining, Whisker Assay

Journal: Science Advances

Article Title: Targeted knockdown of Kv1.3 channels in T lymphocytes corrects the disease manifestations associated with systemic lupus erythematosus

doi: 10.1126/sciadv.abd1471

Figure Lengend Snippet: ( A ) Representative merged confocal images of kidney biopsy sections stained for CD8 (green), Kv1.3 (magenta), GrB (cytotoxicity marker; orange,), and Ki-67 (proliferation marker; blue) in LN, DN, and NK individuals. Scale bar, 25 μm. ( B to D ) Fluorescence intensities (measured as mean gray values) of Kv1.3 (B), GrB (C), and Ki-67 (D) in CD8 + T cells in kidney biopsies from LN, DN, and NK individuals. In (B) to (D), data are presented as box and whisker plots. The data are reported as the median (horizontal line), first (top box), and third (bottom box) quartiles for 675 CD8 + T cells from 10 LN kidneys, 520 cells from 10 DN kidneys, and 36 cells from 10 NK kidney biopsies. Data were analyzed by one-way ANOVA [ P < 0.001 for (B) to (D)]. Post hoc testing was performed by Dunn’s test.

Article Snippet: For CD8 functionality, sections were stained with mouse monoclonal anti-human CD8 antibody (clone 4B1, Novus Biologicals), rabbit monoclonal anti-human Ki-67 (clone EPR3612, Abcam), and guinea pig polyclonal anti-human Kv1.3 (Alomone Labs).

Techniques: Staining, Marker, Fluorescence, Whisker Assay

Journal: Science Advances

Article Title: Targeted knockdown of Kv1.3 channels in T lymphocytes corrects the disease manifestations associated with systemic lupus erythematosus

doi: 10.1126/sciadv.abd1471

Figure Lengend Snippet: ( A ) Schematic of humanized mouse model generation by engraftment of a NSG mouse with PBMCs from either a patient with LN or an HDs. ( B ) Left: Splenic T cell abundance in LN and HDs engrafted, as well as NE, mice measured by flow cytometry 6 to 8 weeks after engraftment. Human-specific antibodies were used to detect the T lymphocyte markers presented in the figure. ( C ) The ratio of CD8 + T cells to CD4 + T cells measured in splenocytes from LN and HDs mice. ( D ) Serum IgG levels (human) measured 6 weeks after engraftment in LN, HDs, and NE mice. ( E ) IHC of CD3 and CD8 expression (brown signal) in kidney tissues harvested from LN, HDs, and NE mice 6 weeks after engraftment. Representative image is shown here. Scale bar, 100 μm. ( F ) Urine protein measured in LN and HDs engrafted, as well as NE, mice. ( G ) Survival in LN, HDs, and NE mice presented as Kaplan-Meier Survival curve. Significance was evaluated by a log-rank test. All experiments were performed 6 to 8 weeks after engraftment. PBMCs from three SLE/LN individuals and two HDs were used for engrafting 4 to 12 mice per group. Bars represent means ± SEM, and each symbol represents an individual mouse. In (B), the CD4, CD8, CD3, and CD45 abundances were compared between the NE, HDs, and LN groups by one-way ANOVA ( P < 0.001 for all groups). Data in (C) were analyzed by Student’s t test, while data in (D) and (F) were analyzed by one-way ANOVA ( P < 0.05) and post hoc testing was performed by Holm-Sidak method.

Article Snippet: For CD8 functionality, sections were stained with mouse monoclonal anti-human CD8 antibody (clone 4B1, Novus Biologicals), rabbit monoclonal anti-human Ki-67 (clone EPR3612, Abcam), and guinea pig polyclonal anti-human Kv1.3 (Alomone Labs).

Techniques: Flow Cytometry, Expressing

Journal: Science Advances

Article Title: Targeted knockdown of Kv1.3 channels in T lymphocytes corrects the disease manifestations associated with systemic lupus erythematosus

doi: 10.1126/sciadv.abd1471

Figure Lengend Snippet: Immune cell population in LN mice on days 2 and 7 after engraftment. PBMCs from two patients with LN were engrafted in four NSG mice, and immune cell populations were profiled on days 2 and 7 by flow cytometry and are presented as percentages of total live cells. Naïve T cells were defined as CD3 + CD45RO − CD38 − FSC intermediate ; Tm cells were defined as CD3 + CD45RO + CD38 − FSc intermediate ; plasma cells were defined as CD3 − CD38 + . Data were analyzed by Student’s t test.

Article Snippet: For CD8 functionality, sections were stained with mouse monoclonal anti-human CD8 antibody (clone 4B1, Novus Biologicals), rabbit monoclonal anti-human Ki-67 (clone EPR3612, Abcam), and guinea pig polyclonal anti-human Kv1.3 (Alomone Labs).

Techniques: Flow Cytometry, Clinical Proteomics

Journal: Science Advances

Article Title: Targeted knockdown of Kv1.3 channels in T lymphocytes corrects the disease manifestations associated with systemic lupus erythematosus

doi: 10.1126/sciadv.abd1471

Figure Lengend Snippet: ( A ) Representative confocal images of kidney and spleen tissues harvested 6 weeks after engraftment from LN mice that were stained for CD8 (yellow), Kv1.3 (magenta), and nuclei [4′,6-diamidino-2-phenylindole (DAPI); cyan]. Scale bar, 50 μm. ( B ) Left: Merged images of CD8 and Kv1.3 channels in the kidney and spleen from LN mice showing Kv1.3 staining in the CD8 + T cells. Right: Fluorescence intensities (measured as mean gray values) of Kv1.3 in CD8 + T cells in the kidneys and spleens from LN mice. Data are presented as box and whisker plots. The data are reported as the median (horizontal line), first (top box), and third (bottom box) quartiles for 134 CD8 + T cells from two LN mice kidneys and 400 CD8 + T cells from two LN mice spleens. Significance was determined by Mann-Whitney rank sum test.

Article Snippet: For CD8 functionality, sections were stained with mouse monoclonal anti-human CD8 antibody (clone 4B1, Novus Biologicals), rabbit monoclonal anti-human Ki-67 (clone EPR3612, Abcam), and guinea pig polyclonal anti-human Kv1.3 (Alomone Labs).

Techniques: Staining, Fluorescence, Whisker Assay, MANN-WHITNEY

Journal: Cancers

Article Title: Murine- and Human-Derived Autologous Organoid/Immune Cell Co-Cultures as Pre-Clinical Models of Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers12123816

Figure Lengend Snippet: Decreased tumor size in mice treated with combinatorial cabozantinib and anti-PD-1 inhibitor. Changes in ( A ) tumor sizes, ( B ) EpCAM/PD-L1+ve organoid death, ( C ) MDSC death/depletion, and ( D ) CTL proliferation in response to chemotherapy and PD-1Inh with or without cabozantinib treatments. Digital spatial analysis demonstrating ( E ) representative ROIs, and ( F ) immune marker protein expression in mouse experimental groups. Protein expression of ( G ) CD8, ( H ) MDSCs, and ( I ) αSMA quantitative RT-PCR, using RNA isolated from mouse tumors, for the expression of ( J ) CD8, ( K ) granzyme, and ( L ) fibronectin. * p < 0.05 compared to untreated; n = 10–20 mice per group.

Article Snippet: Human-derived cultures were immunostained using antibodies specific for HNF-1β (Novus Biologicals, NBP1-89680, Rabbit, 1:50), Sox 9 (Novus Biologicals, NBP2-52943, mouse, 1:250) CK19 (R&D Systems, AF3506, sheep,1:80), PD-L1 (Novus Biologicals, 76769, rabbit, 1:100), CD8a (R&D Systems, MAB1509, mouse, 1:60), Histone (abcam, ab125027, 1:100) or CD44V9 (CosmoBio, LKG-M003, Rat, 1:1000).

Techniques: Marker, Expressing, Quantitative RT-PCR, Isolation

Journal: Cancers

Article Title: Murine- and Human-Derived Autologous Organoid/Immune Cell Co-Cultures as Pre-Clinical Models of Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers12123816

Figure Lengend Snippet: Changes in PMN-MDSC, CD8 and SMA cell compartments in organoids directly derived from mouse tumors in response to experimental treatments. ( A ) Light micrographs of cultured organoids and ( B ) H&E staining of embedded organoids that were derived from mouse tumors in response to experimental treatments. Flow cytometric contour plots demonstrating the changes in ( C ) PMN-MDSC, ( D ) CD8 and SMA cell populations in organoids derived from mouse tumors in response to experimental treatments. Quantification (% cell populations) is shown in ( E ). * p < 0.05 compared to untreated; n = 10 mice per group.

Article Snippet: Human-derived cultures were immunostained using antibodies specific for HNF-1β (Novus Biologicals, NBP1-89680, Rabbit, 1:50), Sox 9 (Novus Biologicals, NBP2-52943, mouse, 1:250) CK19 (R&D Systems, AF3506, sheep,1:80), PD-L1 (Novus Biologicals, 76769, rabbit, 1:100), CD8a (R&D Systems, MAB1509, mouse, 1:60), Histone (abcam, ab125027, 1:100) or CD44V9 (CosmoBio, LKG-M003, Rat, 1:1000).

Techniques: Derivative Assay, Cell Culture, Staining

Journal: Cancers

Article Title: Murine- and Human-Derived Autologous Organoid/Immune Cell Co-Cultures as Pre-Clinical Models of Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers12123816

Figure Lengend Snippet: Analysis of murine-derived autologous pancreatic cancer organoid/immune cell co-cultures. Flow cytometric analysis quantifying the percentage of ( A ) zombie-positive (dead) EpCAM+PD-L1+ tumor, and ( B ) CD8+IFNγ+ and CD8+IL-2+ cells in co-cultures. ( C , D ) CTL proliferation as measured by CFSE T cell proliferation assay. ( E ) Co-cultures were fractionated into EpCAM+, MDSC+, and CD8+ cell populations by magnetic separation. Quantitative RT-PCR was performed using RNA extracted from ( F ) MDSC, (G) CD8, and ( H ) EpCAM fractions. * p < 0.05; n = 4 individual co-cultures per group.

Article Snippet: Human-derived cultures were immunostained using antibodies specific for HNF-1β (Novus Biologicals, NBP1-89680, Rabbit, 1:50), Sox 9 (Novus Biologicals, NBP2-52943, mouse, 1:250) CK19 (R&D Systems, AF3506, sheep,1:80), PD-L1 (Novus Biologicals, 76769, rabbit, 1:100), CD8a (R&D Systems, MAB1509, mouse, 1:60), Histone (abcam, ab125027, 1:100) or CD44V9 (CosmoBio, LKG-M003, Rat, 1:1000).

Techniques: Derivative Assay, Proliferation Assay, Quantitative RT-PCR

Journal: Cancers

Article Title: Murine- and Human-Derived Autologous Organoid/Immune Cell Co-Cultures as Pre-Clinical Models of Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers12123816

Figure Lengend Snippet: Correlation analysis of patient PDAC tumor tissue array using digital spatial profiling. ( A ) Digital spatial analysis demonstrating representative ROIs, and tumor (PanCK, green), stroma (SMA, yellow) and immune (CD68, red) expression in a patient pancreatic ductal adenocarcinoma tumor tissue array. Scatter plots showing ( B ) tumor, stromal and immune cell components. ( C ) Pearson correlation matrix and correlation between ( D ) MDSCs/PD-L1, ( E ) Stroma/CD8, ( F ) Arg1/SMA, ( G ) Arg1/FAPalpha, ( H ) Arg1/fibronectin, ( I ) S100B/SMA, ( J ) S100B/FAPalpha, and ( K ) S100B/fibronectin. was performed. The intensity of the color at the bottom bar indicates the degree of correlation.

Article Snippet: Human-derived cultures were immunostained using antibodies specific for HNF-1β (Novus Biologicals, NBP1-89680, Rabbit, 1:50), Sox 9 (Novus Biologicals, NBP2-52943, mouse, 1:250) CK19 (R&D Systems, AF3506, sheep,1:80), PD-L1 (Novus Biologicals, 76769, rabbit, 1:100), CD8a (R&D Systems, MAB1509, mouse, 1:60), Histone (abcam, ab125027, 1:100) or CD44V9 (CosmoBio, LKG-M003, Rat, 1:1000).

Techniques: Expressing

Journal: Cancers

Article Title: Murine- and Human-Derived Autologous Organoid/Immune Cell Co-Cultures as Pre-Clinical Models of Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers12123816

Figure Lengend Snippet: Analysis of patient-derived autologous PDAC organoid/immune cell co-cultures. ( A ) Light micrographs of representative images of patent-derived immune cells cultured with PDAC organoids. ( B ) Schematic representation of experimental conditions 1–5 used in organoid/immune cell co-cultures. ( C ) Histograms generated from CFSE T cell proliferation assays using co-cultures under treatment conditions 1–5. Percent of ( D ) proliferating CTLs, and ( E ) CD8+perforin+-expressing cells in co-cultures under treatment conditions 1–5. ( F ) Representative contour plots were quantified for changes in EpCAM+PD-L1+ zombie (dead) tumor cells within co-cultures under treatment conditions 1–5. ( G ) Representative contour plots were quantified for changes in arginase 1 expression in PMN-MDSCs within co-cultures under treatment conditions 1–5. * p < 0.05; n = 10 individual patient-derived co-cultures.

Article Snippet: Human-derived cultures were immunostained using antibodies specific for HNF-1β (Novus Biologicals, NBP1-89680, Rabbit, 1:50), Sox 9 (Novus Biologicals, NBP2-52943, mouse, 1:250) CK19 (R&D Systems, AF3506, sheep,1:80), PD-L1 (Novus Biologicals, 76769, rabbit, 1:100), CD8a (R&D Systems, MAB1509, mouse, 1:60), Histone (abcam, ab125027, 1:100) or CD44V9 (CosmoBio, LKG-M003, Rat, 1:1000).

Techniques: Derivative Assay, Cell Culture, Generated, Expressing