Original Article Identification of soluble and membrane- bound isoforms of porcine tumor necrosis factor receptor 2 Introduction TNF pleiotropic activities are mediated by two dis- tinct high affinity receptors, TNFR1 and TNFR2 [1]. Both TNF receptors are co-expressed in most cell types, although in different ratios. TNFR1 is constitutively expressed at moderate levels in most tissues, where it mediates the majority of cytotoxic and pro-inflammatory activities of TNF [1]. On the contrary, TNFR2 is predominantly expressed in immune and endothelial cells and is highly up-regulated by pro-inflammatory stimuli [1]. Uribe-Herranz M, Casinghino SR, Bosch-Presegue´ L, Fodor WL, Costa C. Identification of soluble and membrane-bound isoforms of porcine tumor necrosis factor receptor 2. Xenotransplantation 2011; 18: 131--146. Ó 2011 John Wiley & Sons A/S. Abstract: Background: TNF and its receptors TNF-Receptor 1 (TNFR1, CD120a) and TNF-Receptor 2 (TNFR2, CD120b) have been implicated in the rejection of transplanted cells and organs. Although pig TNFR1 (pTNFR1) is known to mediate the effects of human TNF in a xenogeneic setting, it is unclear whether pig TNFR2 (pTNFR2) could contribute to xenograft rejection. Methods: We have cloned the cDNA of various pTNFR2 variants by reverse transcription--polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. We have characterized the predicted proteins with bioinformatic tools and conducted expression, affinity, and functional studies to investigate their roles. Results: We have identified four isoforms of pTNFR2: one comprising the four cysteine-rich domains (CRD) conserved between species, a shorter variant (pTNFR2DE7-10) encoding for a soluble isoform, an- other with only three CRD due to the lack of exon 4 (pTNFR2DE4), and a fourth variant containing both modifications. Accordingly, multiple mRNA transcripts were observed by northern blotting. Quantitative RT-PCR determined high pTNFR2 expression in lung and immune cells and detected the two alternative splicings in all cells/tissues examined. The full receptor was moderately expressed on the surface of pig cells such as porcine aortic endothelial cells and PK-15 and was regulated by TNF. On the contrary, the membrane-bound pTNFR2DE4 was located only intracellularly. Plasmon resonance studies showed that pTNFR2 binds pig and human TNFa with high affinity, but pTNFR2DE4 interacts poorly with pig TNFa and does not bind to the human cytokine. Moreover, pull-down experiments with the two recombinant soluble isoforms consistently demonstrated that the two bound together and soluble pTNFR2DE4 was able to modulate the TNF inhibitory activity of pTNFR2-GST in a cell-based assay. Conclusion: The pTNFR2 may participate in the process of xenograft rejection and other related events, as well as be used in soluble form to block TNF in this setting. In addition, we have discovered other pTNFR2 isoforms that may affect the pig immune responses and have an impact on rejection of xenografts. Mireia Uribe-Herranz, 1 Sandra R. Casinghino, 2,3 Laia Bosch- PreseguØ, 1 William L. Fodor 2,3 and Cristina Costa 1,2 1 New Therapies of Genes and Transplants Group [Institut d'Investigació Biomdica de Bellvitge] IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain, 2 Department of Molecular Sciences, Alexion Pharmaceuticals Inc, Cheshire, CT, 3 Department of Molecular and Cellular Biology, CT Center for Regenerative Biology, University of Connecticut, Storrs, CT, USA Key words: cytokine -- endothelium -- tumor necrosis factor -- tumor necrosis factor R2 -- Xenotransplantation Abbreviations: Ab, antibody; CRD, cysteine-rich domains; PAC, porcine articular chondrocytes; PCC, porcine costal chondrocytes; PAEC, porcine aortic endothelial cells; PLAD, pre-ligand assembly domain; SLA, swine leukocyte antigen. Address reprint requests to Cristina Costa, Institut d'Investigació Biomdica de Bellvitge (IDIBELL), Hospital Duran i Reynals, Gran Via de L'Hospitalet 199, 08908 L'Hospitalet de Llobregat, Barcelona, Spain (E-mail: ccosta@idibell.cat) Received 3 January 2011; Accepted 15 March 2011 Xenotransplantation 2011: 18: 131--146 Printed in Singapore. All rights reserved doi: 10.1111/j.1399-3089.2011.00634.x Ó 2011 John Wiley & Sons A/S XENOTRANSPLANTATION 131