Figure 6.

Sequential treatment with Gemcitabine and Durvalumab or Pembrolizumab does not improve CD8+ T cell effector phenotype. PancTu1 or Panc89 cells were mono- or co-cultured with either M1- or M2-like Mϕ for 24 h in ultra-low attachment plates to form spheroids. After 24 h, mono- or co-cultures were treated with Gemcitabine (Gem) (10 µg/ml) for 24 h. Afterwards, medium was changed, and cultures were treated with either Durvalumab (D) or Pembrolizumab (P) or their respective isotype controls (10 µg/ml) and simultaneous CD8+ T cell co-culture was started for 24 h. (A) Schematic illustration of experimental set-up. (B) PD-L1 staining of mono-cultured Panc89 or PancTu1 cells as well as M1- or M2-like Mϕ which were left untreated (-) or treated with Gemcitabine (+) for 24 h PD-L1 cell surface level was measured by flow cytometer and data is depicted as MFI ratio of specific staining. Immunofluorescence staining of activation marker (C) CD69, (D) CD25 and (E) PD-1 on cells surfaces of CD8+ T cells cultured with mono-cultured PDAC cell spheroids (dark grey) or co-cultured Panc89 (left panel) or PancTu1 (right panel) spheroids with M1Mϕ (light grey) or M2Mϕ (middle grey) after sequential treatment with Gemcitabine and ICI. Cell surface levels were measured by flow cytometer and data was normalized to the respective IgG control treated cells of the indicated Gemcitabine treated culture conditions. (F) Detection of Granzyme A, Granzyme B, Perforin and IFNγ in supernatants of CD8+ T cells cultured with either mono-cultured (dark grey) Panc89 (left panel) or PancTu1 spheroids (right panel) or with the indicated co-cultures with M1- (light grey) or M2Mϕ (middle grey) spheroids after sequential treatment with Gemcitabine and ICI. Concentrations were measured by multiplex assay and data is normalized to the respective IgG control samples of Gemcitabine treated culture conditions. Mono: 2x10⁴, ratio 3:1: 1.5x10⁴ PDAC cells and 0.5x10⁴ Mϕ; ratio 1:1: 1x10⁴ PDAC cells and 1x10⁴ Mϕ. Not normally distributed data are depicted as median with interquartile range in both directions. Two-way ANOVA with Dunnett’s multiple comparison test comparing all samples with a control of 1. The dashed lines mark an MFI ratio of “1”. * = p<0.05. n=3.