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.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

Foxp3(+) T-regulatory cells (Tregs) are primarily generated in the thymus (tTreg), but also may be generated extrathymically at peripheral sites (pTreg), or induced in cell culture (iTreg) in the presence of transforming growth factor β (TGFβ). A major unresolved issue is how these different populations of Tregs exert their suppressive function in vivo. We have developed novel systems in which the function of Tregs can be evaluated in vivo in normal mice. Our studies demonstrate that one prominent mechanism of action of polyclonal tTregs is to inhibit T-effector cell trafficking to the target organ, while antigen-specific iTregs primarily prevent T-cell priming by acting on antigen-presenting dendritic cells (DCs). Interleukin-10 (IL-10) plays an important role in the suppressive function of antigen-specific iTregs by controlling the expression of MARCH1 and CD83 on the DC. Activated tTregs may mediate infectious tolerance by delivery of cell surface-expressed TGFβ to naive responder T cells to generate pTregs. Manipulation of Treg function will require the ability to differentiate tTregs from pTregs and iTregs. The expression of the transcription factor Helios has proven to be a useful marker for the identification of stable tTregs in both mouse and human.

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tTregs, pTregs, and iTregs: Similarities and Differences

Regulatory T (TReg) cells are crucial for the prevention of fatal autoimmunity in mice and humans. Forkhead box P3 (FOXP3)(+) TReg cells are produced in the thymus and are also generated from conventional CD4(+) T cells in peripheral sites. It has been suggested that FOXP3(+) TReg cells might become unstable under certain inflammatory conditions and might adopt a phenotype that is more characteristic of effector CD4(+) T cells. These suggestions have caused considerable debate in the field and have important implications for the therapeutic use of TReg cells. In this article, Nature Reviews Immunology asks several experts for their views on the plasticity and stability of TReg cells.

The plasticity and stability of regulatory T cells.

CD4(+)T cells are crucial for directing appropriate immune responses during host defense and for the pathogenesis of inflammatory diseases. In addition to the classical biphasic model of differentiation of T-helper 1 (Th1) and Th2 cells, unexpected increases in the numbers of CD4(+)T-cell subsets, including Th17, Th9, T follicular-helper (Tfh) and T-regulatory (Treg) cells, have been recognized. In the present review, we focus on how these various T-helper cell subsets contribute to the pathogenesis of immune-mediated inflammatory diseases. In particular, we focus on multiple sclerosis, psoriasis and asthma as typical model diseases in which multiple T-helper cell subsets have recently been suggested to play a role. We will also discuss various unique sub-populations of T-helper cells that have been identified. First, we will introduce the heterogeneous T-helper cell subsets, which are classified by their simultaneous expression of multiple key transcription factors. We will also introduce different kinds of memory-type Th2 cells, which are involved in the pathogenesis of chronic type-2 immune-related diseases. Finally, we will discuss the molecular mechanisms underlying the generation of the plasticity and heterogeneity of T-helper cell subsets. The latest progress in the study of T-helper cell subsets has forced us to reconsider the etiology of immune-mediated inflammatory diseases beyond the model based on the Th1/Th2 balance. To this end, we propose another model--the pathogenic T-helper population disease-induction model--as a possible mechanism for the induction and/or persistence of immune-mediated inflammatory diseases.

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CD4+ T cell subsets in inflammatory diseases: beyond the Th1/Th2 paradigm

HLA-G is a molecule that was first known to confer protection to the fetus from destruction by the immune system of its mother, thus critically contributing to fetal-maternal tolerance. The first functional finding constituted the basis for HLA-G research and can be summarized as such: HLA-G, membrane-bound or soluble, strongly binds its inhibitory receptors on immune cells (NK, T, B, monocytes/dendritic cells), inhibits the functions of these effectors, and so induces immune inhibition. HLA-G function may therefore be beneficial because when expressed by a fetus or a transplant it protects them from rejection, or deleterious because when expressed by a tumor, it also protects it from antitumor immunity. This is the primary HLA-G function: that of a checkpoint molecule. Great work has been done in the past years to characterize HLA-G itself, its regulation, its functions and mechanisms of action, and its pathological relevance. We will review this here, focusing on transplantation and oncology because these pathological contexts have been studied the most and also because they best represent the two opposite sides of HLA-G: beneficial to be promoted, or deleterious to be blocked.

HLA-G: An Immune Checkpoint Molecule.

c o rr e s p o n d e n c e npg © 2013 Nature America, Inc. All rights reserved. Regulatory T cells: recommendations to simplify the nomenclature To the Editor: Regulatory T cells (T reg cells) have attracted much interest from both basic and clinical immunologists. Although questions remain about their fundamental biology and their clinical potential has yet to be fully realized, considerable advances have been made in the under- standing of the differentiation, homeostasis and function of T reg cells. This new knowledge has led to a substantial increase in the number of Foxp3 + T reg cell subpopulations described in the literature and conse- quently to an increase in the use of new abbreviations and terminology. Furthermore, as the understanding of T reg cell biology has grown, so too has the realization that some aspects of the original terminology are no longer accurate, and its use has become less stringent. At the Third International Conference on Regulatory T Cells and Th Subsets and Clinical Application in Human Diseases held in Shanghai, China, on 13–16 October 2012, a small workshop was convened to discuss T reg cell nomenclature and to develop several recommendations. We hasten to add that this is simply a list of recommendations, and it remains the prerogative of journals to develop their own editorial preferences and for the authors to use the nomenclature they feel best suits their manuscript. However, we support the recommendations noted below. There were three general issues that provided the momentum for this workshop. First, the terms used for the principal Foxp3 + T reg cell populations—those that differentiate in the thymus, those that differ- entiate in the periphery and those generated in vitro—are not ideal, as these are, to some extent, inaccurate, ambiguous and/or uninformative. For example, the widely used term ‘natural T reg cell’ is misleading and ambiguous, as it indicates that all other Foxp3 + T reg cell populations are ‘unnatural’. It also does not convey any useful or accurate information. One feature of these Foxp3 + T reg cell populations that is more informa- tive is the anatomical location of their differentiation. Instead, use of the terms ‘thymus’, ‘periphery’ and ‘in vitro’ provides a clear indica- tion of whence the Foxp3 + T reg cells in question are derived. Thus, we would recommend that ‘natural Foxp3 + T reg cells’ instead be referred to as ‘thymus-derived T reg cells’ (Box 1). Consistent with the rationale above, we would recommend that Foxp3 + T reg cells that differentiate in the periphery be referred to as ‘peripherally derived T reg cells’ rather than ‘induced or adaptive T reg cells’ (Box 1). In this context, we noted that terms used to define a T reg cell subpopulation, such as ‘induced T reg cells’, are often used when the location of their differentiation is unclear. Thus, we would suggest that the newly recommended terms ‘thymus-derived T reg cells’ and ‘peripherally derived T reg cells’ be used only when the anatomical location of their differentiation has been clearly demonstrated. When the origin of the T reg cell being studied is unclear, the general term ‘Foxp3 + T reg cell’ would be more appropriate. Finally, to clearly distinguish between Foxp3 + T reg cell populations that are generated in vivo versus those generated in vitro, we would suggest nature immunology volume 14 number 4 april 2013 that the term ‘in vitro–induced T reg cells’ be used for all Foxp3 + T reg cell populations generated ex vivo, such as those generated through the use of transforming growth factor-b (Box 1). Second, there has been a growing tendency to use terms such as ‘T reg cells’ or ‘iT reg cells’ when confirmation of such identity is lacking. The frequent use of the term ‘T reg cells’ has been a particular problem in studies focusing on human T reg cells, as activated conventional T cells can also express Foxp3. The term ‘T reg cell’ should be used only when it is clear that the cells have (or had) suppressive ability or have a transcriptional, epigenetic and/or protein-expression signature that suggests that the cells in question are, beyond a reasonable doubt, Foxp3 + T reg cells (Box 1). Also, as indicated above, the terms ‘thymus- derived T reg cell’ and ‘peripherally derived T reg cells’ should be used only when the anatomical location of their development has been clearly demonstrated. Third, there has been progressive growth in the development and use of new T reg cell terminology that is likely to lead to more confusion and the further ‘jargonization’ of immunology. Until a new popula- tion has been extensively demonstrated to be unique, distinct from other populations and stable, we would recommend not coining new terms for such subpopulations (Box 1). Instead, we would encourage investigators to identify them through the use of prominent aspects of their expression pattern, such as a transcription factor or cytokine. Although the focus of this Correspondence has been CD4 + Foxp3 + T reg cell populations, many CD4 – Foxp3 – T reg cell populations have been described, and we would similarly recommend that new terms be used to describe these only when their identity and stability have been clearly defined. Box 1 T reg cell nomenclature recommendations 1. ‘Thymus-derived T reg cell (tT reg cell)’ should be used instead of ‘natural T reg cell (nT reg cell)’. 2. ‘Peripherally derived T reg cell (pT reg cell)’ should be used instead of ‘induced or adaptive T reg cell (iT reg cell or aT reg cell)’. 3. ‘In vitro–induced T reg cell (iT reg cell)’ should be used to clearly distinguish between those T reg cell populations generated in vivo versus those generated in vitro. 4. T reg cell terms should be used only when there is definitive evidence justifying their use. 5. The development and use of new T reg cell terminology should be limited, especially for subpopulations.

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Shuiping Jiang

Papers

Regulatory T cells: recommendations to simplify the nomenclature