Nk1.1 fitc

Single-cell suspensions of splenocytes were obtained by mechanic dissociation of the spleen, while the single-cell suspensions of PECs were obtained following peritoneal lavage. RBC lysis was performed for 5 min for the single-cell suspensions of splenocytes. Cells were subsequently stained with a cocktail of antibodies. The following anti-mouse antibodies were used: CD11b FITC (101206, BioLegend), CD11b PE (101207, BioLegend), CD11c APC (117309, BioLegend), CD19 FITC (115505, BioLegend), F4/80 PE (123109, BioLegend), Ly6G Pacific Blue (127611, BioLegend), NK1.1 FITC (108705, BioLegend), and TCR-β FITC (109205, BioLegend). Splenic CD11b⁺ innate immune cells were sorted as CD19^-TCR-β^-NK1.1^-Ly6G^-CD11b⁺, while peritoneal macrophages were sorted as F4/80⁺CD11c^-Ly6G^-CD11b⁺ (Supplementary Figure 13). Splenic CD11b⁺ innate immune cells and peritoneal macrophages were sorted by BD FACSMelody™ (Becton Dickinson) and stimulated with 50 ng/ml LPS for 2 h 45 min at 37°C/5% CO₂ followed by RNA isolation.

The following directly labeled anti-mouse monoclonal antibodies were used for BM cell phenotypical analysis: CD90.2-APC and CD45-PE/Cy7 for lymphoid progenitors, CD61-APC and CD41-FITC for megakaryocytic population, CD71-PE and Ter119-FITC for erythroid precursors, CD11b-PE and Gr1-FITC for granulocytes/monocytes progenitors, Lineage Cocktail (CD3, Gr1, CD11b, CD45R, Ter119)-FITC, Sca1-PE, cKit (CD117)-APC for hematopoietic stem cells, all purchased from BioLegend (BioLegend, San Diego, CA, USA).
The phenotypical analysis of splenocytes was performed using the following anti-mouse antibodies: CD4-PE/Cy5, CD8a-PE (BioLegend) for helper and cytotoxic T cells, CD19 (BioLegend) for B cells, CD11c-PE, I-Ab-FITC, and TLR4 (CD284)-PE/Cy7 (all from BioLegend) for dendritic cells (DCs), and NK1.1-FITC (BioLegend) for NK cells. To detect proliferative cells, Ki67-eFluor660 (eBioscience, San Diego. USA) was used.
Single-cell suspensions of splenocytes or BM cells were incubated with the fluorescently labeled antibodies in PBS containing 1% BSA, at 4°C for 20 min for cell surface staining. For intracellular staining (Ki67), cells were permeabilized using the Foxp3 Fix/Perm Buffer (eBioscience), according to the manufacturer’s instructions. Measurements were performed with a FACSCalibur flow cytometer as described above.

The cells were extracted from lymph nodes, spleen, or colon tissue digested by collagenase. Isolated cells were stained with following antibodies: CD8-PE-CY7 (Thermo Scientific), CD4-APC-Fire™ 750 (BioLegend), IFN-γ-FITC (BioLegend), IL-17-APC (BioLegend), FOXP3-PE (BioLegend), CD19-PE (BioLegend), NK1.1-FITC (BioLegend), CD11B-APC (BioLegend), Gr-1-FITC (BioLegend), F4/80-PE (BioLegend), CD11C-APC-CY7 (BioLegend), MHC-II-PE/Dazzle™ 594 (BioLegend), CD8-APC (BioLegend), Ki67-PerCP-CY5.5 (BioLegend), Annexin V-FITC (BioLegend), CD103-PE (BioLegend), CD69-PE-CY7 (BioLegend), CD44-PE-CY7 (BioLegend), CD62L-FITC (BioLegend), CD107-FITC (BioLegend), IFN-γ-APC (BioLegend), CD107-PE (BioLegend), IFN-γ-PE (BioLegend), CD3-FITC (BioLegend), CD3-APC/Fire™ 750 (BioLegend), CD8-APC (BioLegend), IFN-γ-PE-CY7 (BioLegend), CD69-APC/Fire™ 750 (BioLegend), Tetramer-SIINFEKL-PE (MBL), p-JNK-PE (Cell Signaling Technology), p-IRAK4-Alexa Fluor 488 (Cell Signaling Technology). Intracellular staining was carried out using the Cytofix/Cytoperm kit (BD Pharmingen) following a 4 h restimulation by PMA/ionomycin (Sigma) in the presence of GolgiPlug (BD Pharmingen). Flow cytometric data acquisition was performed on a NovoExpress flow cytometer and analyzed with NovoExpress software (ACEA Biosciences).

Tumors were harvested and resuspended into single-cell solution in PBS as described above. Murine spleens were mashed through a 70 μm filter into a ACK lysing buffer (Gibco, #A1049201) and incubated for 3 minutes. A total of 10 mL of PBS was added after the RBC lysis and the suspension was centrifuged at 1,500 rpm for 5 minutes. The supernatant was discarded and the pellet was resuspended in PBS. After Fc blocking (BioLegend, 101319; 1:100) and Live/Dead staining with zombie NIR (BioLegend, #423105; 1:1,000) was performed during a 10-minute incubation at 4°C, the samples were washed with PBS. Conjugated antibodies including CD45-BV711 (BioLegend, #103147; 1:200), CD4-PE (BioLegend, #100407; 1:100), CD8-Pe-Cy7 (BioLegend, #100721; 1:100), NK1.1-FITC (BioLegend, #108705; 1:100), B220-Pe-Cy5.5 (BioLegend, #103209; 1:100), CD11b-APC (BioLegend, #101211; 1:200), F4/80-BV785 (BioLegend, #123141; 1:200), GR1-Pe Tx red (BD Biosciences, # 562710; 1:200) were incubated with each sample at 4°C in the dark for 20 minutes and washed with FACS buffer (2% FBS in PBS). The samples were analyzed using BD LSRFortessa X-20 flow cytometer. Collected flow cytometry data were analyzed using FlowJo software.