GARP and LAP are selectively expressed on activated FOXP3⁺ Tregs and platelets. (A) Level of FOXP3 and GARP mRNA on fresh (0 h) and activated CD4⁺CD25^– T cells and Tregs (CD25^hi) at 12, 24, 120 h, or 120 h with TGF-β1. Tregs were rested until day 14 (0 h) and restimulated for 18 h. (B) Flow cytometric analysis of surface LAP, GARP and intracellular FOXP3 on fresh (0 h) and activated Tregs (CD25^hi) and CD4⁺CD25^int T cells. (C) Surface staining of LAP and GARP on platelets based on FSC/SSC and CD61 expression. (D) LAP and GARP surface staining of plasmacytoid and myeloid DCs from PBMCs by gating on CD303⁺Lin-1^– and CD1c⁺, respectively. (E) Surface LAP and GARP expression on Tregs after 12 h activation in the absence (none) or presence of monensin or brefeldin A for the last 8 h. Data are representative of 3 independent experiments. Numbers indicate percentage in each quadrant for B–E.

Colocalization of GARP and latent TGF-β1 on the surface of activated Tregs. Tregs activated for 48 h from 3 different donors (D1, D2, D3) were surface-stained with anti-GARP and anti-LAP or isotype controls. The cells were imaged with a Leica SP2-AOBS confocal microscope.

Dual functions of TGF-β in Treg-mediated suppression and infectious tolerance mechanism. (A) Tregs were transfected with siNS, siGARP, or siTGFβ1 and preactivated for 24 h before assessing their suppression of the proliferation of CD4⁺CD25^– T cells stimulated with HLA-DR⁺ APCs and soluble anti-CD3 for 3 days and pulsed with ³H-TdR. The top panel is the FOXP3 expression, and the middle panel is the surface GARP and LAP expression of the 3 Treg populations at the end of the 3-day suppression assay. *, P < 0.05 between the siNS and siGARP or siTGFβ1 Treg suppression. (B) Similarly, CFSE-labeled CD4⁺CD25⁻ responders were activated with HLA-DR⁺ APCs and soluble anti-CD3 for 3 days alone (top row) or in the presence of Tregs transfected with siNS, siGARP, or siTGFβ1 at a ratio of 2:1, 4:1, and 8:1 responder:Treg (bottom rows). (C) CFSE-labeled CD4⁺CD25⁻CD127⁺CD45RA⁺ T cells were stimulated with anti-CD3/CD28 Dynabeads for 5 days alone (top panel) or with 24-h preactivated Tregs transfected with siNS, siGARP, or siTGFβ1 and analyzed for FOXP3 induction in the CFSE-labeled cells. Data are representative of 3 independent experiments. Numbers indicate percentage in each quadrant.

Surface expression of latent TGF-β1 requires GARP association. (A) Tregs were transfected with nonspecific (siNS), TGF-β1 (siTGFβ1), or GARP (siGARP) siRNA and rested in IL-2 culture medium for 24 h before stimulation for 48 h to assess surface expression of GARP and LAP. (B) Tregs transfected with TGF-β1 or GARP siRNA were activated for 48 h then incubated for 30 min at 37 °C with 5 μg/mL mature rhTGF-β1, LAP, LAP + TGF-β1, or latent TGF-β1 and then surface stained for LAP. (C) GARP transfected Jurkat cells were incubated for 30 min at 37 °C without (NONE) or with 5 μg/mL LAP, TGF-β1, TGF-β2, or TGF-β3 alone or mixed with LAP and then stained for surface LAP. Jurkat cells transfected with the RFP vector were used as a control. (D) GARP-transfected Jurkat cells were incubated with 5 μg/mL latent TGF-β1 (LTGFβ1) or preincubated for 20 min at 37 °C with a 10-fold excess of TSP1, RGD, or RGDS peptides before latent TGF-β1 incubation and then stained for surface LAP. (E) Tregs were expanded in vitro for 14 days and then restimulated for 48 h before immunoprecipitation with anti-LAP (left lane) or isotype (right lane) followed by immunoblot with anti-GARP. (F) Dynabeads conjugated to αGARP (Left) or αLAP (Right) were incubated with a mixture of GARP-Fc + TGF-β1 (shaded histogram), GARP-Fc + LAP (dashed histogram), or GARP-Fc + latent TGF-β1 (solid histogram) followed by flow cytometric analysis with PE-labeled anti-hLAP (Left) or anti-hFc (Right). Data are representative of 3 independent experiments for A, B, C, D, and F and 2 for E. Numbers indicate percentage in each quadrant for A–D.

GARP is required for the surface expression of latent TGF-β1. (A) FACS-sorted Tregs (CD25^hi), naïve (CD45RA⁺), and memory (CD45RO⁺) T cells were activated for 5 days in the absence or presence of TGF-β1 then rested for 7 days and restimulated for 48 h without TGF-β1 before analysis of FOXP3 with surface GARP and LAP expressions. (B) Tregs were transfected with nonspecific (siNS) or FOXP3 (siFOXP3) siRNA and cultured for 5 days before activation for 48 h and analysis of FOXP3 with surface GARP and LAP expressions. (C) Naïve T cells were transduced with control (vector) or GARP encoded RFP-expressing lentiviral vectors and expanded for 7 days with anti-CD3/CD28 Dynabeads before restimulation for 48 h to evaluate for surface GARP and LAP (Left) and intracellular FOXP3 expressions among RFP⁺ and RFP⁻ cells (Right). Data are representative of 3 independent experiments. Numbers indicate percentage in each quadrant.