human pd1 duoset  (R&D Systems)

Journal: Cancer research

Article Title: Tumor-localized secretion of soluble PD1 enhances oncolytic virotherapy

doi: 10.1158/0008-5472.CAN-16-1638

Figure Lengend Snippet: (A) B16/F10 cells were either mock infected or infected with MYXV at MOI=10. 24 hours post infection cells were harvested and the total level of PDL1 was analyzed using westernblot (band representing PDL1 is indicated by arrow). Expression of β Actin is shown as a loading control. Data shown is representative is three independent experiments. (B) B16/F10 cells were either mock infected or infected with MYXV at MOI=10. 24 hours post infection cells were harvested and the cell surface expression of PDL1 was analyzed using flowcytometry. Data shown is representative of two independent experiments. (C) C57/B6 mice were injected SQ with 4x105 B16/F10 cells. On days 7, 9, and 11 post injection mice were given IT injection of either saline (n=9) or 1x107 FFU of MYXV (n=9). On day 12, mice were euthanized and the percentage of either CD4+ or CD8+ splenocytes expressing PD1 was determined using flowcytometry. Data represents the summation of four independent experiments. Significance was determined using unpaired students T-Test. (D) C57/B6 mice were injected SQ with either 4x105 B16/F10-wt or B16/F10-PDL1−/− cells. On days 7, 9, and 11 post injection mice were given IT injection of either saline (B16/F10 n=5, B16/F10-PDL1−/− n=5) or 1x107 FFU of MYXV (B16/F10 n=5, B16/F10-PDL1−/− n=14). Animals were then monitored for tumor burden and euthanized when tumors reached 15mm in any direction. Data is representative of two independent experiments. (E) C57/B6 mice were injected SQ with 4x105 B16/F10 cells. On day 7 post injection, mice were randomly separated into one of four cohorts: saline + isotype (n=9), saline + αPD1 antibody (n=8), MYXV + isotype (n=5), MYXV + αPD1 antibody (n=7). Viral injections consisted of 1x107 FFU of MYXV and were given on days 7, 9, and 11. Antibody injections consisted of 100μg/injection and were given on days 7, 11, 14, and 18. Animals were then monitored for tumor burden and euthanized when tumors reached 15mm in any direction. Data is representative >four independent experiments. (F) Example of alopecia in mice on day 70 post-treatment and H&E stain from the belly skin of displayed animal. (G) Average alopecia score from MYXV + αPD1 antibody treated animals (n=7, shown in 1E) and B16/F10-PDL1−/− bearing animals treated with MYXV (n=14, shown in 1D). * denotes significance of p<0.05 for the indicated day and was determined using unpaired students T-Test.

Article Snippet: Secretion of soluble human PD1 was measured using the human PD-1 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) per manufacturer’s recommendations.

Techniques: Infection, Expressing, Injection, Saline, Staining

Journal: Cancer research

Article Title: Tumor-localized secretion of soluble PD1 enhances oncolytic virotherapy

doi: 10.1158/0008-5472.CAN-16-1638

Figure Lengend Snippet: (A) B16/F10 cells were either mock infected or infected with vGFP or vPD1 at MOI=10. 24 hours post infection cells were harvested and the expression of intracellular PD1 was analyzed using westernblot. Expression of β Actin is shown as a loading control. Data shown is representative of two independent experiments. (B) B16/F10 cells were infected with either vGFP or vPD1 at MOI=10. At the indicated times post-infection, supernatant was collected from each infection and frozen. The concentration of soluble human PD1 in each sample was then determined using ELISA. Dotted line indicates approximate detection limit of assay. Data shown is representative of two independent experiments. (C) C57/B6 mice were injected SQ with 4x105 B16/F10 cells. On day 7 post-injection, mice were treated with a single IT injection of either saline or 1x107 FFU of vGFP or vPD1. 48 hours post-treatment, tumors were harvested and stained for either GFP or human PD1 using immunohistochemistry. Data shown is representative of two independent experiments each with n>5. (D) C57/B6 mice were injected SQ with 4x105 B16/F10 cells. On day 7 post-injection, mice were treated with a single IT injection of either saline (n=3) or 1x107 FFU of vGFP (n=3) or vPD1 (n=3). At the indicated times post infection, animals were euthanized and serum as well as the indicated tissues were harvested. Tissues were disassociated over a 40μM mesh and separated into cell and non-cell fractions. The concentration of human PD1 in the non-cell-associated fraction was then determined using ELISA. Dotted line indicates approximate detection limit of assay. Data is representative of two independent experiments.

Article Snippet: Secretion of soluble human PD1 was measured using the human PD-1 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) per manufacturer’s recommendations.

Techniques: Infection, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Injection, Saline, Staining, Immunohistochemistry

Journal: Cancer research

Article Title: Tumor-localized secretion of soluble PD1 enhances oncolytic virotherapy

doi: 10.1158/0008-5472.CAN-16-1638

Figure Lengend Snippet: (A–C) C57/B6 mice were injected SQ with 4x105 B16/F10 cells. On day 7 post injection, mice were randomly separated into one of four cohorts: saline + isotype (n=12), vGFP + isotype (n=10), vGFP + αPD1 antibody (n=17), or vPD1 (n=22). Viral injections consisted of 1x107 FFU of MYXV and were given on days 7, 9, and 11. Antibody injections consisted of 100μg/injection and were given on days 7, 11, 14, and 18. (A) Animals were monitored for tumor burden and euthanized when tumors reached 15mm in any direction. Data represents summation of four independent experiments. Significance was determined using log-rank test. (B) Average overall response rates for each treatment group (defined as the number of animals surviving when the last saline treated animal was euthanized). Significance was determined by averaging response rates from four experiments and analyzing using unpaired students T-Test. (C) Average complete response rates for each treatment group (defined as animals displaying no remaining tumor burden 80 days post treatment). Significance was determined by averaging response rates from four experiments and analyzing using unpaired students T-Test. (D) Average alopecia score from vGFP + αPD1 antibody (n=12) and vPD1 (n=22) treated animals. * denotes significance of p<0.05 for the indicated day and was determined using unpaired students T-Test. (E) Example of alopecia in mice on day 70 post treatment. Abbreviations: OR (overall response), CR (compete response).

Article Snippet: Secretion of soluble human PD1 was measured using the human PD-1 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) per manufacturer’s recommendations.

Techniques: Injection, Saline

Journal: Cancer research

Article Title: Tumor-localized secretion of soluble PD1 enhances oncolytic virotherapy

doi: 10.1158/0008-5472.CAN-16-1638

Figure Lengend Snippet: (A and B) C57/B6 mice were injected SQ with 4x105 B16/F10 cells. On day 7 post injection, mice were randomly separated into one of four cohorts: saline (n=9), vGFP + isotype (n=7), vGFP + αPD1 antibody (n=6), or vPD1 (n=11). Viral injections consisted of 1x107 FFU of MYXV and were given on days 7, 9, and 11. Antibody injections consisted of 100μg/injection and were given on days 7 and 11. On day 12, mice were euthanized and tumors were extracted and disassociated into single cells for analysis by flowcytometry. (A) Percent of tumor cells staining as viable. (B) Percent of viable tumor cells expressing detectable GFP fluorescence. Data represents summation of four independent experiments. Significance was determined using unpaired students T-Test. (C) C57/B6 mice were injected SQ with 4x105 B16/F10 cells. On day 5, animals were split into two groups and given either mock injection or IP injection of depleting antibodies against both CD4 and CD8. Antibody depletion treatment was repeated on day 12. Depleted animals were separated into three cohorts and given IT injections of either: saline (n=5), or 1x107 FFU of vGFP (n=5) or vPD1 (n=5) on days 7, 9, and 11. Non-depleted animals injected IT with either saline (n=5) or 1x107 FFU vPD1 (n=5) were used as controls. Animals were then monitored for tumor burden and euthanized when tumors reached 15mm in any direction. (D) C57/B6 mice were injected SQ with 4x105 B16/F10 cells. On day five, animals were given either mock injection (n=12) or IP injection of depleting antibodies against CD4 (n=10), CD8 (n=10), or NK1.1 (n=15). Antibody depletion treatment was repeated on day 12. Animals were then treated with IT injection of 1x107 FFU of vPD1 on days 7, 9, and 11. Non-depleted animals treated with IT injection of saline (n=8) were included as a control. Animals were then monitored for tumor burden and euthanized when tumors reached 15mm in any direction. Data represents summation of two independent experiments. Significance was determined using log-rank test.

Article Snippet: Secretion of soluble human PD1 was measured using the human PD-1 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) per manufacturer’s recommendations.

Techniques: Injection, Saline, Staining, Expressing, Fluorescence

Journal: Cancer research

Article Title: Tumor-localized secretion of soluble PD1 enhances oncolytic virotherapy

doi: 10.1158/0008-5472.CAN-16-1638

Figure Lengend Snippet: (A–C) C57/B6 mice were injected SQ with 4x105 B16/F10 cells. On day 7, mice were randomly separated into one of four cohorts: saline (n=9), vGFP + isotype (n=7), vGFP + αPD1 antibody (n=6), or vPD1 (n=11). Viral injections consisted of 1x107 FFU and were given on days 7, 9, and 11. Antibody injections consisted of 100μg/injection and were given on days 7 and 11. On day 12, mice were euthanized, tumors disassociated into single cell suspensions, and TIL’s extracted using Histopaque. TILs were then analyzed using flowcytometry. (A) Numbers of CD4+ and CD8+ cells as percentage of total viable cells. (B) Surface expression of the early activation marker CD69 on CD8+ TILs. (C) Surface expression of the early and late activation markers CD69 and CD25 on the surface of CD8+ TILS. Data represents summation of four independent experiments. Significance was determined using unpaired students T-Test.

Article Snippet: Secretion of soluble human PD1 was measured using the human PD-1 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) per manufacturer’s recommendations.

Techniques: Injection, Saline, Expressing, Activation Assay, Marker