Fig. 1. An anti-GARP:TGF-β1 mAb that blocks TGF-β1 activation by mouse Tregs in vitro.

a 293T cells were transfected with constructs encoding HA-tagged mouse GARP, mouse TGF- β1 (i.e. LAP + mature TGF- β1), or both, to induce surface expression of free GARP, free latent TGF- β1, or GARP:TGF- β1 complexes, respectively (Cuende et al.²¹). Transfected cells were stained with the indicated antibodies and analyzed by flow cytometry. Histograms are gated on live cells. b Mouse splenocytes were stimulated (stim) or not with anti-CD3/CD28 coated beads during 24 h, then analyzed by flow cytometry after staining with anti-CD4 and anti-FOXP3 antibodies in the presence (+) or absence (−) of the biotinylated anti-GARP:TGF-β1 clone 58A2, followed by streptavidin coupled to BV421. Histograms in red are gated on CD4⁺FOXP3⁺ cells (Tregs), in blue on CD4⁺Foxp3^- cells (Teff). c Magnetically-sorted CD25⁺ mouse splenocytes were stimulated during 24 h with anti-CD3/CD28 coated beads in the presence or absence of blocking antibodies against active TGF- β (clone 1D11), GARP:TGF- β1 complexes (clone 58A2, mIgG2a WT or FcD), or an isotype control (mIgG2a WT), then analyzed by Western blot with antibodies against β-ACTIN and pSMAD2, as a read-out for active TGF-β1 production. Bar graphs on the bottom show quantification of ECL signals (ratio of pSMAD2/β-ACTIN signals relative to that in cells stimulated in the absence of blocking mAb). Full scans are shown in Supplementary Fig. 14.