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Several recent reports have suggested that in vitro exposure of CD4+T cells to rabbit antithymocyte globulin (rATG), which is commonly used to prevent and treat graft-versus-host disease and allograft rejection, is an effective method to induce CD4+CD25+FOXP3+T regulatory cells (Tregs). We and others, however, have shown that FOXP3 is also expressed in activated T cells. We therefore investigated whether the induction of FOXP3 expression by rATG resulted in a stable population of suppressive Tregs. We found that exposure of peripheral blood mononuclear cells (PBMCs) or conventional T cells to rATG resulted in induction of transient rather than stable expression of CD25 and FOXP3. Furthermore, rATG-treated T effector cells acquired neither an immunosuppressive profile of cytokine production nor suppressive capacity, even at the time of maximal FOXP3 expression. These findings indicate that the notion that rATG can be used to induce Tregs in vitro for cellular therapy in vivo should be re-evaluated.

Forkhead box P3 (FOXP3) is constitutively expressed by CD4+CD25hiregulatory T cells (nTregs). Mutations of FOXP3 cause a severe autoimmune syndrome known as immune dysregulation polyendocrinopathy enteropathy X-linked, in which nTregs are absent or dysfunctional. Whether FOXP3 is essential for both differentiation and function of human nTreg cells remains to be demonstrated. Because FOXP3is an X-linked gene subject to X-chromosome inactivation (XCI), we studied 9 healthy female carriers of FOXP3mutations to investigate the role of wild-type (WT) versus mutated FOXP3 in different cell subsets. Analysis of active WT versus mutated (mut)–FOXP3allele distribution revealed a random pattern of XCI in peripheral blood lymphocytes and in naive and memory CD4+T cells, whereas nTregs expressed only the active WT-FOXP3. These data demonstrate that expression of WT-FOXP3 is indispensable for the presence of a normal nTreg compartment and suggest that FOXP3 is not necessary for effector T-cell differentiation in humans.

Suppression by T regulatory cells (Treg cells) is a major mechanism by which the immune system controls responses to self and nonharmful foreign proteins. Although there are many different types of Treg cells, the best characterized are those that constitutively express cell-surface IL-2Rα (CD25). We investigated whether altered T-cell–receptor (TCR)–mediated signaling in pure populations of ex vivo human CD4+CD25+ Treg cells might underlie their unique phenotype, including hyporesponsiveness to TCR–mediated activation and lack of cytokine production. CD4+CD25+ Treg cells displayed a consistent defect in phosphorylation of AKT at serine 473 and reduced phosphorylation of the AKT substrates FOXO and S6. Restoration of AKT activity via lentiviral-mediated expression of an inducibly active form of the kinase revealed that reduced activity of this pathway was necessary for the suppressive function of CD4+CD25+ Treg cells. These data represent the first demonstration of a causal association between altered signaling and the function of CD4+CD25+ Treg cells. Moreover, we have created the first system allowing inducible abrogation of suppression through manipulation of the suppressor cells. This system will be a powerful tool to further study the mechanism(s) of suppression by CD4+CD25+ Treg cells.

Suppression by T regulatory cells (Treg cells) is a major mechanism by which the immune system controls responses to self and nonharmful foreign proteins. Although there are many different types of Treg cells, the best characterized are those that constitutively express cell-surface IL-2Rα (CD25). We investigated whether altered T-cell–receptor (TCR)–mediated signaling in pure populations of ex vivo human CD4+CD25+Treg cells might underlie their unique phenotype, including hyporesponsiveness to TCR–mediated activation and lack of cytokine production. CD4+CD25+Treg cells displayed a consistent defect in phosphorylation of AKT at serine 473 and reduced phosphorylation of the AKT substrates FOXO and S6. Restoration of AKT activity via lentiviral-mediated expression of an inducibly active form of the kinase revealed that reduced activity of this pathway was necessary for the suppressive function of CD4+CD25+Treg cells. These data represent the first demonstration of a causal association between altered signaling and the function of CD4+CD25+Treg cells. Moreover, we have created the first system allowing inducible abrogation of suppression through manipulation of the suppressor cells. This system will be a powerful tool to further study the mechanism(s) of suppression by CD4+CD25+Treg cells.

Dendritic cells (DCs) are specialized antigen-presenting cells that monitor the antigenic environment and activate naive T cells. The role of DCs is not only to sense danger but also to tolerize the immune system to antigens encountered in the absence of maturation/inflammatory stimuli. Indeed, if a naive T cell encounters its antigen on immature DCs (iDCs), it may differentiate into a T-regulatory (Tr) rather than a T-effector cell. However, little is known about the mechanisms by which iDCs differentiate Tr cells. We developed a standardized and highly reproducible protocol to differentiate Tr cells by repetitive exposure of naive peripheral blood CD4+T cells to allogeneic iDCs. The resultant Tr cells are phenotypically and functionally identical to type 1 Tr (Tr1) cells because their generation requires production of IL-10 by iDCs, and they suppress T-cell responses through an interleukin-10 (IL-10)– and a transforming growth factor β (TGF-β)–dependent mechanism. In addition, Tr1 cells induced by iDCs do not require the presence of CD4+CD25+Tr cells for their generation, nor do they express high constitutive levels of CD25 or the transcription factor FoxP3. Thus, iDCs can drive the differentiation of Tr1 cells and can be used to generate large numbers of alloantigen-specific Tr1 cells for clinical use as a cellular therapy to restore peripheral tolerance.

The high-affinity receptor for interleukin-3 (IL-3) is a complex of the IL-3–binding subunit (?IL-3) and a larger ß chain—ßc, or, in the mouse, ßc or its close relative ßIL-3. There is evidence that the critical event that initiates signaling is not the approximation of the cytoplasmic domains of ?IL-3 and ßIL-3, but is, rather, the formation of a ß-ß homodimer. Many of these studies involved the analyses of receptor chimeras where the cytoplasmic domains were derived from ?IL-3, ßc or ßIL-3, and the extracellular domains were derived from other cytokine receptors, such as the erythropoietin receptor (EpoR). However, evidence that the EpoR may also associate with other receptors clouds the interpretation of these experiments. Therefore, we reevaluated the structure of the functional IL-3R using chimeric receptors with extracellular domains derived not from members of the cytokine-receptor family, but from CD8 or CD16. We show, by expression of these chimeras in Ba/F3 or CTLL-2 cells, that mitogenic signals were only generated by heterodimerization of the cytoplasmic domains of ?IL-3 and ßIL-3. Homodimers of either ?IL-3 or ßIL-3, alone or in combination, were nonfunctional. Furthermore, the ability of heterodimers to stimulate mitogenesis correlated with their ability to induce tyrosine phosphorylation of JAK-2. These data suggest that the physiological activation of the IL-3R involves the generation of simple heterodimers of ?IL-3 and ßIL-3.

Human CD4+CD25+regulatory T cells (TR) have the ability to suppress T cell responses and have recently been shown to play a critical role in peripheral tolerance and regulation of immune responses. CD4+CD25+TRare anergic, phenotypically CD25+brightand express high levels of the transcription factor FOXP3. Peripheral blood TRare rare and must be highly enriched for their suppressor function to be detected in vitro. This, combined with the lack of a unique marker that distinguishes them from activated T cells, makes it difficult to study TRfunction and evaluate their therapeutic potential. Current methods for isolating TRare cumbersome and time-consuming, and generally require three steps: Ficoll™ density centrifugation to isolate mononuclear cells; subsequent immunomagnetic T cell enrichment; and finally FACS sorting. The objective of this study was to develop a more simple technique to isolate highly purified TRfrom whole blood without using FACS sorting. Three steps were reduced to two by replacing the Ficoll™ separation and immunomagnetic T cell enrichment with a single antibody-mediated buoyant density centrifugation (RosetteSep®) to enrich CD4 T cells directly from whole blood. A cocktail of bi-specific antibodies was used to selectively bind unwanted cells to red cells causing them to pellet when centrifuged over Ficoll™. Purified CD4 T cells were recovered at the plasma-Ficoll™ interface and then separated using EasySep® column-free magnetic separation to select CD25 expressing cells. The EasySep® separation conditions were optimized for maximal CD4+CD25+brightT cell purity and recovery. The purified cells were analyzed for CD25, GITR, CD62L, HLA-DR and CTLA-4 expression. FOXP3 mRNA levels in purified CD4+CD25+T cell fractions were compared to corresponding CD4+CD25negT cell fractions using quantitative PCR. As TRare anergic and therefore not responsive to TCR stimulation, the purified CD4+CD25+T cell fractions were assessed for anergy in response to anti-CD3/CD28 coated beads. Suppression activity of purified CD4+CD25+T cells was assessed by measuring their ability to reduce the proliferative response of CD4+CD25negT cells to CD3/CD28 beads. T cell proliferation was quantified by measuring dilution of the fluorescent dye CFSE with flow cytometry. Purified CD4+CD25+T cell suspensions were 94 ± 3% (n=6) CD4+CD25+, 83 ± 7% (n=6) CD4+CD25+bright, 89 ± 2% (n=4) GITR+and CD62L+(92%, 95%; n=2). The purified cell suspensions were also highly enriched for CTLA-4 and HLA-DR expressing cells. FOXP3 mRNA levels in the purified CD4+CD25+T cell fractions from two donors were found to be 64 and 124 times higher than for the corresponding CD4+CD25negT cell fractions. The purified CD4+CD25+T cells displayed low or undetectable proliferative responses (n=4) to CD3/CD28 beads suggesting most cells in the purified fractions were anergic. Mixing purified CD4+CD25+T cells with CD4+CD25negT cells at a ratio of 1:1 almost completely eliminated detectable CD4+CD25negT cell proliferation (95 ± 3 % reduction, n=3), and suppression of proliferation continued to be detected when cells were mixed at a ratio of 1:10 (25 ± 6% reduction, n=3). In conclusion, combining RosetteSep® CD4 T cell enrichment with EasySep® CD25 positive selection yields highly pure CD4+CD25+bright/ FOXP3+TRin less time and with fewer steps than current isolation methods.