(A) A flow cytometric dot plot showing the gating of CD4⁺Foxp3^– and CD4⁺Foxp3⁺ T cells. (B–D) Binding of anti-TNFRSF25 antibody (B and C) or sTL1A (D) to CD4⁺Foxp3^– and CD4⁺Foxp3⁺ T cells. Splenocytes from Foxp3-GFP knockin mice were incubated with anti-FcγRII/III (2.4G2) and then phycoerythrin-conjugated anti-CD4 mAbs. After washing, cells were incubated with affinity-purified biotinylated goat anti-mouse TNFRSF25 IgG (R&D Systems) or normal biotinylated goat IgG for 40 minutes at 4°C. Binding was detected using allophycocyanin-conjugated streptavidin. Histograms are electronically gated on CD4⁺GFP^– or CD4⁺GFP⁺ cells. To detect TNFRSF25 intracellularly, cells were permeabilized using Cytofix/Cytoperm solution (BD Biosciences). For staining with sTL1A, cells were first incubated with anti-FcγRII/III and then phycoerythrin-conjugated anti-CD4 mAbs. After washing, cells were incubated (40 minutes, 4°C) with affinity-purified sTL1A, which consists of an N-terminal, rat CD4 domains 3 and 4 tag, and the extracellular domain of mouse TL1A (6), or with bovine serum albumin as a control. Binding was detected using allophycocyanin-conjugated mouse anti-tag mAb (OX68). Histograms in B–D are representative of 4 independent experiments in which anti-TNFRSF25 antibody or sTL1A were used at 10 μg/ml. (E) Anti-TNFRSF25 antibody binds to full-length TNFRSF25 and the short variant lacking the fourth extracellular cysteine-rich repeat. NIH3T3 cells transduced with an “empty” recombinant retrovirus or recombinant virus that encodes full-length TNFRSF25 or the short TNFRSF25 variant were stained with anti-TNFRSF25 or control antibody as described above.