Cd8 t cell

Sorted WT/IRU CAR-T cells were stimulated with irradiated Naml6-FFLuc-GFP cells for 24 hrs, followed by the enrichment of CD8⁺ T cells (Miltenyi Biotec) (n = 3, 1 biological sample). The purity of CD8⁺ T cells was confirmed by flow cytometry. The total RNA of 1 × 10⁵ isolated CD8⁺ T cells were extracted using Trizol (Invitrogen, Carlsbad) and samples were sent to BGI-Shenzhen following the standard protocol for RNAseq. The fastq files of samples were analysed by STAR (v.2.7.2a) with the reference of GRCh38. The DEGs and enriched pathways were obtained by DEseq2 (v1.34.0) and ClusterProfiler (v4.2.2) respectively. Heatmap of DEGs was visualized by R package pheatmap.

Lymph nodes from Gra6 TN donor mice were harvested and the released cells were negatively selected for CD8 T cells (Miltenyi Biotec). Recipient Thy1.1 (BALB/c) mice received 10⁶ Gra6⁺ CD8 T cells through intravenous injection before infection. Mice were infected orally with five cysts of the ME49 Toxoplasma strain. Cells were harvested at the indicated time‐points during the acute and chronic phases of infection and were processed accordingly.

GYF-21 was isolated from Chinese agarwood and the isolation procedure was described primarily (Huo et al., 2015 (link)). GYF-21 was dissolved at a concentration of 25 mM in dimethyl sulfoxide (DMSO). Cell Counting Kit-8 (CCK-8) was purchased from Beyotime, Co. (Shanghai, China). Lipopolysaccharide (LPS) from Escherichia coli O55: B5 was purchased from Sigma Chemicals, Co. (St. Louis, MO, United States). ELISA kits for determining TNF-α,IL-6, IL-1β, MCP-1, MIP-1α, IFN-γ, and IgG were purchased from R&D Systems (Minneapolis, MN, United States). The antibodies to p65, I-κB, p38, ERK1/2, JNK, STAT1, STAT3, and their phosphorylated forms were purchased from Cell signaling Technology (Beverly, MA, United States). Monoclonal antibodies (mAbs) conjugated to APC, APC, PE, FITC, PE, FITC, FITC, PE, PE-Cy7, Alexa Fluor 647, FITC, and PE (specific for Ly-6G, CD11c, CD11b, CD62L, CD69, CD25, CD80, CD86, CD4, CD8, IFN-γ, and IL-17A), and purified monoclonal antibodies (specific for CD3e, CD28, IFN-γ, and IL-4) were obtained from Becton Dickinson (San Diego, CA, United States). Magnetic bead isolation kit for mouse CD4⁺ T cells, naive CD4⁺ T cells and CD8⁺ T cells were purchased from Miltenyl Biotec (Bergisch Gladbach, Germany). Recombinant mouse GM-CSF, IL-4, IL-12, IFN-γ, IL-6, and TGF-β were purchased from PeproTech (Rocky Hill, NJ, United States).

Immunogenicity of mutated RAS peptides was performed using HLA-matched healthy donors’ autologous PBMCs. Briefly, mature DCs were pulsed with individual mutated RAS epitopes at 25 μg/mL and co-cultured with autologous magnetic-sorted CD8+ T-cells (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in RPMI with 5% human male AB serum, 10 units/mL penicillin–streptomycin, 2 mmol/L L-glutamine, 1% nonessential amino acid, IL-6 (1000 U/mL) and IL-12 (5 ng/mL). IL-2 (30 U/mL) and IL-15 (5 ng/mL) were added every 2 days. CD8+ T-cells underwent another 3 rounds of stimulation with peptide-pulsed CD14+ monocytes on day 7, 14 and 21. Seven days after the last stimulation, CD8+ T-cells were challenged by overnight incubation with peptide-loaded T2 cells or autologous monocytes. As a control, CD8+ T-cells were incubated with T2 cells or monocytes without peptide. Reactivity of the T cells was determined by intracellular IFN-γ staining measured by flow cytometry. In some experiments, cells were also stained with specific tetramers (BioLegend).