Gavin, M.A. et al. Single-cell analysis of normal and FOXP3-mutant human T cells: FOXP3 expression without regulatory T cell development. Proc. Natl. Acad. Sci. USA 103, 6659-6664

Department of Immunology and Howard Hughes Medical Institute, University of Washington, Box 357370, Seattle, WA 98195, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 05/2006; 103(17):6659-64. DOI: 10.1073/pnas.0509484103
Source: PubMed


Forkhead winged-helix transcription factor Foxp3 serves as the dedicated mediator of the genetic program governing CD25+CD4+ regulatory T cell (T(R)) development and function in mice. In humans, its role in mediating T(R) development has been controversial. Furthermore, the fate of T(R) precursors in FOXP3 deficiency has yet to be described. Making use of flow cytometric detection of human FOXP3, we have addressed the relationship between FOXP3 expression and human T(R) development. Unlike murine Foxp3- T cells, a small subset of human CD4+ and CD8+ T cells transiently up-regulated FOXP3 upon in vitro stimulation. Induced FOXP3, however, did not alter cell-surface phenotype or suppress T helper 1 cytokine expression. Furthermore, only ex vivo FOXP3+ T(R) cells persisted after prolonged culture, suggesting that induced FOXP3 did not activate a T(r) developmental program in a significant number of cells. FOXP3 flow cytometry was also used to further characterize several patients exhibiting symptoms of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) with or without FOXP3 mutations. Most patients lacked FOXP3-expressing cells, further solidifying the association between FOXP3 deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. Interestingly, one patient bearing a FOXP3 mutation enabling expression of stable FOXP3(mut) protein exhibited FOXP3(mut)-expressing cells among a subset of highly activated CD4+ T cells. This observation raises the possibility that the severe autoimmunity in FOXP3 deficiency can be attributed, in part, to aggressive T helper cells that have developed from T(R) precursors.

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    • "It is not fully clear whether Foxp3+ Treg cells can lose Foxp3 expression and suppressive function, as well as whether Foxp3+ Treg cells exhibit characteristics of other Th cell subsets. A number of studies in which Foxp3+ Treg cells were adoptively transferred into lymphopenic mice demonstrated that approximately 10–50% of the transferred Treg cells lost Foxp3 expression (14–16). Furthermore, Treg cells from both the periphery and the thymus were converted into Th17 cells upon stimulation with anti-CD3, anti-CD28, and IL-6, demonstrating a degree of plasticity (17). "
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    ABSTRACT: Regulatory T (Treg) cells are essential for normal immune surveillance systems, and their dysfunction leads to development of diseases, such as autoimmune disorders. CD4(+)CD25(+) Treg cells are well-known suppressive cells, which express the transcription factor Foxp3, are indispensable for the maintenance of immune self-tolerance and homeostasis by suppressing aberrant or excessive immune response. Other Foxp3(-) Treg cells include Tr1, Th3, CD8(+)CD28(-/-), and Qa1-restricted T cells; however, the contribution of these Treg cells to self-tolerance, immune homeostasis as well as preventing autoimmunity is not well defined. Here, we discuss the phenotypes and function of Foxp3(+) Treg cells and the potential use of such Treg cells against rheumatoid arthritis (RA). Of note, even though most expanded populations of Foxp3(+) Treg cells exhibit suppressive activity, tissue-associated or antigen-specific Treg cells appear superior in suppressing local autoimmune disorders such as RA. In addition, utilizing tissue-associated Foxp3(+) Treg cells from stem cells may stable Foxp3 expression and avoid induction of a potentially detrimental systemic immunosuppression.
    Frontiers in Oncology 08/2014; 4:209. DOI:10.3389/fonc.2014.00209
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    • "Although FoxP3 generally identifies natural thymus-derived Tregs, adaptive Tregs may or may not express this transcription factor [45]. Studies have also indicated that FoxP3 expression can be induced in human CD4+ effector T cells after activation as a normal consequence of CD4+ T cell activation [42]–[46] and that IFN-γ and IL-2 production are not suppressed in FoxP3+ effector T cells [47], [48], thus bringing into doubt its validity as an exclusive marker of Tregs. The characterisation of the pool of CD25+FoxP3+ Tregs identified post CD25 enrichment following CMVpp65 stimulation and the subsequent loss of FoxP3 expression following short-term culture highlights the need for future studies. "
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    ABSTRACT: Previous studies have demonstrated the effective control of cytomegalovirus (CMV) infections post haematopoietic stem cell transplant through the adoptive transfer of donor derived CMV-specific T cells (CMV-T). Strategies for manufacturing CMV immunotherapies has involved a second leukapheresis or blood draw from the donor, which in the unrelated donor setting is not always possible. We have investigated the feasibility of using an aliquot of the original G-CSF-mobilized graft as a starting material for manufacture of CMV-T and examined the activation marker CD25 as a targeted approach for identification and isolation following CMVpp65 peptide stimulation. CD25+ cells isolated from G-CSF-mobilized apheresis revealed a significant increase in the proportion of FoxP3 expression when compared with conventional non-mobilized CD25+ cells and showed a superior suppressive capacity in a T cell proliferation assay, demonstrating the emergence of a population of Tregs not present in non-mobilized apheresis collections. The expansion of CD25+ CMV-T in short-term culture resulted in a mixed population of CD4+ and CD8+ T cells with CMV-specificity that secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. Furthermore CD25 expanded cells retained their suppressive capacity but did not maintain FoxP3 expression or secrete IL-10. In summary our data indicates that CD25 enrichment post CMV stimulation in G-CSF-mobilized PBMCs results in the simultaneous generation of both a functional population of anti-viral T cells and Tregs thus illustrating a potential single therapeutic strategy for the treatment of both GvHD and CMV reactivation following allogeneic haematopoietic stem cell transplantation. The use of G-CSF-mobilized cells as a starting material for cell therapy manufacture represents a feasible approach to alleviating the many problems incurred with successive donations and procurement of cells from unrelated donors. This approach may therefore simplify the clinical application of adoptive immunotherapy and broaden the approach for manufacturing multi-functional T cells.
    PLoS ONE 01/2014; 9(1):e85911. DOI:10.1371/journal.pone.0085911 · 3.23 Impact Factor
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    • "In contrast, several studies have claimed that this is not the case in humans. They suggest that human CD4+CD25- T effector cells activated through TCR stimulation alone with anti-CD3 and anti-CD28 show transiently upregulated Foxp3 expression and demonstrate that this Foxp3 expression correlates with neither anergy nor suppressive function [35,41-45]. In addition to confirming these previous studies, our results shown in Figure 2A and Figure 3A reveal new aspects involving Foxp3 expression. "
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    ABSTRACT: The peripheral Foxp3(+) Treg pool consists of naturally arising Treg (nTreg) and adaptive Treg cells (iTreg). It is well known that naive CD4(+) T cells can be readily converted to Foxp3(+) iTreg in vitro, and memory CD4(+) T cells are resistant to conversion. In this study, we investigated the induction of Foxp3(+) T cells from various CD4(+) T-cell subsets in human peripheral blood. Though naive CD4(+) T cells were readily converted to Foxp3(+) T cells with TGF-β and IL-2 treatment in vitro, such Foxp3(+) T cells did not express the memory marker CD45RO as do Foxp3(+) T cells induced in the peripheral blood of Hepatitis B Virus (HBV) patients. Interestingly, a subset of human memory CD4(+) T cells, defined as CD62L(+) central memory T cells, could be induced by TGF-β to differentiate into Foxp3(+) T cells. It is well known that Foxp3(+) T cells derived from human CD4(+)CD25(-) T cells in vitro are lack suppressive functions. Our data about the suppressive functions of CD4(+)CD62L(+) central memory T cell-derived Foxp3(+) T cells support this conception, and an epigenetic analysis of these cells showed a similar methylation pattern in the FOXP3 Treg-specific demethylated region as the naive CD4(+) T cell-derived Foxp3(+) T cells. But further research showed that mouse CD4(+) central memory T cells also could be induced to differentiate into Foxp3(+) T cells, such Foxp3(+) T cells could suppress the proliferation of effector T cells. Thus, our study identified CD4(+)CD62L(+) central memory T cells as a novel potential source of iTreg.
    PLoS ONE 10/2013; 8(10):e77322. DOI:10.1371/journal.pone.0077322 · 3.23 Impact Factor
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