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Original Contribution |

Regulatory T Cells Are Reduced During Anti-CD25 Antibody Treatment of Multiple Sclerosis FREE

Unsong Oh, MD; Gregg Blevins, MD; Caitlin Griffith; Nancy Richert, MD, PhD; Dragan Maric, PhD; C. Richard Lee, MD, PhD; Henry McFarland, MD; Steven Jacobson, PhD
[+] Author Affiliations

Author Affiliations: Neuroimmunology Branch, National Institute of Neurological Diseases and Stroke (NINDS) (Drs Oh, Richert, McFarland, and Jacobson), NINDS FACS (fluorescence-activated cell sorter) facility (Dr Maric), and Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (Dr Lee); University of Alberta Medicine and Dentistry, Edmonton, Canada (Dr Blevins); and Grove City College, Grove City, Pennsylvania (Ms Griffith).


Arch Neurol. 2009;66(4):471-479. doi:10.1001/archneurol.2009.16.
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Published online

Objective  Maintenance therapy with anti-CD25 antibody has emerged as a potentially useful treatment for multiple sclerosis (MS). Constitutive CD25 expression on CD4+CD25+ regulatory T cells (Treg) suggests that anti-CD25 antibody treatment may potentially target a subset of T cells that exhibit immune suppressive properties. We examined changes to CD4+CD25+ Treg in patients with MS receiving maintenance anti-CD25 monoclonal antibody treatment to determine the effect of treatment on Treg and, consequently, on immunological tolerance.

Design  Peripheral blood and cerebrospinal fluid samples obtained from a before-and-after trial of anti-CD25 antibody monotherapy were examined to compare baseline and treatment differences in CD4+CD25+ Treg.

Subjects  A total of 15 subjects with MS. One subject was withdrawn owing to an adverse effect.

Results  Sustained reduction of the frequency of CD4+CD25+ Treg was observed during treatment. Anti-CD25 antibody treatment led to evidence of impaired in vivo Treg proliferation and impaired ex vivo Treg suppression. Inflammatory MS activity was substantially reduced with treatment despite reduction of circulating Treg, and there was no correlation between changes in the frequency of Treg and changes in brain inflammatory activity. However, new-onset inflammatory disease, notably dermatitis, was also observed in a number of subjects during treatment.

Conclusion  The reduction in Treg did not negatively affect maintenance of central nervous system tolerance during anti-CD25 antibody treatment. The incidence of new-onset inflammatory disease outside of the central nervous system in a subset of patients, however, warrants further studies to examine the possibility of compartmental differences in the capacity to maintain tolerance in the setting of reduced CD4+CD25+ Treg.

Figures in this Article

The anti-CD25 monoclonal antibody daclizumab targets the α subunit of the high-affinity interleukin (IL)-2 cytokine receptor complex. The upregulation of CD25 following T cell activation and the subsequent IL-2 signaling constitutes a key event in T cell clonal expansion and differentiation. Abnormalities of the IL-2–CD25 cytokine pathway have been reported in a number of immune-mediated diseases including multiple sclerosis (MS) and suggest that CD25 is a potential target for MS immunotherapy. Increased soluble CD25 levels and abnormally high IL-2 responsiveness of autoreactive T cells in subjects with MS implicate an aberrant IL-2/CD25 circuit in the pathogenesis of MS, and constitute the rationale for anti-CD25 antibody treatment to modulate IL-2 signaling in MS.1,2 A number of clinical studies are beginning to demonstrate the immunomodulatory effect of the anti-CD25 monoclonal antibody daclizumab in subjects with MS.3,4

Experimental evidence of the past decade has made increasingly clear that a subset of CD25-expressing CD4+ T cells demonstrate suppressive or regulatory properties and contribute to the maintenance of immunological self-tolerance by their inhibitory influence on autoreactive T cells.5 These CD4+CD25+ regulatory T cells (Treg) are distinguished from conventional activated T cells by constitutive high expression of CD25 and by the expression of Treg lineage specification factor forkhead box P3 (Foxp3).6,7 Conventional activated T cells, by contrast, express intermediate levels of CD25 and lack Foxp3.6,8 Whereas conventional activated T cells coordinate and amplify immune responses, CD4+CD25+ Treg actively suppress immune responses, including those involved in autoimmunity.9 The development of multiorgan inflammatory disease following Treg depletion indicates that CD4+CD25+ Treg make a critical contribution to the maintenance of immunologic self-tolerance.10 The loss or dysfunction of CD4+CD25+ Treg has been implicated in the pathogenesis of a growing number of disorders including systemic lupus erythematosus,11 psoriasis,12 aplastic anemia,13 and MS,14 suggesting a potentially broad relevance with respect to human autoimmune diseases.

The shared expression of CD25 on conventional activated T cells and CD4+CD25+ Treg suggest that both are potentially targeted by anti-CD25 antibody. Based on the knowledge that CD4+CD25+ Treg contribute to maintenance of tolerance, an inhibitory effect on Treg could potentially exacerbate existing inflammatory disease or unmask underlying predilection for new inflammatory disease. We therefore examined the changes to the CD4+CD25+ T cell subsets in subjects with MS undergoing anti-CD25 antibody treatment. In particular, we asked what effect an antihuman CD25 antibody has on CD4+CD25+ Treg, whether changes to CD4+CD25+ Treg affected the immunomodulatory effect of treatment, and whether changes to CD4+CD25+ Treg affected maintenance of overall immunological tolerance.

SAMPLES

Subjects with MS15 were enrolled in an open-label trial of anti-CD25 antibody (daclizumab). Subjects were free of immunomodulatory therapy for 24 weeks prior to enrollment and received intravenous infusion of daclizumab monotherapy (1 mg/kg) every 4 weeks for 54 weeks. Peripheral blood was obtained at baseline and during treatment. Cerebrospinal fluid was obtained at baseline and during treatment. Whole blood was processed immediately for fluorescence-activated cell sorting (FACS) analysis. Peripheral blood mononuclear cells (PBMC) available from 12 subjects were processed by Ficoll-Hypaque density centrifugation and cryopreserved for later use. All treatment samples were obtained just prior to administration of treatment (trough sample). Unless otherwise stated, baseline PBMC samples were compared with treated PBMC samples obtained at month 2.5 (trough sample following third dose). Brain magnetic resonance images were obtained monthly as previously described.3 Informed consent was obtained from each subject. The study was reviewed and approved by the National Institute of Neurological Disorders and Stroke institutional review board.

FLOW CYTOMETRY

The following antibodies were used according to manufacturer's instructions: CD3, CD4, CD8, CD25 (M-A251), CD56, CD127, IL-2, Ki67, and pSTAT5 (signal transduction and activator of transcription 5) antibodies were obtained from BD Biosciences (San Jose, California). The Foxp3 phycoerythrin (PE) or allophycocyanin antibodies were obtained from eBioscience (San Diego, California). The CD25 (Anti-Tac) fluorescein isothiocyanate conjugate was from Immunotech (Westbrook, Maine). The CD25 (7G7) PE was from Ancell (Bayport, Minnesota). The FACS analysis of surface markers were performed on erythrocyte-lysed washed whole blood samples. The PBMC were used for all other analyses. Green Dead Stain (Invitrogen, Carlsbad, California) was used for live or dead cell discrimination with Foxp3 staining. Flow cytometric data was acquired on FACSCalibur (BD Biosciences) and analyzed on FlowJo (TreeStar, Ashland, Oregon).

Treg SUPPRESSION ASSAY

To compare Treg suppression between patients with MS at baseline and healthy donors, CD4+ T cells were purified from PBMC by negative selection magnetic beads (Miltenyi, Auburn, California). Purity was more than 95%. The CD25-PE–labeled CD4+ T cells were then sorted (FACS diva; BD Biosciences) into CD4+CD25high Treg (highest, 3% CD25 expression) and CD4+CD25 responder cells. The Treg coculture suppression assay was performed as previously described,8 with minor modifications. Soluble anti-CD3 (HIT3a, 0.1μg/mL; BD Pharmingen, Franklin Lakes, New Jersy) was used for stimulation. Irradiated (3000 rad) T cell–depleted (MACS [magnetic activated cell sorting] cell separation/anti-CD3 microbeads; Miltenyi) autologous PBMC was used as accessory cells. Cells were plated in triplicate into 96-well plates, incubated at 37°C for 5 days, and pulsed with [3H] thymidine for the final 18 hours of incubation. The percentage of suppression was calculated as %suppression = [1−(cpm of coculture well/cpm of responder well)] × 100, where cpm indicates counts per minute, and the coculture well ratio was 1:1 (responder, Treg). To compare the baseline and treatment Treg suppression, CD4+ T cells were purified from viably cryopreserved PBMC by negative selection magnetic beads. Purified CD4+ T cells were labeled with CD127-PE and CD25-PE/Cy5 antibodies. The CD4+CD25+ CD127low/neg (Treg) and CD4+CD25CD127+ (responder) cells were sorted by FACS and used for Treg coculture suppression assay as described above.

IMMUNOHISTOCHEMISTRY

Formalin-fixed paraffin-embedded tissue sections were prepared on poly-L-lysine coated slides. Immunohistochemistry for CD3 (Dako, Glostrup, Denmark) and Foxp3 (Abcam, Cambridge, Massachusetts) was carried out on consecutive tissue sections and developed with 3,3′-diaminobenzidine, tetrahydrochloride chromogen. A semiquantitative assessment of CD3 and Foxp3 expression was carried out by counting the number of positively stained lymphocytes under an objective microscopy lens at magnification ×40.

STATISTICAL ANALYSIS

Statistical significance was determined by unpaired t test to compare subjects with MS with healthy donors and by paired t test to compare baseline and treatment values. Where appropriate, comparisons were made using general linear model repeated measures analysis of variance. Pearson correlation coefficients were used to analyze relationships between parameters.

BASELINE CD4+CD25+ Treg CHARACTERISTICS IN SUBJECTS WITH MS

To establish pretreatment characteristics of CD4+CD25+ Treg in this cohort of subjects with MS, Treg suppression was measured by an in vitro Treg coculture assay8 (Figure 1A). Most subjects in this cohort demonstrated baseline Treg suppression within the range for healthy donors (P = .27) (Figure 1B). Likewise, the frequency of circulating CD4+Foxp3+ Treg in this cohort of subjects with MS did not differ significantly at baseline compared with that of age-, sex-, and race-matched healthy donors (P = .37) (Figure 1C). Furthermore, as in healthy donors, Foxp3+ cells from subjects with MS were characterized by negative CD69 and low or negative CD127 expression16,17 and demonstrated attenuation of IL-2 production6,18 (Figure 1D).

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Figure 1.

Baseline regulatory T cell (Treg) characteristics in subjects with multiple sclerosis (MS). A, Representative Treg coculture assay [3H] thymidine incorporation data (mean counts per minute [standard deviation]) showing dose-dependent Treg suppression for a subject with MS (86% suppression). Up to 5 × 103 fluorescence-activated cell sorter (FACS) sorted CD4+ CD25high (Treg) cells were titrated in coculture with 5 × 103 CD4+CD25 (responder) cells for 5-day stimulation with anti-CD3 antibody. B, Baseline Treg suppression in subjects with MS (mean [SD], 69.7% [18%]) compared with healthy donors (HD) (mean [SD], 79.7% [13%]; P = .27). C, Frequency of peripheral blood CD4+Foxp3+ cells from subjects with MS (mean [SD], 2.7% [1.6%] of lymphocytes) compared with age-, race-, and sex-matched HD (mean [SD], 3.2% [1.0%] of lymphocytes; P = .37). D, Representative CD4+ gated FACS plots from a subject with MS showing negative CD69 and low or negative CD127 expression in forkhead box P3 (Foxp3+) cells (data representative of 6 subjects with MS and 3 HD), and attenuation of interleukin (IL)-2 production following 6-hour phorbol myristate acetate/ionomycin stimulation (data representative of 4 subjects with MS and 3 HD).

Graphic Jump Location
REDUCTION OF FOXP3+ REGULATORY T CELLS DURING ANTI-CD25 ANTIBODY TREATMENT

Antibody saturation was monitored during the course of anti-CD25 antibody treatment by flow cytometry using 2 fluorochrome-labeled antibodies (anti-Tac and 7G7) that bind noncompeting epitopes on CD25. Complete antibody saturation of CD25, demonstrated by the absence of fluorochrome-labeled anti-Tac binding, was maintained during the course of treatment (Figure 2A, anti-Tac), whereas reduction in the mean total CD25 expression on lymphocytes was less pronounced (13%) but nevertheless statistically significant (P < .001) (Figure 2A, 7G7). Examination of cerebrospinal fluid demonstrated complete antibody saturation of CD25 on cerebrospinal fluid lymphocytes and a 20% decline in mean total CD25 expression during treatment (P = .04) (Figure 2B).

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Figure 2.

Reduction in regulatory T cells (Treg) during anti-CD25 antibody treatment. A, Anti-CD25 antibody saturation and total CD25 expression on lymphocytes over time. Antibody saturation and CD25 expression were evaluated by fluorescence-activated cell sorter (FACS) analysis of whole blood using 2 fluorochrome-labeled antibodies (Anti-Tac and 7G7) directed at noncompeting epitopes on CD25. Complete antibody saturation of CD25 was demonstrated by lack of fluorochrome-labeled Anti-Tac binding. Total CD25 expression (7G7) declined by 13% (P < .001, repeated-measures analysis of variance; n = 12). Months 0 and 0.5 represent the first 2 infusions. Monthly administration followed thereafter, with month 13.5 representing the final infusion. B, Antibody saturation (Anti-Tac) and CD25 expression (7G7) in cerebrospinal fluid (CSF) lymphocytes over time (P = .04; n = 10). C, Histograms show representative signal transduction and activator of transcription 5 phosphorylation (pSTAT5) in peripheral blood mononuclear cells obtained during anti-CD25 antibody treatment (bold line) compared with baseline (thin line) and control (shaded) following 15-minute ex vivo interleukin (IL)-2 exposure. The box plot compares pSTAT5 during treatment compared with baseline (P = .005; n = 7). D, Representative CD4+ gated FACS analysis of CD4+CD25+ forkhead box P3 (Foxp3) conventional activated T cells (upper left quadrants) and CD4+CD25+Foxp3+ Treg (upper right quadrants) at baseline and during treatment. E, Reduced Foxp3 mean fluorescence intensity during treatment compared with baseline (P < .001; n = 12). F, Reduction in frequency of CD4+Foxp3+ cells over time during the course of anti-CD25 antibody treatment (P < .001, repeated-measures analysis of variance; n = 12). The dashed line indicates the end of treatment.

Graphic Jump Location

Signaling of IL-2 was inhibited by anti-CD25 antibody treatment. STAT5 phosphorylation, which mediates downstream IL-2 signaling, was used as a marker of IL-2 signaling. Lymphocytes obtained during treatment demonstrated nearly complete absence of STAT5 phosphorylation in response to low-level (10 U/mL) IL-2. Significant reductions in STAT5 phosphorylation were also observed at higher levels (50 and 100 U/mL) of IL-2 (P = .005) (Figure 2C).

The effect of anti-CD25 antibody treatment on CD4+CD25+ Treg was analyzed by examining Foxp3 as a marker of Treg. Flow cytometric analysis (Figure 2D) demonstrated a reduction in mean fluorescence intensity of Foxp3 expression during treatment compared with baseline (P < .001) (Figure 2E). Reduction in Foxp3 expression at the single cell level during treatment is consistent with previous studies implicating STAT5 as a regulator of Foxp3 gene transcription.19 Furthermore, the frequency of total Foxp3-expressing CD4+ cells were reduced, with approximately 30% reduction in the mean frequency of CD4+Foxp3+ cells observed by month 2.5 and 44% reduction by month 7.5 (P < .001) (Figure 2F). Similar reductions were observed in the frequency of CD4+CD25+Foxp3+ cells (45% reduction; P < .001). Posttreatment samples available from a limited number of subjects demonstrated recovery of Treg frequencies to near baseline levels.

REDUCTION OF Treg PROLIFERATIVE CAPACITY AND IMPAIRED Treg SUPPRESSION BY ANTI-CD25 ANTIBODY TREATMENT

Based on the known role of IL-2 in promoting cell cycle progression in conventional T cells,20 we asked whether altered homeostatic proliferation of Treg could account for the reduction in frequency of Treg during treatment. The effect of anti-CD25 antibody treatment on Treg proliferation was examined using Ki67 expression to estimate the in vivo proliferating fraction,21 determined as the proportion of CD4+Foxp3+ cells expressing Ki67. The PBMC from baseline and treatment were stained ex vivo for intracellular expression of Ki67. Consistent with a previous study demonstrating high in vivo proliferative kinetics of human Treg,22 CD4+Foxp3+ cells demonstrated high Ki67 expression at baseline compared with total CD4+ cells (mean [SD], 11.7% [2.2%] vs 2.13% [0.8%], respectively). Analysis of treatment samples showed a reduction in the Ki67-expressing proliferating Treg fraction (P < .001) (Figure 3A), suggesting impaired homeostatic proliferation of Treg during anti-CD25 antibody treatment.

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Figure 3.

Regulatory T cell (Treg) in vivo proliferation and ex vivo suppression are impaired during anti-CD25 antibody treatment. A, Representative fluorescence-activated cell sorter (FACS) analysis comparing Ki67 expression in forkhead box P3 (Foxp3+) cells at baseline and during anti-CD25 antibody treatment (the percentage of Ki67-expressing cells is in parentheses). The bar graph compares the mean (SD) proportion of CD4+Foxp3+ cells expressing Ki67 at baseline and during treatment (P = .001; n = 12). B, Representative CD4+ gated FACS analysis showing that most CD4+Foxp3+ cells (blue) are CD25+CD127low/neg (polygonal gate) at baseline and remain CD25+CD127low/neg during treatment. The numbers indicate the frequency of cells that are Foxp3+ within the polygonal gate and, in parentheses, the percentage of total lymphocytes that are CD4+Foxp3+ cells. C, Representative [3H] thymidine incorporation data (mean [standard deviation] counts per minute) shown for up to 5 × 103 FACS-sorted Treg (CD4+CD25+CD127low/neg) titrated in coculture with 5 × 103 responder (CD4+CD25CD127+) cells. Closed circles represent cells from baseline and open circles represent cells obtained during anti-CD25 antibody treatment. D, Reduced suppressive capacity (mean [standard deviation] percentage of suppression) of circulating Treg during anti-CD25 antibody treatment (P = .03; n = 4).

Graphic Jump Location

To assess Treg function, coculture suppression assays were performed to compare Treg suppression at baseline and during treatment. Because of altered CD25 expression during treatment, an additional surface marker, CD127, was used to sort Treg, which express low or no CD127 (CD127low/neg).17 The FACS analysis demonstrated that most CD4+Foxp3+ cells were contained within the CD25+CD127low/neg subset at baseline and during treatment (Figure 3B). To determine the effect of anti-CD25 antibody treatment on Treg suppressive capacity, CD4+CD25+CD127low/neg cells were sorted by FACS from PBMC obtained at baseline and during treatment and cocultured with autologous CD4+CD25CD127+ (responder) cells. Treatment samples demonstrated impaired Treg suppression compared with baseline samples (P = .03) (Figure 3C and D), indicating a functional impairment of ex vivo Treg suppression during anti-CD25 antibody treatment.

LACK OF CORRELATION BETWEEN REDUCTION OF Treg AND ACUTE CENTRAL NERVOUS SYSTEM INFLAMMATION

In addition to changes in Treg, anti-CD25 antibody treatment led to significant alteration in conventional activated CD4+CD25+ T cells and CD56bright natural killer (NK) cells. The frequency of conventional activated T cells (CD4+CD25+Foxp3) was reduced during anti-CD25 antibody treatment (P < .001), which corresponded to the contraction of Ki67+ proliferating fraction of conventional activated T cells. In contrast, the proportion of proliferating CD56+ NK cells was increased during anti-CD25 antibody treatment and corresponded to the expansion of CD56bright NK cells observed during the course of therapy (P <.001).

In the setting of simultaneous changes to activated conventional T cell and NK cell compartments, Treg were not necessarily the major determinants of acute central nervous system inflammation. Contrast (gadolinium dithylenetriamine pentaacetic acid [Gd-DTPA]) enhancement of MS lesions on brain magnetic resonance imaging, a marker of MS inflammatory activity, was assessed on a monthly basis. The number of MS lesions demonstrating Gd-DTPA enhancement (Gd-DTPA+ lesions) was significantly reduced during treatment with anti-CD25 antibody (P < .001) (Figure 4A). To assess whether the reduction in Treg had any negative effect on the immunomodulatory effect of anti-CD25 antibody, we analyzed the relationship between changes in the frequency of Treg and brain inflammatory activity. No significant correlations were observed between changes in the frequency of Treg and changes in brain inflammatory activity measured as the total number of Gd-DTPA+ lesions per month (r2 = 0.0171; P = .69) (Figure 4B) or the number of new Gd-DTPA+ lesions per month (r2 = 0.0157; P = .71) when assessed at month 7.5. Analysis of earlier and later time points (months 2.5 and 12.5) yielded similar results (r2 = 0.068 and r2 = 0.0391, respectively, for correlation between change in total number of Gd-DTPA+ cells and Treg).

Place holder to copy figure label and caption
Figure 4.

Lack of correlation between reduction in the frequency of regulatory T cells (Treg) and changes in acute central nervous system inflammatory activity. A, Reduction in brain inflammatory activity indicated by reduction in the total number of contrast-enhancing multiple sclerosis (MS) lesions per month (total number of gadolinium dithylenetriamine pentaacetic acid [Gd-DTPA] lesions) on serial brain magnetic resonance images (P < .001; n = 13). B, Regression analysis of correlation between change in total number of contrast enhancing lesions per month (percentage of reduction in Gd-DTPA+ lesions) and change in the frequency of circulating Treg (percentage of reduction in the number of Treg) at month 7.5 during anti-CD25 antibody treatment (r2 = 0.0171; P = .68, Pearson correlation coefficient). C-F, Analysis of Treg in lesional skin. C, Hematoxylin-eosin (H&E)–stained tissue section (subject MS 9) showing epidermal changes of the skin lesion, characterized by compact hyperkeratosis, acanthosis, and focal spongiosis with exocytosis of lymphocytes (magnification ×20). D, H&E-stained section showing histologic changes in the superficial dermis, characterized by a perivascular chronic inflammatory infiltrate comprised predominantly of lymphocytes. E, Immunohistochemical staining for CD3. F, Immunohistochemical staining for forkhead box P3 (Foxp3).

Graphic Jump Location
NEW-ONSET INFLAMMATORY DERMATITIS AS AN ADVERSE EVENT DURING ANTI-CD25 ANTIBODY TREATMENT

Dermatitis occurred in 3 of 15 individuals who were taking anti-CD25 antibody (Table). The onset of dermatitis occurred during anti-CD25 antibody treatment in 2 subjects and at the end of treatment in one subject who nevertheless still demonstrated more than 80% saturation of CD25 at the onset of dermatitis. An additional subject with a family history of rheumatoid arthritis developed palindromic rheumatism during treatment. Reductions in the frequencies of Foxp3+ cells for subjects who developed dermatitis are shown in the Table. Histologic examination of lesional skin from 2 subjects who developed dermatitis during treatment showed spongiotic to psoriasiform epidermal changes with perivascular lymphocytic inflammatory infiltrate in the subjacent superficial dermis (Figure 4C and D). In situ quantitative detection of Foxp3 in the lesional skin showed that approximately 13% of infiltrating CD3+ cells were Treg (Figure 4E and F).

Table Graphic Jump LocationTable. New-onset Inflammatory Disorders During Anti-CD25 Antibody Treatment

Here we demonstrate that long-term maintenance anti-CD25 antibody treatment led to sustained reduction of CD4+CD25+ Treg in subjects with MS. In contrast to the hypoproliferative nature of Treg in culture, we and others22 find evidence that Treg exhibit high replicative capacity in vivo. Reduced proliferating Treg fraction corresponds to decline in Treg numbers in the setting of impaired IL-2 signaling and suggests that IL-2–supported Treg proliferation accounts for a substantial portion of the human circulating Treg pool. Clinical trials in patients with cancer demonstrated upregulation of Foxp3 and increased frequency of Treg following administration of IL-2.23,24 Our data demonstrates, conversely, that negative perturbation of IL-2 signaling reduces Foxp3 expression and reduces the circulating Treg pool. Collectively, these studies indicate that IL-2 plays a major role in controlling the homeostatic set point for the size of the human circulating Treg pool.

We asked whether reduction in circulating Treg during anti-CD25 antibody treatment negatively affected MS inflammatory activity. Overall brain inflammatory activity was reduced during treatment, suggesting a shift toward tolerance. The lack of a correlation between changes in frequency of Treg and changes in brain inflammatory activity suggests that sustained reduction in circulating Treg did not negatively affect disease activity. One likely explanation is that simultaneous changes in other cell subsets during anti-CD25 antibody treatment countered any negative affect of reduced Treg. Anti-CD25 antibody treatment led to a contraction of the CD4+CD25+ activated conventional T cell fraction and an expansion of CD56bright NK cells. A previous study demonstrated the capacity of CD56bright NK cells to suppress inflammation; expansion of CD56bright NK population is potentially a major determinant of acute brain inflammatory activity during anti-CD25 antibody treatment.25 Alternatively, the lack of correlation between changes in circulating Treg and changes in brain inflammatory activity suggests the possibility that Treg are not a major determinant of acute inflammatory activity in MS. Data from experimental autoimmune encephalomyelitis, an animal model of MS, have not yet reconciled what role CD4+CD25+ Treg play in modulating acute central nervous system inflammation. Loss of Treg appears to confer susceptibility to experimental autoimmune encephalomyelitis in an otherwise resistant strain of mice,26 but central nervous system antigen-specific Treg failed to inhibit central nervous system effector T cells during the acute phase of experimental autoimmune encephalomyelitis, possibly owing to the in situ cytokine milieu, particularly IL-6, that renders effector T cells resistant to Treg suppression.27

Dermatitis occurred in 3 of the 15 subjects receiving treatment. An additional subject with a family history of rheumatoid arthritis developed migratory tenosynovitis during treatment, diagnosed as palindromic rheumatism. The incidence of new-onset inflammatory disease during anti-CD25 antibody treatment raised the possibility that there may be compartmental differences in the capacity to maintain tolerance in the setting of reduced circulating Treg. A recent study showed that CD4+CD25+ Treg contribute to routine immune surveillance and inflammatory response in the human skin.28 Furthermore, the availability of circulating Treg capable of migrating into the skin was shown to be critical to the maintenance of skin-specific tolerance in an animal model, suggesting that the skin may be particularly vulnerable to reduction in circulating Treg.29 Histologic findings from skin biopsies taken from our subjects were relatively nonspecific, but not inconsistent with what has been described in the Foxp3-deficiency syndrome immune dysregulation, polyendocrinopathy, enteropathy, X-linked (syndrome).30 In situ quantitative detection of Foxp3 in the lesional skin showed that approximately 13% of infiltrating CD3+ cells were Treg, which represents a lower frequency of Treg at the site of skin inflammation compared with historical controls.31 Reduction in the frequency of Treg was relatively high (above the cohort mean/median) in subjects who developed new-onset inflammatory disease, but did not reach statistical significance compared with those who did not develop new inflammatory disease on treatment. The relationship between reduction in Treg and new-onset inflammatory disease during anti-CD25 antibody treatment, though suggestive, is inconclusive, and further work is required to determine whether there are compartmental differences in requirements for CD4+CD25+ Treg to maintain organ-specific tolerance.

A functional defect of CD4+CD25+ Treg cells has been reported in subjects with MS.14,32 We found no significant difference in mean Treg suppression between our cohort of subjects with MS and healthy donors, suggesting either that a functional defect of Treg may not be a uniform finding in all subjects with MS or that the differences are on a scale that requires a larger cohort to adequately power such comparisons. Studies comparing Treg suppression in subjects with MS and healthy volunteers suggest that the differences may be age-dependent33 or disease stage–dependent.34

The collective clinical experience with anti-CD25 antibody treatment constitutes a large body of data that demonstrates its safety and efficacy as induction therapy in the prevention of allograft rejection35 and suggest its utility in an array of human disorders.36 The effect of short-term anti-CD25 antibody induction therapy on Treg is likely transient or modified by concomitant use of immunosuppressive agents.37 We now demonstrate that a consequence of long-term maintenance monotherapy with anti-CD25 antibody is a sustained reduction of CD4+CD25+ Treg. Maintenance therapy with anti-CD25 antibody is likely to be a valuable therapeutic option in a number of immune-mediated inflammatory diseases.3,38 Our findings underscore the need to clarify the organ-specific consequences of sustained reduction in human CD4+CD25+ Treg.

Correspondence: Steven Jacobson, PhD, 10 Center Dr, Bldg 10, Room 5C103, Bethesda, MD 20892 (jacobsons@ninds.nih.gov).

Accepted for Publication: September 11, 2008.

Author Contributions: Dr Oh had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Oh, Blevins, and Jacobson. Acquisition of data: Oh, Blevins, Griffith, Richert, Maric, and Lee. Analysis and interpretation of data: Oh, Griffith, Richert, McFarland, and Jacobson. Drafting of the manuscript: Oh, Blevins, Griffith, Richert, and Jacobson. Critical revision of the manuscript for important intellectual content: Oh, Blevins, Maric, Lee, McFarland, and Jacobson. Statistical analysis: Oh. Obtained funding: McFarland and Jacobson. Administrative, technical, and material support: Oh, Griffith, Richert, and McFarland. Study supervision: Jacobson.

Financial Disclosure: Dr McFarland reports being coinventor on National Institutes of Health patents related to the use of daclizumab in multiple sclerosis and, as such, received patent royalty payments.

Funding/Support: This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Neurological Disorders and Stroke.

Additional Contributions: We thank Joan Ohayon, CRNP, for clinical assistance, and Azita Kashani, BS, for technical assistance.

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Valencia  XYarboro  CIllei  GLipsky  PE Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus. J Immunol 2007;178 (4) 2579- 2588
PubMed Link to Article
Sugiyama  HGyulai  RToichi  E  et al.  Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation. J Immunol 2005;174 (1) 164- 173
PubMed Link to Article
Solomou  EERezvani  KMielke  S  et al.  Deficient CD4+ CD25+ FOXP3+ T regulatory cells in acquired aplastic anemia. Blood 2007;110 (5) 1603- 1606
PubMed Link to Article
Viglietta  VBaecher-Allan  CWeiner  HLHafler  DA Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J Exp Med 2004;199 (7) 971- 979
PubMed Link to Article
McDonald  WICompston  AEdan  G  et al.  Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis. Ann Neurol 2001;50 (1) 121- 127
PubMed Link to Article
Seddiki  NSantner-Nanan  BMartinson  J  et al.  Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med 2006;203 (7) 1693- 1700
PubMed Link to Article
Liu  WPutnam  ALXu-Yu  Z  et al.  CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med 2006;203 (7) 1701- 1711
PubMed Link to Article
Bettelli  EDastrange  MOukka  M Foxp3 interacts with nuclear factor of activated T cells and NF-kappa B to repress cytokine gene expression and effector functions of T helper cells. Proc Natl Acad Sci U S A 2005;102 (14) 5138- 5143
PubMed Link to Article
Yao  ZKanno  YKerenyi  M  et al.  Nonredundant roles for Stat5a/b in directly regulating Foxp3. Blood 2007;109 (10) 4368- 4375
PubMed Link to Article
Nourse  JFirpo  EFlanagan  WM  et al.  Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature 1994;372 (6506) 570- 573
PubMed Link to Article
Gerdes  JLemke  HBaisch  HWacker  HHSchwab  UStein  H Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984;133 (4) 1710- 1715
PubMed
Vukmanovic-Stejic  MZhang  YCook  JE  et al.  Human CD4+ CD25hi Foxp3+ regulatory T cells are derived by rapid turnover of memory populations in vivo. J Clin Invest 2006;116 (9) 2423- 2433
PubMed Link to Article
Zorn  ENelson  EAMohseni  M  et al.  IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo. Blood 2006;108 (5) 1571- 1579
PubMed Link to Article
Ahmadzadeh  M Rosenberg  SA IL-2 administration increases CD4+ CD25(hi) Foxp3+ regulatory T cells in cancer patients. Blood 2006;107 (6) 2409- 2414
PubMed Link to Article
Bielekova  BCatalfamo  MReichert-Scrivner  S  et al.  Regulatory CD56(bright) natural killer cells mediate immunomodulatory effects of IL-2Ralpha-targeted therapy (daclizumab) in multiple sclerosis. Proc Natl Acad Sci U S A 2006;103 (15) 5941- 5946
PubMed Link to Article
Reddy  JIlles  ZZhang  X  et al.  Myelin proteolipid protein-specific CD4+CD25+ regulatory cells mediate genetic resistance to experimental autoimmune encephalomyelitis. Proc Natl Acad Sci U S A 2004;101 (43) 15434- 15439
PubMed Link to Article
Korn  TReddy  JGao  W  et al.  Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation. Nat Med 2007;13 (4) 423- 431
PubMed Link to Article
Hirahara  KLiu  LClark  RAYamanaka  KFuhlbrigge  RCKupper  TS The majority of human peripheral blood CD4+CD25highFoxp3+ regulatory T cells bear functional skin-homing receptors. J Immunol 2006;177 (7) 4488- 4494
PubMed Link to Article
Sather  BDTreuting  PPerdue  N  et al.  Altering the distribution of Foxp3(+) regulatory T cells results in tissue-specific inflammatory disease. J Exp Med 2007;204 (6) 1335- 1347
PubMed Link to Article
Nieves  DSPhipps  RPPollock  SJ  et al.  Dermatologic and immunologic findings in the immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. Arch Dermatol 2004;140 (4) 466- 472
PubMed Link to Article
de Boer  OJvan der Loos  CMTeeling  Pvan der Wal  ACTeunissen  MB Immunohistochemical analysis of regulatory T cell markers FOXP3 and GITR on CD4+CD25+ T cells in normal skin and inflammatory dermatoses. J Histochem Cytochem 2007;55 (9) 891- 898
PubMed Link to Article
Haas  JHug  AViehover  A  et al.  Reduced suppressive effect of CD4+CD25high regulatory T cells on the T cell immune response against myelin oligodendrocyte glycoprotein in patients with multiple sclerosis. Eur J Immunol 2005;35 (11) 3343- 3352
PubMed Link to Article
Haas  JFritzsching  BTrubswetter  P  et al.  Prevalence of newly generated naive regulatory T cells (Treg) is critical for Treg suppressive function and determines Treg dysfunction in multiple sclerosis. J Immunol 2007;179 (2) 1322- 1330
PubMed Link to Article
Venken  KHellings  NHensen  K  et al.  Secondary progressive in contrast to relapsing-remitting multiple sclerosis patients show a normal CD4+CD25+ regulatory T-cell function and FOXP3 expression. J Neurosci Res 2006;83 (8) 1432- 1446
PubMed Link to Article
Vincenti  FKirkman  RLight  S  et al. Daclizumab Triple Therapy Study Group, Interleukin-2-receptor blockade with daclizumab to prevent acute rejection in renal transplantation: Daclizumab Triple Therapy Study Group. N Engl J Med 1998;338 (3) 161- 165
PubMed Link to Article
Waldmann  TA Anti-Tac (daclizumab, Zenapax) in the treatment of leukemia, autoimmune diseases, and in the prevention of allograft rejection: a 25-year personal odyssey. J Clin Immunol 2007;27 (1) 1- 18
PubMed Link to Article
Kreijveld  EKoenen  HJKlasen  ISHilbrands  LBJoosten  I Following anti-CD25 treatment, a functional CD4+CD25+ regulatory T-cell pool is present in renal transplant recipients. Am J Transplant 2007;7 (1) 249- 255
PubMed Link to Article
Nussenblatt  RBThompson  DJLi  Z  et al.  Humanized anti-interleukin-2 (IL-2) receptor alpha therapy: long-term results in uveitis patients and preliminary safety and activity data for establishing parameters for subcutaneous administration. J Autoimmun 2003;21 (3) 283- 293
PubMed Link to Article

Figures

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Figure 1.

Baseline regulatory T cell (Treg) characteristics in subjects with multiple sclerosis (MS). A, Representative Treg coculture assay [3H] thymidine incorporation data (mean counts per minute [standard deviation]) showing dose-dependent Treg suppression for a subject with MS (86% suppression). Up to 5 × 103 fluorescence-activated cell sorter (FACS) sorted CD4+ CD25high (Treg) cells were titrated in coculture with 5 × 103 CD4+CD25 (responder) cells for 5-day stimulation with anti-CD3 antibody. B, Baseline Treg suppression in subjects with MS (mean [SD], 69.7% [18%]) compared with healthy donors (HD) (mean [SD], 79.7% [13%]; P = .27). C, Frequency of peripheral blood CD4+Foxp3+ cells from subjects with MS (mean [SD], 2.7% [1.6%] of lymphocytes) compared with age-, race-, and sex-matched HD (mean [SD], 3.2% [1.0%] of lymphocytes; P = .37). D, Representative CD4+ gated FACS plots from a subject with MS showing negative CD69 and low or negative CD127 expression in forkhead box P3 (Foxp3+) cells (data representative of 6 subjects with MS and 3 HD), and attenuation of interleukin (IL)-2 production following 6-hour phorbol myristate acetate/ionomycin stimulation (data representative of 4 subjects with MS and 3 HD).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Reduction in regulatory T cells (Treg) during anti-CD25 antibody treatment. A, Anti-CD25 antibody saturation and total CD25 expression on lymphocytes over time. Antibody saturation and CD25 expression were evaluated by fluorescence-activated cell sorter (FACS) analysis of whole blood using 2 fluorochrome-labeled antibodies (Anti-Tac and 7G7) directed at noncompeting epitopes on CD25. Complete antibody saturation of CD25 was demonstrated by lack of fluorochrome-labeled Anti-Tac binding. Total CD25 expression (7G7) declined by 13% (P < .001, repeated-measures analysis of variance; n = 12). Months 0 and 0.5 represent the first 2 infusions. Monthly administration followed thereafter, with month 13.5 representing the final infusion. B, Antibody saturation (Anti-Tac) and CD25 expression (7G7) in cerebrospinal fluid (CSF) lymphocytes over time (P = .04; n = 10). C, Histograms show representative signal transduction and activator of transcription 5 phosphorylation (pSTAT5) in peripheral blood mononuclear cells obtained during anti-CD25 antibody treatment (bold line) compared with baseline (thin line) and control (shaded) following 15-minute ex vivo interleukin (IL)-2 exposure. The box plot compares pSTAT5 during treatment compared with baseline (P = .005; n = 7). D, Representative CD4+ gated FACS analysis of CD4+CD25+ forkhead box P3 (Foxp3) conventional activated T cells (upper left quadrants) and CD4+CD25+Foxp3+ Treg (upper right quadrants) at baseline and during treatment. E, Reduced Foxp3 mean fluorescence intensity during treatment compared with baseline (P < .001; n = 12). F, Reduction in frequency of CD4+Foxp3+ cells over time during the course of anti-CD25 antibody treatment (P < .001, repeated-measures analysis of variance; n = 12). The dashed line indicates the end of treatment.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Regulatory T cell (Treg) in vivo proliferation and ex vivo suppression are impaired during anti-CD25 antibody treatment. A, Representative fluorescence-activated cell sorter (FACS) analysis comparing Ki67 expression in forkhead box P3 (Foxp3+) cells at baseline and during anti-CD25 antibody treatment (the percentage of Ki67-expressing cells is in parentheses). The bar graph compares the mean (SD) proportion of CD4+Foxp3+ cells expressing Ki67 at baseline and during treatment (P = .001; n = 12). B, Representative CD4+ gated FACS analysis showing that most CD4+Foxp3+ cells (blue) are CD25+CD127low/neg (polygonal gate) at baseline and remain CD25+CD127low/neg during treatment. The numbers indicate the frequency of cells that are Foxp3+ within the polygonal gate and, in parentheses, the percentage of total lymphocytes that are CD4+Foxp3+ cells. C, Representative [3H] thymidine incorporation data (mean [standard deviation] counts per minute) shown for up to 5 × 103 FACS-sorted Treg (CD4+CD25+CD127low/neg) titrated in coculture with 5 × 103 responder (CD4+CD25CD127+) cells. Closed circles represent cells from baseline and open circles represent cells obtained during anti-CD25 antibody treatment. D, Reduced suppressive capacity (mean [standard deviation] percentage of suppression) of circulating Treg during anti-CD25 antibody treatment (P = .03; n = 4).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Lack of correlation between reduction in the frequency of regulatory T cells (Treg) and changes in acute central nervous system inflammatory activity. A, Reduction in brain inflammatory activity indicated by reduction in the total number of contrast-enhancing multiple sclerosis (MS) lesions per month (total number of gadolinium dithylenetriamine pentaacetic acid [Gd-DTPA] lesions) on serial brain magnetic resonance images (P < .001; n = 13). B, Regression analysis of correlation between change in total number of contrast enhancing lesions per month (percentage of reduction in Gd-DTPA+ lesions) and change in the frequency of circulating Treg (percentage of reduction in the number of Treg) at month 7.5 during anti-CD25 antibody treatment (r2 = 0.0171; P = .68, Pearson correlation coefficient). C-F, Analysis of Treg in lesional skin. C, Hematoxylin-eosin (H&E)–stained tissue section (subject MS 9) showing epidermal changes of the skin lesion, characterized by compact hyperkeratosis, acanthosis, and focal spongiosis with exocytosis of lymphocytes (magnification ×20). D, H&E-stained section showing histologic changes in the superficial dermis, characterized by a perivascular chronic inflammatory infiltrate comprised predominantly of lymphocytes. E, Immunohistochemical staining for CD3. F, Immunohistochemical staining for forkhead box P3 (Foxp3).

Graphic Jump Location

Tables

Table Graphic Jump LocationTable. New-onset Inflammatory Disorders During Anti-CD25 Antibody Treatment

References

Waldmann  TA The IL-2/IL-2 receptor system: a target for rational immune intervention. Immunol Today 1993;14 (6) 264- 270
PubMed Link to Article
Zhang  JMarkovic-Plese  SLacet  BRaus  JWeiner  HLHafler  DA Increased frequency of interleukin 2-responsive T cells specific for myelin basic protein and proteolipid protein in peripheral blood and cerebrospinal fluid of patients with multiple sclerosis. J Exp Med 1994;179 (3) 973- 984
PubMed Link to Article
Bielekova  BRichert  NHoward  T  et al.  Humanized anti-CD25 (daclizumab) inhibits disease activity in multiple sclerosis patients failing to respond to interferon beta. Proc Natl Acad Sci U S A 2004;101 (23) 8705- 8708
PubMed Link to Article
Rose  JWBurns  JBBjorklund  JKlein  JWatt  HECarlson  NG Daclizumab phase II trial in relapsing and remitting multiple sclerosis: MRI and clinical results. Neurology 2007;69 (8) 785- 789
PubMed Link to Article
Sakaguchi  S Naturally arising CD4+ regulatory t cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 2004;22531- 562
PubMed Link to Article
Yagi  HNomura  TNakamura  K  et al.  Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells. Int Immunol 2004;16 (11) 1643- 1656
PubMed Link to Article
Fontenot  JDRasmussen  JPWilliams  LMDooley  JLFarr  AGRudensky  AY Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 2005;22 (3) 329- 341
PubMed Link to Article
Baecher-Allan  CBrown  JAFreeman  GJHafler  DA CD4+CD25high regulatory cells in human peripheral blood. J Immunol 2001;167 (3) 1245- 1253
PubMed Link to Article
Shevach  EM Certified professionals: CD4(+)CD25(+) suppressor T cells. J Exp Med 2001;193 (11) F41- F46
PubMed Link to Article
Bennett  CLChristie  JRamsdell  F  et al.  The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 2001;27 (1) 20- 21
PubMed Link to Article
Valencia  XYarboro  CIllei  GLipsky  PE Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus. J Immunol 2007;178 (4) 2579- 2588
PubMed Link to Article
Sugiyama  HGyulai  RToichi  E  et al.  Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation. J Immunol 2005;174 (1) 164- 173
PubMed Link to Article
Solomou  EERezvani  KMielke  S  et al.  Deficient CD4+ CD25+ FOXP3+ T regulatory cells in acquired aplastic anemia. Blood 2007;110 (5) 1603- 1606
PubMed Link to Article
Viglietta  VBaecher-Allan  CWeiner  HLHafler  DA Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J Exp Med 2004;199 (7) 971- 979
PubMed Link to Article
McDonald  WICompston  AEdan  G  et al.  Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis. Ann Neurol 2001;50 (1) 121- 127
PubMed Link to Article
Seddiki  NSantner-Nanan  BMartinson  J  et al.  Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med 2006;203 (7) 1693- 1700
PubMed Link to Article
Liu  WPutnam  ALXu-Yu  Z  et al.  CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med 2006;203 (7) 1701- 1711
PubMed Link to Article
Bettelli  EDastrange  MOukka  M Foxp3 interacts with nuclear factor of activated T cells and NF-kappa B to repress cytokine gene expression and effector functions of T helper cells. Proc Natl Acad Sci U S A 2005;102 (14) 5138- 5143
PubMed Link to Article
Yao  ZKanno  YKerenyi  M  et al.  Nonredundant roles for Stat5a/b in directly regulating Foxp3. Blood 2007;109 (10) 4368- 4375
PubMed Link to Article
Nourse  JFirpo  EFlanagan  WM  et al.  Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature 1994;372 (6506) 570- 573
PubMed Link to Article
Gerdes  JLemke  HBaisch  HWacker  HHSchwab  UStein  H Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984;133 (4) 1710- 1715
PubMed
Vukmanovic-Stejic  MZhang  YCook  JE  et al.  Human CD4+ CD25hi Foxp3+ regulatory T cells are derived by rapid turnover of memory populations in vivo. J Clin Invest 2006;116 (9) 2423- 2433
PubMed Link to Article
Zorn  ENelson  EAMohseni  M  et al.  IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo. Blood 2006;108 (5) 1571- 1579
PubMed Link to Article
Ahmadzadeh  M Rosenberg  SA IL-2 administration increases CD4+ CD25(hi) Foxp3+ regulatory T cells in cancer patients. Blood 2006;107 (6) 2409- 2414
PubMed Link to Article
Bielekova  BCatalfamo  MReichert-Scrivner  S  et al.  Regulatory CD56(bright) natural killer cells mediate immunomodulatory effects of IL-2Ralpha-targeted therapy (daclizumab) in multiple sclerosis. Proc Natl Acad Sci U S A 2006;103 (15) 5941- 5946
PubMed Link to Article
Reddy  JIlles  ZZhang  X  et al.  Myelin proteolipid protein-specific CD4+CD25+ regulatory cells mediate genetic resistance to experimental autoimmune encephalomyelitis. Proc Natl Acad Sci U S A 2004;101 (43) 15434- 15439
PubMed Link to Article
Korn  TReddy  JGao  W  et al.  Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation. Nat Med 2007;13 (4) 423- 431
PubMed Link to Article
Hirahara  KLiu  LClark  RAYamanaka  KFuhlbrigge  RCKupper  TS The majority of human peripheral blood CD4+CD25highFoxp3+ regulatory T cells bear functional skin-homing receptors. J Immunol 2006;177 (7) 4488- 4494
PubMed Link to Article
Sather  BDTreuting  PPerdue  N  et al.  Altering the distribution of Foxp3(+) regulatory T cells results in tissue-specific inflammatory disease. J Exp Med 2007;204 (6) 1335- 1347
PubMed Link to Article
Nieves  DSPhipps  RPPollock  SJ  et al.  Dermatologic and immunologic findings in the immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. Arch Dermatol 2004;140 (4) 466- 472
PubMed Link to Article
de Boer  OJvan der Loos  CMTeeling  Pvan der Wal  ACTeunissen  MB Immunohistochemical analysis of regulatory T cell markers FOXP3 and GITR on CD4+CD25+ T cells in normal skin and inflammatory dermatoses. J Histochem Cytochem 2007;55 (9) 891- 898
PubMed Link to Article
Haas  JHug  AViehover  A  et al.  Reduced suppressive effect of CD4+CD25high regulatory T cells on the T cell immune response against myelin oligodendrocyte glycoprotein in patients with multiple sclerosis. Eur J Immunol 2005;35 (11) 3343- 3352
PubMed Link to Article
Haas  JFritzsching  BTrubswetter  P  et al.  Prevalence of newly generated naive regulatory T cells (Treg) is critical for Treg suppressive function and determines Treg dysfunction in multiple sclerosis. J Immunol 2007;179 (2) 1322- 1330
PubMed Link to Article
Venken  KHellings  NHensen  K  et al.  Secondary progressive in contrast to relapsing-remitting multiple sclerosis patients show a normal CD4+CD25+ regulatory T-cell function and FOXP3 expression. J Neurosci Res 2006;83 (8) 1432- 1446
PubMed Link to Article
Vincenti  FKirkman  RLight  S  et al. Daclizumab Triple Therapy Study Group, Interleukin-2-receptor blockade with daclizumab to prevent acute rejection in renal transplantation: Daclizumab Triple Therapy Study Group. N Engl J Med 1998;338 (3) 161- 165
PubMed Link to Article
Waldmann  TA Anti-Tac (daclizumab, Zenapax) in the treatment of leukemia, autoimmune diseases, and in the prevention of allograft rejection: a 25-year personal odyssey. J Clin Immunol 2007;27 (1) 1- 18
PubMed Link to Article
Kreijveld  EKoenen  HJKlasen  ISHilbrands  LBJoosten  I Following anti-CD25 treatment, a functional CD4+CD25+ regulatory T-cell pool is present in renal transplant recipients. Am J Transplant 2007;7 (1) 249- 255
PubMed Link to Article
Nussenblatt  RBThompson  DJLi  Z  et al.  Humanized anti-interleukin-2 (IL-2) receptor alpha therapy: long-term results in uveitis patients and preliminary safety and activity data for establishing parameters for subcutaneous administration. J Autoimmun 2003;21 (3) 283- 293
PubMed Link to Article

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