Clare Baecher-Allan

CD4+CD25high Regulatory Cells in Human Peripheral Blood

Thymectomy in mice on neonatal day 3 leads to the development of multiorgan autoimmune disease due to loss of a CD+CD25+ T cell regulatory population in their peripheral lymphoid tissues. Here, we report the identification of a CD4+ population of regulatory T cells in the circulation of humans expressing high levels of CD25 that exhibit in vitro characteristics identical with those of the CD4+CD25+ regulatory cells isolated in mice. With TCR cross-linking, CD4+CD25high cells did not proliferate but instead totally inhibited proliferation and cytokine secretion by activated CD4+CD25− responder T cells in a contact-dependent manner. The CD4+CD25high regulatory T cells expressed high levels of CD45RO but not CD45RA, akin to the expression of CD45RBlow on murine CD4+CD25+ regulatory cells. Increasing the strength of signal by providing either costimulation with CD28 cross-linking or the addition of IL-2 to a maximal anti-CD3 stimulus resulted in a modest induction of proliferation and the loss of observable suppression in cocultures of CD4+CD25high regulatory cells and CD4+CD25− responder cells. Whereas higher ratios of CD4+CD25high T cells are required to suppress proliferation if the PD-L1 receptor is blocked, regulatory cell function is shown to persist in the absence of the PD-1/PD-L1 or CTLA-4/B7 pathway. Thus, regulatory CD4 T cells expressing high levels of the IL-2 receptor are present in humans, providing the opportunity to determine whether alterations of these populations of T cells are involved in the induction of human autoimmune disorders.

Loss of Functional Suppression by CD4+CD25+ Regulatory T Cells in Patients with Multiple Sclerosis

CD4+CD25+ regulatory T cells contribute to the maintenance of peripheral tolerance by active suppression because their deletion causes spontaneous autoimmune diseases in mice. Human CD4+ regulatory T cells expressing high levels of CD25 are suppressive in vitro and mimic the activity of murine CD4+CD25+ regulatory T cells. Multiple sclerosis (MS) is an inflammatory disease thought to be mediated by T cells recognizing myelin protein peptides. We hypothesized that altered functions of CD4+CD25hi regulatory T cells play a role in the breakdown of immunologic self-tolerance in patients with MS. Here, we report a significant decrease in the effector function of CD4+CD25hi regulatory T cells from peripheral blood of patients with MS as compared with healthy donors. Differences were also apparent in single cell cloning experiments in which the cloning frequency of CD4+CD25hi T cells was significantly reduced in patients as compared with normal controls. These data are the first to demonstrate alterations of CD4+CD25hi regulatory T cell function in patients with MS.

IL-21 and TGF-beta are required for differentiation of human T(H)17 cells.

The recent discovery of CD4+ T cells characterized by secretion of interleukin (IL)-17 (TH17 cells) and the naturally occurring regulatory FOXP3+ CD4 T cell (nTreg) has had a major impact on our understanding of immune processes not readily explained by the TH1/TH2 paradigm. TH17 and nTreg cells have been implicated in the pathogenesis of human autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease and psoriasis. Our recent data and the work of others demonstrated that transforming growth factor-β (TGF-β) and IL-6 are responsible for the differentiation of naive mouse T cells into TH17 cells, and it has been proposed that IL-23 may have a critical role in stabilization of the TH17 phenotype. A second pathway has been discovered in which a combination of TGF-β and IL-21 is capable of inducing differentiation of mouse TH17 cells in the absence of IL-6 (refs 6–8). However, TGF-β and IL-6 are not capable of differentiating human TH17 cells and it has been suggested that TGF-β may in fact suppress the generation of human TH17 cells. Instead, it has been recently shown that the cytokines IL-1β, IL-6 and IL-23 are capable of driving IL-17 secretion in short-term CD4+ T cell lines isolated from human peripheral blood, although the factors required for differentiation of naive human CD4 to TH17 cells are still unknown. Here we confirm that whereas IL-1β and IL-6 induce IL-17A secretion from human central memory CD4+ T cells, TGF-β and IL-21 uniquely promote the differentiation of human naive CD4+ T cells into TH17 cells accompanied by expression of the transcription factor RORC2. These data will allow the investigation of this new population of TH17 cells in human inflammatory disease.

Isolation and functional characterization of regulatory CD25brightCD4+ T cells from the target organ of patients with rheumatoid arthritis

In the homeostasis of the immune system regulatory cells play a major role. Removal of one group of regulatory cells, the CD25+CD4+ T cells, leads to autoimmune manifestations in experimental animal models, and reintroduction of this population prevents disease. This study addresses the role of such regulatory T cells in humans with an autoimmune disease, where we demonstrate the presence of CD25brightCD4+ T cells in the target organ of patients with active rheumatoid arthritis. The patients displayed an enrichment of CD25brightCD4+ T cells in synovial fluid as compared to peripheral blood. These cells are functional regulatory cells, as they were able to suppress in vitro proliferation of autologous T cells, bothfrom synovial and peripheral blood origin. Although the frequency of CD25brightCD4+ T cells varied between patients, it was found to be constant over time in any one joint during each relapse. Numbers were also comparable in two inflamed knee joints of one and the same patient, emphasizing the symmetry of the disease. In summary, it is striking that in addition to all activated, potentially pathological T cells the synovial fluid from RA patients also contains CD25‐expressing CD4+ T cells with a regulatory capacity.

IL-17-producing human peripheral regulatory T cells retain suppressive function

Although implicated in antagonistic functions, both regulatory T cells (Tregs) and Th17 effector cells play an important role in controlling autoimmune pathogenesis. Paradoxically, recent studies indicate that Tregs have the capacity to produce interleukin-17 (IL-17), although the ability of these cells to retain their suppressive function remains unknown. Here we report that human Tregs within the CD4(+)CD45RA(-)CD25(high)CCR6(+)HLA-DR(-)FoxP3(+) population produce IL-17 when activated in the presence of the proinflammatory cytokines IL-1beta and IL-6, whereas IL-17 secretion was inhibited by TGFbeta. To assess the ability of a single Treg to secrete IL-17 and to suppress in vitro immune function, we isolated clones from this population. We found that IL-17(+)/FoxP3(+) Treg clones retain suppressive function and exhibit the plasticity to secrete IL-17 or suppress depending on the nature of the stimulus provided. IL-17 production by these Treg clones was accompanied by sustained FoxP3 expression and concomitant, but reversible, loss of suppressive activity. Our data demonstrate that at the single cell level a subset of in vitro suppressive FoxP3(+) cells can be driven to secrete IL-17 under inflammatory conditions. These findings suggest a new mechanism by which inflammation can drive Tregs to secrete IL-17, thereby dampening suppression and promoting an inflammatory milieu.

MHC class II expression identifies functionally distinct human regulatory T cells.

It has been known for decades that circulating human CD4 cells can express functional MHC class II molecules that induce T cell nonresponsiveness with Ag presentation. Because there is significant expression of MHC class II (MHC-II) determinants (DR) on a subpopulation CD4+CD25high regulatory T cells (Treg), we examined the function of CD4 cells expressing MHC-DR. We demonstrate that MHC-II expression on human CD4+CD25high T cells identifies a functionally distinct population of Treg that induces early contact-dependent suppression that is associated with high Foxp3 expression. In striking contrast, MHC-II− CD4+CD25high Treg induce early IL-4 and IL-10 secretion and a late Foxp3-associated contact-dependent suppression. The DR expressing CD25high Treg express higher levels of Foxp3 message and protein, compared with the DR−CD25high Treg population. Direct single-cell cloning of CD4+CD25high Treg revealed that, regardless of initial DR expression, ex vivo expression of CD25high, and not DR, predicted which clones would exhibit contact-dependent suppression, high levels of Foxp3 message, and an increased propensity to become constitutive for DR expression. Thus, the direct ex vivo expression of MHC-II in the context of CD25high identifies a mature, functionally distinct regulatory T cell population involved in contact-dependent in vitro suppression.

Inhibition of Human CD4+CD25+high Regulatory T Cell Function

CD4+CD25+high T cells are potent regulators of autoreactive T cells. However, it is unclear how regulatory CD4+CD25+high cells discriminate between desirable inflammatory immune responses to microbial Ags and potentially pathologic responses by autoreactive T cells. In this study, an in vitro model was created that allowed differential activation of regulatory CD4+CD25+high and responder CD4+ T cells. If CD4+CD25+high regulatory cells were strongly activated, they maintained suppressive effector function for only 15 h, while stimulation with weaker TCR stimuli produced regulatory cells that were suppressive until 60 h after activation. In contrast, strongly activated CD4+ responder T cells were resistant to regulation at all time points, while weakly stimulated CD4+ cells were sensitive to suppression until 38 or 60 h after activation depending upon the strength of the stimulus. The extent of suppression mediated by CD4+CD25+high cells also depended on the strength of stimulation in an Ag-specific system. Thus, the stronger the TCR signal, the more rapidly and more completely the responder cells become refractory to suppression.

Identification of T helper type 1–like, Foxp3 + regulatory T cells in human autoimmune disease

CD4+CD25highCD127low/– forkhead box p3 (Foxp3)+ regulatory T cells (Treg cells) possess functional plasticity. Here we describe a higher frequency of T helper type 1 (TH1)-like, interferon-γ (IFN-γ)-secreting Foxp3+ T cells in untreated subjects with relapsing remitting multiple sclerosis (RRMS) as compared to healthy control individuals. In subjects treated with IFN-β, the frequency of IFN-γ+Foxp3+ T cells is similar to that in healthy control subjects. In vitro, human Treg cells from healthy subjects acquire a TH1-like phenotype when cultured in the presence of interleukin-12 (IL-12). TH1-like Treg cells show reduced suppressive activity in vitro, which can partially be reversed by IFN-γ–specific antibodies or by removal of IL-12.

Human regulatory T cells and their role in autoimmune disease

Summary:  As self‐recognition is fundamental to the efficient operation of the immune system, a number of mechanisms have evolved to keep this potential pathologic self‐reactivity in check. Thus, even though the majority of strongly self‐reactive T cells are deleted in the thymus during T‐cell maturation, a number of mature T cells that recognize self‐antigens can be found in the peripheral circulation in healthy individuals as well as in patients with autoimmune disease. These self‐reactive cells are kept in a non‐responsive state in healthy individuals while they appear to be involved in the etiology of a number of autoimmune diseases in patients. The primary role of a relatively recently identified T‐cell population, referred to as natural CD4+CD25+ regulatory T cells, is to modulate the activity of these self‐reactive cells. Although it is still unclear how these regulatory cells function, they can inhibit the activation of other potentially pathologic T cells in in vitro assays. Using such assays, regulatory T cells isolated from patients with a number of autoimmune diseases have been shown to exhibit reduced inhibitory function as compared with those isolated from healthy individuals. In this review, we discuss human natural regulatory T cells, what is known about their function, and their associations with specific autoimmune diseases.

Suppressor T Cells in Human Diseases

Although central and peripheral tolerance are important for the regulation of human immune responses to self- and microbial antigens, an important role of suppressor CD4+ CD25+ T cells is suggested from the recent investigations of human autoimmune diseases and HIV. These new data provide increasing evidence that altered function of CD4+ CD25+ T cells may be an important factor in a wide range of human inflammatory and infectious diseases.