Journal: The Journal of Physiology

Article Title: T cells mediate cell non‐autonomous arterial ageing in mice

doi: 10.1113/jp281698

Figure Lengend Snippet: Figure 1. Anti-CD3 F(ab’)2 fragment treatment results in depletion of T cells in the blood and spleen Blood (100 μL) from young isotype (n = 10), young anti-CD3 (n = 10), old isotype (n = 9) and old anti-CD3 (n = 10) mice was directly stained. Spleens from young isotype (n = 11–14), young anti-CD3 (n = 8–11), old isotype (n = 15–17) and old anti-CD3 (n = 18–19) mice were enzymatically digested and passed through a cell strainer and then stained for CD45 (total leukocytes), CD3 (pan T cells), CD4 and CD8. Percentages of CD3+ cells and the CD4-to-CD8 ratio in (A) blood and (B) spleen were assessed by flow cytometry. A two-way ANOVA was employed to assess the effects of age and anti-CD3 treatment, P values for age, treatment and the age × treatment interaction are inset on each panel. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Following stimulation, cells were labelled with the following anti-mouse antibodies: PE/Dazzle-CD45, Biolegend #109846, APC-CD3, Tonbo #20-0032, FITC-CD4, Tonbo #30-0041 and PE-Cy5-CD8 Tonbo #55-0081.

Techniques: Staining, Flow Cytometry, Standard Deviation

Journal: The Journal of Physiology

Article Title: T cells mediate cell non‐autonomous arterial ageing in mice

doi: 10.1113/jp281698

Figure Lengend Snippet: Figure 2. Aortic T cell accumulation with age and anti-CD3 F(ab’)2 Fragment treatment Single-cell suspensions of thoracic aortas from young isotype (n = 7–12), young anti-CD3 (n = 9–11), old isotype (n = 8–14) and old anti-CD3 (n = 10–19) mice were stained with antibodies against CD45 (total leukocytes), CD3 (pan T cells), CD4, CD8, CD44 (memory) and CD62L (central vs. effector). A, number of total leukocytes per aorta. B, sample aortic CD4 and CD8 flow cytometry plot. C, aortic pan, CD4 and CD8 T cell counts. D, percentage of CD3 cells out of total aortic immune cells. E, aortic CD4:CD8 ratio. F, sample aortic naïve (CD44lo) vs. memory (CD44hi) flow cytometry plots. G, aortic CD8 effector memory T cell proportion and counts. To assess aortic macrophage and B cell accumulation, thoracic aorta single-cell suspensions were stained for CD45 (total leukocytes), CD19 (B cells), CD64 (macrophages), CD11c (exclusion of dendritic cells) and CD206 (M1/M2 macrophage phenotype. H, aortic B cell counts. I, aortic macrophage counts. J, macrophage M1:M2 ratio. A two-way ANOVA was employed to assess the effects of age and anti-CD3 treatment, P values for age, treatment and the age × treatment interaction are inset on each panel. When a significant age × treatment interaction occurred, Tukey’s post hoc test was employed to determine group differences. Significant post hoc test P values are included on the panel with a horizontal line indicating the group comparison. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Following stimulation, cells were labelled with the following anti-mouse antibodies: PE/Dazzle-CD45, Biolegend #109846, APC-CD3, Tonbo #20-0032, FITC-CD4, Tonbo #30-0041 and PE-Cy5-CD8 Tonbo #55-0081.

Techniques: Staining, Flow Cytometry, Comparison, Standard Deviation

Journal: The Journal of Physiology

Article Title: T cells mediate cell non‐autonomous arterial ageing in mice

doi: 10.1113/jp281698

Figure Lengend Snippet: Figure 3. Ageing results in an enhanced proinflammatory phenotype of aortic accumulating T cells Single-cell suspensions of thoracic aortas from young (n = 6) and old (n = 8) mice were activated in vitro and stained for CD45 (total leukocytes), CD3 (pan T cells), CD4, CD8, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. A, sample IFN-γ flow cytometry plots. B, proportion and (C) number of IFN-γ -producing T cells. D, sample TNF-α flow cytometry plots. E, proportion and (F) number of TNF-α-producing T cells. Group differences were assessed with an independent samples t test, P values are included on the panel with a horizontal line indicating the group comparison. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Following stimulation, cells were labelled with the following anti-mouse antibodies: PE/Dazzle-CD45, Biolegend #109846, APC-CD3, Tonbo #20-0032, FITC-CD4, Tonbo #30-0041 and PE-Cy5-CD8 Tonbo #55-0081.

Techniques: In Vitro, Staining, Flow Cytometry, Comparison, Standard Deviation

Journal: The Journal of Physiology

Article Title: T cells mediate cell non‐autonomous arterial ageing in mice

doi: 10.1113/jp281698

Figure Lengend Snippet: Figure 4. T cell depletion reverses age-related increases in large artery stiffness Aortic pulse wave velocity (PWV) was assessed in young (n = 19) and old (n = 25) mice (A) before anti-CD3 treatment and an independent samples t test was used to assess group differences. P values are included on the panel with a horizontal line indicating the group comparison. PWV was assessed before and after anti-CD3 F(ab’)2 fragment treatment in (B) young (n = 10 per group) and (C) old mice (n = 12–13 per group). A repeated measures ANOVA was used to assess the effect of time, treatment and time × treatment interaction, with P values inset on each panel. When a significant age × treatment interaction occurred, Sidak’s post hoc test was employed to determine group differences. Significant post hoc test P values are included on the panel with a vertical line indicating the group comparison. D, gene expression of Nox2, Xanthine Oxidase (Xo), superoxide dismutase isoforms 1–3 (Sod1, 2 & 3) from aortas of young isotype (n = 7), young anti-CD3 (n = 7) old isotype (n = 5) and old anti-CD3-treated (n = 5) mice. Gene expression data are expressed as fold change compared with young isotype calculated using the Ct method. Two-way ANOVA was employed to assess the effects of age and anti-CD3 treatment. P values for age, treatment and the age × treatment interaction are inset on each panel. Data are shown as means ± standard deviation, n represents the number of independent animals in each group.

Article Snippet: Following stimulation, cells were labelled with the following anti-mouse antibodies: PE/Dazzle-CD45, Biolegend #109846, APC-CD3, Tonbo #20-0032, FITC-CD4, Tonbo #30-0041 and PE-Cy5-CD8 Tonbo #55-0081.

Techniques: Comparison, Gene Expression, Standard Deviation

Journal: The Journal of Physiology

Article Title: T cells mediate cell non‐autonomous arterial ageing in mice

doi: 10.1113/jp281698

Figure Lengend Snippet: Figure 5. Mesenteric T cell accumulation with age and anti-CD3 F(ab’)2 Fragment treatment Single-cell suspensions of the mesenteric vascular arcade from young isotype (n = 8–14), young anti-CD3 (n = 10–12), old isotype (n = 9–21) and old anti-CD3 (n = 7–11) mice (excluding lymph nodes) were stained for CD45 (total leukocytes), CD3 (pan T cells), CD4, CD8, CD44 (memory) and CD62L (central vs. effector). A, number of total leukocytes per mesentery. B, proportion of CD3 cells out of all mesenteric leukocytes. C, mesenteric CD3 cell counts. D, mesenteric arcade mass. E, mesenteric CD3 cell counts normalized to tissue mass. F, sample CD4 and CD8 flow cytometry plot. G, mesenteric CD4 (left) and CD8 (right) T cell counts. H, mesenteric CD4:CD8 ratio. I, sample mesenteric naïve (CD44lo) vs. memory (CD44hi) flow cytometry plots. J, proportion of mesenteric (left) and counts (right) of CD8 CD44hi/CD62Llo effector memory T cell counts. To assess mesenteric macrophage and B cell accumulation, mesenteric single-cell suspensions were stained for CD45 (total leukocytes), CD19 (B cells), CD64 (macrophages), CD11c (exclusion of dendritic cells) and CD206 (M1/M2 macrophage phenotype). K, mesenteric B cell counts. L, mesenteric macrophage counts. M, macrophage M1:M2 ratio. Two-way ANOVA was employed to assess the effects of age and anti-CD3 treatment. P values for age, treatment and the age × treatment inter- action are inset on each panel. When a significant age × treatment interaction occurred, Tukey’s post hoc test was employed to determine group differences. Significant post hoc test P values are included on the panel with a horizontal line indicating the group comparison. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Following stimulation, cells were labelled with the following anti-mouse antibodies: PE/Dazzle-CD45, Biolegend #109846, APC-CD3, Tonbo #20-0032, FITC-CD4, Tonbo #30-0041 and PE-Cy5-CD8 Tonbo #55-0081.

Techniques: Staining, Flow Cytometry, Comparison, Standard Deviation

Journal: The Journal of Physiology

Article Title: T cells mediate cell non‐autonomous arterial ageing in mice

doi: 10.1113/jp281698

Figure Lengend Snippet: Figure 6. Ageing results in an enhanced proinflammatory phenotype of mesenteric accumulating T cells To assess cytokine production, mesenteric single-cell suspensions from young (n = 6) and old (n = 8) mice were activated in vitro. Cells were then stained for CD45 (total leukocytes), CD3 (pan T cells), CD4, CD8, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. A, sample IFN-γ flow cytometry plots. B, proportion and C, number of IFN-γ -producing T cells. D, sample TNF-α flow cytometry plots. E, proportion and (F) number of TNF-α-producing T cells. Group differences were assessed with an independent samples t test. P values are included on the panel with a horizontal line indicating the group comparison. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Following stimulation, cells were labelled with the following anti-mouse antibodies: PE/Dazzle-CD45, Biolegend #109846, APC-CD3, Tonbo #20-0032, FITC-CD4, Tonbo #30-0041 and PE-Cy5-CD8 Tonbo #55-0081.

Techniques: In Vitro, Staining, Flow Cytometry, Comparison, Standard Deviation

Journal: The Journal of Physiology

Article Title: T cells mediate cell non‐autonomous arterial ageing in mice

doi: 10.1113/jp281698

Figure Lengend Snippet: Figure 7. T cell depletion results in augmented endothelium-dependent dilatation in mesenteric arteries from old mice Endothelium-dependent dilatation was assessed in second order mesenteric arteries from (A) young isotype control, young anti-CD3, (B) old isotype control and old anti-CD3 mice in response to increasing doses of acetylcholine. C, endothelium-independent dilatation was assessed in second order mesenteric arteries in from young isotype control, young anti-CD3, old isotype control and old anti-CD3 mice in response to increasing doses of sodium nitroprusside. Dose–response curve data are expressed as means ± standard deviation. A repeated measures ANOVA was used to assess the effect of dose, treatment and dose × treatment interaction, with P values inset on each panel. n sizes are in parentheses next to the corresponding group in each panel legend and represent the number of independent animals in each group.

Article Snippet: Following stimulation, cells were labelled with the following anti-mouse antibodies: PE/Dazzle-CD45, Biolegend #109846, APC-CD3, Tonbo #20-0032, FITC-CD4, Tonbo #30-0041 and PE-Cy5-CD8 Tonbo #55-0081.

Techniques: Control, Standard Deviation

Journal: The Journal of Physiology

Article Title: T cells mediate cell non‐autonomous arterial ageing in mice

doi: 10.1113/jp281698

Figure Lengend Snippet: Figure 8. T cell depletion results in augmented nitric oxide bioavailability in mesenteric arteries from old mice Endothelium-dependent dilatation in response to increasing doses of acetylcholine was assessed in second order mesenteric arteries from old isotype control or old mice treated with anti-CD3 F(ab)’2 fragments in the presence or absence of (A) L-NAME, (B) TEMPOL and (C) TEMPOL & L-NAME. Endothelium-dependent dilatation was also assessed in arteries from young isotype control mice or young mice treated with anti-CD3 F(ab)’2 fragments in the presence or absence of (D) L-NAME, (E) TEMPOL and (F) TEMPOL & L-NAME. Dose–response curve data are expressed as means ± standard deviation. A repeated measures ANOVA was used to assess the effect of dose, group and dose × group interaction, with P values inset on each panel. In the case of a significant group effect, Tukey’s post hoc test was used to compare groups with comparison P values indicated on the legend of each figure panel. In the case of a significant dose × group interaction, Tukey’s post hoc test was employed to assess significant main effects of dose with P values reported in the text. n sizes are in parentheses next to the corresponding group in each panel legend and represent the number of independent animals in each group.

Article Snippet: Following stimulation, cells were labelled with the following anti-mouse antibodies: PE/Dazzle-CD45, Biolegend #109846, APC-CD3, Tonbo #20-0032, FITC-CD4, Tonbo #30-0041 and PE-Cy5-CD8 Tonbo #55-0081.

Techniques: Control, Standard Deviation, Comparison

Journal: Journal of Extracellular Vesicles

Article Title: High‐Yield Outer Membrane Vesicles Derived From Probiotics as a Nanoplatform for Precise Treatment and Prophylaxis of Pseudomonas aeruginosa Infection

doi: 10.1002/jev2.70194

Figure Lengend Snippet: In vivo immune response and preventive effect activated by FI‐mOMVs. (a) Schematic diagram of immunization, sample collection and challenge schedule. BALB/c mice were immunized s.c. with the indicated formulations on Days 0, 14, 28 ( n = 8), and a PBS control was set up ( n = 5). (b) Serum was collected 7, 14, 21, 28, 35 and 42 days after initial vaccination, and the FI antigen–specific IgG antibody titre was detected by ELISA. (c–e) Detection of antigen‐specific cellular immune response. The spleen lymphocytes were isolated 6 weeks after the initial immunization. The proliferation levels were detected by CCK8 (c), and the expression levels of IFN‐γ (d) and IL‐4 (e) in the culture supernatant were determined by ELISA. (f, g) Percentage of CD3 + CD4 + T lymphocytes (f), and CD3 + CD8 + T lymphocytes (g) in the spleen on Day 42 after immunization ( n = 8). Mice without immunization were used as controls ( n = 5). (h) Survival curves of the mice infected with PAO1 (1 × 10 8 CFU/mouse, i.n.) after different treatments ( n = 12). (i, j) Quantification of the number of bacteria in the lung (i) and the pathological changes of the lung (j) of the mice infected with a sublethal dose of PAO1 (5 × 10 6 CFU/mouse, i.n.) after different treatments ( n = 6). Scale bar, 200 µm. The data are presented as mean ± SEM. Statistical analysis was performed by a two‐tailed unpaired Student's t ‐test (b–g), log‐rank (Mantel–Cox) test (h), or two‐tailed Mann–Whitney U ‐test (i), and differences were considered significant at p < 0.05. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001.

Article Snippet: The cell suspensions (100 μL) were taken and stained with FITC‐conjugated anti‐mouse CD4 (cat# FITC‐65104, Proteintech, USA), PE‐conjugated anti‐mouse CD8 (cat# PE‐65069, Proteintech, USA) and APC‐conjugated anti‐mouse CD3 (cat# APC‐65077, Proteintech, USA) for 30 min at 37°C in the dark.

Techniques: In Vivo, Control, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Infection, Bacteria, Two Tailed Test, MANN-WHITNEY

Journal: Cancer Communications

Article Title: Metabolic landscape of head and neck squamous cell carcinoma informs a novel kynurenine/Siglec‐15 axis in immune escape

doi: 10.1002/cac2.12545

Figure Lengend Snippet: Kyn induced Siglec‐15‐mediated immune escape. (A) The chromatin immunoprecipitation (ChIP)‐PCR assay was performed using an IgG or AhR antibody after treatment with 200 µmol/L Kyn for 1 h. Two primers targeting the promoter region of SIGLEC15 and CD274 mRNA were used for RT‐qPCR analysis. (B) 293T cells were co‐transfected with SIGLEC15 / CD274 promoter‐luciferase reporter PGL3 for 24 h and treated with the indicated concentration of Kyn for another 6 h, followed by an analysis of luciferase activity. (C) 293T cells were co‐transfected with SIGLEC15 / CD274 promoter‐luciferase reporter PGL3 for 24 h and treated with 200 µmol/L Kyn for the indicated time, followed by an analysis of luciferase activity. (D) 293T cells were co‐transfected with promoter‐luciferase reporter plasmids and siAhR /siScr for 24 h and treated with 200 µmol/L Kyn for 6 h. (E) SCCVII cells were transfected with lentivirus‐ SIGLEC15 , and transfection was confirmed using Western blotting analysis. (F) Vector or SIGLEC15 ‐overexpressing SCCVII cells were subcutaneously injected into C3H/He mice ( n = 5 per group). Tumor volumes were measured once every two days. (G) Tumor weights were measured after mice were euthanized. (H‐J) H&E (H), TUNEL (I), and Ki‐67 (J) staining analyses of tumor tissues in each group. (K‐P) Tumor‐infiltrating lymphocytes (TIL) harvested from xenograft tumors, and the percentages of CD3 + CD8 + (K), Ki‐67 + (L), PD‐1 + (M), IFN‐γ + (N), granzyme B + (O), and perforin + cells (P) were analyzed using flow cytometry. (Q) The percentages of CD8 + in the indicated tumors were analyzed by multiplex immunofluorescence staining. (R) The volume and Ki‐67 expression of tumor were compared in tongue orthotopic transplant models established by vector or SIGLEC15 ‐overexpressing SCCVII cells ( n = 6 mice per group). (S) PD‐1 + CD8 + T cells were compared in tongue orthotopic transplant models ( n = 6 mice per group), based on multiplex immunofluorescence staining. Three regions of interest (ROIs) in each tumor were analyzed and measured. Data are represented as mean ± SEM (A‐D, F‐G and I‐S) based on three independent experiments (A‐D). * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. Abbreviations: Kyn, kynurenine; ChIP, chromatin immunoprecipitation; RT‐qPCR, real‐time quantitative polymerase chain reaction; AhR, aryl hydrocarbon receptor; siScr, siScramble; H&E, hematoxylin and eosin; TUNEL, TdT‐mediated dUTP‐biotin nick end labeling; PD‐1, programmed cell death protein 1; TIL, Tumor‐infiltrating lymphocyte; IFN‐γ, interferon‐gamma; ROI, region of interest; SEM, tandard error of the mean; ns, not significant.

Article Snippet: For TIL analysis, samples were incubated with membrane antibodies containing anti‐mouse CD3 allophycocyanin (APC)‐Cy7, anti‐mouse CD8‐FITC, anti‐mouse PD‐1 phycoerythrin (PE)‐Cy7, and incubated with transcription factor fixation/permeabilization working solution (92550‐00, BioGems) for 30 min at 4°C, and washed with permeabilization buffer (BioGems).

Techniques: Chromatin Immunoprecipitation, Quantitative RT-PCR, Transfection, Luciferase, Concentration Assay, Activity Assay, Western Blot, Plasmid Preparation, Injection, TUNEL Assay, Staining, Flow Cytometry, Multiplex Assay, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction, End Labeling

Journal: Cancer Communications

Article Title: Metabolic landscape of head and neck squamous cell carcinoma informs a novel kynurenine/Siglec‐15 axis in immune escape

doi: 10.1002/cac2.12545

Figure Lengend Snippet: Targeting AhR reversed Kyn‐Siglec‐15‐mediated immune escape in vivo. (A) Mice with SCCVII were treated with Kyn (100 mg/kg) or/and CH‐223191 (10 µmol/L) once every two days for three times ( n = 5 per group). Tumor volumes were measured once every two days. (B) Tumor weights were measured after mice euthanized on day 20 ( n = 5 per group). (C) Western blotting analysis of Siglec‐15 expression after Kyn stimulation in mice. (D‐F) H&E (D), TUNEL (E and F upper), and Ki‐67 (E and F lower) staining analyses of tumor tissues in each group. (G) Concentration of serum TNF‐α and IFN‐γ in mice were measured by ELISA. (H‐M) The percentage of CD3 + CD8 + (H), Ki‐67 + (I), PD‐1 + (J), IFN‐γ + (K), granzyme B + (L), and perforin + cells (M) in cytotoxic T lymphocytes (CTLs) isolated from the indicated tumors were analyzed. (N) Representative images of multiplex immunofluorescence staining of CD3 + (green) and CD8 + (red) are shown, and quantification analysis were performed in tumor tissues ( n = 10 fields of 5 mice per group). Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. Abbreviations: AhR, aryl hydrocarbon receptor; Kyn, kynurenine; H&E, hematoxylin and eosin; TUNEL, TdT‐mediated dUTP‐biotin nick end labeling; TNF‐α, tumor necrosis factor‐alpha; IFN‐γ, interferon‐gamma; ELISA, enzyme‐linked immunosorbent assay; PD‐1, programmed cell death protein 1; CTL, cytotoxic T lymphocyte; DAPI, 4',6‐diamidino‐2‐phenylindole; SEM, tandard error of the mean; ns, not significant.

Article Snippet: For TIL analysis, samples were incubated with membrane antibodies containing anti‐mouse CD3 allophycocyanin (APC)‐Cy7, anti‐mouse CD8‐FITC, anti‐mouse PD‐1 phycoerythrin (PE)‐Cy7, and incubated with transcription factor fixation/permeabilization working solution (92550‐00, BioGems) for 30 min at 4°C, and washed with permeabilization buffer (BioGems).

Techniques: In Vivo, Western Blot, Expressing, TUNEL Assay, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation, Multiplex Assay, Immunofluorescence, End Labeling

Journal: Cancer Communications

Article Title: Metabolic landscape of head and neck squamous cell carcinoma informs a novel kynurenine/Siglec‐15 axis in immune escape

doi: 10.1002/cac2.12545

Figure Lengend Snippet: Siglec‐15 specific siRNA delivered by NH 2 ‐MSN nanoparticles enhances immunotherapy efficacy in vivo. (A) Synthesis routes of NH 2 ‐MSN nanoparticles loaded with siRNA. (B) Macroscopy characterization of NH 2 ‐MSNs with siS15. (C) Transmission electron microscopy images of NH 2 ‐MSNs and NP‐siS15 with indicated ratios. (D‐E) The morphology and Zeta potential of NH 2 ‐MSNs and NP‐siS15. (F) Scanning microscopy analysis of the cellular uptake of NH 2 ‐MSNs with or without the indicated siS15‐Cy5 by SCCVII cells after in vitro treatment for 24 h. (G) Western blotting analysis confirming Siglec‐15 gene‐silencing effect by siRNA released from nanoparticles in SCCVII cells. (H) Schematic diagram of the treatment strategy in SCCVII mouse model. (I) Probability of survival analysis for each group was performed. (J) Analysis of TUNEL and Ki‐67 staining of tumor tissues in each group. (K‐L) The percentage of CD3 + CD8 + (K) and PD‐1 + cells in CTLs (L) of the indicated treatment groups. (M) Analysis of multiplex immunofluorescence staining of CD3 + (green) and CD8 + (red) were shown and quantification analysis were performed in tumor tissues ( n = 10 fields of five mice per group). (N) Schematic diagram of the anti‐PD‐L1 and NP‐siS15 combination treatment strategy in C3H/He subcutaneous tumorigenesis models. (O‐P) Tumor volume and weights were measured and analyzed in the indicated groups. Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. Abbreviations: siS15, Siglec‐15 small interfering RNA; DAPI, 4',6‐diamidino‐2‐phenylindole; AhR, aryl hydrocarbon receptor; NP‐siS15, NH 2 ‐MSN‐si SIGLEC15 ; DAPI, 4',6‐diamidino‐2‐phenylindole; siScr, siScramble; anti‐PD‐1, anti‐programmed cell death protein 1 antibody; TUNEL, TdT‐mediated dUTP‐biotin nick end labeling; CTL, cytotoxic T lymphocyte; anti‐PD‐L1, anti‐programmed death‐ligand 1 antibody; SEM, tandard error of the mean; ns, not significant.

Article Snippet: For TIL analysis, samples were incubated with membrane antibodies containing anti‐mouse CD3 allophycocyanin (APC)‐Cy7, anti‐mouse CD8‐FITC, anti‐mouse PD‐1 phycoerythrin (PE)‐Cy7, and incubated with transcription factor fixation/permeabilization working solution (92550‐00, BioGems) for 30 min at 4°C, and washed with permeabilization buffer (BioGems).

Techniques: In Vivo, Transmission Assay, Electron Microscopy, Zeta Potential Analyzer, Microscopy, In Vitro, Western Blot, TUNEL Assay, Staining, Multiplex Assay, Immunofluorescence, Small Interfering RNA, End Labeling

Journal: International Journal of Biological Sciences

Article Title: CYSLTR1 antagonist inhibits Th17 cell differentiation by regulating the NF-κB signaling for the treatment of psoriasis

doi: 10.7150/ijbs.92514

Figure Lengend Snippet: Montelukast sodium cream ameliorates IMQ-induced psoriasis-like skin lesions in BALB/c mice. (A) A schematic of the treatment using montelukast sodium cream and solution in IMQ-induced psoriasis-like murine model. (B) High expression of CYSLTR1 in IMQ-induced psoriasis-like skin lesions. (C) High mRNA level of CYSLTR1 in skin lesion of patients with psoriasis. (D) IHC of CYSLTR1 staining in skin lesions from psoriasis murine models and psoriasis patients. (E) Multi-color IHC staining of CD11c + CYSLTR1 + and CD3 + CYSLTR1 + cells in skin lesions from patients with psoriasis. (F) The skin lesions, histological features, and body weight, as well as the changes in PASI scores, erythema, scale, and thickness scores of the psoriasis-like mice. n = 7 in Blank group; n = 11 in IMQ + cream base (negative control: NC) group; n = 7 in IMQ + HMS (positive control: PC) group; n = 10 in IMQ + montelukast cream group; n = 11 in IMQ + montelukast intraperitoneal (i. p) injection group. Horizontal bars represent the mean ± standard error of the mean (SEM), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The flow cytometry reagents are as follows: Zombie Aqua™ Fixable Viability Kit (423102, BioLegend), APC-Cy™7 Rat Anti-Mouse CD3 Molecular Complex (560590, BD Biosciences), PE anti-mouse CD3 Antibody (100206, BioLegend), Alexa Fluor® 700 anti-mouse CD4 Antibody (100430, BioLegend), Brilliant Violet 605™ anti-mouse/human CD45R/B220 Antibody (103244, BioLegend), Brilliant Violet 785™ anti-mouse CD25 Antibody (102051, BioLegend), Brilliant Violet 605™ anti-mouse CD69 Antibody (104530, BioLegend), Brilliant Violet 421™ anti-mouse CD103 Antibody (121422, BioLegend), BCL6 Monoclonal Antibody (BCL-DWN), APC (17-5453-82, eBioscience, San Diego, CA, USA), CD278 (ICOS) Monoclonal Antibody (7E.17G9) (16-9942-85, eBioscience, San Diego, CA, USA), PE-Cyanine7 (25-9942-82, eBioscience), PE/Dazzle™ 594 anti-T-bet Antibody (644828, BioLegend), PE Mouse anti-Mouse RORγt (562607, BD Biosciences), Alexa Fluor® 488 anti-mouse/rat/human FOXP3 Antibody (320012, BioLegend), APC anti-mouse/human CD11b Antibody (101212, BioLegend), PerCP/Cyanine5.5 anti-mouse CD11c Antibody (117328, BioLegend), FITC Hamster Anti-Mouse γδ T-Cell Receptor (553177, BD Biosciences), PE/Cyanine7 anti-mouse I-A/I-E Antibody (107630, BioLegend), Brilliant Violet 785™ anti-mouse F4/80 Antibody (123141, BioLegend), and Brilliant Violet 650™ anti-mouse Ly-6G Antibody (127641, BioLegend).

Techniques: Cream, Expressing, Staining, Immunohistochemistry, Negative Control, Positive Control, Injection

Journal: International Journal of Biological Sciences

Article Title: CYSLTR1 antagonist inhibits Th17 cell differentiation by regulating the NF-κB signaling for the treatment of psoriasis

doi: 10.7150/ijbs.92514

Figure Lengend Snippet: Montelukast reduces the levels of inflammatory cells in the epidermis of the psoriasis-like mice. (A) Representative flow cytometry diagrams and statistical analysis of the (B) frequencies and (C) numbers of T-cell subtypes (CD3 + T, CD4 + T, CD8 + T, Th1, Th17, Tfh, and Treg cells) and B220 + cells in the epidermis. (D) Representative flow cytometry diagrams and statistical analysis of the (E) frequencies and (F) numbers of CD3 + /CD4 + TRM, CD4 + /CD8 + γδ T, macrophage, neutrophil, cDC, cDC1 and cDC2 cells in the epidermis. n = 8 in IMQ + cream base (negative control: NC) group; n = 8 in IMQ + montelukast sodium cream group; n = 8 in IMQ + montelukast i. p group. Skin lesions of 8 animals were randomly selected from each group for flow cytometry analysis. Horizontal bars represent the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The flow cytometry reagents are as follows: Zombie Aqua™ Fixable Viability Kit (423102, BioLegend), APC-Cy™7 Rat Anti-Mouse CD3 Molecular Complex (560590, BD Biosciences), PE anti-mouse CD3 Antibody (100206, BioLegend), Alexa Fluor® 700 anti-mouse CD4 Antibody (100430, BioLegend), Brilliant Violet 605™ anti-mouse/human CD45R/B220 Antibody (103244, BioLegend), Brilliant Violet 785™ anti-mouse CD25 Antibody (102051, BioLegend), Brilliant Violet 605™ anti-mouse CD69 Antibody (104530, BioLegend), Brilliant Violet 421™ anti-mouse CD103 Antibody (121422, BioLegend), BCL6 Monoclonal Antibody (BCL-DWN), APC (17-5453-82, eBioscience, San Diego, CA, USA), CD278 (ICOS) Monoclonal Antibody (7E.17G9) (16-9942-85, eBioscience, San Diego, CA, USA), PE-Cyanine7 (25-9942-82, eBioscience), PE/Dazzle™ 594 anti-T-bet Antibody (644828, BioLegend), PE Mouse anti-Mouse RORγt (562607, BD Biosciences), Alexa Fluor® 488 anti-mouse/rat/human FOXP3 Antibody (320012, BioLegend), APC anti-mouse/human CD11b Antibody (101212, BioLegend), PerCP/Cyanine5.5 anti-mouse CD11c Antibody (117328, BioLegend), FITC Hamster Anti-Mouse γδ T-Cell Receptor (553177, BD Biosciences), PE/Cyanine7 anti-mouse I-A/I-E Antibody (107630, BioLegend), Brilliant Violet 785™ anti-mouse F4/80 Antibody (123141, BioLegend), and Brilliant Violet 650™ anti-mouse Ly-6G Antibody (127641, BioLegend).

Techniques: Flow Cytometry, Cream, Negative Control

Journal: Oncology Letters

Article Title: Natural killer cells inhibit pulmonary metastasis of hepatocellular carcinoma in nude mice

doi: 10.3892/ol.2016.4170

Figure Lengend Snippet: Isolation and culture of the NK cells. (A) CD56 + CD3 − cells were identified as the marker of the NK cells. The percentage of the NK cells in peripheral blood mononuclear cells was 10–30%. The NK cell percentage of certain samples was >30%. (B) The purity of the NK cells was >95%. (C) NK cell culture suspensions formed aggregates or clusters (magnification, ×100). NK, natural killer; PE, phycoerythrin; APC, allophycocyanin; CD, cluster of differentiation.

Article Snippet: Flow cytometry was used to examine the expression of the following cell surface proteins: Cluster of differentiation (CD)56 [phycoerythrin (PE)-labeled mouse anti-human CD56 clone B159; BD Biosciences, San Diego, CA, USA], CD3 [allophycocyanin (APC)-labeled mouse anti-human CD3 clone UCHTI; BD Pharmingen, San Diego, CA, USA], NKG2D [fluorescein isothiocyanate (FITC)-labeled anti-human NKG2D clone 1D11; eBioscience, Inc., San Diego, CA, USA], NKB1 (FITC-labeled anti-human NKB1 clone DX9; BD Pharmingen), perforin (FITC Perforin Reagent set containing FITC-labeled mouse anti-human perforin clone δG9 and FITC-mouse immunoglobulin (Ig)G2bκ clone 27–35; BD Pharmingen) and granzyme (FITC-labeled mouse anti-human granzyme clone GB11; BD Pharmingen).

Techniques: Isolation, Marker, Cell Culture

Journal: Nature Communications

Article Title: Re-designing Interleukin-12 to enhance its safety and potential as an anti-tumor immunotherapeutic agent

doi: 10.1038/s41467-017-01385-8

Figure Lengend Snippet: Ad-TD-nsIL-12 is an effective treatment for subcutaneous hamster PaCa. 2 × 10 6 HPD1NR cells were seeded into the right flank of Syrian hamsters. When tumor volumes reached 300 mm 3 , seven hamsters per group were each injected i.t. with 100 µl PBS, 1 × 10 9 PFU Ad-TD-LUC or Ad-TD-nsIL-12/IL-12 on days 0, 2, 4, 6, 8, and 10. a Mean tumor sizes and SEM are shown for each group and compared using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. * p < 0.05, *** p < 0.001. b Kaplan–Meier survival curves were generated and a log-rank (Mantel–Cox) test used to analyze significance. ** p < 0.01, *** p < 0.001. Treatment with both Ad-TD-IL-12 and Ad-TD-nsIL-12 resulted in 100% survival in this model. c Hamsters that had cleared tumors after i.t treatment with Ad-TD-nsIL-12 during efficacy experiments were re-challenged 4 weeks later in the opposite flank with 4 × 10 6 HPD1NR or 5 × 10 6 HaK cells, or were injected i.p with anti-CD3 mAb (500 μg/injection) on the day before re-challenge with HPD1NR cells. In parallel, naive hamsters were injected with 5 × 10 6 HaK cells at the same sites and tumor growth measured and analyzed using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. *** p < 0.001

Article Snippet: Splenocytes and lymph node cells (1 × 10 6 ) were prepared and stained with mAb against Syrian hamster CD3 and mAb against mouse CD4-conjugated with FITC (GK1.5, cross-reactive with Syrian hamster, BD), followed by secondary APC-conjugated rabbit anti-mouse antibody for CD3 staining (BD).

Techniques: Injection, Generated

Journal: Nature Communications

Article Title: Re-designing Interleukin-12 to enhance its safety and potential as an anti-tumor immunotherapeutic agent

doi: 10.1038/s41467-017-01385-8

Figure Lengend Snippet: Ad-TD-nsIL-12 is an effective and safe treatment for orthotopic PaCa in Syrian hamsters. Six days after Hap-T1 cells were seeded into the tail of the pancreas, hamsters were injected i.p. with 500 µl PBS, 1 × 10 9 PFU Ad-TD-LUC, Ad-TD-IL-12 or Ad-TDnsIL-12 ( a ) or 2.5 × 10 9 PFU Ad-TD-LUC, 2.5 × 10 9 PFU Ad-TD-nsIL-12 or 5 × 10 8 PFU Ad-TD-IL-12 ( b ) ( n = 7/group) on days 0, 2, 4, 6, 8, and 10. Kaplan–Meier survival curves were generated. Significance was assessed using the log-rank (Mantel–Cox) test. * p < 0.05, ** p < 0.01. c − h Animals were treated as b ( n = 3/time point/group) and killed on day 3, 7, and 14 after last viral treatments. c Representative images of immunohistochemical staining for Hexon at day 7. Hexon-positive cells were counted in five high-power fields (HPF) from each tumor section (×200). ND: not detected. d Infectious virion recovery from tumor tissue was determined by TICD50 using JH293 cells. e Tumors, lung, and livers were analyzed by qPCR for the copy numbers of the viral E1A gene after treatment with Ad-TD-nsIL-12 at 2.5 × 10 9 PFU/injection on day 0, 2, and 4. The sensitivity of the assay is illustrated by the dotted line. Mean and SEM are shown for each group and compared using an independent t -test. * p < 0.05, ** p < 0.01. f Mean tumor volumes and SEM are shown for each group. Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. * p < 0.05. g Representative images of immunohistochemical staining for CD3 at day 7. Quantitative scores of lymphocyte infiltration within tumors are shown (right panel) from five HPF from each tumor section (×200). The scoring was conducted within the tumor and stroma and necrotic areas were avoided. The extent of positive cells was categorized into the following four grades: 1, <15 cells/HPF; 2, 16–30 cells/HPF; 3, 31–45 cells/HPF; 4, >45 cells/HPF. ND: not detected. h IL-12 expression in sera was detected by ELISA. i Representative histopathology of HE staining of livers after three i.p injections of virus into hamsters bearing orthotopic PaCa tumors ( n = 3/group) on day 0, 2, and 4, using the same dose as in b . Livers were collected one day following the last injection and analyzed using HE staining (×200)

Article Snippet: Splenocytes and lymph node cells (1 × 10 6 ) were prepared and stained with mAb against Syrian hamster CD3 and mAb against mouse CD4-conjugated with FITC (GK1.5, cross-reactive with Syrian hamster, BD), followed by secondary APC-conjugated rabbit anti-mouse antibody for CD3 staining (BD).

Techniques: Injection, Generated, Immunohistochemical staining, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Histopathology

Journal: Nature Communications

Article Title: Re-designing Interleukin-12 to enhance its safety and potential as an anti-tumor immunotherapeutic agent

doi: 10.1038/s41467-017-01385-8

Figure Lengend Snippet: Ad-TD-nsIL-12 efficacy is dependent on hamster CD3+/CD4− immune cell subsets. a , b Syrian hamsters were inoculated subcutaneously with 2 × 10 6 HPD1NR cells. The established tumors were injected i.t. with 1 × 10 9 PFU Ad-TD-nsIL-12 or PBS ( n = 7/group) on day 0, 2, 4, 6, 8, and 10. Control IgG and either mouse anti-hamster CD3 mAb (4F11) a or CD4 mAb b were injected i.p. at doses of 500 μg/injection every fourth day from the day before the viral therapy to the end of the experiment and FACS analysis used to confirm the depletion. Mean tumor volumes and SEM are shown for each group. Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. * p < 0.05, *** p < 0.001. c – g 2 × 10 6 HPD1NR cells were seeded into the right flank of Syrian hamsters. When tumor volumes reached 300 mm 3 , nine hamsters per group were each injected i.t with PBS, 1 × 10 9 PFU Ad-TD-LUC or Ad-TD- IL-12/nsIL-12 on day 0. On days 3, 7, and 21 tumors were collected and processed for IHC. c Representative images of immunohistochemical staining for CD3 and CD4 at day 7 (×200). d − f Quantitative scores of lymphocyte infiltration within tumors. Lymphocytes were counted in 5 HPFs randomly selected from each tumor section (×200). The scoring was conducted within the tumor and stroma; necrotic areas were avoided. The extent of lymphocyte infiltration was categorized into the following four grades: 1, <25 cells/HPF; 2, 25–49 cells/HPF; 3, 50–75 cells/HPF; 4, >75 cells/HPF. Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. * p < 0.05, ** p < 0.01, *** p < 0.001. g Spleens and lymph nodes were collected and analyzed by FACS for CD3 and CD4 expression at the time points shown. Mean expression and SEM is plotted ( n = 3/group). Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Splenocytes and lymph node cells (1 × 10 6 ) were prepared and stained with mAb against Syrian hamster CD3 and mAb against mouse CD4-conjugated with FITC (GK1.5, cross-reactive with Syrian hamster, BD), followed by secondary APC-conjugated rabbit anti-mouse antibody for CD3 staining (BD).

Techniques: Injection, Immunohistochemical staining, Staining, Expressing