Eur. J. Immunol. 2008. 38: 1677–1688 In situations characterised by high responder frequencies such as stringent strain combinations and most likely in clinical transplantation we need powerful protocols for in vitro generation and differentiation of antigen specific regulatory T cells. Our protocol presented here may be one approach to generate such potent Tregs. Recently, Golshayan et al. [44] have presented a protocol of selecting and expanding Treg with indirect allospecificity for a single MHC class I antigen (H2Kb) from a pool of naturally occurring Treg using H2Kb peptide-pulsed immature dendritic cells. The expanded Treg lines could regulate indirect allo- responses of effector CD4+ cells in vivo. In contrast, the expanded Treg could not inhibit direct pathway alloresponses, such that C57BL/6 skin transplants were rejected with the same kinetic as in control mice [44]. These data indicate that Treg with a broader spectrum of alloreactivty is most likely required to control rejection of a fully allogeneic graft. The protocol for generating Treg reported here is aimed at selecting Treg controlling direct pathway immune responses and therefore complements the findings made by Golshayan et al. [44]. The underlying mechanism by which anti-CD4 antibody treatment induces the enrichment/selection of Treg in vitro remains to be investigated. CD4 is an important accessory and costimulatory molecule that augments the signal received by the TCR complex and helps in the activation of the T cells [45]. Blockade of this interaction using anti-CD4 antibodies increases the threshold of activation through the TCR, so that, under these conditions, only cells with high affinity have the capacity to be activated and proliferate [46]. These cells with high affinity/ specificity TCR could be identical with cells that manifest regulatory activity in this study. Indeed, autoreactive CD25+CD4+ regulatory cells can be generated in the thymus by interactions with a single self-peptide. Selection of these cells appears to require a TCR with high affinity for a self peptide as thymocytes with low-affinity TCR do not undergo selection into this pathway [47]. Furthermore, reduced or blocking levels of CD4 expression, can convert T cell responses from stimulatory to inhibitory [48]. We can speculate that using the protocol described here, T cell activation has purposefully become more difficult and only a small percentage of cells can undergo activation and proliferation (regulatory population), while cells that do not receive any signals eventually die by apoptosis (these include the effector popula- tion). Indeed, in our previous studies we could show that anti-CD4 mAb-treated allo-reactive T cells are characterised by a relative resistance to activation-induced cell death (AICD) [49]. Thus, we can conclude that anti-CD4 mAb treatment of allo-reactive T cells facilitates the expansion and differentiation of antigen.specific Treg, which are characterised by a relative resistance to AICD, whereas effector T cells dye by apoptosis. Alternatively, the anti- CD4 antibody may directly deliver a negative signal into the T cells [50] and thereby drive the selection of a population of Treg instead of selecting it from a pre-existing pool. Interestingly, a recent study performed by McFadden et al. [51] has revealed that stimulation with the CD4 ligand IL-16 results in enhanced migration and de novo induction of a regulatory population of CD25+CTLA4+ Immunomodulation 1685 Foxp3+ cells. It is likely that TCR affinity and CD4 expression levels at the cell surface both play equal roles in our protocol. Following this line of thinking, we believe that it may be possible to develop protocols, targeting one or more accessory or costimulatory molecules [11], to generate stringent culture conditions in which only a specific T cell population (regulatory population) can be selected [20]. Our findings may help to understand the mechanisms underlying the function of Treg in vitro and in vivo, which will enable its use the cells for therapeutic strategies in vivo. This protocol, stimulating T cells with alloantigen-presenting cells in the presence of anti-CD4, provides a powerful, simple and fast method of obtaining cells ex vivo with strong regulatory potential but also illustrates the immense tolerogenic and therapeutic potential of non-depleting anti-CD4 antibodies. Mice CBA.Ca (CBA, H2k), C57BL/10 (B10, H2b), BALB/c (H2d), CD52- transgenic CP1-CBA.Ca (H-2k), and CBA.Rag 1–/– (H2k) were bred and housed in the Biomedical Services facility at the John Radcliffe Hospital (Oxford, UK) in accordance with the Animals (Scientific Procedure) Act 1986 of the UK. Sex-matched mice aged 6–12 weeks were used in all experiments. Reagents and monoclonal antibodies The following reagents were used for in vitro assays and flow cytometry. The hybridomas YTS 169.4.2 (anti-CD8) and YTS 177.9 (“YTS 177”, anti-CD4) were kindly provided by Professor Herman Waldmann (Sir William Dunn School of Pathology, Oxford, UK). TIB120 (anti-class II) was obtained from American Type Culture Collection (ATCC) Manassas, VA). RM4–5 (anti-CD4)-cychrome, 16A (anti-CD45RB)-PE, MEL-14 (anti-CD62L)-PE, 7D4 (anti- CD25)-biotin and streptavidin-allophycocyanin were from Phar- Mingen (San Diego, CA). 4B12 (anti-CCR7)-PE and FJK-16s (anti- FoxP3)-APC were from eBioscience (San Diego, CA). CFSE was from Molecular Probes (OR). Cell preparation and purification Purification of T cell subpopulations Single-cell suspensions from spleen and lymph nodes of naive CBA mice were prepared by forcing the organs through a 70-lm nylon mesh. After erythrocyte removal by hypotonic lysis, cell suspen- sions were incubated with YTS169 (anti-CD8, 200 lg/mL) and TIB120 (anti-class II, 100 lg/mL) at 2 108 cells/mL. Cell suspension was incubated with sheep anti-rat-coated Dynabeads f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu