Anti mouse cd3

A flat bottom 96-well plate was coated overnight with anti-mouse CD3 (eBioscience, ThermoFisher, Carlsbad, CA, USA) in PBS (10 µg/mL for IL-2/2 µg/mL for IFN-γ and IL-10 ELISA) or PBS at 4 °C. Isolated memory CD4⁺ T cells in T cell medium (5 × 10⁵ cells/mL) were incubated for 1 h with DMSO, 50 µM, and 100 µM Ned-19 in DMSO at 37 °C and 5% CO₂. Meanwhile, the plate was washed twice with PBS. Thereafter, 1 µg/mL soluble anti-mouse CD28 (eBioscience) was added to those cell suspensions that would be seeded to wells coated with anti-mouse CD3 (for IL-10 and IFN-γ ELISA). Thereafter, the plate was seeded with cells (10⁵ cells/well) and the plate was incubated at 37 °C and 5% CO₂ for 48 h (96 h for IL-10 and IFN-γ ELISA). The supernatant was collected and stored in the freezer at −80 °C until the ELISA was performed. The ELISA was performed using IL-2 Mini ABTS ELISA Development Kit/Murine IL-10 Mini TMB ELISA Development Kit/Murine IFN-γ Standard TMB ELISA Development Kit (all from Peprotech, Carlsbad, CA, USA). TMB one-component HRP Microwell Substrate (Surmodics, Eden Prairie, MN, USA) was used as substrate. The plate was read at 620 nm (for IL-2 ELISA)/450 nm with wavelength correction set at 620 nm (for IL-10 and IFN-γ ELISA) using a CLARIOstar microplate reader from BMG Labtech (Ortenberg, Germany).

Mouse spleens were homogenized into single-cell suspensions by digesting in PBS containing 0.1% BSA and 0.6% Na-citrate, washed, and incubated with 120 Kunitz units of DNase I for 15 min following red blood cell lysis (eBioscience, 00433357)^{12 (link)}. Cells were then filtered through a 30-µm cell strainer to generate a single-cell suspension. CD4⁺ and CD8⁺ T cells were isolated using CD8a (Miltenyi Biotec, 130-104-075) and CD4 (Miltenyi Biotec, 130-117-043) T cell isolation kits, respectively. T cells were maintained at 2.5 × 10⁶ cells/ml in RPMI-1640 medium supplemented with 10% FBS and 1% P/S. T cells were activated using 1.25 µg/ml anti-mouse CD3 (Fisher Scientific, 16-0031-85) and 2 µg/ml anti-mouse CD28 (Fisher Scientific, 16-0281-82) antibody treatment for 2d. Recombinant mouse midkine (R&D Systems, 9760-MD-050) was added, and Ccl4 levels were quantitated by ELISA (R&D Systems, MMB00). Conditioned medium was collected from activated T cells for chemokine assays and microglia co-culture experiments. Cell viability was measured using a commercial WST-1 cell viability assay (Millipore).

4–6-week-old C57BL/6J and Rag1^−/− mouse spleens were homogenized into single cell suspensions by digestion in PBS containing 0.1% BSA and 0.6% sodium citrate. The homogenates were subsequently washed and incubated with 120 Kunitz units of DNase I for 15 min following red blood cell lysis (eBioscience). Cells were then filtered through a 30 µM cell strainer to obtain a single cell suspension. CD4⁺ and CD8⁺ T cells were isolated using CD8a (Miltenyi Biotech) or CD4 (Miltenyi Biotech) T cell isolation kits, respectively. T cells were maintained at 2.5 × 10⁶ cells ml⁻¹ in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/ streptomycin. T cells were activated by 1.25 µg ml⁻¹ anti-mouse CD3 (Fisher Scientific) and 2 µg ml⁻¹ anti-mouse CD28 (Fisher Scientific) antibody treatment for 48 h. T cell conditioned media (TCM) was collected both from non-activated and activated T cells following 22 µM filtration for subsequent chemokine assay, ELISA and co-culture experiments.