Cutting Edge: Active TGF-β1 Released from GARP/TGF-β1 Complexes on the Surface of Stimulated Human B Lymphocytes Increases Class-Switch Recombination and Production of IgA

Abstract

Production of active TGF-β is regulated at a posttranslational level and implies release of the mature cytokine dimer from the inactive, latent TGF-β precursor. There are several cell-type specific mechanisms of TGF-β activation. We identified a new mechanism operating on the surface of human regulatory T cells and involving membrane protein GARP, which binds latent TGF-β1. The paracrine activity of regulatory T cell-derived TGF-β1 contributes to immunosuppression and can be inhibited with anti-GARP Abs. Whether other immune cell types use surface GARP to activate latent TGF-β1 was not known. We show in this study that stimulated, human B lymphocytes produce active TGF-β1 from surface GARP/latent TGF-β1 complexes with isotype switching to IgA production.

FIGURE 1.

Human B cells express surface GARP/latent TGF-β1 complexes after in vitro stimulation. (A) Human PBMCs were labeled with anti-GARP (mAb MHG-6) or isotype control Abs and analyzed by flow cytometry. Dot plots are gated on live cells. (B) CD19⁺ B cells were stimulated or not with anti-IgM/IgG Abs, IL-2, IL-21, sCD40L, and CpG for 24 h. Bar graphs indicate mean GARP mRNA copy numbers normalized to 10⁵ EF-1 mRNA copies (technical duplicates + SD) as determined by quantitative RT-PCR. (C) CD19⁺ B cells stimulated as in (B) were labeled with Abs against free GARP (mAb MHG-5), total GARP (mAb MHG-6), GARP/TGF-β1 complexes (mAb MHG-8), or LAP, then analyzed by flow cytometry. Histograms are gated on live cells. (D) CD20⁺CD27⁻ and CD20⁺CD27⁺ B cells were stimulated or not as in (B) during the indicated number of days, labeled with Abs against total GARP (mAb MHG-6) and analyzed by flow cytometry. Med. FI: median fluorescence intensity. (E) CD20⁺CD27⁻ B cells were exposed during 24 h to the indicated stimuli, then labeled with Abs against total GARP (mAb MHG-6) and analyzed by flow cytometry. Histograms are gated on live cells.

FIGURE 2.

Human B cells produce active TGF-β1 in a GARP-dependent manner. (A) Levels of TGFB1, 2 and 3 mRNA were measured in CD19⁺ B cells stimulated for 24 h with the mixture used in Fig. 1B. Bar graphs indicate mRNA copy numbers normalized to 10⁵ EF-1 mRNA copies (means of duplicates + SD) as determined by quantitative RT-PCR. (B) Total and active TGF-β1 were measured by ELISA in the supernatants of B cells stimulated with the mixture used in Fig. 1B during 4 d in serum-free medium. Means of duplicates + SD. Detection limit: 16 pg/ml. nd, not detected. (C) CD19⁺ B cells were stimulated as in (A). The indicated Abs (10 μg/ml) were added on day 0 and 3 of the cultures, and rTGF-β1 (0.1 ng/ml) on day 3. Cells collected on day 4 were analyzed by Western blot, and luminescence signals were quantified using Bio1D software. Reduction in pSMAD2/β-actin ratios in cells treated with anti-TGF-β or the blocking anti-GARP MHG-8 Ab (both mIgG1s) are shown relative to the cells stimulated in the presence of a mIgG1 isotype control. Reduction in cells treated with the blocking anti-GARP LHG-10 Ab is shown relative to cells treated with the nonblocking anti-GARP LHG-11 (both hIgG1s). Data are representative of three independent experiments.

FIGURE 3.

Blocking active TGF-β1 production in B cells with anti-GARP blocking Abs decreases IgA class switch. (A) CD20⁺CD27⁻ B cells were stimulated or not with anti-IgM, sCD40L, IL-2, IL-21, and CpG for 4 d in the presence of the indicated Abs. IgA1 GLT were quantified by quantitative RT-PCR. rTGF-β1 (0.1 ng/ml) added on day 2 was used as positive control. Bar graphs indicate mean IgA1 GLT per 10⁵ EF-1 copies (technical duplicates + SD). (B and C) Naive B lymphocytes were stimulated as above during 7 d, then labeled with anti-IgA, IgG, and IgD and analyzed by flow cytometry. IgD stainings on stimulated and nonstimulated cells are shown in Supplemental Fig. 2G. rTGF-β1 (0.1 ng/ml) added 3 d after stimulation was used as a positive control. Representative contour plots are gated on live cells (B) and bar graphs indicate the mean percentages of six technical replicates ± SD (C). Selected pairs were compared using one-way ANOVA followed by a Bonferroni multiple comparison test. Data are representative of at least three independent experiments. ****p < 0.0001.