Table 1.
Patient and Tumor Characteristics.
Figure 1.
T cells infiltrate PDA regardless of GM-CSF expression.
A-C) IHC of normal adjacent pancreas, well-differentiated PDA, and poorly differentiated PDA showing dual staining of T cells (CD3+ in brown, arrows) and myeloid cells (CD11b+in purple, arrowheads). Open arrowhead indicates a normal duct. Photomicrographs taken with a 20x objective. D) Quantification of CD3+ and CD11b+ cells in PDA (p = 0.0009). E) Correlation between density of CD3+ and CD11b+ cells in each tumor (r = 0.51; p = 0.0008). F-H) Representative photomicrographs (40x) showing GM-CSF expression in normal pancreas and PDA. I) Distribution of myeloid cells and T cells stratified based upon chromogen intensity for GM-CSF. At least five 40x fields were analyzed per specimen. Scale bars = 25 µm.
Figure 2.
T cells dominate the intratumoral immune cell infiltrate.
A) Representative flow cytometry plots illustrating the typical gating strategy used to identify the viable immune cells in blood and tumor. B) The proportion of live CD45+ cells was calculated for each specimen. C) CD3+ T cells consistently comprised the majority of the viable immune cells in blood and tumor, while CD19+ B cells and non-T or B cells comprise smaller proportions.
Figure 3.
The majority of intratumoral T cells are antigen-experienced memory cells or regulatory cells.
A) Both CD4+ and CD8+ T cells infiltrate PDA. B) Representative plots and summed data of central memory (CM), effector memory (EM), and naïve CD4+ and CD8+ T cell populations in the blood and tumor among live CD3+ T cells. Tumors contained significantly more CD4+ (p<0.0001) and CD8+ (p = 0.0001) EM cells than did the blood, while containing fewer CD4+ CM (p = 0.01), CD4+ naïve (p = 0.0003) and CD8+ naïve (p = 0.0007) T cells. C) After gating on live CD3+ CD4+ T cells, we used CD25 and FOXP3 to identify putative regulatory T cells (p<0.0001).
Figure 4.
Histologic localization of cellular subtypes confirms the flow cytometric analysis of the immune infiltrate in PDA.
A–C) Representative sections of normal pancreas and PDA showing multicolor IHC for CD8+ (brown, arrows) and CD4+ (purple, arrowheads) cells. D) Quantification of CD8+ (p = 0.0001) and CD4+ (p = 0.0005) cells. E–G) Representative sections of normal pancreas and PDA stained for FOXP3. Arrowheads indicate FOXP3+ cells, open arrowhead demonstrates a normal duct. H) Quantification of FOXP3+ cells (p = 0.02). At least five 20x fields were analyzed per specimen per cell type. Scale bars = 25 µm.
Figure 5.
T cells in the tumor microenvironment retain functional capacity to activate inflammation despite the presence of multiple potential regulatory mechanisms.
A) Single-cell tumor suspensions and PBMC isolated from matched blood samples were incubated with PMA/ionomycin for 4 hours prior to staining for multiparameter flow cytometry. Staining for intracellular IFN-γ was positive in a significantly higher proportion of CD8+ T cells in tumor than in blood (p = 0.03), and there was a similar trend among CD4+ T cells (p = 0.05). A small proportion of CD3− cells thought to represent NK cells produced IFN-γ. The ratio of IFN-γ producing CD8+ T cells to Treg in blood and tumor is shown (p = 0.02). B) Th17 cells were identified by gating on live IL-17+CD4+ T cells. (p = 0.0008). C) Plot showed expression of PD-1 by both CD4+ and CD8+ unstimulated T cells (p = 0.003 and 0.02, respectively).
Figure 6.
Multimodal neoadjuvant therapy alters the regulatory cell makeup of the tumor microenvironment.
A) Kaplan-Meier survival of patients in this study. There was a trend towards longer survival among patients undergoing neoadjuvant therapy (p = 0.07). B) Quantification of CD11b+ (p = 0.04) and CD3+cells in neoadjuvant therapy vs. untreated tumors. C) CD4+ cells were unchanged, however CD8+ cells were less frequent after neoadjuvant therapy (p = 0.04). D) Quantification of FOXP3+ cells in treated and untreated tumors (p = 0.002). E) Analysis of ratio of CD8+ to FOXP3+ T cells (p = 0.01) and CD4+ to FOXP3+ T cells (p = 0.01) in treated and untreated tumors.