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,
* Autoimmunity Branch,
Office of the Clinical Director National Institute of Arthritis and Musculoskeletal and Skin Diseases/National Institutes of Health, and
Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research/National Institutes of Health, Bethesda, MD 20892
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Systemic lupus erythematosus (SLE), the prototypical systemic autoimmune disease, is characterized by a wide spectrum of clinical manifestations and abundant production of autoantibodies to nuclear Ags, cell surface molecules, and serum proteins (14, 15). In SLE, it is well recognized that B cells are hyperactive and produce a variety of autoantibodies, resulting in the formation of immune complexes, that play a central role in the effector phase of the disease. Furthermore, it has also become evident that SLE T cells participate in the attack on target cells or tissues through overproduction of proinflammatory cytokines or an increase in cell-to-cell adhesion, ultimately leading to the apoptosis of the target cells (16). One possibility to explain the emergence of autoimmunity in diseases such as SLE could relate to deficient function of Tregs. The deficiency in Treg function could result in increased helper T cell activity or directly in enhanced B cell activity, both of which have been shown to be regulated by Tregs in normal subjects (17, 18). Murine models that lack CD4+CD25+ Tregs develop a systemic autoimmune disease, characterized by gastritis, oophoritis, arthritis, and thyroiditis (5). Interestingly, some animal models lacking Treg also develop glomerulonephritis and increased titers of anti-dsDNA (5, 19), which are hallmarks of SLE.
Initial studies in SLE suggested there was a decrease in circulating CD4+CD25+ T cells in patients with active disease (20, 21), and more recently it was claimed that Treg from active SLE were decreased in number during disease flares but displayed normal in vitro suppressive function (22). Therefore, the potential role of Tregs in SLE remains to be fully delineated.
We have previously reported a reliable system to assess human Treg function in vitro. Together with flow cytometric analysis of cell surface phenotype and determination of FoxP3 expression (7), this has provided an objective means to assess the presence and function of Tregs in human autoimmune diseases. We, therefore, used these approaches to compare the frequency and function of CD4+CD25high Treg from a group of SLE patients and with those from age-matched healthy control subjects. In this study, we found that CD4+CD25high Tregs from active but not inactive SLE patients manifest deficient in vitro suppressive activity. Importantly, this defect is associated with a decrease in FoxP3 mRNA and protein that can be restored after in vitro stimulation. A reversible defect in Treg function may contribute to flares of disease activity in patients with SLE.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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We enrolled 25 patients who were 18 years or older and fulfilled the American College of Rheumatology criteria for the classification of SLE (23, 24), and 40 healthy donors between the ages of 23 and 69 years with no history of autoimmune disease. Disease activity was scored based on the SLE disease activity index (SLEDAI) (25), with one group comprising patients with inactive disease (SLEDAI <3; n = 8) and another group with active SLE (SLEDAI 
3; n = 17), with or without immunosuppressive treatment. We excluded patients with a history of infection within 3 wk and comorbidities, such as diabetes mellitus. Informed consent was provided according to the declaration of Helsinki. The study was approved by the Institutional Review Board of the National Institute of Arthritis and Musculoskeletal and Skin Diseases/National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. Data from some normal controls (n = 20) have been previously reported (7).
Cell culture reagents
X-VIVO 20 medium (BioWhitaker) supplemented with 1% heat-inactivated normal human serum (BioWhitaker), 20 µg/ml gentamicin, 1 µg/ml Fungizone, and 2 mM glutamine (all obtained from Invitrogen Life Technologies) was used for T cell cultures. FBS was obtained from HyClone.
Cytokines
All cytokines used in this study were recombinant human proteins. Final concentrations were as follows: 100 ng/ml GM-CSF, IL-4 and 2 ng/ml TGF
1 (R&D Systems), and 100 U/ml IL-2 (National Cancer Institute, Frederick, MD).
mAbs
For immunostaining, mouse PE-, FITC-, and CyChrome-conjugated mAbs against human CD3 (UCHT 1), CD4 (RPA-T4), CD8 (RPA-T8), CD14 (M5E2), CD25 (M-A251), CD45RA (HI 100), CD45RO (UCHL 1), CD62L (DREG-56), CD80 (L307.4), CD83 (HB15e), CD86 (FUN-1), CD122 (MIK-2), CD127 (hIL7R-M21), CD152 (BNI3.1), HLA-DR (G46-6), CCR4 (1G1), and corresponding mouse isotype controls (all obtained from BD Pharmingen), glucocorticoid-induced tumor factor receptor (GITR)-FITC (110416), TNFRI-FITC (16803), and TNFRII-allophycocyanin (22235.311) (obtained from R&D Systems), and CD25-PE (Beckman Coulter) were used. Cells were stained with FoxP3-allophycocyanin (PCH101; eBioscience) and FoxP3-AlexaF488 (150D; Biolegend) according to the manufacturer’s instructions for fixation and permeabilization, after the cells were stained for surface expression of CD4 and CD25 with CD25-PE and CD4-CyChrome. Anti-CD3 (64.1; Ref. 26) was used for polyclonal activation of T cells.
Cytokine assays
T cells were stimulated with plate-bound anti-CD3 mAb 64.1 (1 µg/well). Cytokine analysis was conducted after a 72-h incubation by analysis of supernatants with commercially available ELISA kits for human IFN-
(BD Pharmingen), according to the manufacturer’s instructions or by the cytometric bead array kit (BD Biosciences).
Cell isolation
CD4+ T cells were enriched from PBMC by negative selection using the AutoMACS (Miltenyi Biotec). Enriched CD4+ T cells were stained with anti-CD4-CyChrome and PE-conjugated anti-CD25 (15 µg/108 cells) for 20 min at 4°C. CD4+CD25– T cells and CD4+CD25high Tregs were purified using a MoFlo high-speed cell sorter (DakoCytomation) to a purity of >98%. In some experiments, CD4+CD25– and CD4+CD25high Tregs were stimulated in vitro before analysis. This was accomplished by culturing them for 3 days in microtiter plates coated with anti-CD3 mAb 64.1 (1 µg/well) in medium containing 100 U/ml IL-2.
TNF preincubation experiments
Purified CD4+CD25– and CD4+CD25high T cells were incubated overnight with TNF at 50 ng/ml in medium supplemented with 1% NHS and 100 U/ml IL-2. Afterward, cells were washed extensively and used in the assays of Treg function.
Flow cytometric analysis
Single-cell suspensions were prepared and stained for 20 min at 4°C with optimal dilutions of each mAb. Expression of cell surface markers was assessed using the flow cytometer (FACSCalibur; BD Biosciences), and data were analyzed using FlowJo software (Tree Star).
Proliferation assays
To assess proliferation, 5 x 104 sorted cells were incubated in X-VIVO-20 medium with 10% FBS in 96-well U-bottom plates coated with anti-CD3 (64.1) at 1 µg/well. For assessment of regulatory properties, 5 x 104 CD4+CD25– T cells were cultured with plate-bound anti-CD3 in 96-well U-bottom plates. Purified autologous CD4+CD25high Tregs were added, usually at a 1:1 ratio if not indicated differently. After 3–4 days of culture, 100 µl of supernatant was removed from each well and used for cytokine detection and 1 µCi of [3H]thymidine (37 KBq/well) was added for an additional 16 h to each well. [3H]Thymidine incorporation was measured using a liquid scintillation counter.
Real-time PCR
Total RNA was isolated from sorted cells using the RNAeasy Mini kit (Qiagen) according to the manufacturer’s instructions. RNA samples were treated with DNase I to remove contaminating genomic DNA and reverse transcribed with Superscript II (Invitrogen Life Technologies). FoxP3 expression was tested using Assays on Demand reagents from Applied Biosystems (Hs00203958 m1). All reported mRNA levels were normalized to the GAPDH mRNA level, where GAPDH = 1.
Statistical analysis
The mean ± SEM thymidine uptake and mean ± SEM cytokine secretion of triplicate cultures were calculated for each experimental condition. The Mann-Whitney U test was used to evaluate possible differences in the CD4+CD25high function following in vitro and TNF stimulation. Percentage of suppression was determined as 1 – (cpm incorporated in the coculture/cpm of responder population alone) x 100%. Correlations between percentage of FoxP3+CD25high cells or percentage of suppression by CD4+CD25high Tregs and SLEDAI scores were assessed by nonparametric Spearman correlation. All statistical tests were performed using StatView software (SAS Institute).
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Results |
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CD4+CD25high Tregs represented 
0.5–3 ± 1% (mean ± SEM) of total CD4+ T cells from healthy donors (n = 40). Because it has been demonstrated that the brightest 2% of the CD25+ population contains most of the Treg (7, 27), the CD25+ brightest subset was studied further. Because we analyzed only the brightest 2% of CD4+CD25+ T cells in both SLE and normal controls, assessment of the comparative number of Tregs could not be undertaken. The surface phenotype of CD4+CD25– and CD4+CD25high Treg subsets among healthy volunteers and patients with active (SLEDAI 3; n = 17) SLE was characterized. As shown in Fig. 1, the CD4+CD25high Treg subset from active SLE patients expressed modestly higher but not significantly different levels of GITR (20 ± 8% (mean ± SEM) vs 15 ± 5% (mean ± SEM) in normal volunteers). An increased expression of TNFRII was also observed by the freshly isolated CD4+CD25high Treg subset from active SLE (30 ± 12%, mean ± SEM) compared with normal individuals (18 ± 5%, mean ± SEM; p < 0.05), whereas patients with inactive SLE had similar expression of TNFRII as normal donors (n = 8; 25 ± 6%, mean ± SEM; p = 0.32). In contrast, TNFRI (CD120a) was practically undetectable in both groups (mean 1.3 ± 0.6%; n = 10). No differences were detected in the CD45RO expression on the CD4+CD25high Treg subset from normal individuals (90 ± 5%, mean ± SEM) and active SLE patients (75 ± 8%, mean ± SEM). Additionally, analysis of CD69 expression on the Treg subset (mean 1.0 ± 0.7%; n = 40) and in SLE (mean 1.2 ± 0.9%; n = 20) confirmed that these cells were not simply contaminated with recently activated CD4+ effector cells, because these would be mainly CD69+. Finally, <25% of CD4+CD25high Tregs in normal donors and patients with active SLE were HLA-DR+ as has been previously reported (27), indicating that CD4+CD25high Tregs in SLE were not enriched in persistently activated T cells. Our data also confirmed previous findings (28) of selective expression of CCR4 on CD4+CD25high Tregs in normal donors (90 ± 7%, mean ± SEM; n = 40) and SLE (85 ± 18%, mean ± SEM; n = 20) compared with CD4+CD25– from normal donors (30 ± 10%, mean ± SEM; n = 40) and inactive SLE patients (20 ± 8%, mean ± SEM; n = 8). These phenotypic characteristics indicate that the cells analyzed were authentic CD4+CD25high Tregs in both normal donors and SLE patients. However, CD4+CD25high Tregs from patients with active SLE exhibited increased expression of GITR and TNFRII, which has been reported in patients with rheumatoid arthritis and after exposure to TNF (7). By scatter characteristics, CD4+CD25high Tregs were not larger or more complex than CD4+CD25– effector cells in either normal donors or SLE patients (data not shown).
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A low proliferative potential is highly characteristic of CD4+CD25high Tregs both in the murine and human systems. The proliferative capacity of freshly isolated CD4+CD25high Tregs from lupus patients to anti-CD3 stimulation was tested. Freshly isolated CD4+CD25high Tregs from active lupus patients showed a somewhat increased proliferative response to immobilized anti-CD3, compared with normal donors, but this increase was not significant (p = 0.27) (Fig. 3). The regulatory properties of CD4+CD25high T cells were investigated by testing their ability to suppress the proliferative responses of CD4+CD25– T cells to immobilized anti-CD3. At a ratio of 1:1, CD4+CD25high Tregs from healthy volunteers inhibited the proliferation of CD4+CD25– T cells by a mean of 80 ± 5% (n = 40; Fig. 3). These data indicate that CD4+CD25high Tregs have a direct suppressive effect on T cells that is independent of APC. However, as shown in Fig. 3, freshly isolated CD4+CD25high Tregs from patients with active SLE exhibited significantly less suppressive activity than those from normal donors (p < 0.005; n = 18). Notably, the functional activity of Tregs was also assessed in the 0.5% of CD4 cells expressing the very brightest levels of CD25. In three individuals with active SLE studied, these cells were hyporesponsive to anti-CD3 stimulation (cpm = 11.0 ± 3.3 x 10, mean ± SEM; n = 3) and also exerted no suppressive function (percentage of suppression = –93.8 ± 95%, mean ± SEM; n = 3). To determine whether the loss of regulatory function in active SLE was explained by a decrease in the intrinsic function of CD4+CD25high Tregs or an increase in the resistance of CD4+CD25– effector T cells to inhibition, we conducted mixing experiments with cells from patients with active SLE and normal controls. Tregs from patients with active SLE failed to suppress the proliferation of autologous CD4+CD25– effector T cells as well as CD4+CD25– effector T cell from healthy controls, whereas CD4+CD25high Tregs from healthy controls readily suppressed the proliferative response of CD4+CD25– effectors from SLE patients (Fig. 4). These data clearly indicate that the primary regulatory defect is in the function of CD4+CD25high Tregs isolated from the circulation of patients with active SLE, and not a resistance of lupus CD4+CD25– effector cells to suppression.
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Constitutive expression of the transcriptional repressor, FoxP3, is characteristic of CD4+CD25high Tregs. As shown in Fig. 7, FoxP3 mRNA levels were significantly diminished in CD4+CD25high Tregs from patients with active SLE. Of note, FoxP3 mRNA increased after in vitro stimulation of Tregs from patients with active SLE. A modest increase in FoxP3 mRNA was also noted in activated CD4+CD25– effector T cells. As noted with the mRNA analysis, the expression of FoxP3 protein also increased in Tregs from patients with active SLE after in vitro activation (up to 60 ± 10%, mean ± SEM). However, we did not note a uniform increase in FoxP3 expression in CD4+CD25– effector cells. In only one of six experiments was a significant increase in FoxP3 expression noted after in vitro stimulation of CD4+CD25– effector cells.
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It was next determined whether CD4+CD25high Tregs from active SLE patients could become suppressive after in vitro activation. Notably, in vitro activation of CD4+CD25high Tregs from active SLE patients restored the capacity of these cells to suppress both proliferation and IFN- secretion (p = 0.036; Fig. 8). Of note, in vitro activation of CD4+CD25high Tregs from lupus patients also increased their hyporesponsiveness to in vitro stimulation (p = 0.05). In vitro activation of CD4+CD25– effector cells did not lead to induction of suppressive activity (data not shown).
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We have previously reported the effect of signaling through TNFRII in downmodulating the suppressive function of CD4+CD25high Treg. Because CD4+CD25high Tregs from active SLE patients failed to display suppressive function ex vivo and expressed increased TNFRII, we therefore examined whether TNF could modulate the action of CD4+CD25high Tregs from patients with active SLE, as we had previously reported with normal donors (7). Whereas activated CD4+CD25high Tregs from healthy and in vitro-activated active SLE subjects were able to suppress the proliferation of CD4+CD25– T cells, they completely lost their regulatory activity when TNFRII was cross-linked (Fig. 9). In contrast, cross-linking TNFRII did not provoke the loss of anergic phenotype of CD4+CD25high Tregs. It was next determined whether the regulatory function of CD4+CD25high Tregs could also be blocked by soluble TNF instead of anti-TNFRII mAb. Again, the addition of soluble TNF to the regulatory assay completely reversed the suppression of the proliferation of CD4+CD25– T cells without influencing the anergic phenotype of CD4+CD25high Tregs (Fig. 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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In this previous report, it was found that SLE patients have significantly lower levels of CD4+CD25+ T cells and CD4+CD25high Tregs compared with normal donors (21). Notably, in a subset of patients, no correlation between the SLEDAI score and the percentage or number of CD4+CD25+ T cells was found, although this analysis was not conducted with CD4+CD25high Tregs. More recently, a study reported a numeric decrease but preserved in vitro function of CD4+CD25high Tregs in active lupus patients (22). A potential explanation to account for the discrepancy between this study and our results is that these investigators analyzed Treg function after activation by allogeneic stimulator cells. It has been reported that after allogeneic stimulation, even inefficient suppressor cells can adequately suppress the proliferation of CD4+CD25– effectors (30 , 31). In a very recent study, defective regulatory function of CD4+CD25+ Tregs was observed in patients with active SLE (32). In this study, the potent mitogen, PHA, was used to assess Treg function. In the current study, the anti-CD3 mAb 64.1, a very robust activation stimulus, was used. Suppression in this model requires fully functional Tregs. Therefore, the current and previous results are consistent with the conclusion that patients with active SLE do not manifest maximal function of Tregs, although some residual activity may persist. Importantly, we documented markedly diminished FoxP3 mRNA and protein in Tregs from subjects with active SLE, consistent with their decreased function.
We found that freshly isolated CD4+CD25high Treg from subjects with active SLE failed to suppress the proliferation of autologous CD4+CD25–, whereas patient’s CD4+CD25– effectors were suppressed by normal donor Treg. We next sought to determine whether CD4+CD25high Tregs from SLE could become suppressive after in vitro activation, because in the murine system this leads to the development of the most potent suppressors (33). Notably, after in vitro activation of Tregs from SLE subjects by culturing them for 3 days with plate-bound anti-CD3 and high doses of IL-2 their suppressive function was restored, because they were able to suppress the proliferation of autologous CD4+CD25– T cells by nearly 85%. In addition, in vitro-activated CD4+CD25high Tregs suppressed the production of IFN- and IL-2 (data not shown) by CD4+CD25– effectors activated with anti-CD3 mAb and their FoxP3 expression increased. Our findings on the restoration of suppressor function of CD4+CD25high Tregs in SLE by in vitro activation with anti-CD3 in the presence of IL-2 is consistent with the fundamental role of IL-2 in maintaining the fitness of CD4+CD25high Tregs in the periphery as has been recently demonstrated (34), although in our system we demonstrated an increase in Treg function not maintenance. We have noted that Treg function can be maintained by IL-2 in vitro (our unpublished data), but IL-2 is not sufficient to induce Treg function (35). The combination of anti-CD3 and IL-2, therefore, may be necessary for up-regulation of Treg function, whereas IL-2 alone may be sufficient to maintain them. Thus, in human SLE, reduced IL-2 generation, as has been previously reported (36), may be a key factor underlying reduced CD4+CD25high Treg.
It was important to document the fact that the CD4+CD25high Tregs in patients with active SLE were not diluted with activated CD25+ effector T cells. Although it is difficult to rule out this possibility completely, the approaches taken and the results obtained make this explanation quite unlikely. First, only the brightest 2% of the CD25+ cells were analyzed to exclude activated T cells that are usually intermediate in their CD25 expression (7, 27). Moreover, in additional studies, analysis of the 0.6% of cells with the very brightest expression of CD25 also showed they were deficient in both FoxP3 expression and suppressive activity. Secondly, by size or phenotype, we could find no evidence that the CD4+CD25high Tregs in patients with active SLE were activated. Thirdly, the CD4+CD25high Tregs were clearly anergic and failed to produce effector cytokines. Although the level of anergy varied somewhat between Tregs from patients with active and inactive SLE, in all experiments proliferation and IFN- production by CD4+CD25high T cells from SLE patients was significantly less than that of CD4+CD25– effector cells. The degree of responsiveness noted by all CD4+CD25high T cells may relate to the extremely potent stimulus used in this analysis. Finally, upon in vitro stimulation, these cells up-regulated expression of FoxP3 and became suppressive. Although FoxP3 can be up-regulated after in vitro activation of CD4+ effector cells, these activated effector cells do not uniformly become suppressive (13). The fact that CD4+CD25high Tregs from active SLE patients reacquire suppressive function following in vitro activation is consistent with the conclusion that they were indeed Tregs that had become functionally inactivated in vivo.
We conducted a series of experiments aimed to unravel the additional mechanisms of impaired Treg function in active SLE. Because the cells were present in the blood but were functionally impaired and their function could be regained after in vitro culture and during disease quiescence, we reasoned that a soluble factor might contribute to the impaired suppressive function. Among the panoply of altered cytokines in SLE, TNF is known to be secreted in excess in human lupus (37), and in different murine models it can contribute to or ameliorate lupus (38). Importantly, we had previously found that TNF can down modulate CD4+CD25high Treg function (7). Therefore, we explored the possibility that TNF might impair Treg function in SLE.
As in rheumatoid arthritis, our results showed an increased constitutive expression of TNFRII in CD4+CD25high Tregs from healthy volunteers and SLE patients. Importantly, patients with active but not inactive SLE exhibited increased expression of TNFRII, consistent with in vivo exposure to TNF. Similar to our findings in RA, we demonstrated that cross-linking TNFRII completely abrogated the suppression exerted by fresh or activated CD4+CD25high Treg from patients with SLE. Similarly, high concentrations of TNF inhibited the function of CD4+CD25high Tregs, consistent with an action mediated by TNFRII (39, 40, 41). These results clearly show that CD4+CD25high Tregs from patients with SLE are sensitive to the modulatory influences of TNF and are consistent with the conclusion that overproduction of TNF may contribute to the defective Treg function in patients with active SLE.
We next sought to determine whether FoxP3 expression correlated with Treg function in SLE. Expression of FoxP3 mRNA and protein was clearly diminished in patients with active but not inactive SLE. In addition, appearance of FoxP3+CD25high Tregs correlated with disease activity as measured by SLEDAI in lupus patients. Moreover, FoxP3 expression was up-regulated in CD4+CD25high Tregs of active SLE patients after in vitro activation, unlike the results noted with CD4+CD25– T cells in which in vivo stimulation up-regulates FoxP3 expression but not suppressive function (13). In CD4+ T cells from SLE patients initially identified by the bright expression of CD25, suppressive function clearly associated with FoxP3 expression. This suggests that a cofactor in CD25highCD4+ T cells, such as B lymphocyte-induced maturation protein 1 (Blimp-1) (42) or gene related in anergy lymphocytes (GRAIL) (43) or an as yet unidentified molecule along with FoxP3 may be essential for their suppressive function.
Finally, we should note that in vitro activation of CD4+CD25– effector cells did not routinely lead to up-regulation of FoxP3 or suppressive activity. Previous investigators have noted that in vitro stimulation can lead to up-regulation of FoxP3 expression (44) but differ as to whether suppressive activity can be induced (13). This may relate to the reagents used to detect FoxP3 because we noted that there may be some possible FoxP3 detected with some available mAb but not others. In addition, aspects of the culture system, such as the mode of stimulation, presence of APCs, or levels of contaminating TGF may alter the results. In the current results, in vitro stimulation of CD4+CD25– effectors did not routinely up-regulate FoxP3 expression or confer suppressive activity. However, the same mode of stimulation clearly increased the regulatory function of CD4+CD25high Tregs from patients with active SLE, consistent with the reversible nature of the regulatory defect in these patients.
In summary, we have provided evidence for a reversible functional defect in the CD4+CD25high Treg in SLE. Excessive TNF production and diminished IL-2 production may contribute to this defect. Based on these results, it becomes conceivable to design strategies to amplify the decreased function of Tregs in patients with active SLE by specific therapeutic interventions currently available, such as low-dose IL-2 or TNF blocking agents.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by the Intramural Research Program, National Institute of Arthritis and Musculoskeletal and Skin Diseases/National Institutes of Health. 
2 Address correspondence and reprint requests to Dr. Xavier Valencia, National Institutes of Health, 10 Center Drive, Room 6D44, Bethesda, MD 20892. E-mail address: xvalencia{at}mail.nih.gov
3 Abbreviations used in this paper: Treg, T regulatory cell; SLE, systemic lupus erythematosus; SLEDAI, SLE disease activity index; GITR, glucocorticoid-induced tumor factor receptor.
Received for publication June 13, 2006. Accepted for publication November 11, 2006.
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References |
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, but not interleukin 10, and are distinct from type 1 T regulatory cells. J. Exp. Med. 196: 1335-1346. 
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M. Bonelli, A. Savitskaya, C.-W. Steiner, E. Rath, J. S. Smolen, and C. Scheinecker Phenotypic and Functional Analysis of CD4+CD25-Foxp3+ T Cells in Patients with Systemic Lupus Erythematosus J. Immunol., February 1, 2009; 182(3): 1689 - 1695. [Abstract] [Full Text] [PDF]
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J. Abe, S. Ueha, J. Suzuki, Y. Tokano, K. Matsushima, and S. Ishikawa Increased Foxp3+ CD4+ Regulatory T Cells with Intact Suppressive Activity but Altered Cellular Localization in Murine Lupus Am. J. Pathol., December 1, 2008; 173(6): 1682 - 1692. [Abstract] [Full Text] [PDF]
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H. Y. Wu, F. J. Quintana, and H. L. Weiner Nasal Anti-CD3 Antibody Ameliorates Lupus by Inducing an IL-10-Secreting CD4+CD25-LAP+ Regulatory T Cell and Is Associated with Down-Regulation of IL-17+CD4+ICOS+CXCR5+ Follicular Helper T Cells J. Immunol., November 1, 2008; 181(9): 6038 - 6050. [Abstract] [Full Text] [PDF]
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B. Matta, P. Jha, P. S. Bora, and N. S. Bora Tolerance to Melanin-Associated Antigen in Autoimmune Uveitis Is Mediated by CD4+CD25+ T-Regulatory Cells Am. J. Pathol., November 1, 2008; 173(5): 1440 - 1454. [Abstract] [Full Text] [PDF]
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J. Kaplan, L Woodworth, K Smith, J Coco, A Vitsky, and J. McPherson Therapeutic benefit of treatment with anti-thymocyte globulin and latent TGF-{beta}1 in the MRL/lpr lupus mouse model Lupus, September 1, 2008; 17(9): 822 - 831. [Abstract] [PDF]
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A. Sharabi and E. Mozes The Suppression of Murine Lupus by a Tolerogenic Peptide Involves Foxp3-Expressing CD8 Cells That Are Required for the Optimal Induction and Function of Foxp3-Expressing CD4 Cells J. Immunol., September 1, 2008; 181(5): 3243 - 3251. [Abstract] [Full Text] [PDF]
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P. F. K. Yong, L. Aslam, M. Y. Karim, and M. A. Khamashta Management of hypogammaglobulinaemia occurring in patients with systemic lupus erythematosus Rheumatology, September 1, 2008; 47(9): 1400 - 1405. [Abstract] [Full Text] [PDF]
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N. Mozaffarian, A. E. Wiedeman, and A. M. Stevens Active systemic lupus erythematosus is associated with failure of antigen-presenting cells to express programmed death ligand-1 Rheumatology, September 1, 2008; 47(9): 1335 - 1341. [Abstract] [Full Text] [PDF]
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 M. Bonelli, A. Savitskaya, K. von Dalwigk, C. W. Steiner, D. Aletaha, J. S. Smolen, and C. Scheinecker Quantitative and qualitative deficiencies of regulatory T cells in patients with systemic lupus erythematosus (SLE) Int. Immunol., July 1, 2008; 20(7): 861 - 868. [Abstract] [Full Text] [PDF]
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B Zhang, X Zhang, F L Tang, L P Zhu, Y Liu, and P E Lipsky Clinical significance of increased CD4+CD25-Foxp3+ T cells in patients with new-onset systemic lupus erythematosus Ann Rheum Dis, July 1, 2008; 67(7): 1037 - 1040. [Abstract] [Full Text] [PDF]
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H.-Y. Lee, Y.-K. Hong, H.-J. Yun, Y.-M. Kim, J.-R. Kim, and W.-H. Yoo Altered frequency and migration capacity of CD4+CD25+ regulatory T cells in systemic lupus erythematosus Rheumatology, June 1, 2008; 47(6): 789 - 794. [Abstract] [Full Text] [PDF]
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A Kuhn and M Bijl Pathogenesis of cutaneous lupus erythematosus Lupus, May 1, 2008; 17(5): 389 - 393. [Abstract] [PDF]
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A La Cava T-regulatory cells in systemic lupus erythematosus Lupus, May 1, 2008; 17(5): 421 - 425. [Abstract] [PDF]
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K. Venken, N. Hellings, T. Broekmans, K. Hensen, J.-L. Rummens, and P. Stinissen Natural Naive CD4+CD25+CD127low Regulatory T Cell (Treg) Development and Function Are Disturbed in Multiple Sclerosis Patients: Recovery of Memory Treg Homeostasis during Disease Progression J. Immunol., May 1, 2008; 180(9): 6411 - 6420. [Abstract] [Full Text] [PDF]
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M Bonelli, K von Dalwigk, A Savitskaya, J S Smolen, and C Scheinecker Foxp3 expression in CD4+ T cells of patients with systemic lupus erythematosus: a comparative phenotypic analysis Ann Rheum Dis, May 1, 2008; 67(5): 664 - 671. [Abstract] [Full Text] [PDF]
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M. Vargas-Rojas, J. Crispin, Y Richaud-Patin, and J Alcocer-Varela Quantitative and qualitative normal regulatory T cells are not capable of inducing suppression in SLE patients due to T-cell resistance Lupus, April 1, 2008; 17(4): 289 - 294. [Abstract] [PDF]
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X. Feng, S. Kajigaya, E. E. Solomou, K. Keyvanfar, X. Xu, N. Raghavachari, P. J. Munson, T. M. Herndon, J. Chen, and N. S. Young Rabbit ATG but not horse ATG promotes expansion of functional CD4+CD25highFOXP3+ regulatory T cells in vitro Blood, April 1, 2008; 111(7): 3675 - 3683. [Abstract] [Full Text] [PDF]
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 A. Rahman and D. A. Isenberg Systemic Lupus Erythematosus N. Engl. J. Med., February 28, 2008; 358(9): 929 - 939. [Full Text] [PDF]
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C. M. Cuda, S. Wan, E. S. Sobel, B. P. Croker, and L. Morel Murine Lupus Susceptibility Locus Sle1a Controls Regulatory T Cell Number and Function through Multiple Mechanisms J. Immunol., December 1, 2007; 179(11): 7439 - 7447. [Abstract] [Full Text] [PDF]
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Q. Lu, A. Wu, L. Tesmer, D. Ray, N. Yousif, and B. Richardson Demethylation of CD40LG on the Inactive X in T Cells from Women with Lupus J. Immunol., November 1, 2007; 179(9): 6352 - 6358. [Abstract] [Full Text] [PDF]
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J. C Crispin, V. Kyttaris, Y.-T. Juang, and G. C Tsokos Systemic lupus erythematosus: new molecular targets Ann Rheum Dis, November 1, 2007; 66(suppl_3): iii65 - iii69. [Abstract] [Full Text] [PDF]
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A. Kessel, H. Ammuri, R. Peri, E. R. Pavlotzky, M. Blank, Y. Shoenfeld, and E. Toubi Intravenous Immunoglobulin Therapy Affects T Regulatory Cells by Increasing Their Suppressive Function J. Immunol., October 15, 2007; 179(8): 5571 - 5575. [Abstract] [Full Text] [PDF]
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 K. Watanabe, P. N. M. Mwinzi, C. L. Black, E. M. O. Muok, D. M. S. Karanja, W. E. Secor, and D. G. Colley T Regulatory Cell Levels Decrease in People Infected With Schistosoma mansoni on Effective Treatment Am J Trop Med Hyg, October 1, 2007; 77(4): 676 - 682. [Abstract] [Full Text] [PDF]
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G. Borsellino, M. Kleinewietfeld, D. Di Mitri, A. Sternjak, A. Diamantini, R. Giometto, S. Hopner, D. Centonze, G. Bernardi, M. L. Dell'Acqua, et al. Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression Blood, August 15, 2007; 110(4): 1225 - 1232. [Abstract] [Full Text] [PDF]
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