Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis

Abstract

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.

Figure 1.

Human CD4⁺CD25^hi T cells mediate suppression of CD4⁺CD25⁻ responder cell proliferation. (A) The regulatory properties of CD4⁺CD25^hi T cells were examined from 21 healthy individuals or (B) from 15 patients with RR MS. CD4⁺CD25⁻ responder (▪) and CD4⁺CD25^hi suppressor (▴) cells (2.5 × 10³ cells/well) were stimulated with plate-bound anti-CD3 (0.1 and 0.5 μg/ml). CD4⁺CD25⁻ T cells were cocultured with CD4⁺CD25^hi T cells at a 1:1 ratio (○). T cell–proliferative responses are expressed as the mean ± SE of triplicate cultures. Culture supernatants were diluted and analyzed to determine the cytokine profile. In the series of experiments shown on the left, cytokine secretion was not measurable. In the subsequent series, IFNγ and IL-10 were detected; their values, representing the mean ± SE of triplicate cultures, are expressed as ng/ml.

Figure 1.

Human CD4⁺CD25^hi T cells mediate suppression of CD4⁺CD25⁻ responder cell proliferation. (A) The regulatory properties of CD4⁺CD25^hi T cells were examined from 21 healthy individuals or (B) from 15 patients with RR MS. CD4⁺CD25⁻ responder (▪) and CD4⁺CD25^hi suppressor (▴) cells (2.5 × 10³ cells/well) were stimulated with plate-bound anti-CD3 (0.1 and 0.5 μg/ml). CD4⁺CD25⁻ T cells were cocultured with CD4⁺CD25^hi T cells at a 1:1 ratio (○). T cell–proliferative responses are expressed as the mean ± SE of triplicate cultures. Culture supernatants were diluted and analyzed to determine the cytokine profile. In the series of experiments shown on the left, cytokine secretion was not measurable. In the subsequent series, IFNγ and IL-10 were detected; their values, representing the mean ± SE of triplicate cultures, are expressed as ng/ml.

Figure 2.

Summary of CD4⁺CD25^hi T cell regulatory function that is altered in patients with MS. The mean percent inhibition of the proliferative response by CD4⁺CD25^hi cells derived from 15 MS patients (○) and 21 healthy controls (▪) was calculated. CD4⁺CD25⁻ and CD4⁺CD25^hi populations were stimulated with plate-bound anti-CD3 mAb, alone or cocultured at varying ratios. The proliferative response was inhibited upon addition of CD4⁺CD25^hi cells to the CD4⁺CD25⁻ responder cells at a 1:1 ratio in normal controls. Regulatory T cells from patients with MS exhibited significantly less suppressor activity. Decreasing the number of CD4⁺CD25^hi T cells (responder/suppressor ratios: 1:1/2 and 1:1/4) resulted in less suppression in all the conditions examined.

Figure 3.

CD4⁺CD25^hi T cells from patients with MS do not inhibit proliferation of responder T cells isolated from either the autologous individual or healthy donors. CD4⁺CD25⁻ responder T cells and CD4⁺CD25^hi cells from MS patients and normal controls were stimulated with plate-bound anti-CD3 at 0.5 μg/ml. 2.5 × 10³ cells/well responder T cells from MS patients were cocultured with the same number of autologous CD4⁺CD25^hi regulatory T cells (1st bar) or with regulatory cells isolated from healthy donors (2nd bar). Conversely, responder T cells from healthy donors were cocultured either with CD4⁺CD25^hi cells derived from the same subject (3rd bar), or with regulatory cells isolated from patients with MS (4th bar).

Figure 4.

The CD62L⁺ subset of CD4⁺CD25^hi regulatory T cells exhibits decreased suppressive function in patients with MS. CD4⁺CD25^hiCD62L⁺ T cells were isolated from seven healthy individuals (left) and three patients with MS (right). Each symbol is representative of a different individual. Total CD4⁺CD25^hi T cells were also isolated from four out of seven healthy subjects and two out of three patients. The percent suppression of coculture proliferation in response to plate-bound anti-CD3 was calculated in each individual. The CD4⁺CD25^hi regulatory T cells derived from healthy controls were able to induce strong inhibition of the proliferative response (55–97% suppression) as shown previously. The CD62L⁺ subset of CD4⁺CD25^hi T cells derived from the same individuals exhibit enhanced suppressive capacity. In contrast, the regulatory cells derived from patients, although depleted of CD62L⁻ activated T cells, show decreased inhibitory function (4–29% suppression) as compared with the suppression observed in healthy individuals.

Figure 5.

The proliferative response in both patients and healthy controls is suppressed in cultures receiving T cell receptor signals of different natures. CD4⁺CD25⁻ responder T cells and CD4⁺CD25^hi cells from six MS patients and six normal controls were stimulated with soluble anti-CD3 and anti-CD28 (5 μg/ml) alone or cocultured at different responder/suppressor ratios (1:1, 1:1/2, and 1:1/4). The average proliferative response was 13,494 cpm in the patient group and 15,974 cpm in the control group. The percent inhibition of coculture proliferation was calculated in patients with MS (○) and healthy controls (▪).

Figure 6.

Reduced ability to generate CD4⁺CD25^hi clones from blood of MS patients. (A) CD4⁺CD25^hi or CD4⁺CD25⁻ T cells from patients with MS and healthy controls were cloned as described in Materials and Methods. The difference in the cloning frequency of the regulatory CD4⁺CD25^hi cells between patients with MS and healthy subjects was highly significant (P < 0.0001), whereas the ability to generate clones from CD4⁺CD25⁻ cells was not different (P = 0.21). (B) T cell clones were tested for dependency on exogenous IL-2 by stimulation with anti-CD3 beads ± IL-2 (data are presented as stimulation indices). The same clones were also tested for their ability to inhibit the proliferation of cocultured responder T cells upon stimulation with anti-CD3 beads. The three representative CD4⁺CD25^hi clones (a–c) shown suppress the proliferation of autologous CD4⁺CD25⁻ T cells, whereas a T cell clone (d) representing a minor population of the clones derived from the CD4⁺CD25^hi population is shown, and all of the CD4⁺CD25⁻ clones (e) did not inhibit proliferation of cocultured T cells.