Isolation and In Vitro Assay for Murine T-Cell Subsets

Spleen and LN cells were harvested from WT or immunized mice. Single cell suspensions were prepared, and RBCs were lysed using Ack lysis buffer (ThermoFisher scientific A1049201). Total CD4⁺ (Miltenyi 130–049-201) and CD8⁺ (Miltenyi 130–049-401) T cells were positively purified using Miltenyi kits following established manufacturer protocol followed by FACS sorting. Similarly, antigen presenting cells were isolated using Miltenyi Pan Dendritic Cell (DC) Isolation Kit (Miltenyi 130–100-875). Post CD4⁺ T cell enrichment, cells were counted and labelled with CellTrace™ Violet Dye (ThermoFisher scientific C34557) according to manufacturer instructions. In vitro proliferation/suppression assays were set up according to previously published protocol^{47 (link)}. Briefly, labelled CD4⁺ T cells were co-cultured either with CD8⁺ T cells (1:1 ratio, 0.25X10⁶ cells/well) or without CD8⁺ T cells in the presence of pan DC’s (0.75X10⁶ cells/well). In some of the suppression experiments cells were pre-included with 10ug/ml of anti-Qa-1b neutralizing antibody (6A8.6F10.1A16, BD Biosciences)^{22 (link),48 (link)}. Cells were cultured in a total volume of 200 ul in a 96-well round bottom plate. The CD4⁺ T cells were stimulated with either MOG_35–55 or not. On day 7, the cells were washed and stained with surface antibodies and analyzed on LSR II (Becton Dickinson).