Figure 3. Kras^G12D-PDEC promote accumulation of immunosuppressive Gr1⁺CD11b⁺ cells in a GM-CSF dependent manner.

(A) Flow cytometry analysis of Lin^negCD34⁺ hematopoietic progenitor cells for the surface markers CD11b and Gr1 following co-culture with GM-CSF or GFP-Kras^G12D-PDEC with or without α-GM-CSF antibody (α-GM). Representative flow cytometry plots and a graph indicating percent of the Gr1⁺CD11b⁺ cells out of total number of live cells are shown. Error bars indicate SD, (n=3).

(B) Quantification of BrdU⁺CD3⁺ T cells treated as indicated. T cells and Gr1⁺CD11b⁺ cells were co-cultured at a 1:1 ratio. Error bars indicate SD, (n=3).

(C) Representative quantification of BrdU⁺CD3⁺ T cells co-cultured with either Gr1⁻ CD11b⁺ or Gr1⁺CD11b⁺ cells isolated from mouse pancreata 8 weeks after injection with GFP-Kras^G12D-PDEC. For proliferation assays, myeloid cells and T cells were cultured at ratios of 1:1, 1:5 or 1:10 respectively. T cells incubated in αCD3-coated wells and in the presence of αCD28 serve as control. Error bars indicate SD, (n=3).

(D) Expression of mouse GM-CSF in the sera of either uninjected mice (control, n=4), or mice with GFP-Kras^G12D-PDEC grafts 8 weeks post-implantation (n=5) was measured using ELISA. Each symbol represents a mouse, mean values for each group are represented by black lines.

(E) Relative expression of GM-CSF mRNA (gray bars, left axis) and protein (black bars, right axis) in GFP-Kras^G12D-PDEC 4 days after infection with lentiviruses containing either scrambled shRNA (scr) or GM-CSF shRNAs (GM-sh1, GM-sh2) was assessed by quantitative RT-PCR and ELISA. Error bars indicate SD, (n=3).

(F) Growth analysis of scr (circles), GM-sh1 (squares) and GM-sh2 GFP-Kras^G12D- PDEC (triangles) was assessed by MTT assay. Error bars indicate SD, (n=3).

(G) Representative flow cytometry plots of pancreatic immune cells for the surface expression of Gr1 and CD11b markers at 4-weeks post-implantation of scr-, GM-sh1- and GM-sh2 GFP-Kras^G12D-PDEC. After gating on the CD45⁺ population, cells were analyzed for the presence of CD11b⁺ and Gr1⁺ populations. The graph shows the percent Gr1⁺CD11b⁺ cells out of total number of live cells sorted from the pancreas.

(H) Quantification of relative abundance of Gr1⁺CD11b⁺ and Gr1⁻CD11b⁺ cell populations is presented as a percent change in CD45⁺ single-positive Gr1⁻CD11b⁺ cells (black bars) and double-positive Gr1⁺CD11b⁺ population (gray bars) relative to scr GFP-Kras^G12D-PDEC. Error bars indicate SD, (n= 6 mice per group). p value: **<0.01; ***<0.001, NS - not significant.