Cd4 pacific blue clone gk1

Spleens were smashed and filtered through a 40 μm gauze (BD Biosciences). Single-cell suspensions were stained for flow cytometry with the following antibodies and analyzed according to the gating strategy shown in (Supplementary Fig. 5b): CD4 Pacific blue (clone GK1.5, Biolegend, dilution 1:800), CD25 PE-cy7 (clone PC61, Biolegend, dilution 1:400), and FoxP3 AlexaFluor647 (clone MF-14, Biolegend, dilution 1:300) were used in combination with a FoxP3 staining kit (eBioscience).

For phenotypical analysis of peripheral leukocytes, spleen samples were dissolved to single cell suspension, erythrocytes were lysed and cell numbers were determined as described previously^{25 (link)}. After blocking the FC-receptor II/III by preincubation with a CD16/CD32-specific Ab (clone 2.4G2; BioXCell), dead cells were stained using the LIVE/DEAD® fixable dead cell stain kit (Invitrogen^TM GmbH). Subsequently, CD4- (PacificBlue; clone GK1.5; BioLegend), CD8α- (HV500; clone 53-6.7; BD Biosciences), CD19- (PE-eFluor610; clone 1D3; eBioscience/Thermo Fisher Scientific), CD69- (FITC; clone H1.2F3; BioLegend) and CD44-specific Ab (APC; clone IM7; BioLegend) were added for fluorochrome-conjugated surface marker staining. For intracellular staining of Foxp3 the Foxp3 Transcription Factor Staining Buffer Set (eBioscience/Thermo Fisher Scientific) was used according to the manufacturer’s instructions. Samples were acquired with an LSRII SORP cytometer (BD Biosciences) and analyzed using FlowJo software version 9.6.4 (Tree Star, Ashland, USA). Blood samples were stained according to the same procedure.