Pe anti mouse cd8a

The mouse splenic suspension was obtained by forcing the spleen through a 200-mesh stainless steel mesh and prepared in PBS. The suspension was centrifuged for 3 min at 4 °C with 900 rpm and then the Tris-NH₄Cl red cell lysis solution was added to remove red blood cells. The solution was centrifuged again and the supernatant was discarded. After gently washed with PBS, the splenic cell pellets were collected. The isolated splenocytes were suspended in sterile PBS and stained with APC anti-mouse CD3ε, FITC anti-mouse CD4 or PE anti-mouse CD8a antibodies (BioLegend Inc, San Diego, CA, USA) for 30 min at 4 °C in the dark, washed with PBS and detected by MACSQuant™ flow cytometer (Miltenyi Biotec Co. Ltd., Bergisch Gladbach, Germany). The data were analyzed with FlowJo software (FlowJo VX, Tree Star Inc., Ashland, OR, USA).

Cell suspensions were counted and incubated in 1% BSA/DPBS for 30 min at 4 °C to block non-specific binding. The cells were incubated with FITC anti-mouse CD45 (157,214, Biolegend), APC-anti-mouse CD3 (100,236, Biolegend), PE/CY5.5 anti-mouse CD4 (100,434, Biolegend), and PE anti-mouse CD8a (100,708, Biolegend) and subsequently permeabilized with a suitable buffer (Biyuntian, China) ; moreover, they were incubated overnight with PE anti-mouse Foxp3 (126,404, Biolegend) and BV421 anti-mouse T-bet (644,815, Biolegend) antibodies at 4 °C for intracellular staining. To analyze cytokine secretion, the cells were stimulated with Monensin (420,701, Biolegend) and a cell activation cocktail (423,301, Biolegend), followed by surface staining, fixation, and permeabilization as described. The cells were the incubated overnight with PE anti-mouse INF-γ (505,808, Biolegend), BV421 anti-mouse IL-4 (504,119, Biolegend), and PE/CY7 anti-mouse IL-17 A (506,922, Biolegend) antibodies at 4 °C. The data was analyzed using Flowjo software (Tree Star Inc., San Carlos, CA).

100 μL of peripheral blood sample was lysed by RBC Lysis Buffer, cells were collected and suspended in Cell Staining Buffer (BioLegend, Cat# 420201). Flow cytometry was performed performed on FACS Aria III (Becton Dickinson) using standard methods. For detection of surface antigens, cells were washed and stained with saturating amounts of antibodies conjugated with fluorescein in the presence of FcR-specific blocking antibody for 20 min on ice. Antibodies used were as follows: Alexa Fluor-488 anti-mouse Gr-1 (Biolegend, Cat# 108417, clone: RB6-8C5, 1:100), PE anti-mouse CD19 (Biolegend, Cat# 152408, clone: 1D3/CD19, 1:100), Alexa Fluor-647 anti-mouse CD3ε (Biolegend, Cat# 100322, clone: 145-2C11, 1:100), PE anti-mouse CD8a (Biolegend, Cat# 100708, clone: 53-6.7, 1:100), Alexa Fluor-488 anti-mouse CD4 (Biolegend, Cat# 100423, clone: GK1.5, 1:100), Alexa Fluor-647 anti-mouse c-Kit (Biolegend, Cat# 105818, clone: 2B8, 1:100), APC anti-mouse CD25 (Biolegend, Cat# 102012, clone: PC61, 1:100), PE/Cy5 anti-mouse/human CD44 (Biolegend, Cat# 103010, clone: PC61, 1:100), Alexa Fluor-647 anti-mouse/human Foxp3 (Biolegend, Cat#320013, clone: 150D, 1:100), PE anti-mouse/human CCR4 (Biolegend, Cat# 131203, clone: 2G12, 1:100). The data were analyzed by FlowJo_V10 software. The gating strategy are shown in Supplementary Fig. 6.

Pancreatic lymphocytes were isolated after pancreas perfusion and cells were stained using PE anti-mouse CD8a and FITC anti-mouse CD69 (Biolegend), as detailed in the ESM Methods. Populations were determined by using BD FACSCanto II and BD FACSDiva software (BD Biosciences).

4- to 6-week-old female BALB/c mice were used for the breast tumor-bearing model. Subcutaneous tumor models were established by inoculating 5 × 10⁵ 4T1 cells into the right flanks of the BALB/c mice. When tumor size reached approximately 100–200 mm³ in volume, the mice were administered physiological saline, PLP (10 mg/kg/d, s.c.) once every 24 h, and TAXOL^® (Harbin Pharmaceutical Group Holding Co., Ltd., Heilongjiang, China, 6 mg/kg, i.p.) and TAXOL^®+PLP once every 4 days. Mice were closely monitored every other day for pain/distress, tumor volume, and body weight. The tumor volume was calculated using the following formula: mm³ = (longest diameter × shortest diameter²)/2. Mice were sacrificed at Day 22. Tumors were removed and weighed, and tumors, spleens, and inguinal lymph nodes were excised for flow cytometer analysis of immune cell subpopulations. Cells were proceeded to be incubated with FITC anti-mouse CD11c, APC anti-mouse CD3, PE anti-mouse CD8a, PerCP/Cyanine5.5 anti-mouse CD4, FITC anti-mouse IFN-γ, PE/Cyanine7 anti-mouse CD11b, and PE anti-mouse F4/80 antibodies (Biolegend, United States) for 30 min at 4°C in the dark. Cellular supernatant was collected for IFN-γ ELISA analysis.

Chloroform, PVA, PLGA, PLL, and poly(I:C) were obtained from Sigma-Aldrich (St Louis, MO, USA). The following individual primary antibodies were purchased: anti-mouse PD-L1 (Cell Signaling, Danvers, MA, USA) and anti-mouse CD8a (eBioscience, San Diego, CA, USA). PE anti-mouse CD8a, FITC anti-mouse CD8a, FITC anti-mouse PD-L1, and APC anti-mouse INF-γ antibodies were obtained from BioLegend (San Diego, CA, USA). Recombinant murine IL-2 was obtained from PeproTech (Cranbury, NJ, USA). All oligoes were purchased from Bioneer Co. (Tae-Geon, Korea) and the sequences of siRNA were as follows: 5′-GCAGUGACCAUCAAGUCCUdTdT-3′ (human sense siPD-L1), 5′-dTdTAGGACUUGAUGGUCACUGC-3′ (human antisense siPD-L1), 5′-CCUACGCCACCAAUUUCGUdTdT-3′ (scrambled sense siRNA), 5′-dTdTGGAUGCGGUGGUUAAAGCA-3′ (scrambled antisense siRNA).