B7-DC (PD-L2) costimulation of CD4+ T-helper 1 response via RGMb

Abstract

The role of B7-DC in T-cell responses remains controversial because both coinhibitory and costimulatory functions have been reported in various experimental systems in vitro and in vivo. In addition to interacting with the coinhibitory receptor PD-1, B7-DC has also been shown to bind repulsive guidance molecule b (RGMb). The functional consequences of the B7-DC/RGMb interaction, however, remain unclear. More than a decade ago, we reported that replacement of a murine B7-DC mutant lysine with serine (K113S) at positive 113 resulted in a loss of binding capacity to PD-1. Nevertheless, K113S remained costimulatory for T cells in vitro, implicating a dual functionality for B7-DC in T-cell responses. Here we show that recombinant K113S protein interacts with RGMb with a similar affinity to wild-type B7-DC. More importantly, K113S costimulates CD4⁺ T-cell responses via RGMb and promotes Th1 polarization. RGMb is expressed on the surface of naive mouse T cells, macrophages, neutrophils and dendritic cells. Finally, K113S/RGMb costimulation suppresses Th2-mediated asthma and ameliorates small airway inflammation and lung pathology in an experimental mouse model. Our findings indicate that RGMb is a costimulatory receptor for B7-DC. These findings from the K113S variant provide not only a possible explanation for the B7-DC-triggered contradictory effects on T-cell responses, but also a novel approach to investigate the B7-DC/PD-1/RGMb axis. Recombinant K113S or its derivatives could potentially be developed as an agonist for RGMb to costimulate the Th1 response without triggering PD-1-mediated T-cell inhibition.

Keywords: B7-DC; K113S; RGMb; Th1/Th2; asthma.

Figure 1

Binding of B7-DC variants to PD-1 or RGMb. (a) CHO cell lines were transfected to overexpress mouse PD-1 (PD-1⁺ CHO) and stained with the indicated concentrations of control Ig (Flag-hIg), B7-DC-hIg or its variants and subsequently analyzed by flow cytometry. (b) 293 cell lines were transfected to overexpress mouse RGMb (RGMb⁺ 293T) and stained with the indicated concentrations of control Ig (Flag-hIg), B7-DC-hIg or its variants and subsequently analyzed by flow cytometry. The second antibody used for staining was mouse anti-human IgG Fc-Alexa Fluor 647. All data are representatives of three or more independent experiments. CHO, Chinese hamster ovary; RGMb, repulsive guidance molecule b.

Figure 2

Binding affinity of B7-DC and its variants to RGMb. (a,b) Biacore analysis of the SPR of B7-DC-hIg (a) or K113S-hIg (b) interactions with mouse RGMb, which was coated on the CM5 biosensor chip at various concentrations (3.906–500 nm). (c, d) Biacore analysis of the SPR of R56S-hIg (c) or E71S-hIg (d) interactions with RGMb, which was coated on the chip at various concentrations (0.1094–7 μm). (e) Response units of the SPR analysis of RGMb interactions with I105A-hIg, D111S-hIg, R101S-hIg and control Flag-hIg at 10 μm. (f) The 293T cell lines were transfected to overexpress mouse RGMb and stained at the indicated concentrations of anti-mRGMb-biotin (secondary antibody SA-APC) and 1 μg B7-DC-hIg or K113S-hIg (secondary antibody goat anti-human Fc-PE) and subsequently analyzed by flow cytometry. All data are representatives of three or more independent experiments. RGMb, repulsive guidance molecule b; SPR, surface plasmon resonance.

Figure 3

Expression of RGMb and B7-DC on mouse myeloid and lymphoid cells. (a) RGMb cell surface expression was examined using a specific polyclonal antibody (R&D Systems) by flow cytometry analysis on AMs, pMφs, BMDCs (immature DCs, blue line; mature DCs, red line), the RAW264.7 macrophage cell line and freshly isolated spleen PMNs, T, B and NK cells. (b) B7-DC cell surface expression was tested by flow cytometry on AMs, pMφs, BMDCs (both immature and mature DCs indicated by the blue and red line, respectively) and the RAW264.7 macrophage cell line. All data are representatives of three or more experiments. AMs, alveolar macrophages; BMDCs, bone marrow-derived dendritic cells; pMφs, peritoneal macrophages.

Figure 4

OVA-induced asthma mouse model and fusion protein levels in BALF and serum. (a) Sensitization and challenge protocol for OVA-induced asthma and treatment. BALB/c mice were inoculated i.p. with 10 μg grade V OVA and 1 mg alum gel in PBS on days 0 and 5. The mice were subsequently challenged by inhalation of 1% OVA in PBS for 30 min on days 11–13. One day before the first OVA immunization, plasmids (Flag-hIg and K113S-hIg) were injected i.v. via the tail vein at 20 μg per mouse in 2 ml PBS within 5–10 s. (b) Flag-hIg and K113S-hIg levels in BALF at day 14 after immunization. (c) Flag-hIg and K113S-hIg levels in sera at the indicated days after hydrodynamic injection. The data represent the mean±s.e.m., and samples were obtained from five individual mice from each group. The data are representatives of at least three independent experiments. BALF, bronchoalveolar lavage fluids; i.p., intraperitoneally; i.v., intravenously; PBS, phosphate-buffered solution.

Figure 5

Effect of K113S variants in an OVA-induced mouse model of asthma. (a) The total cell and eosinophil number in BALF from mice after asthma induction with Flag-hIg and K113S-hIg treatment. (b) Cells in BALF from Flag-hIg or K113S-hIg-treated mice were stained with Diff-Quick dye liquor. (c) Lung histology from mice treated with Flag-hIg or K113S-hIg. The mice were killed on day 14, and the lung tissues were processed and stained with H&E to observe the cell infiltration around the small airway. (d) AHR to methacholine challenge. Mice were treated with Flag-hIg and K113S-hIg and were measured for airway resistance and Penh of the lung. The data represent the mean±s.e.m. and encompass three independent experiments with five mice in each group. NS, no significant difference; *P<0.05; **P<0.01; ***P<0.001. BALF, bronchoalveolar lavage fluids; CFSE, carboxyfluorescein succinimidyl ester; H&E, hematoxylin and eosin.

Figure 6

Effect of K113S on CD4⁺ T-cell costimulation and Th1/Th2 cytokine production. (a, b) Purified CD4⁺ T cells from naive BALB/c mice were labeled with CFSE and stimulated under Th1 differentiation conditions (IL-2 10 ng/ml, IL-12 10 ng/ml and anti-IL-4 10 μg/ml) in the absence (a) or presence (b) of pMφs on pre-coated plates with anti-CD3 at 2.5 μg/ml and soluble anti-CD28 at 1 μg/ml for 3 days. CFSE dilution and IFN-γ intracellular staining were evaluated by flow cytometry. (c) Intracellular IL-2, IFN-γ and IL-4 were assayed by flow cytometry using a specific mAb in CD4 cells from the lung following treatment with Flag-hIg or K113S-hIg during OVA-induced asthma. On day 14 after asthma induction, cells from the BALF were stimulated with PMA/ionomycin/brefeldin A for 4–6 h and collected for flow cytometry analysis. (d) Intracellular IL-5, IL-13 and IL-4 were assayed using a specific mAb by ELISA in BALF and OVA-specific IgE in sera following treatment with Flag-hIg or K113S-hIg during OVA-induced asthma. The data are representative of at least three independent experiments consisting of five mice in each group. *P<0.05. BALF, bronchoalveolar lavage fluids; CFSE, carboxyfluorescein succinimidyl ester; IFN-γ, interferon-γ; NS, no significant difference; PMA, phorbol 12-myristate 13-acetate; pMφs, peritoneal macrophages.