Journal: Translational Oncology

Article Title: Vaccine-induced ErbB (EGFR/HER2)-specific immunity in spontaneous canine cancer

doi: 10.1016/j.tranon.2021.101205

Figure Lengend Snippet: cEGFR p527 immunization mediates CD8 infiltration and antibody deposition into canine tumors. Postmortem bladder cancer tissue from a cEGFR p527 immune dog and tissue from healthy, non-cancerous bladder was examined for infiltration of CD8 T cells and endogenous antibody. A. CD8 isotype control. B. CD8 T cell staining (green). Arrows illustrate presence of CD8+ cells within the tumor tissue. C. & D. Antibody deposition in canine bladder tumor tissue from a cEGFR p527 immunized dog (two different fields). E. & F. Canine IgG deposition in normal canine bladder tissue (two different fields). Magnification is 40x (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Article Snippet: CD8 T cells were stained in tissues using mouse anti-dog CD8 (MAB6709; R&D Systems, Minneapolis, MN), followed by goat anti-mouse IgG ( H + L ) Alexa Fluor 488 (A11017; Life Technologies, Eugene, OR) and counterstaining with DAPI (Molecular Probes; Eugene, OR).

Techniques: Control, Staining

Journal: Science China. Life sciences

Article Title: Total body irradiation of donors can alter the course of tolerance and induce acute rejection in a spontaneous tolerance rat liver transplantation model.

doi: 10.1007/s11427-012-4370-3

Figure Lengend Snippet: Figure 3 Detection of T cell subpopulations in the peripheral blood and CTL-mediated lytic activity. A, There was no difference in the percentage of CD3+ T and CD3+CD4+ T cells between the transplant groups. The number of CD3+CD8+ T cells in the acute rejection group increased significantly (P<0.05), while the number of CD4+/CD8+ cells decreased significantly compared to the other groups (P<0.05). B, The ratio of Foxp3+CD4+CD25+ Tregs and the expression of CD4+CD25+ T cells in the acute rejection group was significantly lower than those in the other groups (P<0.05). C, In the control group, GITR expression was not detectable on T cells. GITR expression on increased T cells from all groups after culture with phorbol ester and ionomycin. The GITR expression level of T cells in the acute rejection group was significantly higher than that in the other groups (P<0.05). D, The spleen CTL-mediated lytic activity of the recipients in the acute rejection group increased by 32.6%, and was significantly higher than those in the other three groups (P<0.05).

Article Snippet: A 50 μL sample of the whole blood obtained after stimulation was incubated with 10 μL of anti-CD3-PerCp and 5 μL of anti-CD8-APC-Cy7 mAbs in the dark at 4°C for 30 min. After haematolysis and washing with PBS, the resuspended cells were incubated in the dark at 4°C for 45 min with 10 μL anti-rat GITR-PE mAb (R&D Systems, Minneapolis, MN, USA) or 10 μL IgG1-PE (Beckman Coulter Inc) as an isotype-control.

Techniques: Activity Assay, Expressing, Control

Journal: Nature communications

Article Title: Monoacylglycerol lipase regulates cannabinoid receptor 2-dependent macrophage activation and cancer progression.

doi: 10.1038/s41467-018-04999-8

Figure Lengend Snippet: Fig. 2 Macrophage MGLL inhibits tumor progression via CD8+T cells. a Six-week-old WT and TgMGLL mice were subcutaneously inoculated with MC-38 cells, and tumor volumes were measured dynamically. This test was performed four times. (n = 10) b The survival time of the MC-38 tumor-bearing mice described in a. The data represent the means ± s.e.ms. (n = 10, ***P < 0.005, Gehan-Breslow-Wilcoxon test). c The growth curves of the subcutaneous MC-38 tumors in Myeloid-mgll-KO mice and the control littermates (fl/fl). (n = 6). d The survival time of the MC-38 tumor-bearing mice described in c. The data represent the means ± s.e.ms. (n = 6, **P < 0.01, Gehan-Breslow-Wilcoxon test). e Incidence of metastasis in lungs and livers of the intravenous tumor model. The 6-week-old C57BL/6 WT or transgenic mice were intravenously injected with MC-38 colorectal cancer cells (5 × 106 cells per mouse) via the tail vein. The mice were sacrificed 2 weeks after tumor inoculation. The lungs and livers were dissected for pathological observation of tumor lesions. Data are means ± s.e.ms. and polled from four individual experiments. Each symbol represents an experimental replicate (n = 8~11). f–h The MC-38 tumor lesions in lungs of WT and TgMGLL mice, as described in e. Representative images of gross anatomy (f) and H&E staining (g) are displayed. The blue dotted lines indicate the tumor lesions. The relative tumor lesions were calculated (h). Scale bars, 200 μm. (n = 8~9). i Macrophage MGLL inhibited tumor growth in a Rag-1 dependent mannaer. MC-38 tumors were inoculated subcutaneously in WT, TgMGLL, Rag1KO or TgMGLL + Rag1KO mice and the tumor volumes were measured dynamically. (n = 5) j mRNA levels of IFNγ in CD8+ or CD4+ T cells that were isolated from the MC-38 tumors inoculated subcutaneously in WT or TgMGLL mice for 3 weeks. (n = 5). k Six-week-old WT or TgMGLL mice were subcutaneously inoculated with MC-38 cells and treated with anti-CD8 antibody or IgG as a control. The tumor volume was measured dynamically. (n = 10). l The survival time of the MC-38 tumor- bearing mice treated as described in k. The data represent the means ± s.e.ms. (n = 10, *P < 0.05, Gehan-Breslow-Wilcoxon test; ns, not significant). Data in a, c and e–k represent the means ± s.e.ms. (*P < 0.05, **P < 0.01, ***P < 0.005; student’s t-test; ns not significant)

Article Snippet: The MC-38 tumor-bearing mice were treated with monoclonal antibodies anti-CD8 (MAB116, Novus Biologicals) or IgG (MAB002, Novus Biologicals) as control.

Techniques: Control, Transgenic Assay, Injection, Staining, Isolation

Journal: Nature communications

Article Title: Monoacylglycerol lipase regulates cannabinoid receptor 2-dependent macrophage activation and cancer progression.

doi: 10.1038/s41467-018-04999-8

Figure Lengend Snippet: Fig. 6 MGLL-CB2 axis regulates tumor progression in mice. a Relative mRNA levels of TNFα, IL-6 and IFNγ in the CD8+ T cells in the MC-38 tumors inoculated subcutaneously in WT, TgMGLL, TgCB2, and TgMGLL+CB2 mice. The data represent the means ± s.e.ms. (n = 5, ***P < 0.005, Student’s t-test; ns not significant). b Six-week-old WT, TgMGLL, TgCB2, and TgMGLL+CB2 mice were subcutaneously inoculated with MC-38 cells, and the tumor volume was measured dynamically. This experiment was repeated three times. The data represent the means ± s.e.ms. (n = 10, **P < 0.01, ***P < 0.005, Student’s t-test). c The survival time of the MC-38 tumor-bearing mice. The mice were described in b. The data represent the means ± s.e.ms. (n = 10, **P < 0.01, ***P < 0.005, Gehan-Breslow-Wilcoxon test). d The incidence of tumor metastasis in MC-38 tumor model. Six-week-old WT, TgMGLL, TgCB2, and TgMGLL

Article Snippet: The MC-38 tumor-bearing mice were treated with monoclonal antibodies anti-CD8 (MAB116, Novus Biologicals) or IgG (MAB002, Novus Biologicals) as control.

Techniques:

Journal: Life (Basel, Switzerland)

Article Title: Diets Differently Regulate Pulmonary Pathogenesis and Immune Signaling in Mice during Acute and Chronic Mycobacterium tuberculosis Infection.

doi: 10.3390/life13010228

Figure Lengend Snippet: Figure 3. Representative immunohistochemistry (IHC) images of the lung sections of mice during acute Mtb infection (30 DPI) stained by CD4+, CD8+, F4/80+, and IFNγ antibodies. (RD_con— uninfected mice fed an RD; MFD_con—uninfected mice fed an MFD; RD_Inf—infected mice fed an RD; and MFD_Inf—infected mice fed an MFD). Magnification-4×; Scale bar–100 µ. IHC sections were graded and presented as a bar graph (see Supplementary Figure S3a).

Article Snippet: The blots were probed with the following primary antibodies: PPARα-specific mouse monoclonal antibody (#MA1-822, ThermoFisher Scientific), Perilipin 1-specific rabbit monoclonal antibody (#9349, Cell Signaling Technology), Phospho-Perilipin 1 (Ser522)-specific mouse monoclonal antibody (#4856, Vala Sciences), F4/80-specific rat monoclonal (#NB600-404, Novus Biologicals), adiponectinspecific mouse monoclonal antibody (#ab22554, Abcam), IFNγ-specific rabbit monoclonal antibody (#ab133566, Abcam), TNFα-specific rabbit polyclonal antibody (#ab6671, Abcam), CD4 specific rabbit polyclonal antibody (#NB100-56457SS, Novus Biologicals), CD8 specific rabbit polyclonal antibody (#NBP2-29475, Novus Biologicals), BNIP3 specific rabbit polyclonal antibody (#44060, Cell signaling Technology), Caspase-3-specific rabbit polyclonal antibody (#9662, Cell signaling Technology), IL-6-specific mouse monoclonal antibody (#66146-1-Ig, Proteintech), and IL-10-specific mouse monoclonal antibody (#60269-1-Ig, Proteintech).

Techniques: Immunohistochemistry, Infection, Staining

Journal: Life (Basel, Switzerland)

Article Title: Diets Differently Regulate Pulmonary Pathogenesis and Immune Signaling in Mice during Acute and Chronic Mycobacterium tuberculosis Infection.

doi: 10.3390/life13010228

Figure Lengend Snippet: Figure 4. MFD alters the level of infiltrated immune cells and inflammatory markers in the lungs during chronic Mtb infection. Immunoblot analysis of (a) immune cells (CD4+, CD8+, and F4/80+) and (b) inflammatory markers (IFNγ, TNFα, and IL-6) in the lung lysates of RD-fed and MFD-fed uninfected and infected C57BL/6 mice during chronic (90 DPI) infection. The error bars represent standard error of the mean. * p < 0.05, ** p < 0.01, and *** p < 0.001 between indicated groups.

Article Snippet: The blots were probed with the following primary antibodies: PPARα-specific mouse monoclonal antibody (#MA1-822, ThermoFisher Scientific), Perilipin 1-specific rabbit monoclonal antibody (#9349, Cell Signaling Technology), Phospho-Perilipin 1 (Ser522)-specific mouse monoclonal antibody (#4856, Vala Sciences), F4/80-specific rat monoclonal (#NB600-404, Novus Biologicals), adiponectinspecific mouse monoclonal antibody (#ab22554, Abcam), IFNγ-specific rabbit monoclonal antibody (#ab133566, Abcam), TNFα-specific rabbit polyclonal antibody (#ab6671, Abcam), CD4 specific rabbit polyclonal antibody (#NB100-56457SS, Novus Biologicals), CD8 specific rabbit polyclonal antibody (#NBP2-29475, Novus Biologicals), BNIP3 specific rabbit polyclonal antibody (#44060, Cell signaling Technology), Caspase-3-specific rabbit polyclonal antibody (#9662, Cell signaling Technology), IL-6-specific mouse monoclonal antibody (#66146-1-Ig, Proteintech), and IL-10-specific mouse monoclonal antibody (#60269-1-Ig, Proteintech).

Techniques: Infection, Western Blot

Journal: Life (Basel, Switzerland)

Article Title: Diets Differently Regulate Pulmonary Pathogenesis and Immune Signaling in Mice during Acute and Chronic Mycobacterium tuberculosis Infection.

doi: 10.3390/life13010228

Figure Lengend Snippet: Figure 6. MFD alters immune signaling in adipose tissue during (a) acute and (b) chronic Mtb infection. Immunoblot analysis of immune cells (CD4, CD8, and F4/80) and inflammatory markers (IFNγ, TNFα, IL-6, and IL-10) in the WAT lysates of RD-fed and MFD-fed uninfected and infected C57BL/6 mice during acute infection (30 DPI) and chronic (3 months) post-infection is shown. The error bars represent standard error of the mean. * p < 0.05, ** p < 0.01, and *** p < 0.001 between indicated groups.

Article Snippet: The blots were probed with the following primary antibodies: PPARα-specific mouse monoclonal antibody (#MA1-822, ThermoFisher Scientific), Perilipin 1-specific rabbit monoclonal antibody (#9349, Cell Signaling Technology), Phospho-Perilipin 1 (Ser522)-specific mouse monoclonal antibody (#4856, Vala Sciences), F4/80-specific rat monoclonal (#NB600-404, Novus Biologicals), adiponectinspecific mouse monoclonal antibody (#ab22554, Abcam), IFNγ-specific rabbit monoclonal antibody (#ab133566, Abcam), TNFα-specific rabbit polyclonal antibody (#ab6671, Abcam), CD4 specific rabbit polyclonal antibody (#NB100-56457SS, Novus Biologicals), CD8 specific rabbit polyclonal antibody (#NBP2-29475, Novus Biologicals), BNIP3 specific rabbit polyclonal antibody (#44060, Cell signaling Technology), Caspase-3-specific rabbit polyclonal antibody (#9662, Cell signaling Technology), IL-6-specific mouse monoclonal antibody (#66146-1-Ig, Proteintech), and IL-10-specific mouse monoclonal antibody (#60269-1-Ig, Proteintech).

Techniques: Infection, Western Blot

Journal: Cell Death & Disease

Article Title: Oncogenic activation of SMYD3-SHCBP1 promotes breast cancer development and is coupled with resistance to immune therapy

doi: 10.1038/s41419-025-07570-8

Figure Lengend Snippet: a The protein levels of Smyd3 and Shcbp1 in normal mouse mammary glands, and tumors initiated with HP5712 cells in 3 months old FVB virgin mice as shown by Western blots. b The protein levels of Smyd3 and Shcbp1 in HP5712 cells expressing sgSmyd3, OE-Smyd3, or OE-Smyd3/sgShcbp1 by Western blots. Representative tumor images ( c ) and tumor weight plots ( d ) at day 32 from FVB virgin mice implanted with HP5712 parental (HP tumors), OE-Smyd3-HP5712, sgSmyd3-HP5712, and OE-Smyd3/sgShcbp1-HP5712 cells at 1X10 6 cells per mammary fat pad (n = 8 mice/group). e The plot of relative spleen weight from the same cohort of mice in c and d . f The protein levels of Smyd3, H3K4me3, and Shcbp1 in tumors initiated with HP5712 parental, OE-Smyd3-HP5712, sgSmyd3-HP5712, and OE-Smyd3/sgShcbp1-HP5712 cells by Western blots. g The protein levels of pMek, pErk, and Kras in tumors initiated with HP5712 parental, OE-Smyd3-HP5712, sgSmyd3-HP5712, and OE-Smyd3/sgShcbp1-HP5712 cells as shown by Western blots. h tSNE visualized immune cells from normal FVB mouse mammary glands (FVB MG, n = 6 mice), tumors implanted with parental HP5712 cells (HP Vector Ctr), HP-OE-Smyd3, HP-OE-Smyd3/sgShcbp1, and HP-sgSmyd3 from FVB mice (n = 6 mice/group) by CyTOF analysis. Quantifications of PMN-MDSCs and M-MDSCs ( i ), CD4+ and CD8 + T cells ( j ) in CD45+ immune cell populations from the same cohorts of mice in h (n = 3 biological independent samples/per group). k , l The WT-T cells were activated with CD3 and CD28 antibody. Co-cultured the activated T cells without or with MDSCs from the mice implanted with HP parental cells, HP cells expressing Smyd3, or sgSmyd3, or OE-Smyd3/sgShcbp1 for 72 hours and examined T cells proliferation status ( k ) and quantifications ( l ) of data in k (n = 3 mice/group). Representative images of CD8+/PD1+ double positive cells in mammary tissues from the mice with the genotypes of HP-Ctr, HP-OE-Smyd3, HP-sgSmyd3, and HP-OE-Smyd3/sgShcbp1 ( m ) and quantifications of CD8+/PD1 double positive cells from mammary tissues ( n ) in these mice (n = 4 mice/group, and at least 20 images for each sample were counted). Scale bar: 20 μm. arrow heads point to PD1/CD8 positive cell. o Expressions of PD1 from the spleen with implantation of parental HP cells, HP cells with OE-Smyd3, HP-sgSmyd3, and HP-OE-Smyd3/sgShcbp1 in FVB mice as determined by qPCR. p Summary of Smyd3-Shcbp1oncogenic signals shape the TIME.

Article Snippet: In Vivo anti-mouse CD8a , BioX Cell , BP0117 , , .

Techniques: Western Blot, Expressing, Plasmid Preparation, Cell Culture

Journal: Cell Death & Disease

Article Title: Oncogenic activation of SMYD3-SHCBP1 promotes breast cancer development and is coupled with resistance to immune therapy

doi: 10.1038/s41419-025-07570-8

Figure Lengend Snippet: Representative tumor images ( a ), and tumor weight plots ( b ) at day 22 from FVB mice implanted with HP5712 cells at 1×10 6 cells per mammary fat pad, and treatment with PBS, PD1, trametinib (Tra), and PD1+Tra (n = 8 mice/group). c The plot of tumor volume in the processes of treatment in ( a , b ). d The plot of relative spleen weight from the same cohort of mice in a , b . Quantifications of PMN-MDSCs and M-MDSCs ( e ), CD4+ and CD8 + T cells ( f ) in CD45+ immune cell populations from the same cohorts of mice in a , b by CyTOF analysis at D12 (n = 3 mice/group). Quantifications of PMN-MDSCs and M-MDSCs ( g ), CD4+ and CD8 + T cells ( h ) in CD45+ immune cell populations from the same cohorts of mice in a , b by CyTOF analysis at D22 (n = 3 mice/group). i , j Tumor images from the HP Ctr mice, HP mice treated with αPD1 and Tra, and HP mice treated with αPD1 and Tra with depletion of T cell using CD8 antibody ( i ). The plot of tumor weight ( j ) in the same cohort of mice in ( i ) (n = 8 mice in each group). The protein levels of Smyd3 and Shcbp1 in tumors initiated with HP5712 cells and treatment with PBS, αPD1, Tra, and αPD1+Tra in FVB mice at D12 ( k ) and D22 ( l ) as shown by Western blots. The protein levels of pMek, pErk, Kras, and Grb2 in tumors initiated with HP5712 cells and treatment with PBS, αPD1, Tra, and αPD1+Tra in FVB mice at D12 ( m ) and D22 ( n ) as shown by Western blots. Expressions of Smyd3 ( o ) and Shcbp1 ( p ) in G600 cells with the treatment of E2, Tra, and E2 together with Tra at 1 hour, 2 hours, 4 hours, and 24 hours as determined by qPCR.

Article Snippet: In Vivo anti-mouse CD8a , BioX Cell , BP0117 , , .

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Oncogenic activation of SMYD3-SHCBP1 promotes breast cancer development and is coupled with resistance to immune therapy

doi: 10.1038/s41419-025-07570-8

Figure Lengend Snippet: Antibodies used for ChIP, IHC, IF, WB and in vivo mouse study.

Article Snippet: In Vivo anti-mouse CD8a , BioX Cell , BP0117 , , .

Techniques: Immunohistochemistry-IF, In Vivo

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