Isolation of Splenocytes, DCs and CD4+ T Cells

Single cell suspensions of splenocytes were generated by rinsing spleens with erythrocyte lysis buffer and washing with PBS supplemented with 2% FCS and 2 mM EDTA. For the isolation of CD11c⁺ DCs, splenocytes were separated from contaminating superparamagnetic splenic red pulp cells (32 (link)) by using the AutoMACS Pro (Miltenyi Biotec, Bergisch Gladbach, Germany) before using MACS CD11c Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s recommendations. CD4⁺ T cells were isolated from splenocytes using the CD4⁺ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol, followed by anti-CD4 staining and cell sorting using an Aria II Cell Sorter (BD Biosciences, Heidelberg, Germany).