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anti-mouse cd8 (53-6.7) pe- cy7 (Thermo Fisher)

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Thermo Fisher anti-mouse cd8 (53-6.7) pe- cy7
Anti Mouse Cd8 (53 6.7) Pe Cy7, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews

anti-mouse cd8 (53-6.7) pe- cy7 - by Bioz Stars, 2026-03

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Journal: Cell

Article Title: Respiratory mucosal delivery of next-generation COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2

doi: 10.1016/j.cell.2022.02.005

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD8 PE-Cy7 (clone 53-6.7) , BD Biosciences , Cat# 552877 RRID: AB_394506.

Techniques: Purification, Recombinant, Staining, Protease Inhibitor, Neutralization, Transfection, Labeling, Software

Journal: eLife

Article Title: Tumor-derived extracellular vesicles regulate tumor-infiltrating regulatory T cells via the inhibitory immunoreceptor CD300a

doi: 10.7554/eLife.61999

Figure Lengend Snippet:

Article Snippet: Antibody , Rat anti-CD8-PE-Cy7 (53–6.7, mouse monoclonal) , BD Bioscience , Cat# 552,877RRID: AB_ 394,506 , FACS (1:5).

Techniques: Purification, Sequencing, Recombinant, Isolation, Software

Journal: Cell host & microbe

Article Title: Toxin-triggered interleukin-1 receptor signaling enables early life discrimination of pathogenic versus commensal skin bacteria

doi: 10.1016/j.chom.2019.10.007

Figure Lengend Snippet: Key Resources Table

Article Snippet: Pe-Cy7 anti-mouse CD8 (clone 53-6.7) , BD Biosciences , Cat# 552877; RRID: AB_394506.

Techniques: Recombinant, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Selection, Staining, Plasmid Preparation, Generated

Iron homeostasis is dynamically regulated during T cell activation. (A) Both unstimulated (D0) and stimulated (D3) CD4 T cells were subjected to ICP-MS analysis. The graph shows the fold change in total iron level [reported in parts per billion (ppb) per 1 x 106 cells] relative to D0. (n=6) (B) Enriched CD4 T cells were stimulated for 3 days and total iron levels were measured in both fresh media (D0) and media collected from cell cultures (D3). (n=3) (C) Splenocytes were stained with 0.02 µM Calcein-AM dye and analyzed via flow cytometry. Representative histograms show the LIP levels in CD4 and CD8 T cells as a function of mean fluorescence intensity (MFI) of Calcein. (n=3) (D) Splenocytes were stimulated with αCD3/αCD28 antibodies after B cell depletion with αCD19 magnetic beads. Representative graphs show the LIP levels in CD4 and CD8 T cells over time post-activation relative to D0. (E) Bar graph illustrates the LIP level in human CD4 T cells at day 5 (D5) relative to that at D0. (n=4) (F) Enriched CD4 T cells were stimulated for up to 3 days and stained for TfR1, ferritin, and Fpn as described in the Materials and Methods. Graphs shows the change in expression of each of the aforementioned proteins relative to D0. (n=3) (G) Bar graphs represent the relative gene expression from both unstimulated and stimulated CD4 T cells over time, which was calculated as described in the Materials and Methods. (n=3). Error bars represent Mean ± SEM. All statistics were performed by comparing each time point to D0. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Activation-induced iron flux controls CD4 T cell proliferation by promoting proper IL-2R signaling and mitochondrial function

doi: 10.4049/jimmunol.1901399

Figure Lengend Snippet: Iron homeostasis is dynamically regulated during T cell activation. (A) Both unstimulated (D0) and stimulated (D3) CD4 T cells were subjected to ICP-MS analysis. The graph shows the fold change in total iron level [reported in parts per billion (ppb) per 1 x 106 cells] relative to D0. (n=6) (B) Enriched CD4 T cells were stimulated for 3 days and total iron levels were measured in both fresh media (D0) and media collected from cell cultures (D3). (n=3) (C) Splenocytes were stained with 0.02 µM Calcein-AM dye and analyzed via flow cytometry. Representative histograms show the LIP levels in CD4 and CD8 T cells as a function of mean fluorescence intensity (MFI) of Calcein. (n=3) (D) Splenocytes were stimulated with αCD3/αCD28 antibodies after B cell depletion with αCD19 magnetic beads. Representative graphs show the LIP levels in CD4 and CD8 T cells over time post-activation relative to D0. (E) Bar graph illustrates the LIP level in human CD4 T cells at day 5 (D5) relative to that at D0. (n=4) (F) Enriched CD4 T cells were stimulated for up to 3 days and stained for TfR1, ferritin, and Fpn as described in the Materials and Methods. Graphs shows the change in expression of each of the aforementioned proteins relative to D0. (n=3) (G) Bar graphs represent the relative gene expression from both unstimulated and stimulated CD4 T cells over time, which was calculated as described in the Materials and Methods. (n=3). Error bars represent Mean ± SEM. All statistics were performed by comparing each time point to D0. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001.

Article Snippet: The fluorescently-conjugated antibodies used for surface and intracellular staining in the presence of anti-FcγR mAb (2.4G2) were: anti-mouse TCR-β (H57–597) Pacific Blue/APC, anti-mouse CD4 (GK1.5) APC-Cy7, anti-mouse CD8 (53–6.7) PE-Cy7, anti-mouse CD71 ( {"type":"entrez-nucleotide","attrs":{"text":"R17217","term_id":"770827","term_text":"R17217"}} R17217 ) FITC/PerCP-Cy5.5, anti-mouse CD25 (PC61.5) PerCP-Cy5.5/PE-Cy7, anti-mouse CD69 (H1.2F3) PE-Cy7, and IL-2 (JES6–5H4) PE (all from eBioscience).

Techniques: Activation Assay, Staining, Flow Cytometry, Fluorescence, Magnetic Beads, Expressing, Gene Expression

T cell proliferation correlates with the downregulation of intracellular iron levels. (A) The bar graph represents total iron levels in CD4 T cells that were freshly isolated or stimulated for 3 days with either 0.25 µg/mL or 5 µg/mL αCD3. (n=3) (B) CD4 T cells were stained with CellTrace Violet (CTV) and stimulated for 3 days with the indicated concentrations of αCD3. Representative histograms show both the change in Calcein signal and cell proliferation as measured by CTV dilutions under each of the indicated stimulation conditions. (n=3) (C) CD4 T cells were stimulated for 3 days with either αCD3 only or both αCD3/αCD28. The bar graph shows relative LIP levels. (n=3) (D) CD4 T cells were stimulated for 3 days in the presence (20 µM) or absence (0 µM) of DFO. The bar graph shows the total iron levels of cells from each of the aforementioned culture conditions. (n=3) (E) CD4 T cells were stained with CTV and stimulated for 3 days in the presence of the indicated concentrations of DFO. Representative histograms show both the change in Calcein signal and cell proliferation as measured by CTV dilutions at each of the indicated DFO concentrations. (n=3) (F) Representative histograms show human CD4 T cell proliferation using CTV dilutions with (+) or without (−) 10 µM of DFO after 4 days of activation. (n=4) (G) The bar graph shows the LIP level in human CD4 T cells at day 4 post-activation with and without DFO. All graphs are cumulative of at least 3 independent experiments. Error bars represent Mean ± SEM. **p<0.005, ***p<0.0005, ****p<0.0001, ns: not significant.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Activation-induced iron flux controls CD4 T cell proliferation by promoting proper IL-2R signaling and mitochondrial function

doi: 10.4049/jimmunol.1901399

Figure Lengend Snippet: T cell proliferation correlates with the downregulation of intracellular iron levels. (A) The bar graph represents total iron levels in CD4 T cells that were freshly isolated or stimulated for 3 days with either 0.25 µg/mL or 5 µg/mL αCD3. (n=3) (B) CD4 T cells were stained with CellTrace Violet (CTV) and stimulated for 3 days with the indicated concentrations of αCD3. Representative histograms show both the change in Calcein signal and cell proliferation as measured by CTV dilutions under each of the indicated stimulation conditions. (n=3) (C) CD4 T cells were stimulated for 3 days with either αCD3 only or both αCD3/αCD28. The bar graph shows relative LIP levels. (n=3) (D) CD4 T cells were stimulated for 3 days in the presence (20 µM) or absence (0 µM) of DFO. The bar graph shows the total iron levels of cells from each of the aforementioned culture conditions. (n=3) (E) CD4 T cells were stained with CTV and stimulated for 3 days in the presence of the indicated concentrations of DFO. Representative histograms show both the change in Calcein signal and cell proliferation as measured by CTV dilutions at each of the indicated DFO concentrations. (n=3) (F) Representative histograms show human CD4 T cell proliferation using CTV dilutions with (+) or without (−) 10 µM of DFO after 4 days of activation. (n=4) (G) The bar graph shows the LIP level in human CD4 T cells at day 4 post-activation with and without DFO. All graphs are cumulative of at least 3 independent experiments. Error bars represent Mean ± SEM. **p<0.005, ***p<0.0005, ****p<0.0001, ns: not significant.

Techniques: Isolation, Staining, Activation Assay

Iron chelation does not affect IL-2 production by activated T cells. (A-C) Enriched CD4 T cells were stimulated for 3 days in the presence (5, 10, 20 µM) or absence (0 µM) of DFO. (A) The graph represents the fold change in mean fluorescence intensity (MFI) of CD25 and CD69 at each of the indicated DFO concentrations relative to untreated cells. Statistics in (A) were performed by comparing each DFO concentration to untreated cells (0 µM). (n=3) (B) Graphs show IL-2 levels (µg/mL) in media collected from CD4 T cells as measured by ELISA. (n=3) (C) Representative dot plots show the % IL-2+ CD4 T cells after 3 days of stimulation followed by re-stimulation with PMA and Ionomycin for 4 h. The bar graph shows the cumulative percentages of IL-2+ cells from 3 independent experiments. (D) Enriched CD4 T cells were stained with CellTrace Violet (CTV) and stimulated in the absence (0 µM) or presence (5, 10, 20 µM) of DFO either with or without 50 units of recombinant IL-2. Representative histograms show cell proliferation at day 3 post-activation as measured by CTV dilutions. (n=3) E) Enriched CD4 T cells were stimulated for 48 h in the presence (10 µM, 20 µM) or absence (0 µM) of DFO and stained for pSTAT5. The pooled graph shows pSTAT5 levels at each treatment condition. Statistics in (E) were performed by comparing DFO-treated conditions to the untreated (0 µM) condition. (n=3). Error bars represent Mean ± SEM. *p<0.05, **p<0.005, ****p<0.0001.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Activation-induced iron flux controls CD4 T cell proliferation by promoting proper IL-2R signaling and mitochondrial function

doi: 10.4049/jimmunol.1901399

Figure Lengend Snippet: Iron chelation does not affect IL-2 production by activated T cells. (A-C) Enriched CD4 T cells were stimulated for 3 days in the presence (5, 10, 20 µM) or absence (0 µM) of DFO. (A) The graph represents the fold change in mean fluorescence intensity (MFI) of CD25 and CD69 at each of the indicated DFO concentrations relative to untreated cells. Statistics in (A) were performed by comparing each DFO concentration to untreated cells (0 µM). (n=3) (B) Graphs show IL-2 levels (µg/mL) in media collected from CD4 T cells as measured by ELISA. (n=3) (C) Representative dot plots show the % IL-2+ CD4 T cells after 3 days of stimulation followed by re-stimulation with PMA and Ionomycin for 4 h. The bar graph shows the cumulative percentages of IL-2+ cells from 3 independent experiments. (D) Enriched CD4 T cells were stained with CellTrace Violet (CTV) and stimulated in the absence (0 µM) or presence (5, 10, 20 µM) of DFO either with or without 50 units of recombinant IL-2. Representative histograms show cell proliferation at day 3 post-activation as measured by CTV dilutions. (n=3) E) Enriched CD4 T cells were stimulated for 48 h in the presence (10 µM, 20 µM) or absence (0 µM) of DFO and stained for pSTAT5. The pooled graph shows pSTAT5 levels at each treatment condition. Statistics in (E) were performed by comparing DFO-treated conditions to the untreated (0 µM) condition. (n=3). Error bars represent Mean ± SEM. *p<0.05, **p<0.005, ****p<0.0001.

Techniques: Fluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Recombinant, Activation Assay

Intracellular iron stores promote T cell proliferation by controlling mitochondrial function. (A) Enriched CD4 T cells were stimulated in the presence or absence of 10 µM DFO for 48 h. Representative histograms show the percentage of cells in the G0/G1, S, and G2 phases of the cell cycle using PI. The bar graph shows the cumulative percentages of cells in each phase either with or without DFO treatment. (n=4) (B) Graphs show the pooled mean fluorescence intensities (MFI) of MitoTracker green, TMRM, or MitoSOX in CD4 T cells stimulated for 3 days in the presence or absence of 10 µM DFO. (n=3) (C-E) CD4 T cells were stimulated either with or without 10 µM DFO. After 24 h of stimulation, 10 µM FAC was added to the DFO-treated cultures. (C) Representative histograms depict cell proliferation of CellTrace Violet (CTV)-stained CD4 T cells at day 3 post-activation. (n=3) (D) The bar graph depicts the cumulative percentages of CD4 T cells in the different phases of the cell cycle after 48 h of culture. (n=4) (E) Bar graphs show the fold change in MFI of MitoTracker, TMRM, and MitoSOX in CD4 T cells after 3 days of stimulation. Data are cumulative of 3 independent experiments. Error bars represent Mean ± SEM. *p<0.05, **p<0.005, ***p<0.0005, ns: not significant.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Activation-induced iron flux controls CD4 T cell proliferation by promoting proper IL-2R signaling and mitochondrial function

doi: 10.4049/jimmunol.1901399

Figure Lengend Snippet: Intracellular iron stores promote T cell proliferation by controlling mitochondrial function. (A) Enriched CD4 T cells were stimulated in the presence or absence of 10 µM DFO for 48 h. Representative histograms show the percentage of cells in the G0/G1, S, and G2 phases of the cell cycle using PI. The bar graph shows the cumulative percentages of cells in each phase either with or without DFO treatment. (n=4) (B) Graphs show the pooled mean fluorescence intensities (MFI) of MitoTracker green, TMRM, or MitoSOX in CD4 T cells stimulated for 3 days in the presence or absence of 10 µM DFO. (n=3) (C-E) CD4 T cells were stimulated either with or without 10 µM DFO. After 24 h of stimulation, 10 µM FAC was added to the DFO-treated cultures. (C) Representative histograms depict cell proliferation of CellTrace Violet (CTV)-stained CD4 T cells at day 3 post-activation. (n=3) (D) The bar graph depicts the cumulative percentages of CD4 T cells in the different phases of the cell cycle after 48 h of culture. (n=4) (E) Bar graphs show the fold change in MFI of MitoTracker, TMRM, and MitoSOX in CD4 T cells after 3 days of stimulation. Data are cumulative of 3 independent experiments. Error bars represent Mean ± SEM. *p<0.05, **p<0.005, ***p<0.0005, ns: not significant.

Techniques: Fluorescence, Staining, Activation Assay

PBMCs (~10 × 106) derived from 20 HLA-A*0201(+) individuals vs. 8 HLA-A*0201(−) HSV-seropositive individuals were stimulated in vitro for 72 hrs with each of 22 groups of equimolar peptides. IFN-γ and CD107a/b were then measured by intracellular staining, as outlined in Materials and Methods. (A) Representative contour plots of percentage of IFN-γ+CD8+ T cells specific to groups of HSV-1 peptides 6, 11 and 16 detected in HLA-A*0201(+) (left panels) and HLA-A*0201(−) (right panels) individuals. (B) Average frequencies of IFN-γ+CD8+ T cells specific to 22 groups of HSV-1 peptides detected in PBMCs from HLA-A*0201(+) (closed circle) and HLA-A*0201(−) (open circle) individuals. (C) Representative contour plots of expression of CD107a/b by CD8+ T cells specific to groups of HSV-1 peptides 6, 11 and 16 detected in HLA-A*0201(+) (left panel) and HLA-A*0201(−) (right panel) individuals. (D) Average frequencies of CD107a/b(+)CD8+ T cells detected in HLA-A*0201(+) (closed circle) and HLA-A*0201(−) (open circle) individuals in response to 22 groups of HSV-1 peptides. (E) Representative dot plots of percentage of dividing CFSE(+)CD8+ T cells from HLA-A*0201(+) (top three rows) and HLA-A*0201(−) individuals (lower three rows) following a 6-days in vitro stimulation with each of the 22 groups of HSV-1 peptides. The results are representative of 2 independent experiments. The nominal P values indicated on panels (B) and (D) show statistical significance between HLA-A*0201(+) and HLA-A*0201(−) individuals. P values are calculated using either the parametric two-sample Student’s t-test or non-parametric Wilcoxon rank sum test, as appropriate. For paired comparisons involving the 22 groups of peptides, we have adjusted for multiple comparisons using the Bonferroni procedure.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Bolstering the Number and Function of HSV-1-Specific CD8 + T EM and T RM cells in Latently Infected Trigeminal Ganglia Reduces Recurrent Ocular Herpes Infection and Disease

doi: 10.4049/jimmunol.1700145

Figure Lengend Snippet: PBMCs (~10 × 106) derived from 20 HLA-A*0201(+) individuals vs. 8 HLA-A*0201(−) HSV-seropositive individuals were stimulated in vitro for 72 hrs with each of 22 groups of equimolar peptides. IFN-γ and CD107a/b were then measured by intracellular staining, as outlined in Materials and Methods. (A) Representative contour plots of percentage of IFN-γ+CD8+ T cells specific to groups of HSV-1 peptides 6, 11 and 16 detected in HLA-A*0201(+) (left panels) and HLA-A*0201(−) (right panels) individuals. (B) Average frequencies of IFN-γ+CD8+ T cells specific to 22 groups of HSV-1 peptides detected in PBMCs from HLA-A*0201(+) (closed circle) and HLA-A*0201(−) (open circle) individuals. (C) Representative contour plots of expression of CD107a/b by CD8+ T cells specific to groups of HSV-1 peptides 6, 11 and 16 detected in HLA-A*0201(+) (left panel) and HLA-A*0201(−) (right panel) individuals. (D) Average frequencies of CD107a/b(+)CD8+ T cells detected in HLA-A*0201(+) (closed circle) and HLA-A*0201(−) (open circle) individuals in response to 22 groups of HSV-1 peptides. (E) Representative dot plots of percentage of dividing CFSE(+)CD8+ T cells from HLA-A*0201(+) (top three rows) and HLA-A*0201(−) individuals (lower three rows) following a 6-days in vitro stimulation with each of the 22 groups of HSV-1 peptides. The results are representative of 2 independent experiments. The nominal P values indicated on panels (B) and (D) show statistical significance between HLA-A*0201(+) and HLA-A*0201(−) individuals. P values are calculated using either the parametric two-sample Student’s t-test or non-parametric Wilcoxon rank sum test, as appropriate. For paired comparisons involving the 22 groups of peptides, we have adjusted for multiple comparisons using the Bonferroni procedure.

Article Snippet: The following anti-mouse antibodies were used to characterize mouse CD8 + T CM , CD8 + T EM , and CD8 + T RM sub-populations: anti-mouse CD8 (clone 53-6.7) PE-Cy7, anti-mouse CD44 (clone IM7) APC-Cy7, anti-mouse CD11a (clone M17/4) FITC, anti-mouse CD103 (clone M290) APC, anti-mouse CD62L (clone MEL-14) A700, anti-mouse CCR7 (clone 4B12) A647, anti-mouse CD69 (clone H1.2F3) PE-Cy7, CD107 a (clone ID4B) FITC, CD107 b (clone M3/84) FITC (BD Pharmingen), anti-mouse IFN-γ (clone XMG1.2) PE-Cy7, (Biolegend).

Techniques: Derivative Assay, In Vitro, Staining, Expressing

PBMCs (~10 × 106) derived from 10 HLA-A*0201(+) individuals vs. 8 HLA-A*0201(−) HSV-seropositive individuals were stimulated in vitro for 72-hours with individual CD8+ T cell epitope peptides (a total of 68 peptides, at 10µg/ml each) derived from Grp. 6, Grp. 11 and Grp. 16 of HSV-1 peptides (see Fig. 1). Representative FACS contour plots showing percentage of IFN-γ+CD8+ T cells detected from one HLA-A*0201(+) individual (top plot) and HLA-A*0201(−) individual (bottom plot) following stimulation with UL9196–204 (A), UL25572–580 (C) and UL43272–280 (E) immunodominant peptides respectively from groups of HSV-1 peptide Grp. 6, Grp. 11 and Grp. 16. Average frequencies of IFN-γ+CD8+ T cells detected in 10 HLA-A*0201(+) individuals (closed circle) vs. 8 HLA-A*0201(−) individuals (open circles) in response to in vitro stimulation with individual peptides from Grp. 6 (B), Grp. 11 (D) and Grp. 16 (F) of HSV-1 peptides. Representative FACS contour plots showing percentage of CD107a/b(+)CD8+ T cells detected from one HLA-A*0201(+) individual (top plot) and HLA-A*0201(−) individual (bottom plot) following stimulation with UL9196–204 (G), UL25572–580 (I) and UL43272–280 (K) immunodominant peptides from Grp. 6, Grp. 11 and Grp. 16 of HSV-1 peptides, respectively. Average frequencies of CD107a/b(+)CD8+ T cells detected in 10 HLA-A*0201(+) individuals (closed circle) vs. 8 HLA-A*0201(−) individuals (open circles) in response to in vitro stimulation with individual peptides from Grp. 6 (H), Grp. 11 (J) and Grp. 16 (L) of HSV-1 peptides. The results are representative of 2 independent experiments. The nominal P values indicated on panels (B), (D), (F), (H), (J) and (L) show statistical significance between HLA-A*0201(+) and HLA-A*0201(−) individuals. P values are calculated using either the parametric two-sample Student’s t-test or non-parametric Wilcoxon rank sum test, as appropriate. For paired comparisons involving individual peptides we have adjusted for multiple comparisons using the Bonferroni procedure.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Bolstering the Number and Function of HSV-1-Specific CD8 + T EM and T RM cells in Latently Infected Trigeminal Ganglia Reduces Recurrent Ocular Herpes Infection and Disease

doi: 10.4049/jimmunol.1700145

Figure Lengend Snippet: PBMCs (~10 × 106) derived from 10 HLA-A*0201(+) individuals vs. 8 HLA-A*0201(−) HSV-seropositive individuals were stimulated in vitro for 72-hours with individual CD8+ T cell epitope peptides (a total of 68 peptides, at 10µg/ml each) derived from Grp. 6, Grp. 11 and Grp. 16 of HSV-1 peptides (see Fig. 1). Representative FACS contour plots showing percentage of IFN-γ+CD8+ T cells detected from one HLA-A*0201(+) individual (top plot) and HLA-A*0201(−) individual (bottom plot) following stimulation with UL9196–204 (A), UL25572–580 (C) and UL43272–280 (E) immunodominant peptides respectively from groups of HSV-1 peptide Grp. 6, Grp. 11 and Grp. 16. Average frequencies of IFN-γ+CD8+ T cells detected in 10 HLA-A*0201(+) individuals (closed circle) vs. 8 HLA-A*0201(−) individuals (open circles) in response to in vitro stimulation with individual peptides from Grp. 6 (B), Grp. 11 (D) and Grp. 16 (F) of HSV-1 peptides. Representative FACS contour plots showing percentage of CD107a/b(+)CD8+ T cells detected from one HLA-A*0201(+) individual (top plot) and HLA-A*0201(−) individual (bottom plot) following stimulation with UL9196–204 (G), UL25572–580 (I) and UL43272–280 (K) immunodominant peptides from Grp. 6, Grp. 11 and Grp. 16 of HSV-1 peptides, respectively. Average frequencies of CD107a/b(+)CD8+ T cells detected in 10 HLA-A*0201(+) individuals (closed circle) vs. 8 HLA-A*0201(−) individuals (open circles) in response to in vitro stimulation with individual peptides from Grp. 6 (H), Grp. 11 (J) and Grp. 16 (L) of HSV-1 peptides. The results are representative of 2 independent experiments. The nominal P values indicated on panels (B), (D), (F), (H), (J) and (L) show statistical significance between HLA-A*0201(+) and HLA-A*0201(−) individuals. P values are calculated using either the parametric two-sample Student’s t-test or non-parametric Wilcoxon rank sum test, as appropriate. For paired comparisons involving individual peptides we have adjusted for multiple comparisons using the Bonferroni procedure.

Techniques: Derivative Assay, In Vitro

(A) The representative FACS plots of tetramer specific CD8+ T cells. Top row shows contour plots of individual tetramer specific CD8+ T cells detected in one HLA-A*0201(+) HSV-1-seropositive ASYMP individual. Middle row shows contour plots of individual tetramer specific CD8+ T cells detected in one HLA-A*0201(+) HSV-1-seropositive SYMP individual. Bottom row shows contour plots of individual tetramer specific CD8+ T cells detected in one HLA-A*0201(−) control individual. (B) Average frequencies of CD8+ T cells specific to ten immunodominant HSV-1 genome-derived epitopes detected in 10 HLA-A*0201(+) ASYMP (closed circle), 8 HLA-A*0201(+) SYMP (open circle) and HLA-A*0201(−) controls (grey filled circles). The nominal P values indicated on panel (B) show statistical significance between SYMP/ASYMP vs. HLA-A*0201(−) individuals. We have used the General Linear Model procedure and compared the least squares means using the Dunnett procedure for multiple comparisons. (C) ImageStram of individual CD8+ T cells derived from one ASYMP vs. SYMP individual, stained with a tetramer specific to the “ASYMP” UL9196–204 epitope (left two panels) or with a tetramer specific to the “SYMP” UL43302–310 epitope (right two panels). (D) Representative FACS plot of CD44highCD62highCD8+ TCM cells and CD44highCD62lowCD8+ TEM cells specific to UL9196–204 epitope detected from one ASYMP individual (left contour plots) vs. one SYMP individual (right contour plots). (E) Average frequency of TCM and TEM cell specific to UL9196–204, UL25572–580, UL43302–310 and UL44400–408 epitopes detected from 10 ASYMP (closed circles) and 8 SYMP individuals (open circles). The nominal P values indicated on panel (E) show statistical significance between SYMP and ASYMP individuals. P values are calculated using either the parametric two-sample Student’s t-test or non-parametric Wilcoxon rank sum test. For paired comparisons involving individual peptides we have adjusted for multiple comparisons using the Bonferroni procedure. (F) Pie charts representing the overall mean number of cumulative CD8+ T cell functions in response to stimulation with individual peptides detected in HLA-A*0201(+) ASYMP individuals (left) vs. HLA-A*0201(+) SYMP individuals (right). The results are representative of 2 independent experiments.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Bolstering the Number and Function of HSV-1-Specific CD8 + T EM and T RM cells in Latently Infected Trigeminal Ganglia Reduces Recurrent Ocular Herpes Infection and Disease

doi: 10.4049/jimmunol.1700145

Figure Lengend Snippet: (A) The representative FACS plots of tetramer specific CD8+ T cells. Top row shows contour plots of individual tetramer specific CD8+ T cells detected in one HLA-A*0201(+) HSV-1-seropositive ASYMP individual. Middle row shows contour plots of individual tetramer specific CD8+ T cells detected in one HLA-A*0201(+) HSV-1-seropositive SYMP individual. Bottom row shows contour plots of individual tetramer specific CD8+ T cells detected in one HLA-A*0201(−) control individual. (B) Average frequencies of CD8+ T cells specific to ten immunodominant HSV-1 genome-derived epitopes detected in 10 HLA-A*0201(+) ASYMP (closed circle), 8 HLA-A*0201(+) SYMP (open circle) and HLA-A*0201(−) controls (grey filled circles). The nominal P values indicated on panel (B) show statistical significance between SYMP/ASYMP vs. HLA-A*0201(−) individuals. We have used the General Linear Model procedure and compared the least squares means using the Dunnett procedure for multiple comparisons. (C) ImageStram of individual CD8+ T cells derived from one ASYMP vs. SYMP individual, stained with a tetramer specific to the “ASYMP” UL9196–204 epitope (left two panels) or with a tetramer specific to the “SYMP” UL43302–310 epitope (right two panels). (D) Representative FACS plot of CD44highCD62highCD8+ TCM cells and CD44highCD62lowCD8+ TEM cells specific to UL9196–204 epitope detected from one ASYMP individual (left contour plots) vs. one SYMP individual (right contour plots). (E) Average frequency of TCM and TEM cell specific to UL9196–204, UL25572–580, UL43302–310 and UL44400–408 epitopes detected from 10 ASYMP (closed circles) and 8 SYMP individuals (open circles). The nominal P values indicated on panel (E) show statistical significance between SYMP and ASYMP individuals. P values are calculated using either the parametric two-sample Student’s t-test or non-parametric Wilcoxon rank sum test. For paired comparisons involving individual peptides we have adjusted for multiple comparisons using the Bonferroni procedure. (F) Pie charts representing the overall mean number of cumulative CD8+ T cell functions in response to stimulation with individual peptides detected in HLA-A*0201(+) ASYMP individuals (left) vs. HLA-A*0201(+) SYMP individuals (right). The results are representative of 2 independent experiments.

Techniques: Derivative Assay, Staining

(A) Schematic diagram of three AAV8 vector constructs expressing one of three different chemokines (i.e. CCL5, CXCL9 or CXCL10) and a GFP both under the neurotropic CamKIIα promoter. (B) Representative FACS plot of frequency of GFP positive neuronal cells (GFP(+)NeuN(+) cells) in TG of mice (n = 10/group) that received topical ocular application of: (1) a rAAV-8-CMV-GFP-CMV-CXCL10 vector (CMV); (1) a rAAV-8-CamKIIα-GFP-CamKIIα-CXCL10 vector (CamKIIα); during HSV-1 latency (i.e. 41 dpi). Bar diagram show average frequency of GFP(+)NeuN(+) cells in TG. (C) HLA-A*0201 Tg mice were ocularly infected with 2 × 105 pfu HSV-1 (strain McKrae). During latency (day 35 post infection), eyes of all mice were exposed to UV-B light. Forty-one dpi, mice (n = 10/group) received topical ocular application of: (1) AAV8-CamKIIα-mCCL5-CamKIIα-eGFP vector (CCL5); (2) AAV8-CamKIIα-mCXCL9-CamKIIα-eGFP (CXCL9); or of (3) CXCL10 (AAV8-CamKIIα-mCXCL10-CamKIIα-eGFP (CXCL10). Eye swabs were collected for 5 days following chemokine administration. On day 46-post infection, mice from all 4 groups were sacrificed and the frequencies of HSV-specific tissue resident memory CD103+CD8+ TRM cells were evaluated in trigeminal ganglia. (D) Representative FACS plot of TG-resident CD8+ T cells (left). Bar diagram of average frequency of TG-resident CD8+ T cells in TG of mice following delivery of three different chemokines (right). (E) Localization of GFP expression in two different types of sensory neurons (A5 and KH10) in TG of mice that received rAAV-8-CamKIIα-GFP-CamKIIα-CXCL10 vector. Mouse TG sections were co-stained using a mAb specific to KH10 (left two panels) or A5 (right two panels) sensory neurons, together with GFP (upper two panels) or with a mAb specific to mouse CD8 (lower two panels). Top two panels show immunofluorescence co-localization of KH10+ neurons with AAV8-GFP detected using a Keyence BZ-X700 fluorescent microscope at 40× magnification and imaged using z-stack. The bottom two panels show immunofluorescence co-localization of KH10 and A5 with CD8+ T cells. (Blue: DAPI, Red: neuron (A5+ or KH10+), Green: GFP-AAV8, Yellow: CD8). Bars in large box = 50 um. Bars in large box = 100 um. The results are representative of 3 independent experiments.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Bolstering the Number and Function of HSV-1-Specific CD8 + T EM and T RM cells in Latently Infected Trigeminal Ganglia Reduces Recurrent Ocular Herpes Infection and Disease

doi: 10.4049/jimmunol.1700145

Figure Lengend Snippet: (A) Schematic diagram of three AAV8 vector constructs expressing one of three different chemokines (i.e. CCL5, CXCL9 or CXCL10) and a GFP both under the neurotropic CamKIIα promoter. (B) Representative FACS plot of frequency of GFP positive neuronal cells (GFP(+)NeuN(+) cells) in TG of mice (n = 10/group) that received topical ocular application of: (1) a rAAV-8-CMV-GFP-CMV-CXCL10 vector (CMV); (1) a rAAV-8-CamKIIα-GFP-CamKIIα-CXCL10 vector (CamKIIα); during HSV-1 latency (i.e. 41 dpi). Bar diagram show average frequency of GFP(+)NeuN(+) cells in TG. (C) HLA-A*0201 Tg mice were ocularly infected with 2 × 105 pfu HSV-1 (strain McKrae). During latency (day 35 post infection), eyes of all mice were exposed to UV-B light. Forty-one dpi, mice (n = 10/group) received topical ocular application of: (1) AAV8-CamKIIα-mCCL5-CamKIIα-eGFP vector (CCL5); (2) AAV8-CamKIIα-mCXCL9-CamKIIα-eGFP (CXCL9); or of (3) CXCL10 (AAV8-CamKIIα-mCXCL10-CamKIIα-eGFP (CXCL10). Eye swabs were collected for 5 days following chemokine administration. On day 46-post infection, mice from all 4 groups were sacrificed and the frequencies of HSV-specific tissue resident memory CD103+CD8+ TRM cells were evaluated in trigeminal ganglia. (D) Representative FACS plot of TG-resident CD8+ T cells (left). Bar diagram of average frequency of TG-resident CD8+ T cells in TG of mice following delivery of three different chemokines (right). (E) Localization of GFP expression in two different types of sensory neurons (A5 and KH10) in TG of mice that received rAAV-8-CamKIIα-GFP-CamKIIα-CXCL10 vector. Mouse TG sections were co-stained using a mAb specific to KH10 (left two panels) or A5 (right two panels) sensory neurons, together with GFP (upper two panels) or with a mAb specific to mouse CD8 (lower two panels). Top two panels show immunofluorescence co-localization of KH10+ neurons with AAV8-GFP detected using a Keyence BZ-X700 fluorescent microscope at 40× magnification and imaged using z-stack. The bottom two panels show immunofluorescence co-localization of KH10 and A5 with CD8+ T cells. (Blue: DAPI, Red: neuron (A5+ or KH10+), Green: GFP-AAV8, Yellow: CD8). Bars in large box = 50 um. Bars in large box = 100 um. The results are representative of 3 independent experiments.

Techniques: Plasmid Preparation, Construct, Expressing, Infection, Staining, Immunofluorescence, Microscopy

(A) Schedule of prime/pull therapeutic vaccination in HSV-1 infected HLA-A*0201 Tg mice. HLA-A*0201 Tg mice (6 – 8 weeks old, n = 30) were ocularly infected using 2 × 105 pfu HSV-1 (strain McKrae). 21-days post-infection, once the latency is well established, mice were divided in three groups (n = 10 each). Group 1 and group 2 were vaccinated with genome-wide derived ASYMP CD8+ T cell epitopes (UL9196–204, UL25572–580 and UL44400–408) along with PADRE CD4+ T helper epitope both mixed with CpG1826 adjuvant. Group 3 mice received adjuvant alone (Mock). Two weeks after the first peptide vaccination (i.e. day 35 post infection), eyes of all three groups of mice were exposed UV-B light to induce reactivation. The eyes of all groups were swabbed daily up to 5-days post UV-B exposure with moist, type 1 calcium alginate swabs. Animals were examined for signs of ocular disease by slit lamp for 30 days. The recurrent herpetic disease was scored according to a standard 0 to 4 scale: 0, no disease; 1, 25%; 2, 50%; 3, 75%; and 4, 100% disease. On day 41 post infection, Group 1 of vaccinated mice (n = 10) was left untreated (Vaccinated) while Group 2 of vaccinated mice (n = 10) received topical ocular application of AAV8-CamKII-mCXCL10-CamKIIa-eGFP neurotropic vector (vaccinated + CXCL10). Eye swabs were collected for five additional days. (B) Top panel shows an eye picture of a mouse vaccinated with ASYMP epitopes + PADRE, without CXCL10 treatment (Vaccinated). Middle panel shows an eye picture of a mouse vaccinated with ASYMP epitopes + PADRE + treatment with CXCL10 chemokine (Vaccinated + CXCL10). Bottom panel shows the eye picture of a mock-vaccinated mouse control (Mock). (C) Average disease scores, on the scale of 0 to 4, detected in all three groups of mice. (D) Virus titer detected in tears of all three groups of mice following UV-B induced reactivation. Five days after CXCL10 treatment (i.e. on day 46 post infection), mice from all groups were scarified and their TG extracted. The frequency, function and phenotype of HSV-specific CD8+ T cells were evaluated in trigeminal ganglia of mice from all 3 groups. (E) Representative FACS plot of the frequency of “ASYMP” UL9196–204 epitope-specific IFN-γ(+)CD8+ T cells; CD107a/b(+)CD8+ T cells; PD-1(+)CD8+ T cells and VISTA(+)CD8+ T cells detected in the TG of Vaccinated; Vaccinated+CXCL10; and Mock-Vaccinated mice. Average percentages (F) and average numbers (G) of “ASYMP” epitopes-specific IFN-γ(+)CD8+ T cells; CD107a/b(+)CD8+ T cells; PD-1(+)CD8+ T cells and VISTA(+)CD8+ T cells detected in the TG of Vaccinated; Vaccinated+CXCL10; and Mock-Vaccinated mice. The nominal P values indicated on panels (C), (D), (F) and (G) show statistical significance between vaccinated and mock-immunized mice. We have used the General Linear Model procedure and compared the least squares means using the Dunnett procedure for multiple comparisons.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Bolstering the Number and Function of HSV-1-Specific CD8 + T EM and T RM cells in Latently Infected Trigeminal Ganglia Reduces Recurrent Ocular Herpes Infection and Disease

doi: 10.4049/jimmunol.1700145

Figure Lengend Snippet: (A) Schedule of prime/pull therapeutic vaccination in HSV-1 infected HLA-A*0201 Tg mice. HLA-A*0201 Tg mice (6 – 8 weeks old, n = 30) were ocularly infected using 2 × 105 pfu HSV-1 (strain McKrae). 21-days post-infection, once the latency is well established, mice were divided in three groups (n = 10 each). Group 1 and group 2 were vaccinated with genome-wide derived ASYMP CD8+ T cell epitopes (UL9196–204, UL25572–580 and UL44400–408) along with PADRE CD4+ T helper epitope both mixed with CpG1826 adjuvant. Group 3 mice received adjuvant alone (Mock). Two weeks after the first peptide vaccination (i.e. day 35 post infection), eyes of all three groups of mice were exposed UV-B light to induce reactivation. The eyes of all groups were swabbed daily up to 5-days post UV-B exposure with moist, type 1 calcium alginate swabs. Animals were examined for signs of ocular disease by slit lamp for 30 days. The recurrent herpetic disease was scored according to a standard 0 to 4 scale: 0, no disease; 1, 25%; 2, 50%; 3, 75%; and 4, 100% disease. On day 41 post infection, Group 1 of vaccinated mice (n = 10) was left untreated (Vaccinated) while Group 2 of vaccinated mice (n = 10) received topical ocular application of AAV8-CamKII-mCXCL10-CamKIIa-eGFP neurotropic vector (vaccinated + CXCL10). Eye swabs were collected for five additional days. (B) Top panel shows an eye picture of a mouse vaccinated with ASYMP epitopes + PADRE, without CXCL10 treatment (Vaccinated). Middle panel shows an eye picture of a mouse vaccinated with ASYMP epitopes + PADRE + treatment with CXCL10 chemokine (Vaccinated + CXCL10). Bottom panel shows the eye picture of a mock-vaccinated mouse control (Mock). (C) Average disease scores, on the scale of 0 to 4, detected in all three groups of mice. (D) Virus titer detected in tears of all three groups of mice following UV-B induced reactivation. Five days after CXCL10 treatment (i.e. on day 46 post infection), mice from all groups were scarified and their TG extracted. The frequency, function and phenotype of HSV-specific CD8+ T cells were evaluated in trigeminal ganglia of mice from all 3 groups. (E) Representative FACS plot of the frequency of “ASYMP” UL9196–204 epitope-specific IFN-γ(+)CD8+ T cells; CD107a/b(+)CD8+ T cells; PD-1(+)CD8+ T cells and VISTA(+)CD8+ T cells detected in the TG of Vaccinated; Vaccinated+CXCL10; and Mock-Vaccinated mice. Average percentages (F) and average numbers (G) of “ASYMP” epitopes-specific IFN-γ(+)CD8+ T cells; CD107a/b(+)CD8+ T cells; PD-1(+)CD8+ T cells and VISTA(+)CD8+ T cells detected in the TG of Vaccinated; Vaccinated+CXCL10; and Mock-Vaccinated mice. The nominal P values indicated on panels (C), (D), (F) and (G) show statistical significance between vaccinated and mock-immunized mice. We have used the General Linear Model procedure and compared the least squares means using the Dunnett procedure for multiple comparisons.

Techniques: Infection, Genome Wide, Derivative Assay, Plasmid Preparation

The trigeminal ganglia were harvested from all groups of mice (vaccinated and non-vaccinated) and single cell suspension from TG were obtained after collagenase treatment for an hour at 37°C and stained for markers of total CD8+ T cells, CD8+ TRM, TCM and TEM cell sub-populations. Positive correlation of the percentage (A) and number (B) of HSV-specific CD8+ T cells in TG with protection against ocular herpes. (C) Representative FACS plot of the frequency of “ASYMP” UL9196–204 epitope-specific CD103+CD8+ TRM cells (top panels); CD44highCD62LhighCD8+ TCM cells (middle panels); and CD44highCD62LlowCD8+ TEM cells (bottom panels) detected in the TG of Vaccinated; Vaccinated+CXCL10; and Mock-Vaccinated mice. Average percentages (D) and average numbers (E) of “ASYMP” epitopes-specific CD103+CD8+ TRM cells (top panels); CD44highCD62LhighCD8+ TCM cells (middle panels); and CD44highCD62LlowCD8+ TEM cells (bottom panels) detected in the TG of Vaccinated; Vaccinated+CXCL10; and Mock-Vaccinated mice. The results are representative of 2 independent experiments. The nominal P values indicated on panels (D) and (E) show statistical significance between vaccinated and mock-immunized mice. We have used the General Linear Model procedure and compared the least squares means using the Dunnett procedure for multiple comparisons.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Bolstering the Number and Function of HSV-1-Specific CD8 + T EM and T RM cells in Latently Infected Trigeminal Ganglia Reduces Recurrent Ocular Herpes Infection and Disease

doi: 10.4049/jimmunol.1700145

Figure Lengend Snippet: The trigeminal ganglia were harvested from all groups of mice (vaccinated and non-vaccinated) and single cell suspension from TG were obtained after collagenase treatment for an hour at 37°C and stained for markers of total CD8+ T cells, CD8+ TRM, TCM and TEM cell sub-populations. Positive correlation of the percentage (A) and number (B) of HSV-specific CD8+ T cells in TG with protection against ocular herpes. (C) Representative FACS plot of the frequency of “ASYMP” UL9196–204 epitope-specific CD103+CD8+ TRM cells (top panels); CD44highCD62LhighCD8+ TCM cells (middle panels); and CD44highCD62LlowCD8+ TEM cells (bottom panels) detected in the TG of Vaccinated; Vaccinated+CXCL10; and Mock-Vaccinated mice. Average percentages (D) and average numbers (E) of “ASYMP” epitopes-specific CD103+CD8+ TRM cells (top panels); CD44highCD62LhighCD8+ TCM cells (middle panels); and CD44highCD62LlowCD8+ TEM cells (bottom panels) detected in the TG of Vaccinated; Vaccinated+CXCL10; and Mock-Vaccinated mice. The results are representative of 2 independent experiments. The nominal P values indicated on panels (D) and (E) show statistical significance between vaccinated and mock-immunized mice. We have used the General Linear Model procedure and compared the least squares means using the Dunnett procedure for multiple comparisons.

Techniques: Staining

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