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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Eur J Immunol. Author manuscript; available in PMC Sep 25, 2009.
Published in final edited form as:
PMCID: PMC2752283
NIHMSID: NIHMS140878

Human Regulatory T cells and Autoimmunity

Abstract

CD4+CD25+ regulatory T cells (Tregs) appear to be critical in regulating immune responses to self-antigens. Treg deficiency is associated with several human autoimmune diseases. Although substantial progress has been made in the study of murine and human Tregs, their fundamental mechanism of action remains unknown. In this review, we discuss phenotype of human natural Tregs, the functional mechanism of these cells, and the role of these Tregs in autoimmune disease.

Keywords: regulatory T cell, autoimmunity, human

While suppressor T cells might have been the so-called “baby thrown out with the bath water” when molecular biology strongly influenced immunology in the 1980s, it was clear to investigators of autoimmune disease that adoptively transferred suppression by T cells could influence these diseases. Here, we will present an overview of T cells as regulators of immune responses from a human species point of view, beginning with a history of T cells as regulatory cells, followed by a discussion of how these cells critically influence human autoimmune disease.

Twenty-five years ago, immunologists determined that two cellular mechanisms of dominant tolerance could mediate peripheral regulation and the phenomena of induced CD4+ T cell non-responsiveness. These two mechanisms were MHC-restricted T cell presentation of antigen to other T cells [1] and suppression via thymically derived ‘suppressor’ T cells [2]. Shortly after the discovery of MHC class II+ CD4+ T cells in human peripheral blood (these cells are present in most mammalian species excluding mice [3]) [4, 5], Kunkel and his colleagues observed that autoimmune patients have a higher percentage of these cells in peripheral blood than healthy patients [6]. Using the newly developed technique of CD4+ T cell cloning, researchers determined that MHC class II+ CD4+ T cell clones induce anergy when they present antigen to other CD4+ T cells [7]. Evidence from these studies was used to support the argument for the existence of anti-idiotypic T cell networks. ‘Suppressor’ T cells, too, were thought to recognize the antigen receptor of autologous CD4+ cells and prohibit activation with specificity directed against the suppressed cell. In the years to follow, the molecular or biochemical processes underlying these mechanisms could not be determined; MHC class II was relegated to a biomarker of late CD4+ T cell activation; many early studies of suppressor T cells were discredited or disproved; and the paradigm of idiotypic networks fell from fashion. Unfortunately, as Rolf Zinkernagel writes, “science (particularly immunology) undergoes periods of democratic, if not demagogic decisions … while populistic or experimentally biased views within science will eventually be corrected, if proven wrong this can take a long time” [8]. Although relegated to the list of immunological “not-so-good ideas” for almost fifteen years, ‘suppressor’ T cells have now been shown to play an essential role in preventing autoimmune disease and immunologists have begun to better characterize the now-called ‘regulatory’ T cells that can mediate suppression of autoimmunity. Not surprisingly, MHC class II+ CD4+ T cell have re-emerged as well, as expression of this immunomodulatory receptor was found to identify a functionally distinct subset of human ‘regulatory’ T cells (Tregs) expressing FoxP3. In this review we discuss recent advances in the characterization of human CD4+CD25high Tregs, with a focus on the impairment of these cells in human autoimmune disease.

Cell surface characterization of human Tregs

Although both murine and human CD4+CD25high Tregs similarly suppress the activation of CD4+CD25− responder T cells (Tresp) in a cell-contact-dependent manner and express the Treg specific lineage specification factor FoxP3, the human Treg population is by far more heterogeneous than that of the mouse, as gauged by both cell surface phenotype and functional capability. Human blood, isolated from an outbred population in a pathogenic environment, contains up to 30% CD4+CD25+ cells; only the 2-4% of the cells with the highest CD25 expression can be considered regulatory [9]. Problematically, there is no consensus as to where the boundary lies between CD25high and CD25intermediate expression, which has hindered both experimental reproducibility and clinical analysis of patient blood, particularly in patients where inflammatory conditions can lead to an increase in CD25 expression by activated T cells. The majority of human Tregs can however be differentiated from recently activated effector cells by CD62L (L-selection) expression because CD4+CD25high Tregs are predominantly CD62L+ (>95%) [9]. Additionally, human Tregs isolated from adult peripheral blood or tonsil are predominantly CD45RO+, CD45RA, and CD45RBlow [10]. For the most part, CD4+CD25high Tregs express a highly differentiated central memory phenotype and share many of the cell surface markers expressed on chronically activated CD4+ T cells.

The CD4+CD25high Treg population is also enriched for many immunomodulatory surface markers, including MHC class II, CD95 (Fas), glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR), and cytotoxic T-lymphocyte associated protein (CTLA-4) that are not Treg specific. The most specific marker for Tregs to date is the nuclear transcription factor FoxP3, the expression of which correlates with suppressive ability. In humans, FoxP3 is expressed strongly in CD4+CD25high T cells and at low levels in activated CD4+ T cells [11], although FoxP3 expression has also been reported in a small fraction of the CD25, CD25intermediate, and CD8+ T cell subsets. Indeed, data from FoxP3-GFP reporter mice has shown that CD25FoxP3GFP+ cells exhibit the same suppressive capacity as CD25+FoxP3GFP+ cells in vitro [12] This evidence, paired with the absence of a unique Treg cell surface marker, suggests the possibly that the CD4+CD25high subset is merely enriched for human Tregs and does not contain the entire Treg population.

Discovery of an alternative marker to CD25 would greatly enhance the identification and purification of human Tregs. The best accepted alternative is lack of cell surface CD127 (IL-7 receptor). FoxP3 expression and suppressive capacity have been detected in CD4+ T cells expressing low levels of CD127 [13, 14]. Certainly, low CD127 expression may be useful in isolating FoxP3+ cells from the CD25+ DN2 and DN3 populations of the thymus. In the periphery, however, the possibility remains that CD127 expression does not discriminate between adaptive Treg cells, such as the Tr1 and Th3 subsets, activated T cells, and thymically-derived Tregs [13, 14].

An alternative marker to CD25 would also be useful for distinguishing tissue-resident Tregs from activated Tresp in inflammatory environments. Ruprect and coworkers have suggested that Tregs from the synovial fluid of patients with juvenile arthritis are CD27+ as well as CD4+CD25+ [15]. This population of Tregs expressed elevated levels of FoxP3 and suppressed Tresp proliferation more efficiently than CD4+CD25+CD27 synovial fluid Tregs. This study did not restrict the Treg pool to the CD25high population, however, so it is difficult to know whether suppressive CD27+ cells are members of this enriched Treg population.

MHC Class II expression delineates a unique subset of human Tregs

One of the most striking heterogeneities of the CD4+CD25high regulatory population is the expression of HLA-DR in approximately one-third of human Tregs [9]. Although presentation of antigen via HLA-DR by CD4+ T cells is associated with a profound degree of anergy, contact-dependent suppression by Tregs is not MHC restricted. Therefore T cell presentation of antigen is not a mechanism of regulation in this cell population; rather, HLA-DR expression identifies a functionally distinct population of what appear to be terminally differentiated human Tregs. FoxP3 expression is significantly higher in the HLA-DR+ Tregs [16]. These cells exhibit earlier kinetics of Tresp suppression than the HLA-DR subset. This suppression is contact-dependent and these cells do not produce IL-10. The action of these cells is unique from that of HLA-DR Tregs, which induce a later, less vigorous suppression of Tresp, and which rely on both cell-contact and IL-10 secretion to inhibit Tresp activation [17] (Figure 1). Suppression by HLA-DR Tregs induce Tresp to secrete IL-10 and therefore cause these cells to become suppressive themselves [16]. Such infectious tolerance is unique to the HLA-DR Treg subset, and may reflect contamination of this population with adaptive Tregs.

Figure 1
CD4+CD25high cells isolated from patients with MS suppress less efficiently than Tregs purified from healthy donors. CD4+CD25 responder T cells and CD4+CD25high cells from six MS patients and six normal controls were stimulated with soluble anti-CD3 ...

Strength of signal and human Treg suppression

Human Tregs must be activated through their TCR in order to be functionally suppressive [18, 19], although once activated, these cells do not need to be viable in co-culture in order to mediate Tresp suppression. The effective outcome of Tresp suppression is also dictated by the quality of T cell stimulation, as the strength of stimuli applied to the Tresp population has a strong influence on whether suppression or proliferation will occur during Treg:Tresp co-culture. Tresp activated in the presence of strong costimulation are refractory to Treg suppression, as are Tresp supplemented with growth promoting cytokines [9, 20]. Furthermore, increasing the strength of TCR signal increases the resistance of Tresp to regulation [20]. This data suggests that human Tregs can only suppress Tresp activated with low signal strength. In highly inflammatory environments, where Tresp are activated with high signal strength, human Tregs are unable to prevent Tresp proliferation and cytokine secretion.

Tregs and autoimmune disease

The inability of human Tregs to suppress strongly activated Tresp in inflammatory environments has major implications in autoimmune disease. Autoreactive T cells isolated from patients with autoimmune disease have a lower threshold of activation when compared to cells isolated from healthy individuals [21]. Patient-derived Tresp, however, have not been shown to be resistant to suppression; instead, our group and others have demonstrated defects in the function of peripheral blood CD4+CD25high Tregs in patients with multiple sclerosis (MS) (Figure 2), type I diabetes (T1D), psoriasis, autoimmune myasthenia gravis and rheumatoid arthritis (RA) [22-26].

Figure 2
HLA-DR+ Tregs and HLA-DR Tregs have distinct mechanisms of suppression. HLA-DR+ Tregs directly inhibit both proliferation and cytokine suppression through a cell-contact dependent mechanism, while HLA-DR Tregs induce secretion of IL-4 ...

The loss of function in autoimmune Tregs has been correlated with decreases in FoxP3 levels. Indeed, the immunodeficiency syndrome IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome), which is associated with autoimmune disease in multiple endocrine organs, is caused by mutations in the foxp3 gene [27]. Impairment of FoxP3 protein expression results in massive inflammatory pathology, in both humans and mouse. Most human autoimmune diseases, however, are complex genetic disorders comprised of multiple common allelic variants that can, in combination with environmental factors, lead to development of a pathologic response. Genome wide association scans have recently been published for patients with MS, T1D, and RA [28, 29]. While these scans have failed to implicate a significant genetic role for foxpP3 in autoimmune disease, the major growth factor receptor for Tregs, the IL-2Rα chain, has been clearly linked to MS and type I diabetes. Thus, these and the other allelic variants such as in the IL-7Rα chain and CD58 may partially regulate Treg function in patients with autoimmune disease.

Conclusions

Despite decades of research, much remains unknown in the human regulatory T cell field. Human immunologists have not yet unraveled the functional mechanism of contact-dependent Tregs; without this understanding, it will be difficult to assess the basis for Treg dysfunction in autoimmune disease. Progression of the field will certainly require the development of new tools to selectively identify and assay homogenous Treg populations. Simple and reproducible methods to isolate and assay Tregs will serve to dampen controversy as well as enable research.

The many years' worth of accumulated data on this topic, however, are not without merit. Perhaps the most valuable understanding gained is that of the distinctive nature of human CD4+ T cell biology. The human system is clearly unique from those of mouse models and this limits the extension of murine findings into clinical application. Markers absent from murine CD4+ T cells, such as HLA-DR, may play a significant role in the biology of human Tregs and they cannot be functionally assayed in mouse models. The possibility remains that man and mouse have evolved divergent mechanisms of tolerance; therefore the understanding of Tregs in human disease will not be achieved by direct application of murine-derived concepts. Human immunologists must redouble their efforts to develop scientist-friendly methods for human study.

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