Thymus-derived CD4 ؉ CD25 ؉ regulatory T cells suppress autoreactive CD4 ؉ and CD8 ؉ T cells and thereby protect from autoimmunity. In animal models, adoptive transfer of CD4 ؉ CD25 ؉ regulatory T cells has been shown to prevent and even cure autoimmune diseases as well as pathogenic alloresponses after solid organ and stem-cell transplantations. We recently described methods for the efficient in vitro expansion of human regulatory T cells for clinical applications. We now demonstrate that only CCR7-and L-selectin (CD62L) - IntroductionSelf-tolerance within the T-cell compartment is primarily ensured by positive and negative selection during T-cell development in the thymus. Autoreactive T cells that escape central deletion are frequently controlled by peripheral tolerance mechanisms, including cell-mediated suppression by CD4 ϩ CD25 ϩ regulatory T (Treg) cells. [1][2][3] Thymic maturation and suppressive function of natural Treg cells depend on expression of the transcriptional repressor forkhead box P3 (FOXP3), as nonsense mutations in this gene result in loss of Treg-cell function and severe autoimmunity in mice and humans. [4][5][6][7][8] Apart from suppression of autoreactive T cells, FOXP3 ϩ CD4 ϩ CD25 ϩ Treg cells also dampen immune responses against infectious pathogens, 9 cancer, 10 and allogeneic organ 11 and stem-cell grafts. 12 Thus, depletion of Treg cells seems a promising strategy to augment immune responsiveness to tumors, chronic infections, and vaccination, while an enhancement of Treg-cell activity is envisaged for the prevention and treatment of T-cellinduced diseases. 13 In animal models, the adoptive transfer of CD4 ϩ CD25 ϩ Treg cells has been shown to protect from type 1 diabetes 14,15 or experimental autoimmune encephalomyelitis 16 and even revert ongoing disease in colitis 17,18 and arthritis. 19 Similarly, adoptively transferred Treg cells protected against rejection and graft-versus-host disease (GVHD) after allogeneic organ transplantation and bone marrow transplantation (BMT), respectively. 11,[20][21][22][23][24][25] In human peripheral blood, natural Treg cells mainly reside within the subpopulation of CD4 ϩ T cells with high CD25 expression levels (CD25 high ), 26 while cells with intermediate CD25 expression (CD25 int ) consist mainly of recently activated and memory T cells, with only 5% to 15% FOXP3 ϩ Treg cells (P.H. and M.E., unpublished results, June 2006). Due to lack of Treg cell-specific surface markers, isolation of CD4 ϩ CD25 high T cells is thus far considered the most promising strategy for the generation of pure Treg-cell products. 27 As they represent only 1% to 3% of peripheral-blood mononuclear cells (PBMCs), we and others recently described methods for the in vitro expansion of Treg cells for future clinical trials. [28][29][30] Cross-linking of stimulating CD3 and CD28 antibodies by beads or Fc receptor-bearing fibroblasts in combination with high-dose interleukin-2 (IL-2) resulted in a 3-to 4-log expansion within 3 to 4 weeks. Cultured cells maintained Treg-c...

The adoptive transfer of donor CD4+CD25+ regulatory T cells has been shown to protect from lethal graft-versus-host disease after allogeneic bone marrow transplantation in murine disease models. Efficient isolation strategies that comply with good manufacturing practice (GMP) guidelines are prerequisites for the clinical application of human CD4+CD25+ regulatory T cells. Here we describe the isolation of CD4+CD25+ T cells with regulatory function from standard leukapheresis products by using a 2-step magnetic cell-separation protocol performed under GMP conditions. The generated cell products contained on average 49.5% CD4+CD25high T cells that phenotypically and functionally represented natural CD4+CD25+ regulatory T cells and showed a suppressive activity comparable to that of CD4+CD25+ regulatory T-cell preparations purified by non-GMP-approved fluorescence-activated cell sorting.

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Graft-versus-host disease (GVHD) is a major complication after allogeneic stem cell transplantation caused by donor T cells in the stem cell graft. Prophylactic treatment of GVHD usually comprises immunosuppressive medication including cyclosporin A (CsA) that blocks calcineurin phosphatase activity and thereby inhibits nuclear factor of activated T cells (NFAT)-mediated cytokine production. CD4+CD25+ natural regulatory T (Treg) cells have been shown to suppress the proliferation and cytokine secretion of conventional CD4+ and CD8+ T cells in vitro and to protect from GVHD lethality in murine models of allogeneic BMT. As the adoptive transfer of human CD4+CD25+ Treg cells as a means of GVHD prophylaxis is currently under investigation, we aimed to explore the influence of CsA on survival, cytokine production and suppressive activity of this T cell subpopulation. To this end, in vitro expanded human CD4+CD25high and CD4+CD25− T cells were stimulated with anti-CD3 and anti-CD28 antibodies and simultaneously exposed to various concentrations of CsA. While T cell death occurred in both in vitro expanded subpopulations at high CsA concentrations of 1000 ng/ml, with only 10 to 20% viable cells after 72h exposure, CD4+CD25high T cells showed significantly enhanced resistance to lower CsA concentrations of 100 ng/ml with still approx. 60% viable cells, as compared to only 20% viability in expanded CD4+CD25− T cells. At this lower concentration, CsA also blocked the proliferation of the two subpopulations in response to anti-CD3/CD28 stimulation, as determined by CFSE-dilution, and inhibited the IL-2 and IFN-γ production of CD4+CD25− T cells as well as the IL-10 production by expanded CD4+CD25high Treg cells, as shown by intracellular staining after stimulation with PMA/ionomycin. Addition of exogenous IL-2 protected both subpopulations from CsA-induced cell death and restored their proliferative capacity but was unable to abrogate the block in cytokine production. Surprisingly, the suppressive activity of in vitro expanded CD4+CD25high Treg cells was neither affected by a pre-incubation of the cells with 100 ng/ml CsA, nor by its presence during the suppression assay. These results indicate that in vitro expanded CD4+CD25+ Treg cells are less sensitive to CsA-induced apoptosis and that their yet unknown mechanism of suppression does not involve the calcineurin pathway.