br min at room temperature After incubation mL M
20 min at room temperature. After incubation, 50 mL 2 M H2SO4 was added to the wells, and absorbance was determined
Figure 8. The DPDL1E Vaccine Decreased the Ratio of PD-1+ Cells and Increased the Ratio of PD-L1+ Cells in Tumors
at 450 nm using SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA).
Lymphocyte Proliferation Assay
C57BL/6 mice (n = 8) were immunized with the DPDL1E or DTT vaccine at 2-week intervals. One week after the third immunization, spleno-cytes were isolated for further study. Briefly, spleens were isolated from mice under sterile con-
dition and ground into single cells. 5 mL red blood cell (RBC) lysis buffer (Sigma-Aldrich, USA) was added, followed by spinning for 5 min. The CCK8 were filtered through a 70-mM filter (BD Lifesciences, USA) and counted. Splenocytes (1 106 /mL in RPMI 1640 medium with 10% FCS) isolated from DPDL1E-immunized mice were cultured in 96-well plate (100 mL/well) and stimulated for 72 h with 50 mg/mL His-PD-L1, 5 mg/mL concanavalin A (Con A; Thermo Fisher Scientific, San Jose, CA, USA), or anti-CD3/CD28 beads (0.5 mg/mL). Splenocytes isolated from DTT-immunized mice were cultured in a 96-well plate and stimulated with 50 mg/mL His-PD-L1 as a negative control. Cell proliferation was measured using a Cell Counting Kit (CCK)-8 kit (Dojindo, Japan). After 72 h stimulation, the supernatant was collected for cytokine detection. The concentrations of TNF-a, IFN-g, and IL-2 were measured using corresponding ELISA kits (Thermo Fisher Scientific, San Jose, CA, USA).
Splenocytes isolated from DPDL1E- or DTT-immunized mice were stained with CFSE (Dojindo, Japan) according to the manufacturer’s instructions and cultured in RPMI 1640 medium containing 10% FCS and 20 units /mL IL-2. The cells were stimulated with His-PD-L1 pro-tein (50 mg/mL) or anti-CD3/CD28 beads (0.5 mg/mL). After 72 h of stimulation, CD8+ and CD4+ T cell proliferation was analyzed by FCM after the cells were stained with anti-CD3-PE, anti-CD8-peridinin chlorophyll protein complex (PerCP)-Cy5.5, and anti-CD4-antigen-presenting cell (APC) (BD Lifesciences, USA).
PD-L1-positive expressed B16-F10 cells were used as target cells, as reported previously.52 Lymphocytes isolated from DPDL1E-immu-nized mice were stimulated for 72 h with a His-PD-L1 protein and
used as effector cells. Effector cells and target cells were mixed at ratio of 100:1, 50:1, or 20:1 and incubated in Corning Life Sciences 96-well plates at 37 C for 4 h. CTL activities were detected using an LDH cytotoxicity detection kit (Biovision, USA).
A single-cell suspension of peripheral blood from tumor-bearing mice was stained with anti-CD3- fluorescein isothiocyanate (FITC), anti-CD8-PerCP-Cy5.5, anti-CD45-APC or anti-CD45-FITC, anti-CD11b-PerCP-Cy5.5, or anti-Gr1-phycoerythrin (PE). All FCM anti-bodies were purchased from BD Biosciences. Immune cells in tumor tissue were purified using tumor immune cell separation fluid (Tianjin Hao Yang, China) and st ained with anti-CD3-FITC, anti-CD8-PerCP-Cy5.5, anti-CD45-APC or anti-CD45-FITC, anti-CD11b-PerCP-Cy5.5, or anti-Gr1-PE. Cells were also stained with anti-CD45-FITC, anti-CD274-APC (anti-PD-L1) or anti-CD3-FITC, anti-CD8-PerCP-Cy5.5, anti-CD279-APC (anti-PD-1), or anti-CD223-PE (anti-LAG3). Cells were analyzed by FCM (Beckman CytoFLEX, USA). Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
Different organs isolated from immunized mice were fixed in 4% phos-phate-buffered formalin for 24 h and embedded in paraffin. The tissue sections were stained with H&E. The cellular composition of mouse blood was analyzed by an automatic hematology analyzer (Mindray, China). For immunohistochemical detection of CD8+ T cell infiltra-tion, tumor tissue sections were stained with anti-CD8a (rabbit mAb, D4W2Z XP, Cell Signaling Technology, USA). The integral op-tical density (IOD) of tumor-infiltrating CD8+ T cells was analyzed by Image Pro-Plus software (Media Cybernetics, Silver Spring, MD, USA).