Short communicationPD-L2 is expressed on activated human T cells and regulates their function
Highlights
• The cosignaling molecule, PD-L2 is expressed on human activated T cell subsets. • The PD-L2 triggering down-regulates (TCR+CD28)-induced cytokine production on human T cells. • A direct targeting of PD-L2 on T cells blocks T cell proliferation.
Introduction
T cell activation requires two signals that are delivered by antigen presenting cells (APCs). The first signal is induced by the T cell receptor (TcR) during antigen recognition. The second signal or costimulatory signal, is provided by adhesion molecules on APCs that bind to costimulatory receptors on T cells. The best well-characterized T cell costimulatory pathway involves the CD28 molecule engaged by two different ligands, B7-1 and B7-2 expressed on APCs (Greenwald et al., 2005). The discovery of additional members of the B7:CD28 family has revealed new costimulatory pathways that provide positive and/or negative second signals to effector T cells. Another «ménage à trois» has been identify corresponding to Programmed death 1 (PD-1) receptor on activated T cells and its two ligands, Programmed death ligand 1 (PD-L1) and Programmed death ligand 2 (PD-L2) on APCs (Greenwald et al., 2005). PD-1 has an important inhibitory function on activated lymphocytes by regulating the immune homeostasis and the maintenance of peripheral tolerance through its interaction with PD-L1 and/or PD-L2.
This T cell costimulatory panorama is even more complex as the ligands of the CD28 or PD-1 cosignaling molecules, normally expressed on APCs, can be also detected on T cells. B7-2 is constitutively expressed on some resting T cells, whereas B7-1 is not present at the resting stage (Taylor et al., 2004). Both B7-1 and B7-2 can be up-regulated on murine or human T cells (Sansom and Hall, 1993, Taylor et al., 2004) and deliver signals into T cells (Paust et al., 2004). PD-L1 is found constitutively expressed on murine T cells and is further up-regulated upon TcR stimulation (Yamazaki et al., 2002), whereas PD-L1 expression is inducible on human T cells (Dong et al., 2003). Autoantibodies against PD-L1 have been detected in sera from patients with rheumatoid arthritis. After immobilization, these antibodies stimulated cytokine production and T cell proliferation (Dong et al., 2003).
Thus, among the CD28/PD-1 ligands, only PD-L2 expression has not been investigated in human T cells. Here, we generate a new monoclonal antibody (mAb) against human PD-L2 and further analyze the PD-L2 protein expression on T cell subsets under different stimulation conditions. As PD-L2 appears to be inducible on CD4+ and CD8+ T cells, we further analyze the effects of PD-L2 mAbs using artificial APCs on activated T cells by cell proliferation and cytokine detection assays.
Section snippets
Generation of anti-human PD-L2 mAbs
Female BALB/c mice were immunized by i.p. injection with 10 μg of human extracellular region of PD-L2/Ig fusion protein with Freund adjuvant. PD-L2 mAbs were generated as previously described (Ghiotto et al., 2010, Serriari et al., 2010). The hybridoma supernatants were screened by cell surface staining of human PD-L2-transfected COS cells (Fig. S1A) and validated with a commercially available PD-L2 mAb (clone 24F.10C12, BioLegend) (Fig. S1B). PD-L2-326.35 clone (mouse, IgG1) was selected as
Inducible expression of PD-L2 in human primary T cells
PD-1 ligands are expressed on APCs. Expression of the first ligand, PD-L1 has been also reported on T cells (Dong et al., 2003, Yamazaki et al., 2002). Here, the potential expression of the second ligand, PD-L2 is investigated on human T cell subsets. We generate a PD-L2 mAb that is used to stain T cells isolated from blood of volunteer donors (see Section 2 and Fig. S1A). Isolated CD4+ and CD8+ T cell subsets were stimulated with plate-bound anti-CD3 plus soluble anti-CD28. Cell aliquots were
Discussion
In this study, we describe for the first time the expression and a functional role of PD-L2 on human T cells. PD-L2 (named also B7-DC) cell surface expression was initially restricted to macrophages and DCs (Latchman et al., 2001). More recently, PD-L2 expression has been detected on resting peritoneal B1 cells (Kaku and Rothstein, 2010) and in solid tumors such as ovarian carcinoma (Okazaki and Honjo, 2007). Here, we demonstrate that PD-L2 is expressed on T cells via TcR signaling (Fig. 1). As
Acknowledgements
We would like to thank Dr. Yves Collette, Dr. Stéphane Audebert and Dr. Jérôme Reboul for helpful discussions and technical supports, also to Dr. François Coulier from the Service informatique of the CRCM for the figure artworks.
Fundings: This work was supported by grants from Institut National de la Santé et de la Recherche Médicale. N. Messal was supported by fellowships from Bourse Franco-Algérienne and Ligue Nationale contre le Cancer. N-E. Serriari was supported by fellowships from Bourse
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Present address: INSERM U925, Faculté de Médecine, F-80036, Amiens, France.