Background Heart transplantation is a lifesaving procedure for patients with end-stage heart failure. P2X7R targeting with periodate-oxidized ATP (oATP) promotes long-term cardiac transplant survival in 80% of murine recipients of a fully mismatched allograft. Long-term survival of cardiac transplants was associated with reduced T cell activation, Th1/Th17 differentiation and inhibition of STAT3 phosphorylation in T cells, thus leading to a reduced transplant infiltrate and coronaropathy. genetic upregulation of the P2X7R pathway was also shown to stimulate Th1/Th17 cell generation. Finally, P2X7R targeting halted the progression of coronaropathy in a murine model of chronic rejection as well. Conclusions P2X7R targeting is a novel clinically relevant strategy to prolong cardiac transplant survival. to T cell activation and IL-2 production14, 19C21 and to XMD8-92 IC50 graft-versus-host disease18. Recently, ATP has been shown to play a major role in Th17 differentiation22 and Treg inhibition23, and Th1 and Th17 immune responses have been associated with organ rejection24, 25. Indeed, natural ATP/P2XsR inhibitors with the ability to hydrolyze ATP are expressed on the cellular plasma membrane (e.g. the ectonucleotidases CD39 and CD73)26. Novel P2X7R inhibitors are available for human use, including periodate-oxidized ATP (oATP), CE224,535, AZD9056 and GSK1482160, thereby rendering P2X7R targeting a potential path to be tested in transplantation27. oATP, a XMD8-92 IC50 small Schiff base molecule, is an irreversible antagonist of P2X7R due to the selective modification of lysine residues that occurs in the vicinity of the ATP-binding XMD8-92 IC50 site28; oATP has been also proposed to exert additional inhibitory effect on the other purinergic receptors20. We aim to unveil the largely unknown role of P2X7R in heart transplantation and to target the ionotropic purinergic receptor P2X7R in order to achieve tolerance towards cardiac transplants. MATERIALS AND METHODS Immunological Methods can be found in Online Supplemental Materials. Patients Cardiac samples were obtained Rabbit polyclonal to PCDHB10 from the right side of the interventricular septum of cardiac transplant recipients at Niguarda Ca Granda Hospital, Milan, Italy. Samples were formalin-fixed, paraffin-embedded, sectioned, and histologically graded by a cardiac pathologist according to the 2005 ISHLT criteria29. Characteristics of patients and immunosuppressive regimen30 are depicted in Table 1. Table 1 Patient characteristics (Data are expressed as MeanSD) Mice C57BL/6, BALB/c, C57BL/6 P2X7?/?, B6.C-H2bm12 (bm12), and C57BL/6 Rag1?/? mice of various ages were obtained from the Jackson Laboratory, Bar Harbor, Maine. ABM TCR-Tg mice have been described previously31 and were maintained as a breeding colony in our animal facility. All mice were cared for and used in accordance with institutional guidelines. Protocols were approved by the Harvard Medical School Animal Care and Use Committee. Interventional studies Mice were injected i.p. with oATP (Medestea srlTurin, Italy) 250 g/day i.p. for 14 days and Colivelin (Tocris, Minneapolis, MN) 7 nM/day for 7 days. assays were performed in the presence of varying concentrations of oATP, NF-449 (P2X1R inhibitor), and 5-BDBD (P2X4R inhibitor) from Tocris Bioscience (Bristol, UK) Heart transplantation Vascularized cardiac allografts were transplanted intra-abdominally using microsurgical techniques as described by Corry et al.32. Rejection was determined as complete cessation of cardiac contractility and was confirmed by direct visualization. Histology and immunohistochemistry Immunohistochemistry was performed with 5-micron-thick formalin-fixed, paraffin-embedded tissue section. Photomicrographs (400x) were taken using an Olympus BX41 microscope (Center Valley, PA). The following primary antibodies were used: anti-Mac2, antiCCD3 (Cell Marque, Rocklin, CA), anti-CD20, anti CD68 (Dako, Glostrup, Denmark), and anti-P2X7 (Alomone Labs, Jerusalem, Israel). As isotype control for anti-P2X7R, normal Rabbit IgG (R&D Systems, Minneapolis, MN) was used. Graft histology was evaluated by an expert pathologist and was quantified as follows: short-term P2X7R targeting prevents cardiac transplant rejection, abrogates the Th1/Th17 immune response and reduces effector T cells in mice We next tested the effect of P2X7R targeting in preventing cardiac transplant rejection using the P2X7R inhibitor oATP. Untreated C57BL/6 mice transplanted with BALB/c hearts (fully mismatched) invariably rejected grafts within 7 days (mean survival time [MST] of 7 days; Figure 2A). P2X7R targeting using short-term treatment of oATP (250 mg oATP i.p. daily for 14 days) induced long-term cardiac.