Journal of Cellular Biochemistry 103:1693–1706 (2008) Immunosuppressive Drug-Free Operational Immune Tolerance in Human Kidney Transplants Recipients: Part II. Non-statistical Gene Microarray Analysis Victor Sivozhelezov, 1,2 Christophe Braud, 3,4 Luca Giacomelli, 1 Eugenia Pechkova, 1,2 Magali Giral, 3 Jean-Paul Soulillou, 3,4 Sophie Brouard, 3,4 and Claudio Nicolini 1,2 * 1 Nanoworld Institute and Eminent Biophysics Chair, University of Genova, Corso Europa 30, Genova 16132, Italy 2 Fondazione Elba, Piazza SS. Apostoli 66, Rome, Italy 3 INSERM U643, Institut de Transplantation Et de la Recherche en Transplantation (ITERT), Nantes F-44093, France 4 Faculte ´ de Me ´decine, Universite ´ de Nantes, Nantes F-44000, France Abstract Kidney transplant is the reference treatment for patients with end-stage renal disease, but patients m develop long-term rejection of the graft. However, some patients do not reject the transplant, but instead are ope tolerant state despite withdrawal of immunosuppressive treatment. In this second article we outline a microarray- identification of key leader genes associated respectively to rejection and to operational tolerance of the kidney tr in humans by utilizing a non/statistical bioinformatic approach based on the identification of ‘‘key genes,’’ either a mostly changing their expression, or having the strongest interconnections. A uniquely informative picture emerg genes controlling the human transplant from the detailed comparison of these findings with the traditional statisti (Tusher et al. [2001] Proc Natl Acad Sci USA 98:5116–5121) analysis of the microarrays and with the clinical study out in the accompanying part I article. J. Cell. Biochem. 103: 1693–1706, 2008. ß 2007 Wiley-Liss, Inc. Key words:gene expression; DNA microarray; bioinformatics; kidney transplant; transplant tolerance Kidney transplant is the reference treatment for patients with end-stage renal disease. The advent of this therapeutic option has notably reduced morbidity and mortality in patients with this disease.Moreover,improvements in the clinical management of transplant recipi- ents have contributed to increase graft survival and to limit the risks of rejection in kidney transplantation by proper pharmacological immunosuppression [Hariharan et al.,2000]. However, such lifelong immunosuppression, which poorly influences long-term chronic transplant dysfunction [Opelz, 1995], may promote tumor growth by a direct effect on tumor cells [Hojo et al., 1999]and may also decrease recipient immune responses to patho- gens, including oncogeneicviruses [Dantal et al., 1998; Soulillou and Giral, 2001]. In humans, immunosuppression withdrawal leads, in most cases, to transplant rejection. Nevertheless, certain rare patients (‘operation- ally tolerant’) maintain stable graft function despite the absence oftreatment,suggesting that a state of nonresponsivenesscan be achieved in clinical transplantation [Strober et al., 2000].This phenomenon can occur in liver transplantation [Thomson et al., 2001; ß 2007 Wiley-Liss, Inc. This article contains supplementary material, which may be viewed at the Journal of Cellular Biochemistry website at http://www.interscience.wiley.com/jpages/0730- 2312/suppmat/index.html. Grant sponsor: FIRB International MIUR (Ministry for Education, University, and Research), Italy; Progreffe foundation, France. *Correspondence to: Claudio Nicolini, Nanoworld Institute and Eminent Biophysics Chair, University of Genova, Italy. E-mail: manuscript@ibf.unige.it Received 19 June 2007; Accepted 3 August 2007 DOI 10.1002/jcb.21557