Compelling evidence suggests that the transcription factor Foxp3 acts as a master switch governing the development and function of CD4⁺ regulatory T cells (Tregs). However, whether transcriptional control of Foxp3 expression itself contributes to the development of a stable Treg lineage has thus far not been investigated. We here identified an evolutionarily conserved region within the foxp3 locus upstream of exon-1 possessing transcriptional activity. Bisulphite sequencing and chromatin immunoprecipitation revealed complete demethylation of CpG motifs as well as histone modifications within the conserved region in ex vivo isolated Foxp3⁺CD25⁺CD4⁺ Tregs, but not in naïve CD25⁻CD4⁺ T cells. Partial DNA demethylation is already found within developing Foxp3⁺ thymocytes; however, Tregs induced by TGF-β in vitro display only incomplete demethylation despite high Foxp3 expression. In contrast to natural Tregs, these TGF-β–induced Foxp3⁺ Tregs lose both Foxp3 expression and suppressive activity upon restimulation in the absence of TGF-β. Our data suggest that expression of Foxp3 must be stabilized by epigenetic modification to allow the development of a permanent suppressor cell lineage, a finding of significant importance for therapeutic applications involving induction or transfer of Tregs and for the understanding of long-term cell lineage decisions. Regulatory T cells play a pivotal role in the maintenance of self-tolerance within the immune system by preventing autoimmunity or excessive activation of the T cells that respond to pathogens (naïve and effector T cells). They differentiate within the thymus, but can also be de novo induced in the rest of the body. Mechanisms determining development of a stable regulatory T cell lineage are unknown. Our study provides evidence for a critical role of epigenetic modifications in the locus coding for the forkhead transcription factor Foxp3, which acts as a master switch controlling regulatory T cell development and function: An evolutionarily conserved region within the non-coding part of the gene contains CpG motifs, which are completely demethylated in regulatory T cells, but methylated in naïve and effector T cells, whereas we observed an inverse occurrence of acetylated histones, another epigenetic chromatin modification. Regulatory T cells induced in vitro—which, in contrast to natural regulatory T cells, do not display a stable regulatory T cell phenotype—display only incomplete DNA demethylation despite high Foxp3 expression. Our data suggest that expression of Foxp3 must be stabilized by epigenetic modification to result in a permanent suppressor cell lineage, a finding of significant importance for therapeutic applications involving induction or transfer of regulatory T cells and for the understanding of long-term cell lineage decisions.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.

International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis

1,25‐dihydroxyvitamin D₃ (calcitriol) regulates immune responses, e.g., inhibits expression of IgE by B cells and enhances expression of IL‐10 by dendritic cells and T cells. We report here that activation of human B cells by B cell receptor, CD40 and IL‐4 signals induces expression of the gene for 25‐hydroxyvitamin‐D3–1α‐hydroxylase (CYP1α). Accordingly, these B cells generate and secrete significant amounts of calcitriol. In activated B cells calcitriol induces expression of the genes Cyp24, encoding a vitamin D hydroxylase, and Trpv6, encoding a calcium selective channel protein. Calcitriol enhances IL‐10 expression of activated B cells more than threefold, both by recruiting the vitamin D receptor to the promoter of Il‐10, and to lesser extent by modulation of calcium‐dependent signaling. The molecular link in activated B cells between vitamin D signaling, expression of IgE and IL‐10, and their ability to produce calcitriol from its precursor, suggest that pro‐vitamin D (25‐hydroxyvitamin D₃) can be used as a modulator of allergic immune responses.Supporting Information for this article is available at http://www.wiley‐vch.de/contents/jc_2040/2008/38216_s.pdf