It has become clear that defects in different populations of regulatory T cells contribute to the induction of autoimmune diseases in animal models. Here, we examined whether a similar defect in regulatory CD4+CD25hi T cells occurs in humans with MS. We observed a significant reduction in the effector functions of this regulatory T cell population in subjects with MS compared with healthy donors. These data are among the first clear demonstrations of the functional defect in CD4+CD25hi regulatory T cells in a human autoimmune disease.
An important aspect of these investigations was the measurement of regulatory T cell function as opposed to simple phenotypic measurement of CD4+
T cell frequency. Unlike 6-wk-old mice raised in clean facilities, whose total CD4+
T cell population manifests regulatory properties, humans are exposed to myriad infections and show a significant population of CD4+
T cells that do not exhibit regulatory function (24
). Thus, it was critical to examine the functional state of the regulatory T cells expressing high levels of CD25. We have demonstrated previously that the strength of signal delivered through the TCR of target T cells is one factor determining whether regulatory CD4+
T cells can suppress the responder T cell proliferation (24
). Thus, to properly examine the function of regulatory CD4+
T cells, we used several different strengths of stimulatory signals in these experiments. We observed that the strong signal provided by maximal concentration of plate-bound anti-CD3 mAb similarly abrogated suppression in both patient and control cocultures. In contrast, lower concentrations of plate-bound anti-CD3 delivered a signal that resulted in the appearance of a significant defect in the suppressive function of this subset of regulatory cells derived from patients with MS.
The use of different stimulatory conditions allowed us to reveal alterations in the regulatory function of CD4+CD25hi T cells while still demonstrating that they are CD25+ regulatory T cells as opposed to activated responder cells expressing CD25. Stimulation of cultures with soluble anti-CD3 and anti-CD28, which have previously shown to be the most permissive for enabling coculture suppression, gave equivalent levels of suppression in patients and control subjects when cocultured at 1:1 ratios. In contrast, the stimulation provided by plate-bound anti-CD3 at 0.1 and 0.5 μg/ml resulted in a threefold decrease of suppression by CD4+CD25hi cells derived from patients with MS as compared with normal controls. Previous experiments showed that delivering qualitatively different T cell receptor signals to responder T cells, such as that provided by self-antigens as compared with microbial antigens, results in a greater sensitivity to suppression. Thus, the present findings may help explain defects in suppression of autoreactive T cells in autoimmune patients as compared with T cells stimulated by microbial antigens during infections.
Because it was of interest to examine the regulatory function of CD4+CD25high T regulatory cells in an antigen-specific system, we attempted to induce antigen-specific responses to myelin basic protein (MBP) in this in vitro system. However, as expected with the low frequency of MBP-specific CD4+CD25− effector T cells and the low number of CD4+CD25high T regulatory cells that can be isolated by FACS®, it was not possible to directly measure suppression of autoantigen-specific T cells in patients. As the frequency of MBP-specific T cells appears to be in the range of 1/106 cells, the majority of the effector T cells that in our assay are cultured at 1–2 × 104 cells/well do not respond to the antigen.
An important control to note in all these experiments is the anergy or lack of thymidine incorporation resulting from stimulation of CD4+CD25hi T cells cultured alone. This anergy indicates that CD4+CD25hi T cells isolated from patients with MS are not CD25+-activated T cells because such cells would not exhibit regulatory activity, but rather enhance proliferation. It was critical to determine whether the decrease in T cell regulatory function observed in patients with MS was due to a defect in the CD4+CD25hi T cell subset or whether the responder CD4+CD25− T cells were refractory to suppression. By performing comixing experiments, we could clearly demonstrate that the defect lies in the CD4+CD25hi T cell function, as opposed to enhanced responder T cell resistance in patients with MS.
In mice thymectomized on day 3 of life, a spontaneous autoimmune disease develops that does not involve inflammation in the CNS (5
). This raises the question as to whether CD4+
T cells can be involved in murine models of CNS autoimmunity, reflecting the observations made here in patients with MS. Thus, it is of interest that in a mouse model of MS, MOG35–55
-specific experimental autoimmune encephalomyelitis, a similar population of CD4+
T cells, has been shown able to protect from both the onset and the progression of autoimmune demyelination induced by either active MOG35–55
immunization or adoptive transfer of autoreactive T cells (26
). These data provide further evidence that CD4+
regulatory T cells can migrate into the CNS to mediate immune responses.
The cell surface marker CD62L was used to further characterize the CD4+
regulatory subset derived from both healthy individuals and patients with MS. In mice, the expression of this molecule on CD4+
T cells has been shown to confer a greater suppressive ability (27
). We demonstrated that in healthy subjects, both CD4+
and total CD4+
regulatory T cell populations exhibit similar abilities to suppress the proliferative response with anti-CD3 stimulation. In contrast, the regulatory T cells isolated from patients with MS, although expressing high levels of CD62L, were still unable to inhibit the responder cell proliferation, thus confirming a defect in the function of regulatory T cells derived from patients with MS.
The relative inability to generate CD4+CD25hi clones from patient blood may suggest a mechanism underlying the defective regulatory T cell function. One possibility is that CD4+CD25hi cells have undergone clonal exhaustion in vivo in the attempt to down-modulate the ongoing autoimmune response in patients. Another possibility is that their expansion is abnormally inhibited by other factors present in the single-cell cloning cultures. We can speculate that altered sensitivity of these cells to stimulation could also negatively affect their ability to exert regulatory functions. Although other factors, such as IL-6 secretion, may abrogate the regulatory function of CD4+CD25+ T cells, they may also adversely affect the growth of these cells. However, because there was no correlation between IL-6 cytokine secretion and the loss of T cell regulatory function, alterations in IL-6 secretion in patients with MS does not appear to be the underlying mechanism for the loss of suppressor function by CD4+CD25hi T cells. These and other possibilities are currently under investigation.
Although in vitro measurement of biologic function in patients with autoimmune diseases will always be correlative, these in vitro experiments, based on in vitro and in vivo experimentation in mouse models of autoimmune disease, provide the first definitive evidence for a defect in regulatory T cell function in a human autoimmune disease. Ultimately, monitoring the effects of immunomodulatory drugs on this regulatory T cell subset will help defining their pathogenic role in MS and other human autoimmune diseases.