Tcrβ fitc

After the mice were sacrificed, the tumors were collected and digested in dissociation buffer [mixture of deoxyribonuclease (50 μg mL^–1), hyaluronidase (100 μg mL^–1), and collagenase IV (2 mg mL^–1)] at 37 °C for 1 h with gentle shaking. The cell suspension was passed through a 70 μm cell strainer and dispersed in PBS buffer to form single-cell suspension. For T lymphocytes detection, the single-cell suspension was stained with antibodies against CD3, CD4 and CD8 to mark the helper (CD3⁺ and CD4⁺) and cytotoxic (CD3⁺ and CD8⁺) T cells. The tumor-cell suspension was stained with antibodies against CD11b, F4/80, CD86, and CD206 to analyze M1-like (CD11⁺ and CD86⁺) and M2-like (F4/80⁺ and CD206⁺) macrophages. The mice’s blood and spleen were also collected for lymphocyte extraction using lymphocyte isolation kit (Solarbio) according to the manufacturer’s manual. NK cells (CD3⁻, CD49b⁺), γδ T cells (CD3⁺, TCRβ⁻), and memory T cells (CD8⁺, CD44^high, CD62L^low) were analyzed using flow cytometer. The anti-mouse of CD3-PE (clone: 17A2), CD4-APC-cy7 (clone: GK1.5), CD8a-FITC (clone: 53-6.7), CD11b-PE (clone: M1/70), F4/80-APC (clone: BM8), CD86-PE-Cy7 (clone: GL-1), CD206-FITC (clone: C068C2), CD49b-APC (clone: HMα2), TCRβ-FITC (clone: H57-597), CD44-PE (clone: IM7), and CD62L-APC-Cy7 (clone: MEL-14) were purchased from Biolegend.

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.

BSLs purified from perfused brain tissue of moribund C57BL/6 mice on day 6 post-infection were stained with TCRβ-FITC, CD3ε-PE, CD4-PerCP, CD4-PerCP/Cy5.5, CD8-APC/Cy7, CD11b-APC, CD14-PE/Cy7, and F4/80-Brilliant Violet 421™ antibodies purchased from Biolegend (San Diego, CA) and fixable viability dye eFluor^®506 (eBioscience, San Diego, CA). An Amnis^® ImageStream^®X MKII (Amnis Merck-Millipore, Seattle, WA) equipped with five lasers (355, 405, 488, 561, and 642nm) was used for acquisition. A minimum of 30,000 events were collected at 40X magnification using the INSPIRE^® software (Amnis Merck-Millipore, Seattle, WA). Analysis was performed using IDEAS® 6.1 software (Amnis Merck-Millipore, Seattle, WA). Focused events were first gated using the Gradient RMS feature. Singlets were then gated by aggregate discrimination using aspect ratio and area. Analysis was then performed on gated live CD11b^highCD14⁺F480⁺TCRβ⁺CD3ε−CD4−CD8− cells.