Figure 1. PD1 blockade enhances MYXV treatment of established melanoma.

(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.