Anti-CD4-Induced Long-Term Acceptance of Rat Liver Allografts in a High-Responder Model Is Not Based on Pure Immunosuppression K. Kohlhaw, U. Sack, I. Lehmann, G. Drews, R. Schwarz, T. Hartwig, A. Tannapfel, F. Berr, M. Oertel, U. Marx, M. Lehmann, C. Witterkind, J. Hauss, and F. Emmrich I N ALLOANTIGEN recognition and consecutive induc- tion of a rejection process, T-helper cells expressing the CD4 molecule on theircell surface play a central role. Presence of CD4 1 cells was elementary for the induction of the rejection process 1 in experimental models. Monoclonal anti-CD4 antibodies (mab) have been shown to have not only an immunosuppressive, but also an immunomodulative effectby inducing donor-specific tolerance in a high-re- sponder renal 2 or heart 3 transplantmodels.Recently we could show thatthe anti-CD4 mab RIB5/2 wasable to induce donor-specific tolerance of a transplanted liver in a high-responder liver transplant model [Dark Agouti (DA) 3 Lewis (LEW)]. 4 Although thisanti-CD4 mab issug- gested to be nondepleting in renal and hearttransplant models, 5 in the liver transplant model its behavior seems to be different since after RIB5/2 therapy and liver transplan- tation,CD4 1 cells were eliminated from the peripheral blood for several days. 4 To learn more about the potency of the anti-CD4 mab RIB5/2 in the liver transplant model, the aim of this study was to evaluate the impact of an additional blockade of a costimulatory pathway (LFA-1) together with an insufficient dose of the anti-CD4 mab. Furthermore, we tried to discriminate between immunomodulation and im- munosuppression. MATERIALS AND METHODS Livers ofDA rats (Haarlan Corp),;250 g (RT1a), were trans- planted orthotopically into LEW rats (Medizinisch-Experimen- telles Zentrum, Leipzig) ;250 g (RT1I), according to the tech- nique of Kamada and Calne 6 (arterialized, bile duct anastomosis stented with a plastic tube). An antibiotic prophylaxis was per- formed perioperatively with Terramycinr for 5 days. Water and food were given ad libitum. RIB5/2 [20 mg/kg body weight (BW)] or 1 mg/kg BW anti-LFA-1mab (WT.1, Pharmingen) were applied intraperitoneallywithoutfurther immunosuppression. Group I received anti-CD4 alone at days 1 and 2 (n 5 7), group II only at day 0 (n 5 7), group III anti-CD4 and anti-LFA-1 at days 0 and 1 (n 5 7), and group IV RIB5/2 atdays 5 and 6 to discriminate between immunosuppressive and immunomodulatory effects ( n 5 7). Controls(n 5 9) did not receive any immunosuppression. Serum transaminases and surface and activation markers of mono- nuclear blood cells were examined by FACS [CD3 1 , CD4 1 , CD8 1 , and NK cells,expression of RT1B (MHC class II), TFR (trans- ferrin receptor) and CD11a/b]. Liver core biopsies were taken at days 7 and 14. All investigations were performed with regard to GMP and GLP conditions with permission of the local government of Saxony (TVV 4/98). RESULTS The additional blockade of a costimulatory pathway (here, LFA-1) with the monoclonal antibody WT.1 was unable either to induce long-term graft acceptance or to enhance the effectof an insufficientdose of the anti-CD4 mab RIB5/2. Median survival time was 18 days (RIB5/2 1 day) vs 14 days (RIB5/2 1 day and anti-LFA-1 2 days, Fig 1) and therefore comparable to those achieved with anti-LFA-1 mab alone. 7 Animals died with histologically proven hepatic allograftrejection diagnosed in the biopsy performed on day 7 and/or in the autopsy. Additional blockade of a signal 2 pathway is not able to enhance the effect of an insufficient blockade of signal 1. Therefore, both monoclonal antibod- ies do not work synergistically. The delayed application of the anti-CD4 mab RIB5/2 starting on day 5 after hepatic transplantation resulted in a slightbut not sufficientprolongation ofmedian survival time (12 days) when compared to untreated controls (9 days, Fig 2). All animals died with clinical signs of rejection (increasing hyperbilirubinemia and serum transaminases, heavy weight loss). By postmortal histology, in four of seven animals of the delayed treatment group, a rejection could be found besides extensive liver cell necrosis in absence of vascular thrombosis. With regard to the effect on the CD4 1 cells, the delayed application of the anti-CD4 mab RIB5/2 starting on day 5 From the Klinik und Poliklinik fu¨ r Chirurgie II (K.K., G.D., R.S., T.H., J.H.), Institut fu¨ rPathologie (A.T.,C.W.), Institute fu¨ r Klinische Immunologie (U.S., I.L., U.M., F.E.), Medizinische Klinik (F.B., M.D.), Universita¨ t Leipzig, Leipzig, Germany, and Institute fu¨ r Biochemie (M.L.), Universita¨ t Rostock, Rostock, Germany. This work was supported by the Interdisziplina¨ ires Zentrum fu¨ r Klinische Forschung (IZKF) of the Universita¨ t Leipzig. Address reprint requests to Dr K. Kohlhaw, Klinik fu¨ r Abdom- inal Transplants und Gefa Bchirurgie,University of Leipzig, Liebigstr. 20a, D-04103 Leipzig, Germany. © 2001 by Elsevier Science Inc. 0041-1345/01/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0041-1345(01)01931-5 Transplantation Proceedings, 33, 2173–2175 (2001) 2173