Suppl. Fig. 4-
Systemic IL-12 expression generates a significant increase in total leukocyte
number in spleen. WT C57BL/6 and Foxp3-EGFP C57BL/6 mice were subcutaneously
inoculated with 1 x 106 B16 cells. When solid tumors were visible, mice were
hydrodynamically injected with 1g of an empty vector (control) or IL-12
cDNA. Seven days after hydrodynamic injection spleens were harvested and
cells were stained with Zombie Acqua Dye, CD4, CD8, B220, CD11b and Gr-1
antibodies for flow cytometry analysis. Samples were acquired in a BD LSR
Fortessa cytometer. Foxp3 expression was analyzed in the CD4 population based
on GFP detection. Total cell numbers were calculated and plotted in each
control vs IL-12 cDNA-treated mice. Data is the result of 3 independent
experiments with 3-5 mice per group. Statistical analysis was performed using
Student t test. *p<0.05, ***p<0.001. Suppl. Fig. 5-
Systemic IL-12 expression generates different total cell numbers of leucocytes
and cell subsets between tumor draining (dLN) and non-draining lymph nodes
(ndLN). WT C57BL/6 and Foxp3-EGFP C57BL/6 mice were subcutaneously inoculated
with 1 x 106 B16 cells. When solid tumors were visible, mice were
hydrodynamically injected with 1g of an empty vector (control) or IL-12 cDNA.
Seven days after hydrodynamic injection both inguinal lymph nodes were taken,
harvested, counted, and stained with Zombie Acqua Dye, CD3, CD4, CD8, B220,
CD11b and Gr-1 antibodies for flow cytometry analysis. Foxp3 expression was
analyzed in the CD4 population based on GFP detection. Samples were acquired in
a BD LSR Fortessa cytometer. Total cell numbers were calculated and plotted in
each control vs IL-12 cDNA-treated mice. Data represent the pool of 3
independent experiments with 3-5 mice per group. Statistical analysis was
performed using student t test. *p<0.05, ***p<0.001 represents the
statistical difference between control and IL-12 mice for each tissue analyzed.
Suppl. Fig. 6-
Systemic IL-12 expression induces a positive effector versus regulatory ratio
in OLS. WT C57BL/6 and Foxp3-EGFP C57BL/6 mice were subcutaneously inoculated
with 1 x 106 B16 cells. When solid tumors were visible, mice were
hydrodynamically injected with 1g of an empty vector (control) or IL-12 cDNA.
Seven days after hydrodynamic injection spleens and tumor-draining and
non-draining inguinal lymph nodes were taken, harvested, counted, and stained
with Zombie Acqua Dye, CD3, CD4 and CD8 antibodies for flow cytometry analysis.
Foxp3 expression was analyzed in the CD4 population based on GFP detection.
Samples were acquired in a BD LSR Fortessa cytometer. Graphs show the ratio of
total cell number of CD8+ T cells to total CD4+ T cells and CD8+ T cells to
Tregs (CD3+CD4+Foxp3+) cells respectively.
Data represent the pool of 2 independent experiments with 3-5 mice per group.
Statistical analysis was performed using student t test. *p<0.05,
***p<0.001, ****p<0.0001 represents the statistical difference between
control and IL-12 mice for each tissue analyzed.
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