Figure 1.
Surface LAP/TGF-β expression on mouse activated CD4 T cells.
(A) BALB/c CD4 T cells were stimulated with plate-bound anti-CD3/anti-CD28 for 2 days and rested for 1 day. The cells were surface stained with anti-LAP mAbs using PE-labeled secondary antibodies, then intracellularly stained with anti-Foxp3-Alexa Fluor647. Staining with representative clones, anti-LAP mAbs TW7-16B4 and TW7-20B9, and anti-latent TGF-β/pro-TGF-β mAb TW7-28G11, are shown. (B) Activated BALB/c CD4 T cells were stained with anti-LAP TW7-20B9 (surface) and anti-Foxp3 (intracellular) (left), with anti-GARP (surface) and anti-Foxp3 (intracellular), or anti-LAP TW7-20B9 (surface) and GARP (surface) (right).
Figure 2.
Surface LAP/TGF-β expression on mouse unstimulated CD4 T cells and time course analysis.
(A) Freshly prepared BALB/c CD4 T cells were surface stained with PE-conjugated anti-LAP/TGF-β mAbs (TW7-16B4, TW7-20B9, or TW7-28G11), anti-CD25-FITC, anti-CD4-APC, and 7-AAD. CD4+7-AAD− cells were gated. (B) BALB/c CD4 T cells were stimulated with plate-bound anti-CD3/anti-CD28 for two days, and then split in 10% FBS-IMDM containing 100 U/ml IL-2. The cells were surface stained with PE-conjugated anti-LAP TW7-20B9 followed by anti-Foxp3-Alexa Fluor647 (intracellular staining) (upper panels), or with APC-conjugated anti-LAP TW7-20B9 and GARP-PE (lower panels).
Figure 3.
Induction of surface LAP by Foxp3 transduction.
BALB/c CD4+CD25− T cells were stimulated with plate-bound anti-CD3/anti-CD28 and retrovirally transduced with pMCs-Foxp3-IRES-GFP vector. The cells were re-stimulated with plate-bound anti-CD3/anti-CD28 for 14 hrs and transferred to uncoated wells. 2 days after re-stimulation, the cells were stained with anti-LAP TW7-16B4 or TW7-20B9 using anti-mouse IgG1-APC secondary antibody.
Figure 4.
Induction of surface LAP by TGF-β.
(A) BALB/c CD4+CD25− T cells were stimulated with plate-bound anti-CD3/anti-CD28 without (upper panels) or with 10 ng/ml recombinant TGF-β (lower panels) for 2 days and rested for 2 days. The cells were surface stained with anti-LAP TW7-16B4 or TW7-20B9, or anti-latent TGF-β/pro-TGF-β TW7-28G11 using goat anti-mouse Ig-PE secondary antibody, then fixed, and intracellularly stained with anti-Foxp3-Alexa Fluor647. (B) BALB/c CD4+CD25− T cells were stimulated with/without TGF-β, and then surface stained with ACP-conjugated anti-LAP TW7-20B9 and GARP-PE, followed by intracellular staining with anti-Foxp3-Alexa Fluor488. Foxp3− and Foxp3+ cells populations were gated and plotted by LAP and GARP expression.