Cd8α b220 mhc 2 microbeads

Splenic APCs from WT or p110δ^KD mice were isolated by negative selection using CD90.2 microbeads (Miltenyi Biotec, Auburn, CA). Splenic APCs were incubated overnight with CBL (50 ng/ml), and, after washing several times to remove extracellular antigen, APC were co-cultured with negatively-selected CD4⁺ T cells (CD8α/B220/MHC II microbeads, Miltenyi Biotec, Auburn, CA) from WT or Il10^-/- mice at a 3:2 ratio (APC:T cell) for 72 hours. For antigen specific studies, APCs were incubated overnight with LPS (10 ng/ml) and OVA peptide (323-339, 5 μM) (13 (link)). After washing to remove extracellular antigen, APC were co-cultured with negatively-selected CD4⁺ T cells (CD8α/B220/MHC II microbeads, Miltenyi Biotec, Auburn, CA) from mice expressing a transgenic TCR that recognizes OVA epitope residues 323-339 (OT-II mice) at a 3:2 ratio (APC:T cell) for 72 hours. CD4⁺ T cells were analyzed for intracellular cytokine expression (IFN-γ and IL-17A) by flow cytometry.

T cell mediated colitis was induced in Rag1^-/- and Rag1^-/-/p110δ^KD (RKO/δ^KD) mice at 8 weeks of age as described previously (18 (link)). CD4⁺ T cells were isolated by negative selection (CD8α/B220/MHC II microbeads, Miltenyi Biotec, Auburn, CA) and stained with FITC-conjugated anti-CD4 (Clone GK1.5, eBioscience, San Diego, CA) and PE-conjugated anti-CD45RB (Clone 16A, BD Pharmingen, San Jose, CA). CD4⁺ T cells were sorted into CD45RB^high and CD45RB^low populations using a MoFlo™ XDP Cell Sorter (Beckman Coulter, Brea, CA). Mice were i.p. injected with 4 × 10⁵ CD4⁺CD45RB^high cells admixed with 2 × 10⁵ CD4⁺CD45RB^low cells as described (18 (link)). Clinical scores were assigned as described (19 (link)).