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BioLegend pe labeled anti murine cd8
Transfecting bystander cells leads to activation of antigen-specific naïve <t>CD8</t> + T cells. SIINFEKL-specific OT-1 T cells were co-cultured with DCs and transfected fibroblasts and the amount of IFN-γ secretion was determined by ELISA. Experiments
Pe Labeled Anti Murine Cd8, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pe labeled anti murine cd8/product/BioLegend
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pe labeled anti murine cd8 - by Bioz Stars, 2021-09
99/100 stars

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1) Product Images from "Polymer-Mediated DNA Vaccine Delivery via Bystander Cells Requires a Proper Balance between Transfection Efficiency and Cytotoxicity"

Article Title: Polymer-Mediated DNA Vaccine Delivery via Bystander Cells Requires a Proper Balance between Transfection Efficiency and Cytotoxicity

Journal: Journal of Controlled Release

doi: 10.1016/j.jconrel.2011.08.037

Transfecting bystander cells leads to activation of antigen-specific naïve CD8 + T cells. SIINFEKL-specific OT-1 T cells were co-cultured with DCs and transfected fibroblasts and the amount of IFN-γ secretion was determined by ELISA. Experiments
Figure Legend Snippet: Transfecting bystander cells leads to activation of antigen-specific naïve CD8 + T cells. SIINFEKL-specific OT-1 T cells were co-cultured with DCs and transfected fibroblasts and the amount of IFN-γ secretion was determined by ELISA. Experiments

Techniques Used: Activation Assay, Cell Culture, Transfection, Enzyme-linked Immunosorbent Assay

Schematic illustration of a co-culture system for the study of polymer-mediated DNA vaccine delivery to bystander cells. The process involves bystander cells (fibroblasts), dendritic cells (DCs), and antigen-specific CD8 + T cells, mixed sequentially and
Figure Legend Snippet: Schematic illustration of a co-culture system for the study of polymer-mediated DNA vaccine delivery to bystander cells. The process involves bystander cells (fibroblasts), dendritic cells (DCs), and antigen-specific CD8 + T cells, mixed sequentially and

Techniques Used: Co-Culture Assay

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other:

Article Title: Circulating myeloid cells invade the central nervous system to mediate cachexia during pancreatic cancer
Article Snippet: The following anti-mouse antibodies were used, with clone, fluorophore, and dilution indicated in parenthesis: CD3 (17A2, PE, 1:100), CD3 (17A2, APC/Cy7, 1:400), CD4 (RM4-5, APC, 1:100), CD8 (53–6.7, APC/Cy7, 1:800), CD11b (M1/70, APC, 1:800), CD11b (M1/70, FITC, 1:200), CD19 (6D5,BV650, 1:33), CD45 (30-F11, PerCP/Cy5.5, 1:400), CD45 (30-F11, APC/Cy7, 1:400), CX3CR1 (SAO11F11, PE/Cy7, 1:100), Dec205 (NLDC-145, APC,1:100), CD115 (AFS98, APC/CY7, 1:400), Ly6C (HK1.4, PerCP, 1:100), Ly6G (1A8, PE/Cy7, 1:800), NK1.1 (PK136, BV785, 1:800), CCR2 (SA203G11, AF647, 1:100).

Flow Cytometry:

Article Title: Therapeutic Mucosal Vaccination of Herpes Simplex Virus 2-Infected Guinea Pigs with Ribonucleotide Reductase 2 (RR2) Protein Boosts Antiviral Neutralizing Antibodies and Local Tissue-Resident CD4+ and CD8+ TRM Cells Associated with Protection against Recurrent Genital Herpes
Article Snippet: .. The following anti-human antibodies were used for flow cytometry assays: CD3 (clone SK7) phycoerythrin (PE)-Cy7, CD4 (clone DE54), CD8 (clone SK1), CD45RA FITC, CD62L allophycocyanin, IFN-γ Alexa Fluor 647, granzyme B (clone GB11) A647, CD107a (clone H4A3) fluorescein isothiocyanate (FITC), CD107b (clone H4B4) FITC (BioLegend, San Diego, CA), and granzyme K (clone G3H69). ..

Article Title: Arthritis Flares Mediated by Tissue Resident Memory T Cells in the Joint
Article Snippet: .. The following antibodies were used for the analysis of murine synovium with flow cytometry or cell sorting: Alexa Fluor 700® anti-CD3 (Clone 17A2, BioLegend Cat# 100216, RRID:AB_493697), APC anti-CD3 (Clone 17A2, BioLegend Cat# 100236, RRID:AB_2561456), FITC anti-CD45 (Clone 30-F11, BD Pharmingen Cat# 561088), FITC anti-CD45.1 (Clone A20, BioLegend Cat# 110706, RRID:AB_313495), PE anti-CD45.2 (Clone 104, BioLegend Cat# 109808, RRID:AB_313445), FITC anti-CD4 (Clone GK15, BioLegend Cat# 100406, RRID:AB_312691), APC/Cyanine7 anti-CD4 (Clone GK1.5, BioLegend Cat# 100414, RRID:AB_312699), Brilliant Violet™ 650 anti-CD8a (Clone 53-6.7, (BioLegend Cat# 100742, RRID:AB_2563056), PerCP/Cyanine5.5 anti-CD8a (Clone 53-6.7, BioLegend Cat# 100734, RRID:AB_2075238), Brilliant Violet™ 605 anti-CD44 (Clone IM7, BioLegend Cat# 103047, RRID:AB_2562451), APC anti-CD62L (Clone MEL-14, BioLegend Cat# 104412, RRID:AB_313099), Pacific Blue™ anti-CD69 (Clone H1.2F3, BioLegend Cat# 104524, RRID:AB_2074979), PE/Cyanine7 anti-CD103 (Clone 2E7, BioLegend Cat# 121426, RRID:AB_2563691), PE anti-CD103 (Clone 2E7, BioLegend Cat# 121406, RRID:AB_1133989), PE anti-ST2 (Clone DIH4, BioLegend Cat# 146608, RRID:AB_2728177), PE anti-CD19 (Clone eBio1D3, Thermo Fisher Scientific Cat# 12-0193-82, RRID:AB_657659), APC anti-CD11b (Clone M1/70, BioLegend Cat# 101212, RRID:AB_312795), Brilliant Violet™ 650 anti-Ly6G (Clone 1A8, BioLegend Cat# 127641, RRID:AB_2565881), Brilliant Violet™ 421 anti-NK1.1 (Clone PK136, BioLegend Cat# 108731, RRID:AB_10895916). ..

Cytometry:

Article Title: Therapeutic Mucosal Vaccination of Herpes Simplex Virus 2-Infected Guinea Pigs with Ribonucleotide Reductase 2 (RR2) Protein Boosts Antiviral Neutralizing Antibodies and Local Tissue-Resident CD4+ and CD8+ TRM Cells Associated with Protection against Recurrent Genital Herpes
Article Snippet: .. The following anti-human antibodies were used for flow cytometry assays: CD3 (clone SK7) phycoerythrin (PE)-Cy7, CD4 (clone DE54), CD8 (clone SK1), CD45RA FITC, CD62L allophycocyanin, IFN-γ Alexa Fluor 647, granzyme B (clone GB11) A647, CD107a (clone H4A3) fluorescein isothiocyanate (FITC), CD107b (clone H4B4) FITC (BioLegend, San Diego, CA), and granzyme K (clone G3H69). ..

Staining:

Article Title: Polymer-Mediated DNA Vaccine Delivery via Bystander Cells Requires a Proper Balance between Transfection Efficiency and Cytotoxicity
Article Snippet: .. An aliquot of cells was stained with PE-labeled anti-murine CD8 (BioLegend) and PerCP Cy 5.5-labeled anti-murine CD90 (CD90.1) (BioLegend). ..

FACS:

Article Title: Arthritis Flares Mediated by Tissue Resident Memory T Cells in the Joint
Article Snippet: .. The following antibodies were used for the analysis of murine synovium with flow cytometry or cell sorting: Alexa Fluor 700® anti-CD3 (Clone 17A2, BioLegend Cat# 100216, RRID:AB_493697), APC anti-CD3 (Clone 17A2, BioLegend Cat# 100236, RRID:AB_2561456), FITC anti-CD45 (Clone 30-F11, BD Pharmingen Cat# 561088), FITC anti-CD45.1 (Clone A20, BioLegend Cat# 110706, RRID:AB_313495), PE anti-CD45.2 (Clone 104, BioLegend Cat# 109808, RRID:AB_313445), FITC anti-CD4 (Clone GK15, BioLegend Cat# 100406, RRID:AB_312691), APC/Cyanine7 anti-CD4 (Clone GK1.5, BioLegend Cat# 100414, RRID:AB_312699), Brilliant Violet™ 650 anti-CD8a (Clone 53-6.7, (BioLegend Cat# 100742, RRID:AB_2563056), PerCP/Cyanine5.5 anti-CD8a (Clone 53-6.7, BioLegend Cat# 100734, RRID:AB_2075238), Brilliant Violet™ 605 anti-CD44 (Clone IM7, BioLegend Cat# 103047, RRID:AB_2562451), APC anti-CD62L (Clone MEL-14, BioLegend Cat# 104412, RRID:AB_313099), Pacific Blue™ anti-CD69 (Clone H1.2F3, BioLegend Cat# 104524, RRID:AB_2074979), PE/Cyanine7 anti-CD103 (Clone 2E7, BioLegend Cat# 121426, RRID:AB_2563691), PE anti-CD103 (Clone 2E7, BioLegend Cat# 121406, RRID:AB_1133989), PE anti-ST2 (Clone DIH4, BioLegend Cat# 146608, RRID:AB_2728177), PE anti-CD19 (Clone eBio1D3, Thermo Fisher Scientific Cat# 12-0193-82, RRID:AB_657659), APC anti-CD11b (Clone M1/70, BioLegend Cat# 101212, RRID:AB_312795), Brilliant Violet™ 650 anti-Ly6G (Clone 1A8, BioLegend Cat# 127641, RRID:AB_2565881), Brilliant Violet™ 421 anti-NK1.1 (Clone PK136, BioLegend Cat# 108731, RRID:AB_10895916). ..

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    BioLegend pe labeled anti murine cd8
    Transfecting bystander cells leads to activation of antigen-specific naïve <t>CD8</t> + T cells. SIINFEKL-specific OT-1 T cells were co-cultured with DCs and transfected fibroblasts and the amount of IFN-γ secretion was determined by ELISA. Experiments
    Pe Labeled Anti Murine Cd8, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pe labeled anti murine cd8/product/BioLegend
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pe labeled anti murine cd8 - by Bioz Stars, 2021-09
    99/100 stars
      Buy from Supplier

    99
    BioLegend pe labeled anti cd8a
    In vivo study showing tumor specific <t>CD8</t> + IFN-γ + T cell induction and tumor prevention effects with a DC-based cancer vaccine using the RPS3 protein as adjuvant
    Pe Labeled Anti Cd8a, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pe labeled anti cd8a/product/BioLegend
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pe labeled anti cd8a - by Bioz Stars, 2021-09
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    99
    BioLegend cd8
    CD3xPDL1 BiTE significantly extends the survival time of humanized NSG mice reconstituted with human PBMC and carrying established metastatic human melanoma C8161 NSG mice were inoculated subcutaneously in the right flank with 1×10 6 human C8161 melanoma cells on day 0. On day 7 when tumors were palpable, mice were either untreated or administered 1×10 7 human PBMC iv in the tail vein and 0.2ng (8ng/kg) CD3xPDL1 BiTE iv in the retro-orbital sinus. On days 8, 9, 10, 11, and 32 mice were given additional iv injections of 0.2μg BiTE. A . Moribund, euthanized control mouse (tumor + PBMC, no BiTE) showing metastases in the lymph nodes and lungs on day 57. B. Kaplan-Meier plot showing survival. Data are pooled from three independent experiments. C . Representative flow cytometry profiles of splenocytes of moribund/dead BiTE-treated and untreated mice stained for human T cells (CD3, CD4, and <t>CD8</t> mAbs), or mouse MDSC (Gr1 and CD11b mAbs). D . Total numbers and ratio of human T cells and mouse MDSC in the spleens of moribund/dead BiTE-treated and control mice of panel B. Data are pooled from 2-3 mice per group.
    Cd8, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cd8/product/BioLegend
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    Price from $9.99 to $1999.99
    cd8 - by Bioz Stars, 2021-09
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    Transfecting bystander cells leads to activation of antigen-specific naïve CD8 + T cells. SIINFEKL-specific OT-1 T cells were co-cultured with DCs and transfected fibroblasts and the amount of IFN-γ secretion was determined by ELISA. Experiments

    Journal: Journal of Controlled Release

    Article Title: Polymer-Mediated DNA Vaccine Delivery via Bystander Cells Requires a Proper Balance between Transfection Efficiency and Cytotoxicity

    doi: 10.1016/j.jconrel.2011.08.037

    Figure Lengend Snippet: Transfecting bystander cells leads to activation of antigen-specific naïve CD8 + T cells. SIINFEKL-specific OT-1 T cells were co-cultured with DCs and transfected fibroblasts and the amount of IFN-γ secretion was determined by ELISA. Experiments

    Article Snippet: An aliquot of cells was stained with PE-labeled anti-murine CD8 (BioLegend) and PerCP Cy 5.5-labeled anti-murine CD90 (CD90.1) (BioLegend).

    Techniques: Activation Assay, Cell Culture, Transfection, Enzyme-linked Immunosorbent Assay

    Schematic illustration of a co-culture system for the study of polymer-mediated DNA vaccine delivery to bystander cells. The process involves bystander cells (fibroblasts), dendritic cells (DCs), and antigen-specific CD8 + T cells, mixed sequentially and

    Journal: Journal of Controlled Release

    Article Title: Polymer-Mediated DNA Vaccine Delivery via Bystander Cells Requires a Proper Balance between Transfection Efficiency and Cytotoxicity

    doi: 10.1016/j.jconrel.2011.08.037

    Figure Lengend Snippet: Schematic illustration of a co-culture system for the study of polymer-mediated DNA vaccine delivery to bystander cells. The process involves bystander cells (fibroblasts), dendritic cells (DCs), and antigen-specific CD8 + T cells, mixed sequentially and

    Article Snippet: An aliquot of cells was stained with PE-labeled anti-murine CD8 (BioLegend) and PerCP Cy 5.5-labeled anti-murine CD90 (CD90.1) (BioLegend).

    Techniques: Co-Culture Assay

    In vivo study showing tumor specific CD8 + IFN-γ + T cell induction and tumor prevention effects with a DC-based cancer vaccine using the RPS3 protein as adjuvant

    Journal: Journal for Immunotherapy of Cancer

    Article Title: A novel TLR4 binding protein, 40S ribosomal protein S3, has potential utility as an adjuvant in a dendritic cell-based vaccine

    doi: 10.1186/s40425-019-0539-7

    Figure Lengend Snippet: In vivo study showing tumor specific CD8 + IFN-γ + T cell induction and tumor prevention effects with a DC-based cancer vaccine using the RPS3 protein as adjuvant

    Article Snippet: To assess CD8+ T cell generation in vivo, splenocytes from treated or vaccinated mice were stained with PE-labeled anti-CD8a (BioLegend) and FITC-labeled anti-IFN-γ (BioLegend).

    Techniques: In Vivo

    CD3xPDL1 BiTE significantly extends the survival time of humanized NSG mice reconstituted with human PBMC and carrying established metastatic human melanoma C8161 NSG mice were inoculated subcutaneously in the right flank with 1×10 6 human C8161 melanoma cells on day 0. On day 7 when tumors were palpable, mice were either untreated or administered 1×10 7 human PBMC iv in the tail vein and 0.2ng (8ng/kg) CD3xPDL1 BiTE iv in the retro-orbital sinus. On days 8, 9, 10, 11, and 32 mice were given additional iv injections of 0.2μg BiTE. A . Moribund, euthanized control mouse (tumor + PBMC, no BiTE) showing metastases in the lymph nodes and lungs on day 57. B. Kaplan-Meier plot showing survival. Data are pooled from three independent experiments. C . Representative flow cytometry profiles of splenocytes of moribund/dead BiTE-treated and untreated mice stained for human T cells (CD3, CD4, and CD8 mAbs), or mouse MDSC (Gr1 and CD11b mAbs). D . Total numbers and ratio of human T cells and mouse MDSC in the spleens of moribund/dead BiTE-treated and control mice of panel B. Data are pooled from 2-3 mice per group.

    Journal: Oncotarget

    Article Title: CD3xPDL1 bi-specific T cell engager (BiTE) simultaneously activates T cells and NKT cells, kills PDL1+ tumor cells, and extends the survival of tumor-bearing humanized mice

    doi: 10.18632/oncotarget.19865

    Figure Lengend Snippet: CD3xPDL1 BiTE significantly extends the survival time of humanized NSG mice reconstituted with human PBMC and carrying established metastatic human melanoma C8161 NSG mice were inoculated subcutaneously in the right flank with 1×10 6 human C8161 melanoma cells on day 0. On day 7 when tumors were palpable, mice were either untreated or administered 1×10 7 human PBMC iv in the tail vein and 0.2ng (8ng/kg) CD3xPDL1 BiTE iv in the retro-orbital sinus. On days 8, 9, 10, 11, and 32 mice were given additional iv injections of 0.2μg BiTE. A . Moribund, euthanized control mouse (tumor + PBMC, no BiTE) showing metastases in the lymph nodes and lungs on day 57. B. Kaplan-Meier plot showing survival. Data are pooled from three independent experiments. C . Representative flow cytometry profiles of splenocytes of moribund/dead BiTE-treated and untreated mice stained for human T cells (CD3, CD4, and CD8 mAbs), or mouse MDSC (Gr1 and CD11b mAbs). D . Total numbers and ratio of human T cells and mouse MDSC in the spleens of moribund/dead BiTE-treated and control mice of panel B. Data are pooled from 2-3 mice per group.

    Article Snippet: Antibodies to human PDL1 (CD274; clone 29E.2A3; BV421 and PE-Cy7), PD1 (clone EH12.2H7, PE-Cy7), CD3 (clone OKT3, FITC, PE, APC), CD3 (clone UCHT1, BV421), CD4 (clone OKT4, PB, BV510, APC, APC-Cy7, PE-Cy7), CD8 (clone SK1, APC), CD8a (clone HIT8a, PE), CD69 (clone FN50, AlexaF488), HLA-DR (clone L243, BV421), CD14 (clone HCD14, FITC), CD15 (clone HI98, PE), CD11b (clone ICRF44, APC), CD56 (clone HCD56, FITC), LEAF purified anti-human CD3 (clone OKT3), LEAF-purified anti-human PDL1 (clone 29E.2A3) were from BioLegend.

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Staining

    CD3xPDL1 BiTE activates cancer patients’ PBMC that are cytotoxic for PDL1 + lung cancer cells A . PBMC from a SCLC patient were stained for MDSC (CD11b, CD14, CD15, HLA-DR). CD11b + HLA-DR - cells were gated and analyzed by flow cytometry for CD14 + CD15 - M-MDSC and CD15 + CD14 - PMN-MDSC. Data are representative of one of three SCLC patients. B . SCLC patient's PBMC were labeled with fluorescently-tagged antibodies to CD3, CD4 and CD8, and the gated CD3 + cells were analyzed for percent CD3 + CD4 + and CD3 + CD8 + cells. Data are for one of three SCLC patients. C . PBMC from the SCLC patients were incubated ± CellTrace violet-labeled PDL1 + H358 or PDL1 - MEL1011 tumor cells ± BiTE at a 20:1 ratio of PBMC:tumor cells. Cells were stained with 7AAD and the CellTrace violet stained tumor cells were gated and analyzed for % dead cells. The ratio of CD8 + plus CD4 + T cells to tumor cells is 2.3:1. Data are pooled from two independent experiments with PBMC from two individual patients. D . PBMC from a NSCLC patient were stained for MDSC. CD11b + HLA-DR - cells were gated and analyzed for CD14 + CD15 - M-MDSC and CD15 + CD14 - PMN-MDSC. E . NSCLC patient's PBMC were labeled with fluorescently-tagged antibodies to CD3, CD4 and CD8, and the CD3 gated cells were analyzed for percent CD3 + CD4 + and CD3 + CD8 + cells. F . PBMC from the NSCLC patient were incubated ± CellTrace violet-labeled H358 tumor cells ± BiTE at a 20:1 ratio of PBMC:tumor cells. Cells were stained with 7AAD and the CellTrace violet stained tumor cells were gated and analyzed for % dead cells.

    Journal: Oncotarget

    Article Title: CD3xPDL1 bi-specific T cell engager (BiTE) simultaneously activates T cells and NKT cells, kills PDL1+ tumor cells, and extends the survival of tumor-bearing humanized mice

    doi: 10.18632/oncotarget.19865

    Figure Lengend Snippet: CD3xPDL1 BiTE activates cancer patients’ PBMC that are cytotoxic for PDL1 + lung cancer cells A . PBMC from a SCLC patient were stained for MDSC (CD11b, CD14, CD15, HLA-DR). CD11b + HLA-DR - cells were gated and analyzed by flow cytometry for CD14 + CD15 - M-MDSC and CD15 + CD14 - PMN-MDSC. Data are representative of one of three SCLC patients. B . SCLC patient's PBMC were labeled with fluorescently-tagged antibodies to CD3, CD4 and CD8, and the gated CD3 + cells were analyzed for percent CD3 + CD4 + and CD3 + CD8 + cells. Data are for one of three SCLC patients. C . PBMC from the SCLC patients were incubated ± CellTrace violet-labeled PDL1 + H358 or PDL1 - MEL1011 tumor cells ± BiTE at a 20:1 ratio of PBMC:tumor cells. Cells were stained with 7AAD and the CellTrace violet stained tumor cells were gated and analyzed for % dead cells. The ratio of CD8 + plus CD4 + T cells to tumor cells is 2.3:1. Data are pooled from two independent experiments with PBMC from two individual patients. D . PBMC from a NSCLC patient were stained for MDSC. CD11b + HLA-DR - cells were gated and analyzed for CD14 + CD15 - M-MDSC and CD15 + CD14 - PMN-MDSC. E . NSCLC patient's PBMC were labeled with fluorescently-tagged antibodies to CD3, CD4 and CD8, and the CD3 gated cells were analyzed for percent CD3 + CD4 + and CD3 + CD8 + cells. F . PBMC from the NSCLC patient were incubated ± CellTrace violet-labeled H358 tumor cells ± BiTE at a 20:1 ratio of PBMC:tumor cells. Cells were stained with 7AAD and the CellTrace violet stained tumor cells were gated and analyzed for % dead cells.

    Article Snippet: Antibodies to human PDL1 (CD274; clone 29E.2A3; BV421 and PE-Cy7), PD1 (clone EH12.2H7, PE-Cy7), CD3 (clone OKT3, FITC, PE, APC), CD3 (clone UCHT1, BV421), CD4 (clone OKT4, PB, BV510, APC, APC-Cy7, PE-Cy7), CD8 (clone SK1, APC), CD8a (clone HIT8a, PE), CD69 (clone FN50, AlexaF488), HLA-DR (clone L243, BV421), CD14 (clone HCD14, FITC), CD15 (clone HI98, PE), CD11b (clone ICRF44, APC), CD56 (clone HCD56, FITC), LEAF purified anti-human CD3 (clone OKT3), LEAF-purified anti-human PDL1 (clone 29E.2A3) were from BioLegend.

    Techniques: Staining, Flow Cytometry, Cytometry, Labeling, Incubation

    CD3xPDL1 BiTE simultaneously and specifically binds to PDL1 + human tumor cells and CD3+ human T cells A . BiTE binds to tumor cell-expressed PDL1. PDL1 + C8161 melanoma cells and PDL1 - MEL1011 cells were stained with fluorescently-tagged antibodies to PDL1 to determine their level of PDL1 expression (left panels). C8161 and MEL1011 cells were incubated with the CD3xPDL1 BiTE or an irrelevant recombinant protein (TROY-Fc) and then stained with a fluorescently-tagged anti-his mAb to detect BiTE binding (right panels). B . BiTE binds to T cell-expressed CD3. Peripheral blood mononuclear cells (PBMC) from healthy human donors were stained with fluorescently-tagged antibodies to CD4 and CD8 followed by addition of BiTE or irrelevant recombinant protein, and then fluorescently tagged antibody to his to detect BiTE binding. To ascertain that the BiTE specifically binds to CD3, aliquots of PBMC were first blocked with antibodies to CD3 to prevent BiTE binding. C . BiTE simultaneously binds CD3 and PDL1. PBMC were stained for CD4 and CD8 as in panel B, and then incubated with either BiTE or an irrelevant protein followed by soluble PDL1 (sPDL1). PDL1 binding was detected with a fluorescently-tagged antibody to PDL1. Data are representative of more than three independent experiments with at least 3 batches of BiTE.

    Journal: Oncotarget

    Article Title: CD3xPDL1 bi-specific T cell engager (BiTE) simultaneously activates T cells and NKT cells, kills PDL1+ tumor cells, and extends the survival of tumor-bearing humanized mice

    doi: 10.18632/oncotarget.19865

    Figure Lengend Snippet: CD3xPDL1 BiTE simultaneously and specifically binds to PDL1 + human tumor cells and CD3+ human T cells A . BiTE binds to tumor cell-expressed PDL1. PDL1 + C8161 melanoma cells and PDL1 - MEL1011 cells were stained with fluorescently-tagged antibodies to PDL1 to determine their level of PDL1 expression (left panels). C8161 and MEL1011 cells were incubated with the CD3xPDL1 BiTE or an irrelevant recombinant protein (TROY-Fc) and then stained with a fluorescently-tagged anti-his mAb to detect BiTE binding (right panels). B . BiTE binds to T cell-expressed CD3. Peripheral blood mononuclear cells (PBMC) from healthy human donors were stained with fluorescently-tagged antibodies to CD4 and CD8 followed by addition of BiTE or irrelevant recombinant protein, and then fluorescently tagged antibody to his to detect BiTE binding. To ascertain that the BiTE specifically binds to CD3, aliquots of PBMC were first blocked with antibodies to CD3 to prevent BiTE binding. C . BiTE simultaneously binds CD3 and PDL1. PBMC were stained for CD4 and CD8 as in panel B, and then incubated with either BiTE or an irrelevant protein followed by soluble PDL1 (sPDL1). PDL1 binding was detected with a fluorescently-tagged antibody to PDL1. Data are representative of more than three independent experiments with at least 3 batches of BiTE.

    Article Snippet: Antibodies to human PDL1 (CD274; clone 29E.2A3; BV421 and PE-Cy7), PD1 (clone EH12.2H7, PE-Cy7), CD3 (clone OKT3, FITC, PE, APC), CD3 (clone UCHT1, BV421), CD4 (clone OKT4, PB, BV510, APC, APC-Cy7, PE-Cy7), CD8 (clone SK1, APC), CD8a (clone HIT8a, PE), CD69 (clone FN50, AlexaF488), HLA-DR (clone L243, BV421), CD14 (clone HCD14, FITC), CD15 (clone HI98, PE), CD11b (clone ICRF44, APC), CD56 (clone HCD56, FITC), LEAF purified anti-human CD3 (clone OKT3), LEAF-purified anti-human PDL1 (clone 29E.2A3) were from BioLegend.

    Techniques: Staining, Expressing, Incubation, Recombinant, Binding Assay

    CD3xPDL1 BiTE activates T cells that are cytotoxic for PDL1 + tumor cells A . Healthy donor PBMC were co-cultured with C8161 tumor cells (ratio 20:1 PBMC:tumor cells) and with CD3xPDL1 BiTE (200 ng/mL) or an irrelevant protein for 48 hrs and the supernatants were analyzed for IFNγ by ELISA. Data are representative of one of two independent experiments. B . PBMC were incubated for 72 hrs with CellTrace Violet-labelled PDL1 + C8161 melanoma cells ± BiTE, and then labeled with fluorescently-coupled antibodies to CD4, CD8, and CD69 or CD25. Violet + tumor cells were gated out and CD4 + and CD8 + T cells were gated and analyzed for the activation markers CD69 and CD25. Data are representative of 3 independent experiments. C . CellTrace Violet-stained CD3 purified PBMC were co-cultured for four days with or without BiTE or tumor, and the gated violet + cells assessed by flow cytometry for proliferation. Data are representative of one of two independent experiments. D . C8161 cells were stained with CellTrace Violet and incubated at varying ratios with healthy donor PBMC ± BiTE. Following 72 hrs of incubation, cells were stained with the viability dye 7AAD. % dead tumor cells = [dead tumor cells (violet + 7AAD + )/total tumor cells (violet + )] x 100%. Values are the average of triplicates per sample. E . C8161 and MEL1011 melanoma cells were stained with fluorescent antibodies to PDL1 (29E.2A3 mAb) or an isotype control mAb and analyzed by flow cytometry. PBMC from healthy donors were incubated ± CellTrace Violet-labeled C8161 or MEL1011 tumor cells ± BiTE and analyzed for % dead tumor cells. PBMC:tumor cell ratio is 20:1. CD8:tumor cell ratio is ~4:1. Values are the average + SE of 7 and 5 independent experiments for C8161 and MEL1011, respectively. For panels A and E, values with asterisks are significantly different from all other values.

    Journal: Oncotarget

    Article Title: CD3xPDL1 bi-specific T cell engager (BiTE) simultaneously activates T cells and NKT cells, kills PDL1+ tumor cells, and extends the survival of tumor-bearing humanized mice

    doi: 10.18632/oncotarget.19865

    Figure Lengend Snippet: CD3xPDL1 BiTE activates T cells that are cytotoxic for PDL1 + tumor cells A . Healthy donor PBMC were co-cultured with C8161 tumor cells (ratio 20:1 PBMC:tumor cells) and with CD3xPDL1 BiTE (200 ng/mL) or an irrelevant protein for 48 hrs and the supernatants were analyzed for IFNγ by ELISA. Data are representative of one of two independent experiments. B . PBMC were incubated for 72 hrs with CellTrace Violet-labelled PDL1 + C8161 melanoma cells ± BiTE, and then labeled with fluorescently-coupled antibodies to CD4, CD8, and CD69 or CD25. Violet + tumor cells were gated out and CD4 + and CD8 + T cells were gated and analyzed for the activation markers CD69 and CD25. Data are representative of 3 independent experiments. C . CellTrace Violet-stained CD3 purified PBMC were co-cultured for four days with or without BiTE or tumor, and the gated violet + cells assessed by flow cytometry for proliferation. Data are representative of one of two independent experiments. D . C8161 cells were stained with CellTrace Violet and incubated at varying ratios with healthy donor PBMC ± BiTE. Following 72 hrs of incubation, cells were stained with the viability dye 7AAD. % dead tumor cells = [dead tumor cells (violet + 7AAD + )/total tumor cells (violet + )] x 100%. Values are the average of triplicates per sample. E . C8161 and MEL1011 melanoma cells were stained with fluorescent antibodies to PDL1 (29E.2A3 mAb) or an isotype control mAb and analyzed by flow cytometry. PBMC from healthy donors were incubated ± CellTrace Violet-labeled C8161 or MEL1011 tumor cells ± BiTE and analyzed for % dead tumor cells. PBMC:tumor cell ratio is 20:1. CD8:tumor cell ratio is ~4:1. Values are the average + SE of 7 and 5 independent experiments for C8161 and MEL1011, respectively. For panels A and E, values with asterisks are significantly different from all other values.

    Article Snippet: Antibodies to human PDL1 (CD274; clone 29E.2A3; BV421 and PE-Cy7), PD1 (clone EH12.2H7, PE-Cy7), CD3 (clone OKT3, FITC, PE, APC), CD3 (clone UCHT1, BV421), CD4 (clone OKT4, PB, BV510, APC, APC-Cy7, PE-Cy7), CD8 (clone SK1, APC), CD8a (clone HIT8a, PE), CD69 (clone FN50, AlexaF488), HLA-DR (clone L243, BV421), CD14 (clone HCD14, FITC), CD15 (clone HI98, PE), CD11b (clone ICRF44, APC), CD56 (clone HCD56, FITC), LEAF purified anti-human CD3 (clone OKT3), LEAF-purified anti-human PDL1 (clone 29E.2A3) were from BioLegend.

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Labeling, Activation Assay, Staining, Purification, Flow Cytometry, Cytometry

    CD3xPDL1 BiTE activates both CD4 + and CD8 + cytotoxic T cells and NKT cells A . PBMC from healthy human donors were either undepleted, or depleted for CD4 + , CD8 + , or CD4 + plus CD8 + T cells. Depleted populations were stained with fluorescently tagged antibodies to CD3, CD4, and CD8, and the CD3 + cells gated and analyzed for CD4 and CD8 expression. B . Violet-labeled PDL1 + H358 tumor cells were incubated with the depleted or not depleted PBMC ± BiTE (200ng/mL), and analyzed for % dead tumor cells. PBMC:tumor cell ratio is 20:1 for undepleted and depleted populations. For the undepleted samples the ratio of CD8 and CD4 T cells to tumor was 5.45:1 and 8.04:1, respectively. In the CD8-depleted and CD4-depleted samples the ratio of CD4:tumor was 16.08:1 and the ratio of CD8:tumor was 11.52:1. Data are the average of three independent experiments. C . The cells of panel B were stained with fluorescent antibodies to CD4, CD8, and CD107a and the CD4 and CD8 cells gated and analyzed for CD107a expression. Data are representative of one of two independent experiments. D . Violet-labeled ± C8161 tumor cells were incubated with magnetic bead purified CD3 + cells (PBMC) or purified CD3 + CD56 + NKT cells ± BiTE (200ng/mL). Data are representative of three independent experiments. Values with * are significantly different from values without *.

    Journal: Oncotarget

    Article Title: CD3xPDL1 bi-specific T cell engager (BiTE) simultaneously activates T cells and NKT cells, kills PDL1+ tumor cells, and extends the survival of tumor-bearing humanized mice

    doi: 10.18632/oncotarget.19865

    Figure Lengend Snippet: CD3xPDL1 BiTE activates both CD4 + and CD8 + cytotoxic T cells and NKT cells A . PBMC from healthy human donors were either undepleted, or depleted for CD4 + , CD8 + , or CD4 + plus CD8 + T cells. Depleted populations were stained with fluorescently tagged antibodies to CD3, CD4, and CD8, and the CD3 + cells gated and analyzed for CD4 and CD8 expression. B . Violet-labeled PDL1 + H358 tumor cells were incubated with the depleted or not depleted PBMC ± BiTE (200ng/mL), and analyzed for % dead tumor cells. PBMC:tumor cell ratio is 20:1 for undepleted and depleted populations. For the undepleted samples the ratio of CD8 and CD4 T cells to tumor was 5.45:1 and 8.04:1, respectively. In the CD8-depleted and CD4-depleted samples the ratio of CD4:tumor was 16.08:1 and the ratio of CD8:tumor was 11.52:1. Data are the average of three independent experiments. C . The cells of panel B were stained with fluorescent antibodies to CD4, CD8, and CD107a and the CD4 and CD8 cells gated and analyzed for CD107a expression. Data are representative of one of two independent experiments. D . Violet-labeled ± C8161 tumor cells were incubated with magnetic bead purified CD3 + cells (PBMC) or purified CD3 + CD56 + NKT cells ± BiTE (200ng/mL). Data are representative of three independent experiments. Values with * are significantly different from values without *.

    Article Snippet: Antibodies to human PDL1 (CD274; clone 29E.2A3; BV421 and PE-Cy7), PD1 (clone EH12.2H7, PE-Cy7), CD3 (clone OKT3, FITC, PE, APC), CD3 (clone UCHT1, BV421), CD4 (clone OKT4, PB, BV510, APC, APC-Cy7, PE-Cy7), CD8 (clone SK1, APC), CD8a (clone HIT8a, PE), CD69 (clone FN50, AlexaF488), HLA-DR (clone L243, BV421), CD14 (clone HCD14, FITC), CD15 (clone HI98, PE), CD11b (clone ICRF44, APC), CD56 (clone HCD56, FITC), LEAF purified anti-human CD3 (clone OKT3), LEAF-purified anti-human PDL1 (clone 29E.2A3) were from BioLegend.

    Techniques: Staining, Expressing, Labeling, Incubation, Purification

    Effect of CD4 and CD8 depletion on RR2 induced protection against recurrent genital disease. (A) Timeline of HSV-2 infection, IVAG and IM immunization, and CD4 + and CD8 + T-cell depletion. (B to E) Representative FACS data for the frequencies of CD4 + and CD8 + T cells detected in the spleen (SPL) (B and C) and VM (D and E) are shown. (F) Cumulative vaginal lesions in the RR2- and dl 5-29-vaccinated groups following CD4 + or CD8 + T-cell depletion. (G) Representative images of genital lesions in guinea pigs vaccinated with either the RR2 protein or dl 5-29 and depleted from either CD4 + or CD8 + cells. The indicated P values show statistical significance between CD4 + and CD8 + T-cell-depleted groups and the mock-depleted group. *, P

    Journal: Journal of Virology

    Article Title: Therapeutic Mucosal Vaccination of Herpes Simplex Virus 2-Infected Guinea Pigs with Ribonucleotide Reductase 2 (RR2) Protein Boosts Antiviral Neutralizing Antibodies and Local Tissue-Resident CD4+ and CD8+ TRM Cells Associated with Protection against Recurrent Genital Herpes

    doi: 10.1128/JVI.02309-18

    Figure Lengend Snippet: Effect of CD4 and CD8 depletion on RR2 induced protection against recurrent genital disease. (A) Timeline of HSV-2 infection, IVAG and IM immunization, and CD4 + and CD8 + T-cell depletion. (B to E) Representative FACS data for the frequencies of CD4 + and CD8 + T cells detected in the spleen (SPL) (B and C) and VM (D and E) are shown. (F) Cumulative vaginal lesions in the RR2- and dl 5-29-vaccinated groups following CD4 + or CD8 + T-cell depletion. (G) Representative images of genital lesions in guinea pigs vaccinated with either the RR2 protein or dl 5-29 and depleted from either CD4 + or CD8 + cells. The indicated P values show statistical significance between CD4 + and CD8 + T-cell-depleted groups and the mock-depleted group. *, P

    Article Snippet: The following anti-human antibodies were used for flow cytometry assays: CD3 (clone SK7) phycoerythrin (PE)-Cy7, CD4 (clone DE54), CD8 (clone SK1), CD45RA FITC, CD62L allophycocyanin, IFN-γ Alexa Fluor 647, granzyme B (clone GB11) A647, CD107a (clone H4A3) fluorescein isothiocyanate (FITC), CD107b (clone H4B4) FITC (BioLegend, San Diego, CA), and granzyme K (clone G3H69).

    Techniques: Infection, FACS

    Function of vaginal mucosa-resident CD4 + and CD8 + T cells from HSV-2-infected guinea pigs following therapeutic vaccination with gD, RR2, VP22, gB, VP16, VP13/14, VP11/12, and UL25 proteins. (A) Enumeration of IFN-γ spot-forming units from guinea pigs immunized with various antigens. Representative wells of IFN-γ-producing cells measured ex vivo by ELISpot assay from VM cells (5 × 10 5 cells/well) and stimulated for 48 h with 10 μg of various HSV-2 proteins are depicted. The number at the top of each image of representative ELISpot wells represents the number of IFN-γ-producing spot-forming T cells per one million total vaginal mucosal cells. (B) Average data for IFN-γ-producing cells measured by ELISpot assay from the VM of four guinea pigs/per group, either ex vivo or after 48 h of in vitro stimulation with the indicated immunizing HSV-2 protein. Vaginal mucosa cell suspensions plated 10 5 cells/well and either left unstimulated (Un-Stim) or stimulated with 10 μg (Stim) with the indicated HSV-2 proteins. IFN-γ production is depicted by the number of spot-forming cells per one million VM cells of HSV-2 infected vaccinated and mock-vaccinated control groups. (C) Frequencies of vaginal mucosa-resident CRTAM + CD4 + T cells (top row) and of CRTAM + CD8 + T cells (bottom row). VM cell suspensions were stained with a MAb specific to the activation marker CRTAM, CD4, or CD8 and analyzed by FACS. The numbers at the top of each dot plot indicate the percentages of CRTAM + CD4 + T cells (top row) and CRTAM + CD8 + T cells (bottom row) per vaginal mucosal total cells, as determined by FACS. (D) Proliferative responses of vaginal mucosa-resident CFSE + CD4 + T cells (top row) and CFSE + CD8 + T cells (bottom row). VM cell suspensions were labeled with CFSE (2.5 × 10 6 cells/well) either alone (mock) or in the presence of the indicated immunizing HSV-2 protein, and proliferative cells were quantified by FACS. The indicated P values show statistical significance between HSV-2-infected vaccinated and mock-vaccinated control groups. *, P

    Journal: Journal of Virology

    Article Title: Therapeutic Mucosal Vaccination of Herpes Simplex Virus 2-Infected Guinea Pigs with Ribonucleotide Reductase 2 (RR2) Protein Boosts Antiviral Neutralizing Antibodies and Local Tissue-Resident CD4+ and CD8+ TRM Cells Associated with Protection against Recurrent Genital Herpes

    doi: 10.1128/JVI.02309-18

    Figure Lengend Snippet: Function of vaginal mucosa-resident CD4 + and CD8 + T cells from HSV-2-infected guinea pigs following therapeutic vaccination with gD, RR2, VP22, gB, VP16, VP13/14, VP11/12, and UL25 proteins. (A) Enumeration of IFN-γ spot-forming units from guinea pigs immunized with various antigens. Representative wells of IFN-γ-producing cells measured ex vivo by ELISpot assay from VM cells (5 × 10 5 cells/well) and stimulated for 48 h with 10 μg of various HSV-2 proteins are depicted. The number at the top of each image of representative ELISpot wells represents the number of IFN-γ-producing spot-forming T cells per one million total vaginal mucosal cells. (B) Average data for IFN-γ-producing cells measured by ELISpot assay from the VM of four guinea pigs/per group, either ex vivo or after 48 h of in vitro stimulation with the indicated immunizing HSV-2 protein. Vaginal mucosa cell suspensions plated 10 5 cells/well and either left unstimulated (Un-Stim) or stimulated with 10 μg (Stim) with the indicated HSV-2 proteins. IFN-γ production is depicted by the number of spot-forming cells per one million VM cells of HSV-2 infected vaccinated and mock-vaccinated control groups. (C) Frequencies of vaginal mucosa-resident CRTAM + CD4 + T cells (top row) and of CRTAM + CD8 + T cells (bottom row). VM cell suspensions were stained with a MAb specific to the activation marker CRTAM, CD4, or CD8 and analyzed by FACS. The numbers at the top of each dot plot indicate the percentages of CRTAM + CD4 + T cells (top row) and CRTAM + CD8 + T cells (bottom row) per vaginal mucosal total cells, as determined by FACS. (D) Proliferative responses of vaginal mucosa-resident CFSE + CD4 + T cells (top row) and CFSE + CD8 + T cells (bottom row). VM cell suspensions were labeled with CFSE (2.5 × 10 6 cells/well) either alone (mock) or in the presence of the indicated immunizing HSV-2 protein, and proliferative cells were quantified by FACS. The indicated P values show statistical significance between HSV-2-infected vaccinated and mock-vaccinated control groups. *, P

    Article Snippet: The following anti-human antibodies were used for flow cytometry assays: CD3 (clone SK7) phycoerythrin (PE)-Cy7, CD4 (clone DE54), CD8 (clone SK1), CD45RA FITC, CD62L allophycocyanin, IFN-γ Alexa Fluor 647, granzyme B (clone GB11) A647, CD107a (clone H4A3) fluorescein isothiocyanate (FITC), CD107b (clone H4B4) FITC (BioLegend, San Diego, CA), and granzyme K (clone G3H69).

    Techniques: Infection, Ex Vivo, Enzyme-linked Immunospot, In Vitro, Staining, Activation Assay, Marker, FACS, Labeling

    Frequency of CD4 + and CD8 + T cells in the vaginal mucosae of HSV-2-infected guinea pigs following therapeutic vaccination with gD, RR2, VP22, gB, VP16, VP13/14, VP11/12, and UL25 proteins. (A) Representative FACS data for the frequencies of CD4 + and CD8 + T cells detected in the VM of vaccinated and mock-vaccinated animals. (B) Quantification of VM-resident CD4 + and CD8 + T cells following therapeutic vaccination with various HSV-2 Antigens. Average frequencies and absolute numbers of CD4 + T cells (top graphs) and CD8 + T cells (bottom graphs) were determined using FACS in the VM of vaccinated and mock-vaccinated animals. Cells were analyzed using a BD FACSCalibur flow cytometry system with a total of 4 × 10 5 events. Density plots showing the percentage of CD4 + T cells and CD8 + T cells are representative of results from two independent experiments. The indicated P values, obtained by 2-way ANOVA for repeated measures followed by Bonferroni’s posttest for significance, show statistical significance between HSV-2-vaccinated and mock-vaccinated control groups. *, P

    Journal: Journal of Virology

    Article Title: Therapeutic Mucosal Vaccination of Herpes Simplex Virus 2-Infected Guinea Pigs with Ribonucleotide Reductase 2 (RR2) Protein Boosts Antiviral Neutralizing Antibodies and Local Tissue-Resident CD4+ and CD8+ TRM Cells Associated with Protection against Recurrent Genital Herpes

    doi: 10.1128/JVI.02309-18

    Figure Lengend Snippet: Frequency of CD4 + and CD8 + T cells in the vaginal mucosae of HSV-2-infected guinea pigs following therapeutic vaccination with gD, RR2, VP22, gB, VP16, VP13/14, VP11/12, and UL25 proteins. (A) Representative FACS data for the frequencies of CD4 + and CD8 + T cells detected in the VM of vaccinated and mock-vaccinated animals. (B) Quantification of VM-resident CD4 + and CD8 + T cells following therapeutic vaccination with various HSV-2 Antigens. Average frequencies and absolute numbers of CD4 + T cells (top graphs) and CD8 + T cells (bottom graphs) were determined using FACS in the VM of vaccinated and mock-vaccinated animals. Cells were analyzed using a BD FACSCalibur flow cytometry system with a total of 4 × 10 5 events. Density plots showing the percentage of CD4 + T cells and CD8 + T cells are representative of results from two independent experiments. The indicated P values, obtained by 2-way ANOVA for repeated measures followed by Bonferroni’s posttest for significance, show statistical significance between HSV-2-vaccinated and mock-vaccinated control groups. *, P

    Article Snippet: The following anti-human antibodies were used for flow cytometry assays: CD3 (clone SK7) phycoerythrin (PE)-Cy7, CD4 (clone DE54), CD8 (clone SK1), CD45RA FITC, CD62L allophycocyanin, IFN-γ Alexa Fluor 647, granzyme B (clone GB11) A647, CD107a (clone H4A3) fluorescein isothiocyanate (FITC), CD107b (clone H4B4) FITC (BioLegend, San Diego, CA), and granzyme K (clone G3H69).

    Techniques: Infection, FACS, Flow Cytometry, Cytometry

    CD4 + and CD8 + T-cell responses against HSV-2 antigens detected from symptomatic and asymptomatic patients. (A) Average intensity of recognition of ORFs expressing HSV-2 proteins by T cells from HSV-2-seropositive symptomatic ( n = 10) and asymptomatic individuals ( n = 10), as tested by IFN-γ production. The names of HSV-2 ORFs driving two representative positive responses are indicated. (B) VP11/12- and VP13/14-specific CFSE + proliferative T cells from symptomatic and asymptomatic individuals represented in panel A. (C and D) Representative analyses of HSV-2 VP11/12- and VP13/14-specific IFN-γ-producing CD4 + T cells (C) and CD8 + T cells (D). The top portion shows representative analysis from the one symptomatic and one asymptomatic individual, and the bottom portion shows averages from the 10 symptomatic and 10 asymptomatic individuals. Representative results are from three independent experiments. The P value compares the results from 10 symptomatic and 10 asymptomatic individuals. Values were calculated using the ANOVA two-tail test.

    Journal: Journal of Virology

    Article Title: Therapeutic Mucosal Vaccination of Herpes Simplex Virus 2-Infected Guinea Pigs with Ribonucleotide Reductase 2 (RR2) Protein Boosts Antiviral Neutralizing Antibodies and Local Tissue-Resident CD4+ and CD8+ TRM Cells Associated with Protection against Recurrent Genital Herpes

    doi: 10.1128/JVI.02309-18

    Figure Lengend Snippet: CD4 + and CD8 + T-cell responses against HSV-2 antigens detected from symptomatic and asymptomatic patients. (A) Average intensity of recognition of ORFs expressing HSV-2 proteins by T cells from HSV-2-seropositive symptomatic ( n = 10) and asymptomatic individuals ( n = 10), as tested by IFN-γ production. The names of HSV-2 ORFs driving two representative positive responses are indicated. (B) VP11/12- and VP13/14-specific CFSE + proliferative T cells from symptomatic and asymptomatic individuals represented in panel A. (C and D) Representative analyses of HSV-2 VP11/12- and VP13/14-specific IFN-γ-producing CD4 + T cells (C) and CD8 + T cells (D). The top portion shows representative analysis from the one symptomatic and one asymptomatic individual, and the bottom portion shows averages from the 10 symptomatic and 10 asymptomatic individuals. Representative results are from three independent experiments. The P value compares the results from 10 symptomatic and 10 asymptomatic individuals. Values were calculated using the ANOVA two-tail test.

    Article Snippet: The following anti-human antibodies were used for flow cytometry assays: CD3 (clone SK7) phycoerythrin (PE)-Cy7, CD4 (clone DE54), CD8 (clone SK1), CD45RA FITC, CD62L allophycocyanin, IFN-γ Alexa Fluor 647, granzyme B (clone GB11) A647, CD107a (clone H4A3) fluorescein isothiocyanate (FITC), CD107b (clone H4B4) FITC (BioLegend, San Diego, CA), and granzyme K (clone G3H69).

    Techniques: Expressing

    Intravaginal versus intramuscular immunization of HSV-2-infected guinea pigs with RR2 protein. (A) Timeline of HSV-2 infection, intravaginal (IVAG) and intramuscular (IM) immunization, and immunological and virological analyses. Guinea pigs ( n = 30) were infected intravaginally with 5 × 10 5 PFU of HSV-2 (MS strain). Once acute infection was resolved, latently infected animals were randomly divided into four groups ( n = 10) and then vaccinated with the RR2 protein emulsified in alum plus CpG adjuvant either IVAG or IM on days 15 and 25. Replication-defective HSV-2 dl 5-29 mutant vaccine delivered IVAG was used as a positive control. Mock-vaccinated guinea pigs, which received IVAG alum plus CpG adjuvants alone, were used as a negative control. (B) Representative images of genital lesions in guinea pigs vaccinated with RR2 protein either IVAG or IM are shown, along with representative images of dl 5-29-vaccinated and mock-vaccinated animals. (C) Cumulative vaginal lesions (left panel) and cumulative positive days with recurrences (right) following IVAG versus IM vaccinations. (D) HSV-2 DNA copy numbers detected in DRG on day 80 p.i. following vaccination by either the IVAG or IM route are shown. (E) Representative FACS data (left panels) and average frequencies and numbers (right panels) of CD4 + T cells and CD8 + T cells in guinea pigs vaccinated with RR2 protein IVAG or IM are shown. (F) Representative FACS data (left plots) and average frequencies and numbers (right graphs) of IFN-γ + CD4 + T cells and IFN-γ + CD8 + T cells vaccinated IVAG or IM. The indicated P values show statistical significance between the groups of animals vaccinated IVAG versus IM. *, P

    Journal: Journal of Virology

    Article Title: Therapeutic Mucosal Vaccination of Herpes Simplex Virus 2-Infected Guinea Pigs with Ribonucleotide Reductase 2 (RR2) Protein Boosts Antiviral Neutralizing Antibodies and Local Tissue-Resident CD4+ and CD8+ TRM Cells Associated with Protection against Recurrent Genital Herpes

    doi: 10.1128/JVI.02309-18

    Figure Lengend Snippet: Intravaginal versus intramuscular immunization of HSV-2-infected guinea pigs with RR2 protein. (A) Timeline of HSV-2 infection, intravaginal (IVAG) and intramuscular (IM) immunization, and immunological and virological analyses. Guinea pigs ( n = 30) were infected intravaginally with 5 × 10 5 PFU of HSV-2 (MS strain). Once acute infection was resolved, latently infected animals were randomly divided into four groups ( n = 10) and then vaccinated with the RR2 protein emulsified in alum plus CpG adjuvant either IVAG or IM on days 15 and 25. Replication-defective HSV-2 dl 5-29 mutant vaccine delivered IVAG was used as a positive control. Mock-vaccinated guinea pigs, which received IVAG alum plus CpG adjuvants alone, were used as a negative control. (B) Representative images of genital lesions in guinea pigs vaccinated with RR2 protein either IVAG or IM are shown, along with representative images of dl 5-29-vaccinated and mock-vaccinated animals. (C) Cumulative vaginal lesions (left panel) and cumulative positive days with recurrences (right) following IVAG versus IM vaccinations. (D) HSV-2 DNA copy numbers detected in DRG on day 80 p.i. following vaccination by either the IVAG or IM route are shown. (E) Representative FACS data (left panels) and average frequencies and numbers (right panels) of CD4 + T cells and CD8 + T cells in guinea pigs vaccinated with RR2 protein IVAG or IM are shown. (F) Representative FACS data (left plots) and average frequencies and numbers (right graphs) of IFN-γ + CD4 + T cells and IFN-γ + CD8 + T cells vaccinated IVAG or IM. The indicated P values show statistical significance between the groups of animals vaccinated IVAG versus IM. *, P

    Article Snippet: The following anti-human antibodies were used for flow cytometry assays: CD3 (clone SK7) phycoerythrin (PE)-Cy7, CD4 (clone DE54), CD8 (clone SK1), CD45RA FITC, CD62L allophycocyanin, IFN-γ Alexa Fluor 647, granzyme B (clone GB11) A647, CD107a (clone H4A3) fluorescein isothiocyanate (FITC), CD107b (clone H4B4) FITC (BioLegend, San Diego, CA), and granzyme K (clone G3H69).

    Techniques: Infection, Mass Spectrometry, Mutagenesis, Positive Control, Negative Control, FACS

    Correlates of protection with function and exhaustion of local vaginal mucosa-resident CD8 + T cells following RR2 protein therapeutic vaccination of HSV-2-infected guinea pigs. (A) Timeline of HSV-2 infection, IVAG and intramuscular (IM) immunization, and immunological and virological analyses. Once acute infection was resolved, latently infected animals were randomly divided into three groups ( n = 11) and then vaccinated twice intramuscularly, on days 15 and 25 postinfection, with either RR2 protein emulsified in alum plus CpG adjuvants (group 1) or the replication-defective dl 5-29 vaccine (positive control; group 2). Mock-vaccinated guinea pigs, which received adjuvant alone, were used as a negative control (group 3). (B) Cumulative virus shedding detected RR2-vaccinated, dl5-29 -vaccinated and mock-vaccinated controls is shown. (C) Cumulative vaginal lesions (left) and cumulative number of days with recurrent genital lesions (right) detected for RR2-vaccinated, dl 5-29-vaccinated, and mock-vaccinated controls are shown. (D) Representative images of genital lesions in guinea pigs vaccinated with RR2 protein or dl 5-29 vaccine and mock-vaccinated animals. (E) Frequencies of CD4 + and CD8 + T cells detected by FACS in the VM tissue of RR2-vaccinated animals. (F) Frequencies of functional CRTAM + CD8 + T cells (top row of plots and graphs) and of CFSE + CD8 + T cells (bottom row of plots and graphs) and IFN-γ-producing cells, enumerated ex vivo by ELISpot assay (bottom portion) in the VM of RR2-vaccinated and dl 5-29-vaccinated animals compared to those in mock-vaccinated animals. (G) Frequencies of exhausted PD-1 + CD8 + T cells (top row of plots and graphs) and TIM-3 + CD8 + T cells (bottom row of plots and graphs) detected in the VM of RR2-vaccinated and dl 5-29-vaccinated animals compared to those in mock-vaccinated animals. The indicated P values show statistical significance between RR2-vaccinated and mock-vaccinated control groups. *, P

    Journal: Journal of Virology

    Article Title: Therapeutic Mucosal Vaccination of Herpes Simplex Virus 2-Infected Guinea Pigs with Ribonucleotide Reductase 2 (RR2) Protein Boosts Antiviral Neutralizing Antibodies and Local Tissue-Resident CD4+ and CD8+ TRM Cells Associated with Protection against Recurrent Genital Herpes

    doi: 10.1128/JVI.02309-18

    Figure Lengend Snippet: Correlates of protection with function and exhaustion of local vaginal mucosa-resident CD8 + T cells following RR2 protein therapeutic vaccination of HSV-2-infected guinea pigs. (A) Timeline of HSV-2 infection, IVAG and intramuscular (IM) immunization, and immunological and virological analyses. Once acute infection was resolved, latently infected animals were randomly divided into three groups ( n = 11) and then vaccinated twice intramuscularly, on days 15 and 25 postinfection, with either RR2 protein emulsified in alum plus CpG adjuvants (group 1) or the replication-defective dl 5-29 vaccine (positive control; group 2). Mock-vaccinated guinea pigs, which received adjuvant alone, were used as a negative control (group 3). (B) Cumulative virus shedding detected RR2-vaccinated, dl5-29 -vaccinated and mock-vaccinated controls is shown. (C) Cumulative vaginal lesions (left) and cumulative number of days with recurrent genital lesions (right) detected for RR2-vaccinated, dl 5-29-vaccinated, and mock-vaccinated controls are shown. (D) Representative images of genital lesions in guinea pigs vaccinated with RR2 protein or dl 5-29 vaccine and mock-vaccinated animals. (E) Frequencies of CD4 + and CD8 + T cells detected by FACS in the VM tissue of RR2-vaccinated animals. (F) Frequencies of functional CRTAM + CD8 + T cells (top row of plots and graphs) and of CFSE + CD8 + T cells (bottom row of plots and graphs) and IFN-γ-producing cells, enumerated ex vivo by ELISpot assay (bottom portion) in the VM of RR2-vaccinated and dl 5-29-vaccinated animals compared to those in mock-vaccinated animals. (G) Frequencies of exhausted PD-1 + CD8 + T cells (top row of plots and graphs) and TIM-3 + CD8 + T cells (bottom row of plots and graphs) detected in the VM of RR2-vaccinated and dl 5-29-vaccinated animals compared to those in mock-vaccinated animals. The indicated P values show statistical significance between RR2-vaccinated and mock-vaccinated control groups. *, P

    Article Snippet: The following anti-human antibodies were used for flow cytometry assays: CD3 (clone SK7) phycoerythrin (PE)-Cy7, CD4 (clone DE54), CD8 (clone SK1), CD45RA FITC, CD62L allophycocyanin, IFN-γ Alexa Fluor 647, granzyme B (clone GB11) A647, CD107a (clone H4A3) fluorescein isothiocyanate (FITC), CD107b (clone H4B4) FITC (BioLegend, San Diego, CA), and granzyme K (clone G3H69).

    Techniques: Infection, Positive Control, Negative Control, FACS, Functional Assay, Ex Vivo, Enzyme-linked Immunospot