Published April 5, 2004
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`⫹
`⫹
`CD25
`Loss of Functional Suppression by CD4
`T Cells in Patients with Multiple Sclerosis
`
` Regulatory
`
`Vissia Viglietta, Clare Baecher-Allan, Howard L. Weiner, and David A. Hafler
`
`Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women’s Hospital,
`Harvard Medical School, Boston, MA 02115
`
`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
`⫹
` regulatory T cells expressing high levels of CD25 are suppressive in vitro and mimic the
`CD4
`⫹
`⫹
`CD25
` regulatory T cells. Multiple sclerosis (MS) is an inflammatory
`activity of murine CD4
`disease thought to be mediated by T cells recognizing myelin protein peptides. We hypothe-
`⫹
`CD25
` regulatory T cells play a role in the breakdown of
`sized that altered functions of CD4
`hi
`immunologic self-tolerance in patients with MS. Here, we report a significant decrease in the
`⫹
`CD25
` regulatory T cells from peripheral blood of patients with MS
`effector function of CD4
`hi
`as compared with healthy donors. Differences were also apparent in single cell cloning experiments
`⫹
`CD25
` T cells was significantly reduced in patients
`in which the cloning frequency of CD4
`hi
`as compared with normal controls. These data are the first to demonstrate alterations of
`⫹
`CD25
` regulatory T cell function in patients with MS.
`CD4
`hi
`
`Key words:
`
`tolerance • autoreactive T cells • autoimmune disease • L-selectin
`
`Introduction
`Clonal deletion of self-reactive T cells in the thymus
`and induction of T cell anergy do not alone explain the
`maintenance of immunologic self-tolerance, as potentially
`pathogenic auto-reactive T cells are present in the periphery
`of healthy individuals (1, 2). Thus, other regulatory mech-
`anisms exist to prevent autoreactive T cells from causing
`immune disorders. Active suppression by regulatory T
`cells plays a key role in the control of self-antigen–reactive
`T cells and the induction of peripheral tolerance in vivo
`(3, 4). Seminal experiments performed by Sakaguchi et al.
`⫹
`⫹
`CD25
` suppressor
`have shown that depletion of CD4
`cells results in the onset of systemic autoimmune diseases
`in mice (5). Furthermore, cotransfer of these cells with
`⫹
`⫺
`CD25
` cells prevents the development of experi-
`CD4
`mentally induced autoimmune diseases such as coli-
`tis, gastritis, insulin-dependent autoimmune diabetes, and
`thyroiditis (6–10). We and others have recently described
`⫹
`CD25
` regulatory T cells in human
`a population of CD4
`hi
`⫹
`CD25
`peripheral blood and thymus (11–13). Human CD4
`hi
`⫹
`⫹
`T cells, similar to the mouse CD4
`CD25
` suppressor
`
`V. Viglietta and C. Baecher-Allan contributed equally to this work.
`Address correspondence to David A. Hafler, Laboratory of Molecular
`Immunology, Center for Neurologic Diseases, Brigham and Women’s
`Hospital, Harvard Medical School, 77 Ave. Louis Pasteur, Boston,
`MA 02115. Phone: (617) 525-5330; Fax: (617) 525-5333; email:
`dhafler@rics.bwh.harvard.edu
`
`cells, are anergic to in vitro antigenic stimulation and
`strongly suppress the proliferation of responder T cells
`⫹
`⫹
`CD25
` T cells are among the
`upon coculture (14). CD4
`best-characterized immunoregulatory subsets shown to pre-
`vent activation and effector function of activated responder
`T cells (15).
`Multiple sclerosis (MS) is a chronic inflammatory disease
`characterized by lymphocyte infiltration and inflammation
`of the central nervous system (CNS) white matter. T cells
`recognizing myelin protein peptides are likely involved in
`the pathogenesis of the disease (16–18). Although autore-
`active T cells are present in healthy individuals and patients
`with autoimmune disorders, autoreactive T cells found in
`patients with autoimmune disease are more easily acti-
`vated as compared with those from normal subjects (19–
`22). This finding led us to hypothesize that either deficient
`⫹
`CD25
`generation or reduced effector function of CD4
`hi
`T cells play a role in regulating the autoimmune response
`in patients with MS. Thus, we compared the frequency
`⫹
`CD25
` T regulatory cells derived
`and function of CD4
`hi
`from a group of untreated patients who have relapsing/
`remitting (RR) MS with those from age matched healthy
`control subjects.
`
`Abbreviations used in this paper: CNS, central nervous system; MBP, myelin
`
`basic protein; MS, multiple sclerosis; RR, relapsing/remitting.
`
`971
`
`
` The Rockefeller University Press • 0022-1007/2004/04/971/9 $8.00
`J. Exp. Med.
`Volume 199, Number 7, April 5, 2004 971–979
`http://www.jem.org/cgi/doi/10.1084/jem.20031579
`
`The Journal of Experimental Medicine
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`Page 1 of 9
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`YEDA EXHIBIT NO. 2072
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`Published April 5, 2004
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`Materials and Methods
`Subjects.
`We enrolled 15 patients in this study with definite
`MS upon informed consent. This work was approved by the In-
`stitutional Review Board at the Brigham and Women’s Hospi-
`tal. The patients were between the ages of 25 and 57 yr, and all
`had RR disease with Kurtzke Expanded Disability Status Scale
`scores between 0 and 2.5. Patients were not treated with any im-
`munomodulatory drugs and did not receive corticosteroids
`within 3 mo of study entry. 21 normal healthy donors between
`the ages of 24 and 57 yr and with no history of autoimmune dis-
`eases were also enrolled in the study. Three healthy subjects be-
`tween the ages of 20–30 received the influenza virus vaccina-
`tion, and blood was drawn at the time points outlined in the
`Results section.
`Cell Culture Reagents.
`Cells were cultured in RPMI 1640
`-glutamine,
`medium supplemented with 5 mM Hepes, 2 nM
`l
`␮
`g/ml streptomycin (BioWhittaker),
`100 U/ml penicillin, 100
`0.5 mM sodium pyruvate, 0.05 mM of nonessential amino acids
`(GIBCO BRL), and 5% human AB serum (Omega Scientific
`Inc.) in U-bottom 96-well plates (CoStar).
`
`⫺
`⫹
`⫹
`Cell Purification.
` T
`CD25
` and CD4
`CD25
`Human CD4
`hi
`cells were separated on a FACSVantage™ SE (BD Biosciences) cell
`sorter. PBMCs were isolated by Ficoll-Paque (Amersham Bio-
`⫻
` 10
` cells for
`sciences) gradient centrifugation. Approximately 2
`8
`each experiment were incubated for 1 h in the culture medium,
`␮
`l anti–CD4-CyChrome (cat. no. 555348, IgG1; BD
`with 300
`Biosciences), anti–CD25-PE (cat. no. IM0479, IgG2a; Immuno-
`tech), and a cocktail of anti-CD14 (cat. no. 30544X, IgG2a), anti-
`␮
`l anti-CD116 (cat. no.
`CD32 (cat. no. 30934X, IgG2b), and 20
`18774B, IgM), all FITC labeled (BD Biosciences). Control
` cells each) were also stained with the aforemen-
`PBMCs (10
`6
`tioned antibodies in combination with different mouse IgGs
`(IgG2a PE-labeled cat. no. 55574; IgG1 CyChrome-labeled cat.
`no. 555750; IgG1 FITC-labeled cat. no. 555748; IgG2a FITC-
`labeled cat. no. 555573, and IgG2b FITC-labeled cat. no. 555742;
`all purchased from BD Biosciences). Lymphocytes were washed
`and sorted according to their forward and side scatter properties,
`excluding large activated cells. T cell–depleted accessory cells were
`obtained by negative selection of PBMCs incubated with anti-
`CD2–coated beads (111.13; Dynal) and irradiated at 3,300 rad.
`
`Figure 1. Human CD4⫹CD25hi T cells mediate
`suppression of CD4⫹CD25⫺ responder cell proliferation.
`(A) The regulatory properties of CD4⫹CD25hi T cells
`were examined from 21 healthy individuals or (B) from
`15 patients with RR MS. CD4⫹CD25⫺ responder (䊏)
`and CD4⫹CD25hi suppressor (䉱) cells (2.5 ⫻ 103 cells/
`well) were stimulated with plate-bound anti-CD3 (0.1
`and 0.5 ␮g/ml). CD4⫹CD25⫺ T cells were cocultured
`with CD4⫹CD25hi T cells at a 1:1 ratio (䊊). 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 secre-
`tion was not measurable. In the subsequent series,
`IFN␥ and IL-10 were detected; their values, representing
`the mean ⫾ SE of triplicate cultures, are expressed as
`ng/ml.
`
`972
`
`Loss of CD4
`+
`
`CD25
`+
`
` Regulatory T Cell Function in Human Autoimmune Disease
`
`Page 2 of 9
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`YEDA EXHIBIT NO. 2072
`MYLAN PHARM. v YEDA
`IPR2015-00643
`
`

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`Published April 5, 2004
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`⫹
`⫺
`⫹
`Proliferation Assay.
` (responders) and CD4
`CD25
`CD4
`⫻
`CD25
` (suppressors) T cells (2.5
` 10
`cells/well) were stimu-
`3
`hi
`lated with plate-bound anti-CD3 (clone UCHT1; BD Bio-
`sciences) used at three different concentrations (0.1, 0.5, and
`␮
`⬚
`g/ml, coated in PBS for 2–4 h at 37
`C), or soluble anti-
`2.5
`␮
`g/ml) together with soluble anti-
`CD3 (clone HIT3a at 5
`␮
`g/ml) in U-bottom 96-well plates.
`CD28 (clone 28.2 at 5
`⫹
`⫺
`CD25
` T cells were also cocultured with decreasing num-
`CD4
`⫹
`CD25
` T cells (responder/suppressor ratios: 1:1,
`bers of CD4
`hi
`1:1/2, 1:1/4, and 1:1/8). All cells were cultured in a final volume
`␮
`l in the presence of 10
` T cell–depleted accessory cells/
`of 200
`4
`␮
`l of supernatant was removed
`well. After 5 d of culture, 100
`␮
`Ci
`from each well and used for cytokine detection, and 1
`H]thymidine (NEN Life Science Products) was added to each
`[
`3
`well. The cells were harvested after 16 h; cpm/well was deter-
`mined by scintillation counting (PerkinElmer). Mixing experi-
`ments were performed in the presence of fixed accessory cells au-
`⫹
`⫺
`CD25
` responder T cells.
`tologous to the CD4
`Measurement of Cytokine Level.
`The supernatants removed
`H]thymidine addition were diluted and
`from each well before [
`3
`analyzed to determine the cytokine profile by ELISA using Im-
`mulon 4 ELISA plates (Dynex Technologies). Antibody pairs
`␥
` were purchased from Endogen. Abs for
`and standards for IFN
`IL-13 and IL-10 were purchased from BD Biosciences. Avidin–
`peroxidase conjugate (Sigma-Aldrich) and tetramethylbenzidine
`peroxidase substrate (Kirkegaard and Perry Laboratories) were
`used to develop the assay. For IL-6 detection, the Human In-
`flammatory Cytometric Bead Array kit obtained from BD Bio-
`sciences was used.
`
`⫹
` Clones.
`CD25
`Generation of CD4
`PBMCs were stained
`hi
`with the aforementioned antibodies for flow cytometry. The
`⫹
`⫹
`⫺
`CD25
` and CD4
`CD25
` cells were single cell sorted
`CD4
`hi
`into 96-well plates. The cells were stimulated (modification of
`method described by Levings et al.; reference 23) with PHA
`␮
`g/ml, autologous PBMCs (6,000 rad) were
`(Boehringer) at 0.05
`⫻
` 10
` cells/well, JY cells (10,000 rad) were stim-
`stimulated at 3
`4
`cells/well, and IL-2 (Teceleukin; National Cancer
`ulated at 10
`4
`Institute) were stimulated at 100 U/ml. Half the medium was re-
`placed at day 7, and every 3–4 d thereafter it was replaced with
`fresh medium containing 100 U/ml IL-2. The cultures were re-
`␮
`g/ml PHA, a mixture of irradiated autolo-
`stimulated with 0.05
`⫻
`⫻
` 10
` cells/well) and allogeneic (1–3
` 10
` cells/well)
`gous (1–3
`4
`4
`PBMCs, and 100 U/ml IL-2. After 2–3 wk, growing clones were
`tested for regulatory activity as defined by suppression of cocul-
`tured anti-CD3–mediated T cell proliferation and the cloned cell
`dependence on exogenous IL-2 for proliferation. With these ex-
`periments, we further confirmed the data from Levings et al.
`⫹
`CD25
`showing the possibility of single cell cloning of CD4
`hi
`regulatory cells. Clones were analyzed in cocultures containing
`⫻
`⫻
`⫹
`⫺
` 10
` autologous CD4
`CD25
` T cells or 4
` 10
` PBMCs/well
`4
`3
`3
`⫻
` 10
` cloned cells/well. Stimulation was provided by the
`and 2
`3
`addition of magnetic beads with covalently attached anti-CD3
`␮
`g/10
`(UCHT1, bound to tosyl-activated magnetic beads at 1
`7
`beads; Dynal Biotech Inc.). The beads were added to the cultures
`⫻
` 10
` cells/well.
`[H]Thymidine was added to the cultures
`at 1.5
`4
`3
`after 4 d of stimulation. Percent suppression was determined as
`⫺
` (cpm incorporated in the coculture)/cpm of responder pop-
`1
`ulation alone) ⫻ 100%.
`
`973
`
`Viglietta et al.
`
`Figure 1 (continued)
`
`Page 3 of 9
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`YEDA EXHIBIT NO. 2072
`MYLAN PHARM. v YEDA
`IPR2015-00643
`
`

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`Published April 5, 2004
`
`Downloaded from
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`jem.rupress.org
`
` on November 4, 2014
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`Statistical Analysis. The mean ⫾ SE thymidine uptake and cy-
`tokine secretion of triplicate cultures was calculated for each ex-
`perimental condition. The response of CD4⫹CD25⫺ T cells were
`normalized to 100 to calculate the percent suppression resulting
`from the addition of CD4⫹CD25hi to the cultures. The Mann-
`Whitney test was used to evaluate possible differences in the
`CD4⫹CD25hi function between patients with MS and healthy do-
`nors. The number of wells that produced growth from the clon-
`ing of cells derived from patients with MS and healthy controls
`was compared using hierarchical logistic regression analysis.
`
`Results
`CD4⫹CD25hi T Cells Are Present with the Same Frequency
`in Healthy Donors and Patients with MS. We stained whole
`mononuclear cells from freshly drawn human blood
`with different combination of anti-CD4–CyChrome, anti-
`CD25–PE, and a cocktail of FITC-labeled anti-CD14,
`anti-CD32, and anti-CD116 antibodies. The cells were
`gated on lymphocytes via their forward and side scatter fea-
`tures, and all FITC-labeled cells were negatively selected
`during sorting. Human peripheral blood contains a hetero-
`geneous population of CD4⫹CD25⫹ T cells that express
`either moderate levels of CD25 consisting of nonregulatory
`T cells or high levels of CD25 that exhibit regulatory func-
`tion (24). As there are no other known cell surface markers
`able to identify regulatory T cells ex vivo, we used CD25
`expression to discriminate regulatory T cells in humans.
`We analyzed the mean fluorescent intensity of the CD25⫹
`population in both patients with MS and control subjects
`and found no differences between the two groups. Simi-
`larly, no differences in the frequency of CD4⫹CD25hi T
`cells were found between patients and healthy controls;
`ⵑ10% of CD4 T cells express the ␣-chain of IL-2 receptor
`CD25, whereas only 1–2% of the CD4 T cells are CD25hi
`in both subject groups. The mean of frequency was 1.4% ⫾
`0.3 in the healthy control group and 1.2% ⫾ 0.2 in the MS
`patient group; no statistically significant differences were
`found between the two populations (P ⫽ 0.21).
`CD4⫹CD25hi Regulatory T Cells Display Impaired Function
`in Patients with MS. Because it was critical to examine
`the regulatory T cell function, we isolated highly pure
`CD4⫹CD25hi regulatory and CD4⫹CD25⫺ responder cell
`
`populations by high speed flow cytometric sorting. CD4⫹
`CD25⫺ responder cells from both patients with MS and
`healthy individuals responded similarly in a dose-dependent
`fashion to varying concentration of plate-bound anti-CD3
`mAb. CD4⫹CD25hi T cells isolated from both groups were
`anergic to stimulation at all doses of plate-bound anti-CD3,
`indicating that CD4⫹CD25hi T cells isolated from patients
`with MS do exhibit this regulatory property.
`To quantitate their regulatory function, CD4⫹CD25hi T
`cells were cocultured with autologous responder cells (2.5 ⫻
`103 cells/well) at different ratios (responder/suppressor
`ratios: 1:1, 1:1/2, 1:1/4, and 1:1/8). As reported previously
`in healthy individuals, CD4⫹CD25hi T cells consistently
`suppressed proliferation at a 1:1 ratio (Figs. 1 A and 2). In-
`creasing the ratio of responder/suppressor T cells resulted
`in less suppression (Fig. 2). In striking contrast, the regula-
`tory CD4⫹CD25hi T cells isolated from the circulation
`of patients with MS (while similar in frequency as com-
`pared with healthy controls) poorly inhibited responder
`CD4⫹CD25⫺ T cell proliferation (Figs. 1 B and 2). Be-
`cause we have shown previously that increased strength of
`signal inhibits regulation (24), we stimulated cocultures of
`regulatory and responder T cells from patients with MS and
`healthy controls with a maximal concentration (2.5 ␮g/ml)
`of plate-bound anti-CD3 mAb. As predicted, the CD4⫹
`CD25hi regulatory T cells no longer suppressed the prolif-
`eration of responder T cells (Fig. 2).
`We also examined the production of cytokines in all
`the cultures and the ability to inhibit their secretion by
`CD4⫹CD25hi T cells cocultured with CD4⫹CD25⫺ re-
`sponder cells. The secretion of the Th1 cytokine IFN␥, as
`would result from the activation of destructive, autoreactive
`T cells, was suppressed in CD4⫹CD25hi T cell cocultures
`from healthy controls, but not in cocultures derived from pa-
`tients with MS (Fig. 1, A and B). Because there was no de-
`tectable secretion of IL-13 at the cell numbers used in these
`experiments, those data are unpublished. IL-10 was variably
`secreted, predominantly by the CD4⫹CD25⫺ T cells. The
`secretion of IL-10 was often reduced upon coculture with
`CD4⫹CD25hi T cells, excluding a potential role of this cyto-
`kine in mediating this regulatory suppressor function (Fig. 1,
`A and B). In this ex vivo model of suppression, blocking IL-
`
`of
`Summary
`2.
`Figure
`CD4⫹CD25hi T cell regulatory
`function that is altered in patients
`with MS. The mean percent inhi-
`bition of the proliferative response
`by CD4⫹CD25hi cells derived
`from 15 MS patients (䊊) and
`21 healthy controls (䊏) was
`calculated. CD4⫹CD25⫺ and
`CD4⫹CD25hi populations were
`stimulated with plate-bound
`anti-CD3 mAb, alone or cocul-
`tured at varying ratios. The pro-
`liferative response was inhibited
`upon addition of CD4⫹CD25hi 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⫹CD25hi T cells (responder/suppressor ratios: 1:1/2 and 1:1/4) resulted in
`less suppression in all the conditions examined.
`
`974
`
`Loss of CD4+CD25+ Regulatory T Cell Function in Human Autoimmune Disease
`
`Page 4 of 9
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`YEDA EXHIBIT NO. 2072
`MYLAN PHARM. v YEDA
`IPR2015-00643
`
`

`
`Published April 5, 2004
`
`Downloaded from
`
`jem.rupress.org
`
` on November 4, 2014
`
`the autologous and the converse target cells isolated from
`either healthy subjects or patients with MS. Regulatory T
`cells from patients with MS could not suppress the prolifer-
`ative response of target responder T cells from either pa-
`tients or healthy controls (suppression ⱕ23%). In contrast,
`in the reciprocal experiments, regulatory CD4⫹CD25hi T
`cells from healthy controls suppressed the proliferative re-
`sponse of target CD4⫹CD25⫺ T cells derived from both
`controls and patients with MS (suppression ⱖ78%; Fig. 3).
`These data indicate that the primary regulatory defect is in
`the function of CD4⫹CD25hi T cells isolated from the cir-
`culation of patients with MS.
`CD62L Expression on CD4⫹CD25hi Regulatory T Cells.
`Although there were no differences in the frequency of
`CD4⫹CD25hi T cells or in their proliferation or cytokine
`secretion in response to different stimuli between healthy
`subjects and patients with MS, it was important to deter-
`mine whether an increase in the frequency of activated
`CD4⫹ T cells in the circulation of patients with MS may be
`diluting the regulatory CD4⫹CD25hi T cells. Therefore,
`we further restricted the isolation of T regulatory cells by
`sorting only those CD4⫹CD25hi T cells that expressed
`CD62L because L-selectin expression is down-regulated
`upon activation. We isolated CD4⫹CD25hiCD62L⫹ and
`total CD4⫹CD25hi regulatory T cells from healthy subjects
`and patients with MS and found that, whereas in the
`healthy controls both populations were able to suppress the
`proliferative response to anti-CD3 stimulation, neither
`population of regulatory cells isolated from patients with
`MS was able to inhibit the proliferation of the CD4⫹
`CD25⫺ responder population (Fig. 4). The fact that the
`CD4⫹CD25hiCD62L⫹ T cells isolated from patients are
`depleted of the potentially activated CD62L⫺ T cells yet
`still unable to suppress indicates that the impaired regula-
`tory T cell function in patients with MS is not due to con-
`tamination by activated T cells.
`In Vivo Immunization Did Not Alter CD4⫹CD25hi Regula-
`tory T Cell Function. To further rule out the possibility
`that impaired suppressive function observed in patients
`with MS was due to an increase in the frequency of acti-
`vated CD4⫹ T cells in the circulation or due to an altered
`activation state of the responder cells, we examined the
`
`Figure 3. CD4⫹CD25hi 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⫹CD25hi cells from MS patients and normal controls were stimulated
`with plate-bound anti-CD3 at 0.5 ␮g/ml. 2.5 ⫻ 103 cells/well responder T
`cells from MS patients were cocultured with the same number of autologous
`CD4⫹CD25hi 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⫹CD25hi cells derived
`from the same subject (3rd bar), or with regulatory cells isolated from
`patients with MS (4th bar).
`
`10 or TGF␤ does not result in loss of suppressor function by
`CD4⫹CD25⫹ regulatory T cells (unpublished data).
`Recent studies have shown that the presence of IL-6 can
`contribute to the loss of CD4⫹CD25⫹ suppression (25).
`We measured the levels of IL-6 secretion in the cultures of
`five healthy individuals and four patients with MS to inves-
`tigate its potential role in the lack of suppression observed
`with patient-derived cells. We found that IL-6 levels did
`not correlate with the presence or absence of CD4⫹CD25hi
`T cell–mediated suppression.
`It was also important to examine whether the loss of reg-
`ulatory function was due to a decrease in CD4⫹CD25hi T
`cell function or an increase in the resistance of activated
`CD4⫹CD25⫺ responder T cells to inhibition. Thus, we
`performed mixing experiments in which patient and con-
`trol regulatory CD4⫹CD25hi T cells were cocultured with
`
`Figure 4. The CD62L⫹ subset of CD4⫹CD25hi
`regulatory T cells exhibits decreased suppressive function
`in patients with MS. CD4⫹CD25hiCD62L⫹ T cells
`were isolated from seven healthy individuals (left) and
`three patients with MS (right). Each symbol is repre-
`sentative of a different individual. Total CD4⫹CD25hi
`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 re-
`sponse to plate-bound anti-CD3 was calculated in
`each individual. The CD4⫹CD25hi 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⫹CD25hi 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.
`
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`compared with plate-bound anti-CD3 and, thus, was more
`significantly inhibited upon coculture with CD4⫹CD25hi.
`It was of interest to examine whether differences in sup-
`pressor function by CD4⫹CD25hi cells between patients
`with MS and control subjects would be observed with
`qualitative changes in TCR signaling. Cocultures of regu-
`latory and responder T cells from patients and controls
`were stimulated with soluble anti-CD3 and anti-CD28. As
`expected, the magnitude of the proliferative response was
`significantly lower than that obtained with plate-bound
`anti-CD3 from the same individual. The CD4⫹CD25⫺ T
`cells isolated from patients and controls responded similarly
`to this stimulus. Yet, in contrast to plate-bound anti-CD3,
`the qualitatively different TCR signal delivered by soluble
`anti-CD3 and anti-CD28 allowed the appearance of sup-
`pressor function by cocultured CD4⫹CD25hi regulatory T
`cells derived from patients with MS, particularly at higher
`ratio of regulatory T cells in the culture (Fig. 5).
`
`Published April 5, 2004
`
`regulatory function of CD4⫹CD25hi T cells derived from
`healthy subjects recently immunized with the influenza vi-
`rus vaccine. The regulatory T cells from three subjects ex-
`amined after vaccination showed intact suppressive ability
`(93, 84, and 65% suppression of proliferation). One indi-
`vidual was examined both before and after vaccination and
`demonstrated no differences in the suppressive ability (68%
`suppression before and 65% after vaccination). These data
`provide evidence that an ongoing immune response does
`not affect the intrinsic ability of CD4⫹CD25hi regulatory T
`cells to function.
`CD4⫹CD25hi Regulatory T Cells in Other Autoimmune
`Diseases. We examined the regulatory activity of CD4⫹
`CD25hi T cells isolated from a small series of patients with
`other autoimmune diseases to confirm our hypothesis that
`the changes in function of these regulatory CD4⫹CD25hi T
`cells would not be specific to a single autoimmune disease.
`The frequency of CD4⫹CD25hi T cells in this control
`group was 1.4% ⫾ 0.4; no differences were found between
`these patients and the subjects with MS (P ⫽ 0.44) or the
`healthy individuals (P ⫽ 0.35). The regulatory cells derived
`from patients with thyroiditis and psoriasis showed reduced
`suppressive capacity (7 and 19%, respectively), whereas the
`regulatory activity of cells from the patients with inflam-
`matory bowel disease and ankylosing spondylitis showed
`strong suppressive activity (97 and 68%, respectively).
`These preliminary data suggest the need for a more exten-
`sive analysis of the role of the regulatory T cell subset in
`other autoimmune diseases and are consistent with our hy-
`pothesis that dysfunctions in regulatory T cells may be
`viewed as one of many risk factors in human autoimmune
`disorders.
`The Nature of TCR Signaling Determines Suppressor Func-
`tion. We have shown previously that a qualitatively dif-
`ferent stimulus, such as soluble anti-CD3 and anti-CD28,
`induced lower levels of responder T cell proliferation as
`
`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⫹CD25hi cells from six MS
`patients and six normal controls were stimulated with soluble anti-CD3
`and anti-CD28 (5 ␮g/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 (䊊) and healthy controls (䊏).
`
`Figure 6. Reduced ability to generate CD4⫹CD25hi clones from blood
`of MS patients. (A) CD4⫹CD25hi 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⫹CD25hi 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 cocul-
`tured responder T cells upon stimulation with anti-CD3 beads. The three
`representative CD4⫹CD25hi 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⫹CD25hi population
`is shown, and all of the CD4⫹CD25⫺ clones (e) did not inhibit proliferation
`of cocultured T cells.
`
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`T cells while still demonstrating that they are CD25⫹ regu-
`latory T cells as opposed to activated responder cells ex-
`pressing 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 sub-
`jects when cocultured at 1:1 ratios. In contrast, the stimula-
`tion provided by plate-bound anti-CD3 at 0.1 and 0.5 ␮g/
`ml resulted in a threefold decrease of suppression by
`CD4⫹CD25hi cells derived from patients with MS as com-
`pared 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 anti-
`gen-specific system, we attempted to induce antigen-spe-
`cific 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 re-
`spond 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 be-
`cause such cells would not exhibit regulatory activity, but
`rather enhance proliferation. It was critical to determine
`whether the decrease in T cell regulatory function ob-
`served 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 dem-
`onstrate that the defect lies in the CD4⫹CD25hi T cell
`function, as opposed to enhanced responder T cell resis-
`tance in patients with MS.
`In mice thymectomized on day 3 of life, a spontaneous
`autoimmune disease develops that does not involve in-
`flammation in the CNS (5). This raises the question as to
`whether CD4⫹CD25⫹ 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⫹CD25⫹ T cells, has been shown able to protect
`from both the onset and the progression of autoimmune
`demyelination induced by either active MOG35–55 immu-
`
`Published April 5, 2004
`
`Low CD4⫹CD25hi Cloning Frequency in Patients with MS.
`To examine potential mechanisms for the loss of reg-
`ulatory T cell function, we directly single cell sorted
`CD4⫹CD25hi and CD4⫹CD25⫺ cells from patients
`and healthy subjects and analyzed their frequency and
`functional activity. Although the cloning frequency of
`CD4⫹CD25⫺ T cells was similar in patients with MS and
`control subjects, the cloning frequency of CD4⫹CD25hi
`cells was significantly lower in patients with MS as com-
`pared with normal control subjects (Fig. 6 A). Due to the
`slow growth of the clones, we tested their regulatory ac-
`tivity after a second round of PHA/IL-2 stimulation. We
`examined IL-2–dependent TCR-mediated proliferation
`and inhibition of T cell proliferation as two major charac-
`teristics of regulatory T cells (Fig. 6 B). By these criteria,
`60% of the CD4⫹CD25hi clones generated from healthy
`controls and 17% of the clones derived from patients with
`MS exhibited regulatory activity.
`
`Discussion
`It has become clear that defects in different populations
`of regulatory T cells contribute to the induction of autoim-
`mune 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 signi