465/30, 520/30, 575/70, and 660/50 nm band-pass filters and nondescanned detectors to generate second harmonic signals
(collagen fibers) and 4-color images. All the images were acquired at least 50mm below the tumor capsule. ZEN software was
used for analysis.
In Vitro T Cell Activation Assays
For T cell activation assays, spleen CD4+or CD8+T cells (5x10^4 ) were labeled with CFSE (eBioscience) and plated alone or with PDA-
infiltratinggdT cells, MDSC, or TAMs (5:1 ratio) in 96 well plates coated with anti-CD3 (145-2C11, 10mg/ml) and anti-CD28 (37.51;
10 mg/ml, both Biolegend). After 72 hr,abT cells were harvested and analyzed by flow cytometry. In selected experiments, cells
were treated with a neutralizing mAb directed against PD-L1 (10F.9G2, 10mg/ml; BioXCell).
QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical Analysis
Data is presented as mean±standard error. Survival was measured according to the Kaplan-Meier method. The sample size for each
experiment, n, is included in the results section and the associated figure legend. Statistical significance was determined by the
Student’s t test and the Wilcoxon test using GraphPad Prism 6 (GraphPad Software, La Jolla, CA). P-values < 0.05 were considered
significant. P values for each experiment are also included in the associated figure legends.
e5 Cell 166 , 1485–1499.e1–e5, September 8, 2016