Journal: Journal of experimental & clinical cancer research : CR

Article Title: Regnase-1 downregulation promotes pancreatic cancer through myeloid-derived suppressor cell-mediated evasion of anticancer immunity.

doi: 10.1186/s13046-023-02831-w

Figure Lengend Snippet: Fig. 6 Suppression of CTLs by CD11b+ MDSCs is responsible for the acceleration of tumor progression by Regnase-1 downregulation. A-D Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1 KO murine pancreatic cancer cells. Representative macro images of pancreatic tumors (A). Relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (B) (N = 6 per group). Representative images of HE (C, left panel) and CD8a immunostaining (C, right panel) and the number of CD8-positive cells (C, right) (N = 6 per group). Dot plots of CD3+CD8+ cells evaluated by flow cytometry (D, left) and the proportion of CD8 + cells among CD45+ cells (D, right) (N = 3 per group). E–H Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1-KO murine pancreatic cancer cells with or without depletion of CD8+ cells upon anti-CD8a antibody or IgG treatment. Experimental schematic (E). Dot plots of CD3+ and CD8.+ cells in WT or Regnase-1-KO syngeneic tumors upon anti-CD8a antibody or IgG treatment evaluated by flow cytometry (F). Tumor weights (G) (N = 6 per group). The relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (H) (N = 6 per group). Student’s t test was used to evaluate differences between 2 groups. One-way ANOVA with Tukey’s post hoc test was used to compare differences among 4 groups. *P < 0.05, scale bars: 100 μm (insets)

Article Snippet: BE0061, a fully neutralizing mAb recognizing CD8a, and control IgG were obtained from Bioxcell.

Techniques: Immunostaining, Flow Cytometry

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

Journal:

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8??

doi: 10.1128/JVI.79.17.11422-11433.2005

Figure Lengend Snippet: Flow cytometric analysis of Nef-mediated receptor down-regulation and internalization in retrovirally transduced SupT1 cells. SupT1 cells were transduced with an inducible NA-7.ER construct. At time zero, 4-hydroxytamoxifen (1 μM) was added to the culture medium. (A) Percent down-regulation was calculated, as described in the Materials and Methods. CD4-allophycocyanin (solid line) and CD8β-phycoerythrin (dashed line) were measured as a function of time. (B) The figure shows the percentage of CD4, CD8αβ, and CD28 molecules internalized by HIV-1 NA-7.ER, calculated as described in Materials and Methods. In each graph, Nef-positive (EGFP expressing Nef, solid line) and Nef-negative (EGFP not expressing Nef, dashed line) cells are depicted. The EGFP ranges used for calculation are indicated in Fig. ​Fig.1A1A.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8β primary antibody (5F2 [ 15 ], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Transduction, Construct, Expressing

Journal:

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8??

doi: 10.1128/JVI.79.17.11422-11433.2005

Figure Lengend Snippet: Mutations in the CD8 β-chain and their effect on endocytosis and down-regulation. Daudi cells were cotransduced with CD8α, wild-type or mutant CD8β, and control or wild-type Nef. (A and C) Alignment of amino acid sequences of wild-type (210*) and mutant CD8 β-chain cytoplasmic tails. An asterisk indicates a stop codon. (A) The bar chart shows the percent down-regulation of (mutant) CD8αβ by HIV-1 Nef alleles NA-7, LAI, and NL4-3. In both B and D the percentage of (mutant) CD8αβ molecules internalized by wild-type HIV-1 NA-7 is shown, including in both the same data for a Nef-negative construct as a control (wild-type Nef−). (C) The bar chart represents the percentage of (mutant) CD8αβ down-regulation after transduction with either control virus or wild-type Nef NL4-3. Each bar represents a (mutant) CD8 β-chain, as indicated by the changed amino acid sequence compared with CD8αβ. Percent down-regulation and internalization were calculated as described in Materials and Methods, using the ranges indicated in Fig. ​Fig.1A.1A. In A, B, and D, mean values are shown and standard deviations are calculated from the data generated from three independent experiments. In B the results for NL4-3 are representative of the results with HIV-1 alleles NA-7 and LAI.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8β primary antibody (5F2 [ 15 ], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Mutagenesis, Construct, Transduction, Sequencing, Generated

Journal:

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8??

doi: 10.1128/JVI.79.17.11422-11433.2005

Figure Lengend Snippet: Chimeric constructs. Daudi cells were retrovirally transduced with the CD8α(EC-TM)-CD8α(IC) chimera (cyt tail) or a CD8α(EC-TM)-CD8β(IC) chimera (cyt tail), using bicistronic constructs with ΔNGFR as the reporter. Bivariate dot plots are gated on ΔNGFR-positive cells, at day 2 after transduction of these cells with control virus, HIV-1 Nef (NA-7 allele), and SIV Nef (mac239), using bicistronic constructs with EGFP as the reporter. CD8α-phycoerythrin versus EGFP expression is shown.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8β primary antibody (5F2 [ 15 ], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Construct, Transduction, Expressing

Journal:

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8??

doi: 10.1128/JVI.79.17.11422-11433.2005

Figure Lengend Snippet: Confocal images of 293T cells. Nef.EGFP was detected by direct fluorescence (green, left panels) and CD8β.HA or CD8β by monoclonal antibodies as indicated in Materials and Methods (red, middle panels). Nuclei were visualized by DAPI staining (blue). Right panels show the merged images from Nef.EGFP and CD8β. Areas of colocalization of Nef.EGFP/CD8β are shown in yellow. As indicated, the upper panels show cells expressing wild-type LAI, the middle panels show the LLAA mutant, and alower panels show the PPAA mutant. Scale bars represent 5 μm.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8β primary antibody (5F2 [ 15 ], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Fluorescence, Staining, Expressing, Mutagenesis