Rm134l

A shaved area on the back and both surfaces of both ears of mice were painted with 150 µl and 20 µl of 0.5% FITC isomer I, respectively. Twenty-four hours later, inguinal, axillary and submaxillary LNs were harvested and pooled. LN single-cell suspensions were prepared and incubated with anti-mouse CD16/CD32 mAb for 15 minutes on ice. The cells were then stained with APC-conjugated MHC-class II mAb, PE-Cy7-conjugated anti-mouse CD11c mAb, and PE-conjugated mAb for CD86 (GL-1, Biolegend), CD40 (3/23, Biolegend) or OX40L (RM134L, Biolegend) for 20 minutes on ice. The expression levels of CD86, CD40 and OX40L on 7-AAD-negative MHC Class II^hi CD11c⁺ FITC⁺ cells were determined using a FACS Verse (BD Biosciences), and data analyses were performed using FlowJo software (Tree Star Inc.).

For the alloantigen-specific system, 10×10⁴ CD4⁺Foxp3⁻ cells derived from Foxp3^RFP x IL-10^GFP double reporter mice (C57BL/6 background) or Foxp3^hCD2 (BALB/c background) were plated with either mTECs (APC to T cell ratio 1:50 if not indicated otherwise) or DCs (APC to T cell ratio 1:10 if not indicated otherwise) in presence of 100 ng/ml recombinant mouse IL-2 (R&D systems) for six days. For in vitro blocking experiments, titrated amounts of anti-CD40 (HM40-3, Biolegend), anti-CD70 (FR70, Biolegend), anti-CD137 (TKS-1, Biolegend) and anti-OX40L (RM134L, Biolegend) was used. At the end of the cultures, allo-iTregs were either directly phenotypically analyzed or sorted as CD4⁺CD90⁺Foxp3⁺ cells for subsequent analyses on FACS Aria II using the Foxp3 reporter molecules RFP or hCD2.