Cd4 apc rm4 5

Spleen and LNs were digested in HBBS (Invitrogen) containing 10% FBS and 0.2 mg/ml collagenase IV (Sigma-Aldrich) for 15 minutes at 37°C. After filtration using 70μm cell strainer (Fisherbrand) to obtain a single cell suspension, red blood cells (RBC) in the spleen were lysed with RBC lysis buffer (eBioscience) for 3 minutes. Brains were similarly digested by a Percoll gradient was used for the isolation of hematopoietic cells previous to the RBC lysis. Briefly, for the brain single cell suspensions were resuspended in a 37% Percoll (GE Healthcare) and underlayed by a 60% Percoll solution. The samples were then centrifuged at 600g for 20 minutes. Hearts were digested in 1mg/ml collagenase IV in 10% FBS HBSS for 30 min at 37°C and similarly filtered through a 70 μM cell strainer and RBC were lysed for 3 min.
The gating strategy used for the flow cytometry analysis is shown in the Supplementary Fig. 6. Samples were stained with: CD45 (30-F11) APC-Alexa780, CD8 (53-6.7)-PerCPCy5.5, CD4 (RM4-5)-APC and eFluro450, CD3e (145-2C11)-PE, H2-Kb (AF6-88.5.5.3)-APC, H2-Kd (SF1-1.1.1)-PE, CD25 (PC6.1)-PerCP-Cy5.5, FoxP3 (FJK-16s)-APC, CD11b (M1/70)-PerCP-Cy5.5, Gr1 (RB6-8C5) PE from eBioscience. DAPI (Sigma-Aldrich) was used to stain dead cells. LSR-Fortessa and LSR-II (BD) were used to acquire the samples and FlowJo® was used to analyze the data.