Cd8 apc

Single-cell suspensions of the lung tissues were prepared by cutting them into small pieces followed by incubation in Dulbecco’s Modified Eagle Media (DMEM) containing 0.18 mg/mL Collagenase Type I (Sigma, St. Louis, MO, USA), 0.02 mg/mL DNase I (Sigma, St. Louis, MO, USA) for 1 h at 37 °C under constant rotation, followed by being mechanically passed through a 100 μm and 70 μm cell strainer sequentially. Erythrocytes were lysed using RBC lysis buffer (155 mM NH₄Cl, 12 mM NaHCO₃, 0.1 mM EDTA). Cells were then counted and subjected to flow cytometry. Lymphoid and myeloid compartments were investigated in the lung samples of mice on various intervention diets. Antibodies used for flow cytometry analysis were as follows: CD64-PeCy7 (Clone X54-5/7.1), Ly6C-PerCPCy5.5 (Clone AL-21), CD11b-V450 (Clone M1/70), MHCII-APC (Clone M5/114.15.2), CD103-PE (Clone M290), CD11c-A700 (Clone HL3), SiglecF-APCCy7 (Clone E5-2440), Ly6G-FITC (Clone 1A8), PD-1-FITC (Clone 29F.1A12), CD4-BV510 (Clone RM4-5), CD44-PE (Clone IM7), NK1.1-APCCy7 (Clone PK136), CD3-A700 (Clone 500A2), CD62L-V450 (Clone MEL-14), CD19-PerCPCy5.5 (Clone 1D3), CD8-APC (Clone 53-6.7), and KLRG1-BV786 (Clone 2F1) purchased from BD (Biosciences, Johannesburg, SA) and eBioscience (ThermoFisher, Johannesburg, SA) [29 (link),30 (link)].

For T-cell activation assays, lymphocytes in mixed splenocyte suspensions were counted using a haemocytometer and 2 × 10⁵ cells plated per 96 well, in complete RPMI media (RPMI with 10% heat inactivated (HI) foetal bovine serum (FBS), 100 U/mL penicillin and streptomycin, and 5 µM 2-mercaptoethanol (all Invitrogen)) supplemented with or without soluble 2 µg/mL anti-CD3e (clone 145-2C11) and 2 µg/mL anti-CD28 (clone 37.51), to activate T cells. Cells were incubated for 24, 48, and 72 h in a humidified 37 °C, 5% CO₂ incubator. To assay for activation markers, cells were analysed by flow cytometry with antibodies to CD4-PE, CD8-APC, CD25-PECy7, and CD69-FITC (clone H1.2F3, eBiosciences). See Supplementary Fig. 1d and e for representative FACS plots. For cytokine analysis, CD4⁺ T cell from spleen were FACS sorted and plated as above. Cells were incubated for 24 and 48 h, and then supernatants collected for cytokine analysis using ELISA assays for IL-2, IFN-γ, as per the manufacturer’s instructions (eBioscience).

To examine the effect of CS exposure on the population of immune cells infiltrated in colonic LP. LP cells were separated into single cells using gentle MACS^TM dissociator. Single cells were stained to identify myeloid cells, CD4 T cells, CD8 T cells, B cells, macrophages, and dendritic cells respectively. Live leukocytes were identified based on CD45 gate. The following antibodies were used: CD11b-APC (17-0112-81, e-bioscience), CD4-FITC (53-0041-82, e-bioscience), CD8-APC (17-0081-81, e-bioscience), B220-PE (553089, BD Pharmingen), F4/80-PE (123110, BioLegend), CD11c-PE (12-0114-82, e-bioscience), and CD45-FITC (103108, BioLegend). The stained cells were detected by a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA).

T cells were washed with PBS containing 2% FBS prior to the addition of antibodies. Cells were stained with fluorophore-conjugated antibodies specific for mouse CD3-AF700 (BD Biosciences, San Jose, CA, USA), CD8-APC (eBioscience, Waltham, MA), CD27-PE (BD Biosciences, San Jose, CA, USA), and CD44-BV421 (BD Biosciences, San Jose, CA, USA) in PBS with 2% FBS for 15 min at 4 °C. Cells were then fixed in 4% paraformaldehyde for 10 min at 4 °C and washed twice in PBS with 2% FBS. Flow cytometry was performed on a Canto II flow cytometry system (BD Biosciences, San Jose, CA) 32 (link).

Tumors were minced into thin pieces and were dissociated in DNase I (1 U/ml; Roche, Indianapolis, IN, USA), collagenase D (1 mg/ml; Roche), and 0.125% trypsin-EDTA (Gibco) in pre-warmed DMEM (Welgene) for an hour at 37 °C with gentle agitation. The tissues were mechanically dissociated on a 100 μm nylon mesh strainer. Next, the single cells were passed through a 40 μm nylon mesh strainer. Spleens were also mechanically dissociated on 40 μm nylon mesh strainer. RBCs were lysed for 5 minutes in 1X Pharmlyse buffer.
Cells were stained with fluorescently tagged antibodies. All data were detected by a FACSCalibur cytometric system and analyzed by FlowJo software. The following fluorophore-labeled antibodies were purchased from e-bioscience (San Diego, CA, USA): CD45-FITC, CD3-FITC, CD4-PE, CD4-FITC, CD8-APC, CD11b-APC, CD11c-PE, CD25-PE, CD25-APC, B220-FITC, CD19-PE, CD86-APC. Foxp3-Alexa Fluor647 was purchased from BD bioscience, and F4/80-PE and CD206-APC were obtained from Biolegend (San Diego, CA, USA).