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B lymphocytes confer immune tolerance via cell surface GARP-TGF-β complex
Caroline H. Wallace, … , Bei Liu, Zihai Li
Caroline H. Wallace, … , Bei Liu, Zihai Li
Published April 5, 2018
Citation Information: JCI Insight. 2018;3(7):e99863. https://doi.org/10.1172/jci.insight.99863.
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Research Article Immunology

B lymphocytes confer immune tolerance via cell surface GARP-TGF-β complex

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Abstract

GARP, a cell surface docking receptor for binding and activating latent TGF-β, is highly expressed by platelets and activated Tregs. While GARP is implicated in immune invasion in cancer, the roles of the GARP-TGF-β axis in systemic autoimmune diseases are unknown. Although B cells do not express GARP at baseline, we found that the GARP-TGF-β complex is induced on activated human and mouse B cells by ligands for multiple TLRs, including TLR4, TLR7, and TLR9. GARP overexpression on B cells inhibited their proliferation, induced IgA class-switching, and dampened T cell–independent antibody production. In contrast, B cell–specific deletion of GARP-encoding gene Lrrc32 in mice led to development of systemic autoimmune diseases spontaneously as well as worsening of pristane-induced lupus-like disease. Canonical TGF-β signaling more readily upregulates GARP in Peyer patch B cells than in splenic B cells. Furthermore, we demonstrated that B cells are required for the induction of oral tolerance of T cell–dependent antigens via GARP. Our studies reveal for the first time to our knowledge that cell surface GARP-TGF-β is an important checkpoint for regulating B cell peripheral tolerance, highlighting a mechanism of autoimmune disease pathogenesis.

Authors

Caroline H. Wallace, Bill X. Wu, Mohammad Salem, Ephraim A. Ansa-Addo, Alessandra Metelli, Shaoli Sun, Gary Gilkeson, Mark J. Shlomchik, Bei Liu, Zihai Li

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Figure 1

Multiple TLR ligands induce GARP expression on both human and mouse B cells.

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Multiple TLR ligands induce GARP expression on both human and mouse B ce...
Splenic B cells were isolated from WT mouse spleens using CD19+ magnetic beads. (A) Cells were stimulated with mouse anti-μ (15 μg/ml), LPS, R848, or CpG for 120 hours. Flow cytometry was used at 24-hour intervals to measure GARP+LAP+ expression on live B cells. Numbers represent the percentage of cells in the GARP+LAP+ quadrant over the entire CD19+ B cell population. Flow plots are representative of 3 independent experiments. A representative baseline flow plot is also shown. (B) Quantification of GARP and LAP expression (n = 4 biological replicates). MFI, mean fluorescent intensity of GARP. Statistical analysis was performed by 2-way ANOVA; ***P < 0.001. (C) Immunoblot of GARP in the whole-cell lysate of untreated (UT) WT B cells or after stimulation with the indicated conditions for 72 hours. Representative of 3 immunoblots. (D) Primary WT and GARP-KO splenic B cells were cultured with LPS, Poly I:C, or IL-1β plus Poly I:C for 72 hours. Cells were stained for GARP and LAP and analyzed by flow cytometry. Representative of 3 independent experiments. (E) Phenotypic analysis of LPS-treated (48 hours) GARP– and GARP+ B cells by flow cytometry. Histogram plots are representative of n = 3 biological repeats and 2 independent experiments. Black lines denote GARP- cells, red lines denote GARP+ cells; shaded areas denote isotype. Numbers represent mean fluorescent intensity (MFI). (F) Human B cells were isolated from normal subjects using human anti-CD19+ magnetic beads. Cells were freshly analyzed or cultured with human anti-μ, R848, or CpG for 72 hours. GARP+LAP+ levels were analyzed by flow cytometry. Representative of 3 independent experiments. (G) Quantification of GARP+LAP+ expression in 3 biological replicates from healthy donors. Each data point represents an individual donor. Statistical analysis was performed by 2-tailed t test (E) and 1-way ANOVA with Tukey’s multiple comparisons (G); *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent SD.

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