Human CD25high Tregs: isolation by beads versus by FACS sorting.
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Antibody Specificity↗
Biomedical subjects
Clare Baecher-Allan
Publications and source records attributed to Clare Baecher-Allan.
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+ CD25(high) regulatory T cells (Treg), we examined the function of CD4 cells expressing MHC-DR. We demonstrate that MHC-II expression on human CD4+ CD25(high) 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+ CD25(high) Treg induce early IL-4 and IL-10 secretion and a late Foxp3-associated contact-dependent suppression. The DR expressing CD25(high) Treg express higher levels of Foxp3 message and protein, compared with the DR- CD25(high) Treg population. Direct single-cell cloning of CD4+ CD25(high) Treg revealed that, regardless of initial DR expression, ex vivo expression of CD25(high), 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 CD25(high) identifies a mature, functionally distinct regulatory T cell population involved in contact-dependent in vitro suppression.
Antigen Presentation↗
Immune regulation in tumor-bearing hosts.
A wealth of data indicates that tumor immunity directed against a wide variety of malignancies is suppressed in cancer patients. Recent studies have explored the role of 'natural' CD4(+)CD25(+) regulatory T cells (Tregs) in the suppression of tumor immunity in cancer patients. It is now clear, using multiple phenotypic and functional criteria, that the frequency of Tregs is increased in the peripheral blood of cancer patients as well as within the tumor microenvironment. Human Tregs with specificity for tumor-associated antigens have recently been identified, and murine studies have demonstrated that vaccination with tumor-associated antigens can expand Tregs, posing a challenge to cancer vaccine strategies. However, a variety of approaches, including depletion of Tregs or modulation of their activity in vivo, might soon enhance the efficacy of existing cancer vaccines directed against a variety of malignancies.
Animals↗
Human regulatory T cells and their role in autoimmune disease.
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.
Animals↗
Regulatory cells and human cancer.
While there is now little debate about the existence or relevance of regulatory populations of T cells to a variety of human diseases, including cancer, there is considerable debate about the ontogeny, phenotype, and mechanisms of action of given regulatory T cell populations. This review will limit itself to discussion to two distinct populations of CD4+ regulatory T cells: T cells co-expressing the CD25 receptor (Tregs), and type 1 regulatory (Tr1) T cells. Attention will be focused on the definition and role of these regulatory T cell populations in human cancers.
Humans↗
Functional analysis of highly defined, FACS-isolated populations of human regulatory CD4+ CD25+ T cells.
The importance of CD4+ CD25+ regulatory T cells (Treg) in maintaining immune homeostasis has been directly demonstrated in vivo by their manipulation in a number of autoimmune disease models in the mouse. In the study of human regulatory cells, we have found that the cells that consistently demonstrate the in vitro regulatory activity most similar to that described for murine cells in vitro are best identified by restricting the isolation of CD25+ CD4 T cells to those cells expressing only the highest levels of CD25, representing approximately 2-3% of total CD4 T cells. Thus, it is the CD4+ CD25high subset that exhibits the in vitro characteristics that are identical to the CD4+ CD25+ regulatory cells initially characterized in mice. Furthermore, the cells expressing medium to low levels of CD25 not only do not exhibit suppressive activity directly ex vivo, but also actually contain a significant proportion of CD62L- CD4 T cells which are believed to be in vivo activated T cells. Due to the inherent difficulties in using CD25 as a marker for the purification of Treg cells, the finding that selection of the CD25high subset of CD4+ CD25+ T cells minimizes the co-isolation of contaminating activated CD4 T cells is important for future studies of these Treg cells in human disease. In order to perform these studies, we first had to establish a highly reproducible 'micro in vitro co-culture' assay system to enable the functional analysis of high-purity, but low-yield regulatory populations derived from FACS sorting. With this system in place, we are poised to dissect the potential heterogeneity of mechanisms employed by highly specific subpopulations of CD4+ CD25+ cells.
Antigens, CD↗
Multiple sclerosis.
Multiple sclerosis (MS) is a complex genetic disease associated with inflammation in the central nervous system (CNS) white matter and is thought to be mediated by autoimmune processes. Clonal expansion of B cells, their antibody products, and T cells, hallmarks of inflammation in the CNS, are found in MS. The association of the disease with major histocompatibility complex genes, the inflammatory white matter infiltrates, similarities with animal models, and the observation that MS can be treated with immunomodulatory and immunosuppressive therapies support the hypothesis that autoimmunity plays a major role in the disease pathology. This review discusses the immunopathology of MS with particular focus given to regulatory T cells and the role of B cells and antibodies, immunomodulatory therapeutics, and finally new directions in MS research, particularly new methods to define the molecular pathology of human disease with high-throughput examination of germline DNA haplotypes, RNA expression, and protein structures that will allow the generation of a new series of hypotheses that can be tested to develop better understandings and therapies for this disease.
Animals↗
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.
Arthritis, Rheumatoid↗
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.
Adult↗
Human CD4+CD25+ regulatory T cells.
In this report, we review studies of human CD4+CD25+ regulatory T cells (T-reg). Although lagging a few years behind the discovery of these cells in the mouse, the equivalent population of CD4+CD25+ regulatory T cells has also been isolated from human peripheral blood, thymus, lymph nodes and cord blood. In general, the characteristics of this T cell subset are strikingly similar between mouse and man. In the recent explosion of research reports on human CD4+CD25+ cells, although the majority of the characteristics ascribed to these cells appear to be consistent, contrasting results have been found primarily in regards to potential involvement of TGFbeta and production of IL-10. One explanation for this variability may reside in the fact that markedly different techniques are used to isolate human CD4+CD25+ T-reg cells and thus may result in the comparison of T-reg populations that differ in cellular composition and/or activation state. Another potential explanation for differences in human T-reg function may rest on the extreme variability of the culture conditions and TCR stimuli that have been used to test the functional properties of these cells in vitro. The strength of the TCR signal provided to the culture greatly affects the functional outcome of the co-culture and can result in the difference between suppression and full activation. Surprisingly, it appears that stronger stimulation has a greater and more rapid effect on the T-resp cell than on the T-reg cell as it causes T-resp cells to quickly become resistant to suppression. Thus, the details of in vitro culture conditions may at least partially account for disparate findings in regard to the functional characterization of human CD4+CD25+ cells. Here we review the evidence regarding the identification of human CD4+CD25+ regulatory T cells and their possible mechanism(s) of function.
Antigens, CD↗
CD4+CD25+ regulatory cells from human peripheral blood express very high levels of CD25 ex vivo.
Selective isolation of only those CD4+ T cells that display the highest levels of CD25 by FACS results in a highly homogeneous regulatory population as defined by functional activity and the expression of multiple surface antigens. Thus greater than 98% of CD4+CD25high cells express CD45RO in the absence of CD45RA expression. Upon TCR stimulation CD4+CD25high cells are both anergic and tolerogenic as they inhibit proliferation and cytokine secretion by activated CD4+CD25- responder T cells in a contact-dependent manner. In contrast, CD4+ cells that express lower levels of CD25 are more heterogeneous in their levels of expression of CD45RO, HLA-DR and CD122, and do not exhibit anergic or suppressive characteristics. Providing either CD28 co-stimulation or IL2 to a maximal anti-CD3 stimulus results in a modest induction of proliferation and the loss of observable suppression by CD4+CD25high regulatory cells. Unlike CTLA4 blockade, blocking the interaction of PD-1 with its ligand PD-L1 affects the level of suppression. However, since this reduction in suppression by alphaPD-L1 can be overcome by increasing the number of CD4+CD25high T cells in the co-culture assay, the mechanism of CD4-CD25high regulation can proceed in the absence of PD-1/PD-L1 interactions, although it is not as efficient.
Antigens, CD↗
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 CD25(bright)CD4(+) T cells in the target organ of patients with active rheumatoid arthritis. The patients displayed an enrichment of CD25(bright)CD4(+) 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, both from synovial and peripheral blood origin. Although the frequency of CD25(bright)CD4(+) 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.
Adult↗
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.
Antigens↗