6 f). provides particularly promising targets for malignancy immunotherapy (Topalian et al., 2012). There is considerable evidence that PD-L2 inhibits immunity by binding to the PD-1 co-inhibitory receptor (Latchman et al., 2001; Zhang et al., 2006). However, several studies have shown that PD-L2 can function to stimulate T cell proliferation and cytokine production, even in PD-1Cdeficient T cells or with PD-L2 mutants that did not bind to PD-1 (Liu et al., 2003; Shin et al., 2003; Wang et al., 2003). These findings suggest that PD-L2 may function through a receptor other than PD-1. Most studies using blocking mAbs show a dominant role for PD-L1 in inhibiting immune responses; Darbufelone mesylate however, PD-L2 plays Darbufelone mesylate a dominant role in responses such as airway hypersensitivity, experimental allergic conjunctivitis and nematode contamination (Ritprajak et al., 2012). In some situations, PD-L2 dominance may be explained by preferential PD-L2 up-regulation by IL-4, but other instances may be explained by the binding of PD-L2 to a receptor other than PD-1. Here, we demonstrate that PD-L2 binds to a second receptor, repulsive guidance molecule b (RGMb). RGMb, also known as DRAGON, is usually a member of the RGM family which consists of RGMa, RGMb, and RGMc/hemojuvelin (Severyn et al., 2009). RGMs are glycosylphosphatidylinositol-anchored membrane proteins that bind bone morphogenetic proteins (BMPs) and neogenin (Conrad et al., 2010). RGMs do not directly transmission but can act as co-receptors that modulate BMP signaling (Samad et al., 2005). RGMb is usually expressed and functions in the nervous system (Severyn et al., 2009). In addition, RGMb expression is usually observed in macrophages and other cells of the immune system (Xia et al., 2011). However, the function of RGMb in the immune system is only beginning to emerge (Galligan et al., 2007; Xia et al., 2011). RGMb-deficient mice have an early lethal phenotype (Xia et al., 2011). Here, we characterize RGMb binding to PD-L2 and identify RGMb protein expression in mouse hematopoietic cells and human malignancy cell lines. Based on the crucial role of PD-L2 in lung immune regulation (Akbari et al., 2010; Singh et al., 2011) and RGMb expression in the lung, we investigated the function of RGMb and PD-L2 in respiratory tolerance. Blockade of PD-L2 and RGMb conversation prevented the development of respiratory tolerance. RESULTS RGMb binds to PD-L2, but not to PD-L1 or other related molecules We recognized RGMb as a novel binding partner for PD-L2 using COS cell expression cloning with PD-L2-Ig fusion protein. Using circulation cytometry with stably transfected 300 cells and Ig fusion proteins, we found that mRGMb binds to mPD-L2 but not mPD-L1 or other proteins of the B7 family (Fig. 1, a and b). ELISA with purified proteins showed that mRGMb binds to mPD-L2 and hPD-L2, and that hRGMb binds to hPD-L2 and mPD-L2 (Fig. 1 c and not depicted). Thus, the RGMbCPD-L2 conversation occurs in both mice and humans. Further studies showed that PD-L2 does not bind to RGMa or RGMc Rabbit Polyclonal to MARK3 (Fig. 1 d). Biacore data showed that PD-L2 bound to RGMb with a similar affinity as to PD-1, = 2; *, P < 0.05; **, P < 0.01. Regular one-way ANOVA Darbufelone mesylate followed by Dunnetts multiple comparison test. (g) RGMb expression in lung AM and IM by Western blotting as in d. All data are representative of two or more experiments. Western blotting showed positive RGMb expression in cells from spleen, thymus, purified splenic CD4+, and CD8+ T cells from naive mice (Fig. 4 d). Cell surface RGMb expression was not detectable in main hematopoietic cells by circulation cytometry with PE-conjugated RGMb mAb 9D1 (unpublished data). RGMb mRNA and protein levels were not up-regulated in T cells by CD3 and/or CD28 activation (unpublished data), suggesting that RGMb is not a T cell activation molecule. Intracellular circulation cytometry staining using PE-conjugated.