Eur. J. Immunol. 2008. 38: 1677–1688 DOI 10.1002/eji.200737562 Immunomodulation 1677 Anti-CD4-mediated selection of Treg in vitro – in vitro suppression does not predict in vivo capacity to prevent graft rejection Vanessa Oliveira*1, Birgit Sawitzki*1,2, Stephanie Chapman1, Christine Appelt2, Inga Gebuhr2, Joanna Wieckiewicz1, Elaine Long1 and Kathryn J. Wood1 1 Transplantation Research Immunology Group, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, UK 2 Institute of Medical Immunology, Charit, Berlin, Germany Regulatory T cells (Treg) have been shown to play a role in the prevention of autoimmune diseases and transplant rejection. Based on an established protocol known to generate alloantigen reactive Treg in vivo, we have developed a strategy for the in vitro selection of Treg. Stimulation of unfractionated CD4+ T cells from naive CBA.Ca (H2k) mice with C57BL/ 10 (H2b) splenocytes in the presence of an anti-CD4 antibody, YTS 177, resulted in the selection of Treg able to inhibit proliferation of naive T cells. In vivo, the cells were able to prevent rejection of 80% C57BL/10 skin grafts when co-transferred to CBA.Rag–/– mice together with naive CD45RBhighCD4+ cells. Purification of CD62L+CD25+CD4+ cells from the cultures enriched for cells with regulatory activity; as now 100% survival of C57BL/10 skin grafts was achieved. Furthermore, differentiation of Treg could be also achieved when using purified CD25–CD4+ naive T cells as a starting population. Interestingly, further in vitro expansion resulted in a partial loss of CD4+ cells expressing both CD62L and CD25 and abrogation of their regulatory activity in vivo. This study shows that alloantigen stimulation in the presence of anti-CD4 in vitro provides a simple and effective strategy to generate alloreactive Treg. Key words: Anti-CD4 IFN-c Transplantation Treg Introduction Naturally occurring regulatory T cells (nTreg) are produced in the thymus during normal T cell development [1] and are involved in the maintenance of self-tolerance [2–5] and development of tolerance to allogeneic transplants [6–8]. Despite its broad spectrum of reactivity, the frequency of nTreg responding to foreign antigens is low and unable to provide protection against grafts mismatched for multiple major and minor histocompat- ibility antigens following transplantation [9, 10]. On the contrary, Treg induced in response to antigen (iTreg) are generated from naive T cells under defined conditions in vivo and in vitro [2, Correspondence: Birgit Sawitzki e-mail: birgit.sawitzki@charite.de 11–19]. For therapeutic purposes, it may be possible to harness both the potential of nTreg as well as to generate T cells with regulatory activity to defined antigens, such as alloantigens, ex vivo. Repeated stimulation of unfractionated CD4+ cells in the presence of IL-10 or using a combination of immunosuppressive drugs induces the selection of Treg in vitro. These induced Treg are able to prevent colitis or central nervous system inflammation in animal models [20–22]. Pre-treatment of naive CBA mice with a non-depleting anti- CD4 mAb (YTS 177) and donor-specific (B10) transfusion enables the development of alloantigen-reactive CD4+ Treg, which render the recipient tolerant to a subsequent challenge with an allogeneic * These authors contributed equally to this work. f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu