Journal: Journal of Nanobiotechnology

Article Title: Silver nanoparticle induced immunogenic cell death can improve immunotherapy

doi: 10.1186/s12951-024-02951-1

Figure Lengend Snippet: Therapeutic effects of Ag-citrate-5 nm on Renca tumors and syngergy with anti-PD1 ICB. ( A ) Relative photon flux (radiance) of Renca-luc + tumors either injected ( a ) peritumorally with PBS (100 µl/animal; control), ( b ) peritumorally with Ag citrate-5 nm (20 µg/mouse), ( c ) intraperitoneally with an anti-PD-1 antibody (150 µg/animal). The mice in the combination groups were injected peritumorally with Ag citrate-5 nm (20 µg/mouse) and intraperitoneally with anti-PD-1 antibody (150 µg/animal). The combination groups and the IT group were in total injected intraperitoneally with anti-PD-1 three times (boost) with an interval of 4 days between injections. Further control groups include (1) intraperitoneal administration of anti-CD8 (150 µg/ml), (2) anti-PD1 + anti-CD8, (3) Ag citrate-5 nm + anti-CD8 or Ag citrate-5 nm + anti-PD1 + anti-CD8. Representative bioluminescence images of mice with Renca Luc + flank tumors in different treatment groups. These groups also include treatment with anti-CD8 antibody either as monotherapy, or in combination with NPs, anti-PD1 or both where anti-CD8 antibody (150 µg/animal) was administered intraperitoneally three times with an interval of 4 days between injections. ( B ) Tumor flux of Renca Luc + cells in photon per flux. Subcutaneous Renca tumors were treated with higher concentration of Ag-citrate-5 nm (50 µg/mouse) and Anti-PD1 (200 µg/mouse). Representative bioluminescence images of mice with Renca Luc + flank tumors in different treatment groups. Representative image of the tumors at the final timepoint. ( C ) Bar graphs displaying the level of caspase and elastase at the tumor as measured through non-invasive optical imaging. Representative fluorescence images of mice with Renca Luc + flank tumors in different treatment groups. ( D ) Bar graphs showing the cancer cell-selective calreticulin translocation to the cell surface as evaluated through ImageStream based flow cytometry upon treatment with the various conditions. The results are presented as the mean of the animals/group ± SEM. The level of significance was indicated when appropriate (*: p < 0.05; **: p < 0.01; ***: p < 0.001)

Article Snippet: Anti-CD8 was based on monoclonal antibody against CD8 alpha ( In vivo MAb anti-mouse CD8α, Biocell).

Techniques: Injection, Control, Concentration Assay, Optical Imaging, Fluorescence, Translocation Assay, Flow Cytometry

Journal: Journal of Nanobiotechnology

Article Title: Silver nanoparticle induced immunogenic cell death can improve immunotherapy

doi: 10.1186/s12951-024-02951-1

Figure Lengend Snippet: Effect of Ag-citrate-5 nm, with or without IT, on immune cell activation in the spleen and tumor tissue. Representative fluorescence images of tumor tissue section obtained from Renca luc+ tumors treated with ( A ) PBS, ( B ) Ag-citrate-5 nm monotherapy, ( C ) anti-PD1 monotherapy or ( D ) combination therapy. The images reveal tissue sections stained for F4/80 (green, macrophages), CD8 (CD8 + T cells, red) and counterstained with DAPI (cell nuclei, blue). Scale bars of 100 μm are indicated in the bottom left corner. Bar graphs displaying the levels of ( E ) CD4+ T cells in the spleen, ( F ) CD4 + CD38+ active T cells in the spleen, ( G ) CD4 + CD69+ active T cells in the spleen or ( H ) CD8+ T cells in the spleen. The results are presented as the normalized mean + SEM in percentages related to the control group (PBS =100%). The level of significance was indicated when appropriate (*: p <0.05; **: p < 0.01; ***: p <0.001; ****: p <0.0001). I ) Representative H&E stained images of kidney (top row), lung (middle row) and liver (bottom row) tissue sections of tumor-bearing mice treated with the respective agents indicated at the top

Article Snippet: Anti-CD8 was based on monoclonal antibody against CD8 alpha ( In vivo MAb anti-mouse CD8α, Biocell).

Techniques: Activation Assay, Fluorescence, Staining, Control

Journal: Cancer immunology research

Article Title: The Tumor Microenvironment Regulates Sensitivity of Murine Lung Tumors to PD-1/PD-L1 Antibody Blockade

doi: 10.1158/2326-6066.CIR-16-0365

Figure Lengend Snippet: CMT167 subcutaneous tumors induce a T-cell response that is skewed toward CD8+ T cells and are less sensitive than CMT167 orthotopic tumors to anti-PD-1/PD-L1 therapy. (A) CMT167 subcutaneous tumor-bearing mice were injected with anti-PD-1, anti-PD-L1, or isotype control antibody (n = 10 for IgG2a and anti-PD-1 groups, n = 5 for IgG2b and anti-PD-L1 groups). CMT167 orthotopic tumor-bearing lungs (n = 9) and CMT167 subcutaneous tumors (n = 7) were analyzed by flow cytometry for (B) CD8+ T cells, (C) CD4+ T cells, (D) CD4:CD8 ratio, (E) PD-1 expression on CD8+ T cells, (F) PD-1 expression on CD4+ T cells, and (G) Foxp3 expression in CD4+ T cells. Statistically significant differences are indicated as determined by Student unpaired t test; ** = P < 0.01, *** = P < 0.001. (H) PD-L1 expression was analyzed by flow cytometry in CMT167 subcutaneous tumors. Similar results were obtained in five independent mice.

Article Snippet: Rat monoclonal anti-mouse CD8 (clone 53-6.72) and rat IgG2a (clone 2A3) were purchased from BioXCell (Lebanon, NH).

Techniques: Injection, Control, Flow Cytometry, Expressing

Journal: Cancer immunology research

Article Title: The Tumor Microenvironment Regulates Sensitivity of Murine Lung Tumors to PD-1/PD-L1 Antibody Blockade

doi: 10.1158/2326-6066.CIR-16-0365

Figure Lengend Snippet: Both CMT167 orthotopic lung tumors and LLC orthotopic lung tumors generate an adaptive immune response and induce PD-1 expression on T cells. Tissue sections from CMT167 (representative image shown in Fig. 5A) and LLC (representative image shown in Fig. 5B) tumor-bearing mice were stained for CD3 (FITC, green) and nuclei were stained with DAPI (blue). Magnification x20. (C) Quantification of tumor-infiltrating lymphocytes in CMT167 tumors (n = 9) and LLC tumors (n = 6). (D) Effects of CD8+ T cell immunodepletion on growth of CMT167 (n = 7 each group) and LLC orthotopic tumors (n = 6 each group). CMT167 (n = 9, same mice as in Fig. 3B–F) or LLC tumor-bearing lungs (n = 9) were analyzed by flow cytometry and compared with lungs from naive mice (n = 8) for (E) CD8+ T cells, (F) CD4+ T cells, (G) PD-1 expression on CD8+ T cells, and (H) PD-1 expression on CD4+ T cells. Statistically significant differences are indicated as determined by Student unpaired t test (C-D) or by one-way ANOVA (E-H); * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Article Snippet: Rat monoclonal anti-mouse CD8 (clone 53-6.72) and rat IgG2a (clone 2A3) were purchased from BioXCell (Lebanon, NH).

Techniques: Expressing, Staining, Immunodepletion, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Induction of SARS-CoV-2 Protein S-Specific CD8+ T Cells in the Lungs of gp96-Ig-S Vaccinated Mice

doi: 10.3389/fimmu.2020.602254

Figure Lengend Snippet: Secreted gp96-Ig-S vaccine induces CD8+ TEM and TRM responses in the lungs. Equivalent number of AD100-gp96-Ig-S vaccine cells that produce 200 ng/ml gp96-Ig or PBS were injected by s.c. route in C56Bl/6 mice. 5 days later, mice were sacrificed and spleen, lungs, and BAL were isolated and (A) frequency of CD4+ and CD8+ T cells; (B) naive (N) CD44-CD62L+, CM CD44+CD62L+ and EM CD44+CD62L- CD8+ T cells; and (C) TRM CD69+ cells were determined by flow cytometry after staining the cells with antibodies against the following surface markers: CD45, CD3, CD4, CD8, CD44, CD62L, and CD69 antibodies. Bar graph shows percentage of CD4+ and CD8+ cells within CD3+ cells or CD8+ T cell memory subset within CD8+ T cells. Data represent at least two technical replicates with three to six independent biological replicates per group. *p<0.05, **p<0.01. (A, B) Mann-Whitney tests were used to compare two experimental groups. To compare >2 experimental groups, Kruskal-Wallis ANOVA with Dunn’s multiple comparison tests were applied). BAL, bronchoalveolar lavage; CM, central memory; EM, effector memory; TEM, T cell effector memory.

Article Snippet: Cells were labelled with LIVE/DEADTM Fixable Violet—Dead Cell Stain Kit (Invitrogen, Carlsbad, CA, USA) and then stained with the following antibody cocktail: APCCy7 CD45: Clone:2D1; AF700 CD3: Clone: 17A2; PECy7 CD4: Clone : RM4-5; FITC CD8: Clone: KT15 (ProImmune, Oxford, UK) or PerCP CD8: Clone:53-6.7; PE Dazzle CD69: Clone:H1.2F3; BV 605 CD44: Clone : IM7; BV510 CD62L: Clone: MEL-14; PerCP/Cy5.5 CXCR6: Clone: SA051D1.

Techniques: Injection, Isolation, Flow Cytometry, Staining, MANN-WHITNEY, Comparison

Journal: Frontiers in Immunology

Article Title: Induction of SARS-CoV-2 Protein S-Specific CD8+ T Cells in the Lungs of gp96-Ig-S Vaccinated Mice

doi: 10.3389/fimmu.2020.602254

Figure Lengend Snippet: Secreted gp96-Ig-S vaccine induces protein S specific CD8+ and CD4+ T cells in the spleen and lung tissue. Five days after the vaccination of C57Bl6 mice, splenocytes and lung cells were isolated from vaccinated and control mice (PBS) and in vitro restimulated with S1 and S2 overlapping peptides from SARS-CoV-2 protein in the presence of protein transport inhibitor, brefeldin A for the last 5 h of culture. After 20 h of culture, ICS was performed to quantify protein S-specific CD8+ and CD4+ T-cell responses. Cytokine expression in the presence of no peptides was considered background and it was subtracted from the responses measured from peptide pool stimulated samples for each individual mouse. (A, B) CD8+ T cells from spleen and lungs expressing IFNγ, TNFα, and IL-2 in response to S1 and S2 peptide pool; (C, D) CD4+ T cells from spleen and lungs expressing IFNγ, TNFα, and IL-2 in response to S1 and S2 peptide pool; (E) Representative dot plot of gated CD8+ T cells in lungs expressing indicated cytokines (IFNγ; IL-2, and TNFα) in vaccinated and non-vaccinated (PBS, control) HLA-A2 mice at day 5 (F) Proportion of antigen (protein S)-experienced CD8+ and CD4+ T cells isolated from spleen and lung tissue expressing IFNγ, TNFα, or IL-2 after o/n stimulation with S1 + S2 peptides. Pie charts corresponding to cytokine profiles of CD8+ and CD4+ T cells isolated from spleen and lung tissue; (G) Pie charts corresponding to cytokine profiles of CD8+ CD4+ T cells isolated from spleen and lung tissue after o/n stimulation with S1 + S2 peptides. Assessment of the mean proportion of cells making any combination of one to three cytokines (IFN-γ, TNFα, IL-2). Data represent at least two technical replicates with three to six independent biologic replicates per group. *p<0.05, ***p<0.001. Kruskal-Wallis ANOVA with Dunn’s multiple comparisons tests were applied. Asterisks (*) above or inside the column denote significant differences between indicated T cells producing cytokines in vaccine versus control (PBS) at 0.05 alpha level. ANOVA, analysis of variance; ICS, intracellular cytokine staining; IFN, interferon; IL, interleukin; PBS, phosphate-buffered saline; TNF, tumor necrosis factor.

Article Snippet: Cells were labelled with LIVE/DEADTM Fixable Violet—Dead Cell Stain Kit (Invitrogen, Carlsbad, CA, USA) and then stained with the following antibody cocktail: APCCy7 CD45: Clone:2D1; AF700 CD3: Clone: 17A2; PECy7 CD4: Clone : RM4-5; FITC CD8: Clone: KT15 (ProImmune, Oxford, UK) or PerCP CD8: Clone:53-6.7; PE Dazzle CD69: Clone:H1.2F3; BV 605 CD44: Clone : IM7; BV510 CD62L: Clone: MEL-14; PerCP/Cy5.5 CXCR6: Clone: SA051D1.

Techniques: Isolation, Control, In Vitro, Expressing, Staining, Saline

Journal: Frontiers in Immunology

Article Title: Induction of SARS-CoV-2 Protein S-Specific CD8+ T Cells in the Lungs of gp96-Ig-S Vaccinated Mice

doi: 10.3389/fimmu.2020.602254

Figure Lengend Snippet: Secreted Gp96-Ig-S vaccine induces S1- and S2-specific CD8 + CD69 + CXCR6 + cells in the spleen, lung tissue, and BAL. Five and 30 days after the vaccination of HLA-A2.1 transgenic mice, splenocytes, lung cells and BAL were isolated form vaccinated and control mice (PBS). Cells were stained with HLA-A2 pentamer containing FIAGLIAIV and YLQPRTFLL peptides, followed by surface staining for CD45, CD3, CD4, CD8, CD69, CXCR6. (A, B) Bar graphs represent percentage of the pentamer positive cells within CD8+ T cells; (C) Representative zebra plots of gated CD8+ T cells expressing indicated pentamer-specific TCR+ CD8+ T cells in vaccinated and non-vaccinated HLA-A2.1 mice at day 5 (D) Bar graphs represent percentage of CD69+, CD69-, and CXCR6+ cells within YQL-pentamer positive cells at days 5 and 30; (Data represent at least two technical replicates with three to six independent biologic replicates per group. *p<0.05, **p<0.01, ***p<0.001. Kruskal-Wallis ANOVA with Dunn’s multiple comparisons tests were applied. Asterisks (*) inside the column denote significant differences between indicated pentamer+CD8+ T cells in the vaccinated group and control (PBS) (A, B) at 0.05 alpha level. ANOVA, analysis of variance; BAL, bronchoalveolar lavage; PBS, phosphate-buffered saline.

Article Snippet: Cells were labelled with LIVE/DEADTM Fixable Violet—Dead Cell Stain Kit (Invitrogen, Carlsbad, CA, USA) and then stained with the following antibody cocktail: APCCy7 CD45: Clone:2D1; AF700 CD3: Clone: 17A2; PECy7 CD4: Clone : RM4-5; FITC CD8: Clone: KT15 (ProImmune, Oxford, UK) or PerCP CD8: Clone:53-6.7; PE Dazzle CD69: Clone:H1.2F3; BV 605 CD44: Clone : IM7; BV510 CD62L: Clone: MEL-14; PerCP/Cy5.5 CXCR6: Clone: SA051D1.

Techniques: Transgenic Assay, Isolation, Control, Staining, Expressing, Saline

Journal: Frontiers in Immunology

Article Title: Induction of SARS-CoV-2 Protein S-Specific CD8+ T Cells in the Lungs of gp96-Ig-S Vaccinated Mice

doi: 10.3389/fimmu.2020.602254

Figure Lengend Snippet: Secreted gp96-Ig-S vaccine induces protein S specific memory CD8+ and CD4+ T cells in the spleen and lungs. Thirty days after the vaccination of HLA-A2.1 transgenic mice, splenocytes and lung cells were isolated from vaccinated and control mice (PBS) and in vitro restimulated with S1 and S2 overlapping peptides from SARS-CoV-2 protein in the presence of protein transport inhibitor, brefeldin A for the last 5 h of culture. After 20 h of culture, ICS was performed to quantify protein S-specific CD8+ and CD4+ T-cell responses. Cytokine expression in the presence of no peptides was considered background and it was subtracted from the responses measured from peptide pool stimulated samples for each individual mouse. (A, B) CD8+ T cells from spleen and lungs expressing IFNγ, TNFα, and IL-2 in response to S1 and S2 peptide pool; (D, E) CD4+ T cells from spleen and lungs expressing IFNγ, TNFα, and IL-2 in response to S1 and S2 peptide pool; (C) Proportion of antigen (protein S)-experienced CD8+ and CD4+ T cells isolated from spleen and lung tissue expressing IFNγ, TNFα, or IL-2 after o/n stimulation with S1 + S2 peptides. Pie charts corresponding to cytokine profiles of CD8+ and CD4+ T cells isolated from spleen and lung tissue; (F) 5 days after primary and secondary vaccination of HLA-A2.1 transgenic mice, splenocytes, lung cells and BAL were isolated form vaccinated and control mice (PBS). Time of primary and secondary vaccination is indicated with black arrows. Cells were stained with HLA-A2 pentamer containing YLQPRTFLL peptide, followed by surface staining for CD45, CD3, CD4, CD8. (G) BAL was analyzed 5 and 30 days after primary and secondary vaccination and frequency of HLA-A2 pentamer positive cells was determined. Graphs represent percentage of the pentamer positive cells within CD8+ T cells in individual mice; Data represent at least two technical replicates with three to six independent biologic replicates per group. *p<0.05, **p<0.01, ***p<0.001, ns, not significant. Kruskal-Wallis ANOVA with Dunn’s multiple comparisons tests were applied. Asterisks (*) above or inside the column denote significant differences between indicated T cells producing cytokines in vaccine versus control (PBS) at 0.05 alpha level. ANOVA, analysis of variance; ICS, intracellular cytokine staining; IFN, interferon; IL, interleukin; PBS, phosphate-buffered saline; TNF, tumor necrosis factor.

Article Snippet: Cells were labelled with LIVE/DEADTM Fixable Violet—Dead Cell Stain Kit (Invitrogen, Carlsbad, CA, USA) and then stained with the following antibody cocktail: APCCy7 CD45: Clone:2D1; AF700 CD3: Clone: 17A2; PECy7 CD4: Clone : RM4-5; FITC CD8: Clone: KT15 (ProImmune, Oxford, UK) or PerCP CD8: Clone:53-6.7; PE Dazzle CD69: Clone:H1.2F3; BV 605 CD44: Clone : IM7; BV510 CD62L: Clone: MEL-14; PerCP/Cy5.5 CXCR6: Clone: SA051D1.

Techniques: Transgenic Assay, Isolation, Control, In Vitro, Expressing, Staining, Saline

Journal: Physiological Reports

Article Title: Cyclophosphamide treatment for hypertension and renal injury in an experimental model of systemic lupus erythematosus

doi: 10.14814/phy2.14059

Figure Lengend Snippet: Impact of cyclophosphamide (CYC) on circulating immune cells. Flow cytometry was performed on cryopreserved peripheral blood leukocytes to determine the impact of CYC treatment on lymphoid and myeloid cell populations. (A) Representative scatter plot of peripheral blood leukocytes. (B) Data suggest that CYC treatment reduced CD45R + B cells compared to vehicle treatment in systemic lupus erythematosus (SLE) mice (15.87 ± 8.49% vs. 26.96 ± 4.72%, P = 0.06). (C) There was no significant difference in the percentage of circulating CD3 + CD4 + T cells between groups or (D) the percentage of CD3 + CD8 + T cells.(E) The percentage of circulating neutrophils was significantly increased in CYC‐treated SLE mice compared to vehicle‐treated mice (39.26 ± 4.92 vs. 20.58 ± 6.01, * P < 0.05). (F) There was no significant different in the percentage of circulating monocytes between groups. ○ SLE Vehicle ( n = 8), and ● SLE CYC ( n = 10).

Article Snippet: The following antibodies were diluted with wash buffer (50 μL/sample; 1:100 dilution), and placed in a single tube‐containing sample to measure the relative percentages of circulating lymphocyte populations: CD3e‐PE‐Cy7 (clone 145‐2C11), CD4‐FITC (clone GK1.5), CD8‐PerCP‐Cy (clone 53‐6.7), and CD45R‐Alexa Fluor (clone RA3‐6B2) (BD Biosciences).

Techniques: Flow Cytometry

Journal: Physiological Reports

Article Title: Cyclophosphamide treatment for hypertension and renal injury in an experimental model of systemic lupus erythematosus

doi: 10.14814/phy2.14059

Figure Lengend Snippet: Impact of cyclophosphamide (CYC) on renal lymphocyte infiltration. (A) Renal CD45R + B cells were significantly increased in systemic lupus erythematosus (SLE) vehicle‐treated mice compared to all other treatment groups (* P < 0.05) (B) Renal CD3 + CD4 + T cells were significantly increased in SLE vehicle‐treated mice compared to all other treatment groups (* P < 0.05). (C) Renal CD3 + CD8 + T cells were not significantly different in response to CYC treatment in control or SLE mice. □ Control Vehicle ( n = 5) ■ Control CYC ( n = 5), ○ SLE Vehicle ( n = 5), and ● SLE CYC ( n = 5).

Article Snippet: The following antibodies were diluted with wash buffer (50 μL/sample; 1:100 dilution), and placed in a single tube‐containing sample to measure the relative percentages of circulating lymphocyte populations: CD3e‐PE‐Cy7 (clone 145‐2C11), CD4‐FITC (clone GK1.5), CD8‐PerCP‐Cy (clone 53‐6.7), and CD45R‐Alexa Fluor (clone RA3‐6B2) (BD Biosciences).

Techniques: