Identification, Detection, and In Vitro Characterization of Cynomolgus Monkey Natural Killer Cells in Delayed Xenograft Rejection of hDAF Transgenic Porcine Renal Xenografts D. Quan, C. Bravery, G. Chavez, A. Richards, G. Cruz, L. Copeman, C. Atkinson, B. Holmes, H. Davies, E. Cozzi, and D. White T HE DEVELOPMENT of animals transgenic for com- plement regulatory proteins have circumvented the development of hyperacute rejection and resulted in pro- longed survival in discordant xenotransplantation. 1 How- ever, acute vascular rejection (AVR) or delayed xenograft rejection, the next hurdle to overcome, remains a formida- ble challenge. From histopathologic data, AVR is thought to be primarily antibody mediated with type II endothelial activation being the initiating event. AVR is characterized by intravascular fibrin deposition and microthrombi, which ultimately progresses to frank necrosis. In rodent models of AVR, macrophages and natural killer (NK) cells have been found to be the predominant cellular infiltrate. 2–6 However, there is little in vivo data to support the role of NK cells in large animal xenograft rejection. The purpose of this study was to determine the presence of NK cells in the hDAF pig-to-cynomolgus monkey model. Data describing the iso- lation and functional characterization of cynomolgus mon- key NK cells are also presented. MATERIALS AND METHODS AND RESULTS Validation of Monoclonal Antibody Crossreactivity A panel of commercially available human monoclonal antibodies were screened by FACS analysis to determine their crossreactivity with cynomolgus lymphocytes. Antibodies with the specificity to the following antigens were tested: CD16, CD56, CD57, CD94, CD158a, CD161, and KIR. Only CD16 (GO22 [Becton-Dickinson, New Jersey] and 3G8 [Research Diagnostics Inc, New Jersey]) antibodies were found to crossreact with 12% to 18% of cynomol- gus monkey lymphocytes, the expected proportion of NK cells in the peripheral blood lymphocytes of monkeys. 7,8 Triple Immunofluorescent Histology Transgenic pig-to-cynomolgus monkey kidney transplants were performed with different immunosuppressive regimens. Grafts that were studied were chosen based on their histology at the time of necropsy. Three-color immunofluorescent histology was carried out using monoclonal antibodies to CD16 (3G8; Becton-Dickin- son), CD3 (FN18 FN18; BPRC, The Netherlands), and CD14 (MøP9; Becton-Dickinson). Cells staining positive for CD16 and negative for CD3 and CD14 were identified as NK cells. Grafts demonstrating no evidence of AVR or those explanted because of technical failure showed one or less NK cells per high power field (/HPF). Increased number of infiltrating NK cells were found in grafts with AVR. Grafts surviving 3, 15, 24, 28, 34, and 51 days demonstrated 3.6, 2.5, 13.4, 11.5, 5, and 4.7 NK cells /HPF respectively. Reverse-Transcriptase Polymerase Chain Reaction of Cynomolgus Monkey CD94 To confirm the presence of NK cells, CD94, found primarily on NK cells, was detected in the mRNA of grafts exhibiting AVR by reverse-transcriptase polymerase chain reaction (RT-PCR). Prim- ers were designed to span conserved sequences in human and murine CD94 (GenBank). Sequencing of the RT-PCR product shows greater than 90% homology to human CD94. CD94 mRNA was not detected in grafts lost to technical failure nor in grafts without histologic rejection. CD94 transcripts were detected in grafts with AVR and correlated with the immunofluorescent histology results. Isolation of Cynomolgus Monkey NK Cells and Functional Characterization Natural killer cells from cynomolgus monkey peripheral blood mononuclear cells were isolated by negative selection using the following antibodies for depletion: CD3 (FN18; BPRC), CD14 (MøP9; Becton-Dickinson), and CD20 (H299; Coulter, Fullerton, Calif). The isolated population was shown to consist of less than 1% CD3, CD4, CD14, and CD20 positive cells by FACS analysis. Following stimulation with recombinant human Interleukin-2 (IL-2) (Sigma, St Louis, Mo) for 48 hours, more than 70% were CD16 positive and more than 90% were CD8+/CD3-. Similar to enriched human NK cells, unstimulated cynomolgus NK cells demonstrated lysis of K562 cells (ATCC; Virginia): 35% at 40:1 effector to target ratio by 51 Cr release assay. Daudi cells (ATCC) were resistant to lysis: 10% at 40:1 effector to target ratio by 51 Cr release assay. Stimulation with recombinant human IL-2 and IL-15 (Sigma, St Louis, Mo) for 48 hours resulted in a significantly elevated lysis of Daudi cells: 35% at 20:1 effector to target ratio. From Imutran, A Novartis Pharma AG Co (D.Q., C.B., G.C., A.R., G.C., L.C., C.A., B.H., H.D., E.C., and D.W.) and the Department of Surgery, Addenbrookeı´s Hospital, University of Cambridge Clinical School, Cambridge, United Kingdom (D.Q.). Address reprint requests to Dr D Quan, London Health Sci- ences Center, Department of Surgery, 339 Windermere Road, London, Ontario, Canada N6A 5A5. 0041-1345/00/$–see front matter © 2000 by Elsevier Science Inc. PII S0041-1345(00)01046-0 655 Avenue of the Americas, New York, NY 10010 936 Transplantation Proceedings, 32, 936–937 (2000)