Cd3e clone 145 2c11

Bone marrow cells were isolated from femurs, tibiae and iliac crests with PBS supplemented with 2% FCS and strained through 70 μm meshes. Red cells were lysed by resuspending the cells in 10 mL red cell lysis buffer (130-094-183, MACS Miltenyi Biotec) for 10 min at room temperature. After centrifugation, the cell pellet was resuspended in PBS supplemented with 2% FCS and nucleated cells were counted with 3% acetic acid on a Vi-Cell XR cell viability counter (Beckman Coulter). 10 × 10⁶ bone marrow cells were resuspended in 200 μL of PBS supplemented with 2% FCS containing the following antibody solution: FITC-conjugated lineage cocktail with antibodies against CD4 (clone H129.19, BD PharMingen), CD3e (clone 145-2C11, eBioscience), Ly-6G/Gr-1 (clone RB6-8C5, eBioscience), CD11b/Mac-1 (clone M1/70, BD PharMingen), CD45R/ B220 (clone RA3-6B2, BD PharMingen), FcεR1α (clone MAR-1, eBioscience), CD8a (clone 53-6.7, BD PharMingen), CD11c (clone N418, eBioscience), TER-119 (clone Ter119, BD PharMingen); c-Kit (PerCP-Cy5.5, clone 2B8, eBioscience), Sca-1 (PE-Cy7, clone D7, eBioscience), Flt3 (PE, clone A2F10, eBioscience), CD34 (eFluor660, clone RAM34, eBioscience), CD16/32 (BV421, clone 93, BioLegend) and Il-7R⍺ (BV605, clone A7R34, BioLegend).

For single-cell suspensions, white blood cells were obtained from blood after lysis of red blood cells with NH₄Cl. Lung white blood cells were obtained from density gradient centrifuged lung cell suspensions (Percoll 40% and 72%, GE Healthcare). Lymph node cells were extracted from both inguinal lymph nodes. For cytospin preparations, single-cell suspensions were spun on a glass slide and stained with H&E. For flow cytometry, cell suspensions were incubated with anti-CD16/anti-CD32 (Miltenyi Biotec) and stained with the following monoclonal antibodies: CD3e (clone 145-2C11), CD8a (53-6.7), CD25 (PC61.5), CD49b (DX5), CD69 (H1.2F3), CD122 (5H4) (eBioscience, BD Biosciences or BioLegend). Dead cells were excluded using propidium iodide (PI) or Zombie Aqua Fixable Viability Dye (BioLegend). Samples were run with a FACSCalibur using CELLQuest software or an LSR Fortessa X-20 using FACSDiva software (all BD Biosciences). Live singlet cells (PI^- or ZombieAqua^-) were analyzed with FlowJo (Version 10).

Splenocytes were collected from 25-week-old ApoE^–/– mice euthanized after 16 weeks of high-cholesterol diet feeding consisting of 0.15% cholesterol and 21% fat (TD.88137, Envigo). RBC-lysed splenocytes were incubated with 20 μg/mL P210-PAM in complete RPMI 1640 medium for 48 hours, then stained with CD3e (clone 145-2C11, eBioscience), CD4 (clone GK1.5, BD Bioscience), CD8b (clone H35-17.2, eBioscience), CD44 (clone IM7, eBioscience), and CD62L (clone MEL-I4, eBioscience) antibodies for T effector/memory cell profiling using flow cytometry.

Male BALB/c mice aged 6‐8 weeks old were purchased from the Laboratory Animal Center of the Chinese Academy of Medical Sciences (SCXK‐jing‐2014‐0004), Beijing, China. The mice were housed in cages in a temperature‐controlled room under a 12‐hour light/dark cycle for at least 7 days before experiments.
Recombinant murine IL‐38 and anti–IL‐38 antibody were obtained from R&D Systems, Minneapolis, MN. CD4⁺CD25⁺ T cell isolation kit was purchased from MiltenyiBiotec. LPS was purchased from Sigma‐Aldrich, St. Louis, MO. Carboxyfluoresceinsuccinimidyl ester (CFSE) cell proliferation kit was obtained from Invitrogen. Enzyme‐linked immunosorbent assay (ELISA) kits for mouse IL‐10, TGF‐β1, IL‐2, IL‐4 and interferon (IFN)‐γ were obtained from ExCell Biology. Antimouse antibodies against Foxp3 [FJK‐16s, conjugated to phycoerythrin (PE)‐cyanine7], CD152 (CTLA‐4) [UC10‐4B9, conjugated to allophycocyanin (APC)], CD4 [RM4‐5, conjugated to fluorescein isothiocyanate (FITC)], CD25 (PC61.5, conjugated to PE), CD3e (clone 145‐2C11) or CD28 (clone 37.51) were obtained from eBioscience. Anti‐CD25 antibody (purified antimouse CD25 antibody, clone PC61) and rat IgG1 were purchased from Biolegend.