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Published as doi: 10.1096/fj.08-110650.
 (The FASEB Journal. 2008;22:3500-3508.)
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CD4+CD25+FoxP3+PD1– regulatory T cells in acute and stable relapsing-remitting multiple sclerosis and their modulation by therapy

Marina Saresella*, Ivana Marventano*, Renato Longhi{ddagger}, Francesca Lissoni§, Daria Trabattoni§, Laura Mendozzi{dagger}, Domenico Caputo{dagger} and Mario Clerici*,||,1

* Laboratory of Molecular Medicine and Biotechnology and

{dagger} Multiple Sclerosis Unit, Don C. Gnocchi ONLUS Foundation, IRCCS, Milan, Italy;

{ddagger} ICRM-CNR, Milan, Italy; and

§ DISP LITA Vialba and

|| Department of Biomedical Sciences and Technologies, University of Milan, Milan, Italy

1 Correspondence: Department of Biomedical Sciences and Technologies, University of Milan, Via Fratelli Cervi 93, 20090 Segrate (Milan), Italy. E-mail: mario.clerici{at}unimi.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The intracellular expression of the programmed death receptor 1 (PD1) identifies a subset of naive Treg cells with enhanced suppressive ability; antigen stimulation results in the surface expression of PD1. Because the role of Treg impairments in multiple sclerosis (MS) is still contradictory, we analyzed naive PD1– and PD1+ Treg cells in peripheral blood and cerebrospinal fluid (CSF) of relapsing-remitting multiple sclerosis (RR-MS) patients and of healthy control subjects. Results showed that 1) CSF PD1– Treg cells were significantly augmented in MS patients; 2) PD1– Treg cells were significantly increased in the peripheral blood of patients with stable disease (SMS) compared to those with acute (AMS) disease, and in patients responding to glatiramer acetate (COPA) compared to AMS- and COPA-unresponsive patients; and 3) PD1+ Treg cells were similar in CSF and peripheral blood of all groups analyzed. PD1– Treg cells were not increased in the peripheral blood of interferon-β (IFNβ) -responsive patients, but the suppressive ability of Treg cells was significantly higher in SMS and in COPA- or IFNβ-responsive compared to AMS- and COPA-unresponsive individuals. The data herein suggest that PD1– Treg cells play a pivotal role in MS and offer a biological explanation for disease relapse and for the mechanism associated with response to COPA and IFNβ.—Saresella, M., Marventano, I., Longhi, R., Lissoni, F., Trabattoni, D., Mendozzi, L., Caputo, D., Clerici, M. CD4+CD25+FoxP3+PD1– regulatory T cells in acute and stable relapsing-remitting multiple sclerosis and their modulation by therapy.


Key Words: immunology • T lymphocytes • glatiramer acetate • copaxone • neurology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
REGULATORY T (Treg) CELLS ARE pivotal agents in the regulation of tolerance by dampening harmful autoimmune T cells in the periphery (1 2 3) . As a consequence, loss of Treg function appears to be a fundamental factor in autoimmunity (4) . Quantitative and qualitative analyses of Treg cells have been performed in autoimmune diseases in the attempt to shed light on the pathological impairments associated with these conditions. Results showed that Treg lymphocytes are defective in most autoimmune diseases, including rheumatoid arthritis, lupus erythematosus systemicus, and type 1 diabetes (5 6 7) . Therapeutic strategies that could augment the number and the activity of Treg cells are thus being considered for treatment of these pathologies.

One of the autoimmune diseases in which Treg cells are actively investigated is multiple sclerosis (MS), a chronic neurological disease with a complex genetic background that is characterized by multifocal inflammation and damage involving the myelin sheath (8) . MS patients present with a variety of clinical patterns, including acute and stable forms (9 , 10) . Although the etiology of MS is still unclear, an immunopathologic mechanism, mainly mediated by the activation of cell-mediated immunity, was suggested to be responsible for the inflammation of the central nervous system (CNS) white matter that is characteristic of MS (11 , 12) . Molecular mimicry with cross-reacting and yet undefined epitopes is likely to be the initial trigger (13 , 14) , leading to the activation of CD4+ TH1 lymphocytes, TH17 cells, CD8+ T lymphocytes, and B lymphocytes as well as myeloid dendritic cells (15 , 16) . All these cell types are thus believed to be part of the pathogenesis cascade that leads to the destruction of the myelin sheath. Recent observations have also revealed that, next to this inflammatory component, MS is accompanied by diffuse microglial activation throughout the white matter and axonal damages; these subtle degenerative processes seem to play a pivotal role in disease progression (17) .

Treg cells are characterized by a number of markers, including CD4, CD25, and the transcription factor FoxP3 (18 , 19) . Other surface markers shown to be expressed by Treg cells include CD127, CD39, and CTLA-4 (20 21 22 23 24) . An universal consensus on the phenotypic classification of these cells nevertheless has not yet been reached; rather, it is slowly emerging that the cells defined as Treg include different functional and phenotypic subpopulations. One such subpopulation has recently been described by Raimondi et al. (25) , who showed that CD4+/CD25+/FoxP3+ Treg cells can be subclassified based on the surface expression of programmed death receptor 1 (PD1). Briefly, PD1 is retained within intracellular compartments in naive Treg, whereas this protein is expressed on cell surface upon activation. PD1– Treg cells are the majority of Treg cells circulating in healthy humans and are endowed with strong suppressive properties. The mechanisms used by PD1– Treg cells to suppress immune responses are not fully clarified but are likely to involve both cell-cell interaction and suppression mediated by the production of IL-10 and transforming growth factor (TGF) -β, as is the case with Treg cells as a whole.

Contradictory results were reported when Treg cells were analyzed in MS patients, and very limited data are available on PD1– Treg cells in this disease (26 , 27) . We therefore designed a study in which we compared these cells in peripheral blood of relapsing-remitting MS patients with either acute (AMS) or stable (SMS) disease. A possible influence on naive Treg cells of glatiramer acetate [Copaxone (COPA); Sanofi-Aventis, Paris, France] or interferon-β 1a/1b (IFNβ), the most widely used therapies for MS, was also analyzed in patients who either responded or did not respond to therapy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and controls
One hundred and two patients with MS diagnosed by clinical and laboratory parameters and followed by the Centro Sclerosi Multipla, Don Gnocchi Foundation, in Milan, Italy, were included in the study. These patients (63 females and 39 males) were affected by relapsing-remitting (RR) -MS with or without sequelae. The disease had been clinically stable in 15 patients for at least 6 months before the study period; these patients (median age=39 yr; range=25–53 yr; 10 females and 5 males) were classified as patients with SMS. The diagnosis of SMS was confirmed by brain and spinal cord magnetic resonance imaging (MRI) with gadolinium: MRI showed no areas of enhancement at the time of enrollment. Median disease duration was 12.5 yr (range: 2–23 yr); the median Kurtze Expanded Disability Status Scale (EDSS) (28) score was 2.0 (range: 0.0–4.0). Twelve RR-MS patients (median age=37 yr, range=27–52 yr, 11 females and 1 male) were undergoing clinical relapses of the disease and were classified as patients with AMS. AMS patients were examined and blood samples were drawn within 7 d after onset of the acute episode and always before starting steroid-based therapy. MRI scans performed during the acute phases showed enhancing lesions in all AMS patients. None of the patients had received immunosuppressive drugs in the year prior to the study period. Thirty-eight RR-MS patients (median age=40 yr, range=21–57 yr, 19 females and 19 male) with median EDSS score 2.0 (range: 1–5) were successfully treated with IFNβ-1{alpha} (Rebif, Serono, Darmstadt, Germany, 22–44 µg subcutaneously 3x/wk, or Avonex, Biogen, Cambridge, MA, USA, 30 µg intramuscularly 1x/wk) or IFNβ-1β (Betaferon; Schering, Berlin, Germany, 250 µg/ml subcutaneously 3x/wk). Thirty-seven RR-MS patients (median age=37 yr, range=24–58 yr, 23 females and 14 males) (median EDSS score 2.0; range: 1–5) received COPA (20 mg/ml subcutaneously daily). COPA resulted in a significant reduction of relapse rate in 32 patients; 5 patients did not respond to therapy (increase of EDSS score >1, and/or clinical exacerbation, and/or new MRI lesions). All patients gave informed consent according to a protocol approved by the local ethics committee of Don Gnocchi Foundation.

Blood sample and cerebrospinal fluid (CSF) collection and cell separation
Whole blood was collected by venopuncture in vacutainer tubes containing ethylenediaminetetraacetic acid (Becton Dickinson & Co., Rutherford, NJ, USA). Peripheral blood mononuclear cells (PBMCs) were separated on lymphocyte separation medium (Organon Teknika Corp., Durham, NC, USA), washed twice in PBS, and the number of viable leukocytes was determined by trypan blue exclusion. Fifteen milliliters of CSF was obtained by lumbar puncture from 10 more patients.

Peptide synthesis
A total of 31 human leukocyte antigen (HLA) I restricted and of 7 HLA II restricted promiscuous peptides partially overlapping and spanning the whole myelin basic protein (MBP) were synthesized, using Fmoc chemistry. Peptide purity was >70%, as assayed by HPLC, and composition was verified by mass spectrometry (29) . Lyophilized peptides were dissolved at 25 mg/ml in dimethyl sulfoxide or sterile water to prepare the peptide pool at 10 µg/ml final concentration and stored at –20°C until use.

Cell cultures
PBMCs resuspended (3x106/ml) in RPMI 1640 (Life Tecnologies, Grand Island, NY, USA) supplemented with 10% heat-inactivated human antibody (Sigma, St. Louis, MO, USA), 2 mM L-glutamine (Sigma) and 1% penicillin (Sigma) were either unstimulated or stimulated with MBP peptide pools (10 µg/ml) at 37°C in a humidified 5% CO2 atmosphere for 4 days.

Monoclonal antibodies (mAbs)
A monoclonal antihuman CD3 antibody (UCHT 1) (mouse IgG1) was used in the cell-stimulation assays. The following mAbs were used for cytofluorometric analysis: phycoerythrin-cyanin-7 (PC7) -labeled anti-CD4 (clone SFCI12T4D11) (mouse IgG1), phycoerythrin-Texas red (ECD) -labeled anti-CD25 (clone B1.49.9) (mouse IgG2a) (Beckman-Coulter, Fullerton, CA, USA), and fluorescein isothiocyanate (FITC) -labeled anti-PD-1 (clone MIH4) (mouse-IgG1) (eBioscience, San Diego, CA, USA) for surface staining; intracellular PD-1 and FOXP3 proteins were detected by phycoerythrin (PE) -labeled anti-PD-1 and phycoerythrin-cyanin-5 (PC5) -labeled anti-FOXP3 (clone PCH101) (rat anti-IgG2a) (eBioscience). Isotype-matched mouse mAbs were PC7-IgG1, ECD-IgG2a (Beckman-Coulter), FITC and PE-IgG1, and isotype-matched rat mAb (PC5-IgG2a) (eBioscience). Cells were prepared, stained, and analyzed using a Beckman-Coulter Cytomics FC-500 flow cytometer.

Flow staining for PD-1 and FoxP3 of CD4+CD25bright
Freshly isolated or cultured PBMCs were washed and incubated with anti-CD4 and anti-CD25 and anti-PD-1 for 30 min at 4°C. PBMCs were then washed, and the intracellular costaining of PD-1 and FoxP3 was conducted using the FoxP3 staining protocol (eBioscience). Intracellular or surface costaining of PD-1 and intracellular FoxP3 was performed on CD4+CD25bright gated T cell by a flow cytometer. Representative dot plot and histograms are shown in Fig. 1 . CSF was processed at 4°C immediately after spinal tap. Cells were centrifuged after collection of 15 ml of CSF and stained using the same protocol.


Figure 2
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  		Figure 2. PD1– Treg and PD1+ Treg lymphocytes in RR-MS patients and HCs: peripheral blood. PD1– Treg lymphocytes (CD4+25bright FoxP3+PD1–) (A) and PD1+ Treg lymphocytes (CD4+25bright FoxP3+PD1+) (B) in the peripheral blood of patients affected by RR-MS and HCs. The boxes stretch from the 25th to the 75th percentile; the lines across the boxes indicate the median values; the lines stretching from the boxes indicate interquartile ranges.

Cell purification
CD4+ T cells were isolated using a negative CD4+ T cell isolation kit (EasySep, StemCell Technologies, Grenoble, France). CD4+ CD25bright (Treg) and CD4+CD25– (Tresp) cells were separated using the ALTRA EPICS cell sorter (Beckman-Coulter). Freshly isolated CD4+ T cells were stained for 40 min at 4°C with human CD25-specific PC5-labeled antibodies. Sort gates were restricted to the CD4+CD25bright and CD4+CD25–.

Suppression assays
To assess the functional activity of Treg cells, CD4+ and CD4+CD25– cells were labeled with 1 µM of carboxyl fluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR, USA). We resuspended 1 x 105 autologous mytomicin-C-treated (25 µg/ml) PBMCs plus 2 x 104 CD4+CD25– cells in the absence or presence of 2 x 104 CD4+CD25bright Treg cells in 200 µl RPMI 1640 medium and stimulated with either coated anti-CD3 (10 µg/ml) or with MBP peptide pool (10 µg/ml). Cultures were set up as duplicates in U-bottom wells (Costar, Cambridge, MA, USA) and incubated at 37°C in a humidified atmosphere with 5% CO2. After 5 days the cells were harvested, and the CFSE signal of gated lymphocytes was analyzed by flow cytometry. The suppressive capacity of Treg cells toward responder cells in coculture (Tresp-Treg ratio 1:1) was expressed as the relative inhibition of the percentage of CFSElow cells [proliferating 100x(1–%CFSElow CD4+CD25– in coculture/%CFSElow CD4+CD25– T cells alone)] for CFSE-based measurement of proliferation (30) .

Data analysis
Data were summarized according to standard statistical tests; nonparametric descriptive statistics were preferred to avoid assuming a definite theoretical distribution for each data set. Nonparametric tests were performed to evaluate differences between the MS patients divided according to their clinical status (i.e., SMS and AMS) or therapy (COPA and IFNβ). For each variable, a Kruskal-Wallis ANOVA was performed. If this analysis showed significant differences between the groups of individuals, nonparametric subsets (Mann-Whitney U tests) were then used to examine differences between the MS patients classified according to clinical status or therapy. All P values were two sided.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PD1– and PD1+ Treg lymphocytes in CSF and peripheral blood
CD4+CD25bright FoxP3+PD1– as well as CD4+CD25bright FoxP3+PD1+ Treg lymphocytes were analyzed in the peripheral blood of 27 patients affected by RR-MS and 15 age- and sex-matched healthy controls (HCs). These cells were also measured in the CSF of 5 additional RR-MS patients and 5 individuals with noninflammatory diseases. None of the patients had received immunosuppressive drugs in the year prior to the study period; these individuals were not undergoing any therapy at the time of the study. PD1– and PD1+ Treg cells were quantified in freshly isolated PMBCs. Both subpopulations of Treg lymphocytes were comparable in the peripheral blood of MS patients and HCs (Fig. 2 ). In contrast, PD1–, but not PD1+, Treg cells were significantly increased in the CSF of MS patients compared to individuals with noninflammatory conditions. These data are shown in Fig. 3 .


Figure 3
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  		Figure 3. PD1– Treg and PD1+ Treg lymphocytes in RR-MS patients and HCs: CSF. Top panels: PD1– Treg lymphocytes (CD4+25bright FoxP3+ PD1–). Bottom panels: PD1+ Treg lymphocytes (CD4+25bright FoxP3+ PD1+). Data obtained in CSF of patients affected by RR-MS or other, noninflammatory, neurological disease (OND) are shown. In panels A and D, the boxes stretch from the 25th to the 75th percentile; the lines across the boxes indicate the median values; the lines stretching from the boxes indicate interquartile ranges. Statistical significance is shown. Representative results obtained in representative RR-MS (B, E) and OND patients (C, F) are shown. Insets present the median, interquartile (i.q.) range, and min-max values observed in the 2 groups (B, C, E, F).

PD1– and PD1+ Treg lymphocytes in peripheral blood of MS patients with acute or stable disease
The 27 RR-MS patients were subsequently subdivided in patients with either stable (SMS; n=15) or acute (AMS; n=12) disease based on clinical parameters and on the absence or presence of enhancing lesions, as determined by brain and spinal cord MRI with gadolinium. This subdivision revealed that PD1– Treg lymphocytes were significantly augmented in peripheral blood of SMS patients compared to either AMS patients (median percentages: SMS=2.5%; AMS=0.6%; P<0.001) or HCs (1.1%; SMS vs. HC; P = 0.005); no differences were seen between AMS patients and HCs. In contrast with these results, the percentage of PD1+ Treg lymphocytes was comparable in the peripheral blood of SMS, AMS, and HC subjects. These data are presented in Fig. 4 .


Figure 4
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  		Figure 4. PD1– Treg and PD1+ Treg lymphocytes in peripheral blood of SMS or AMS patients and HCs. Top panels: PD1– Treg lymphocytes (CD4+25bright FoxP3+ PD1–). Bottom panels: PD1+ Treg lymphocytes (CD4+25bright FoxP3+ PD1+). Data obtained in peripheral blood of patients affected by either SMS or AMS and HCs are shown. In panels A and E, the boxes stretch from the 25th to the 75th percentile; the lines across the boxes indicate the median values; the lines stretching from the boxes indicate interquartile ranges. Statistical significance is shown. Representative results obtained in a SMS patient (B, F), an AMS patient (C, G), and an HC subject (D, H) are shown. Insets present the median, i.q. range, and min-max values observed in the 2 groups (B–D, F–H).

PD1– and PD1+ lymphocytes in peripheral blood of MS patients undergoing therapy with COPA or IFNβ
PD1– and PD1+ Treg lymphocytes were next analyzed in the peripheral blood of MS patients undergoing therapy with either COPA (n=37) or IFNβ (n=38). Whereas all the IFNβ-treated patients responded to therapy as determined by clinical and instrumental criteria, COPA was clinically efficacious in 32/37 patients. Also in this case, PD1– and PD1+ Treg lymphocytes were quantified in freshly isolated PMBCs. Results showed that PD1– Treg lymphocytes were significantly augmented in COPA-responsive patients compared both to COPA-unresponsive (median percentage of PD1– Treg: COPA responsive=1.7%; COPA unresponsive=0.35%; P=0.035) and AMS patients (P=0.028). Interestingly, nevertheless, PD1– Treg lymphocytes in COPA-responsive patients were still significantly reduced compared to what was observed in the peripheral blood of SMS patients (P=0.033). Clinical response to IFNβ was apparently not associated with an up-regulation of PD1– Treg lymphocytes. Thus, the percentage of PD1– Treg lymphocytes in the peripheral blood of IFNβ-responsive individuals (0.8%) was similar to what observed in AMS and in COPA-unreponsive patients and was reduced compared to both SMS (P=0.005) and COPA-responsive (P=0.059) patients. These results, shown in Fig. 5 , suggest that effectiveness of COPA, but not that of IFNβ, might be associated with an ability of this compound to up-regulate PD1– Treg. Again, no differences were detected in the peripheral blood of any of the groups studied when PD1+ Treg lymphocytes were analyzed.


Figure 5
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  		Figure 5. PD1– Treg and PD1+ Treg lymphocytes in peripheral blood of RR-MS patients treated with either COPA or IFNβ and in HCs. A) PD1– Treg lymphocytes (CD4+25bright FoxP3+PD1–) in the peripheral blood of patients with either SMS or AMS and HCs. Results obtained in patients responding (COPA) or nonresponding (NR) to therapy with glatiramer acetate and of individuals responding to therapy with IFNβ are also presented. B) PD1+ Treg lymphocytes (CD4+25bright FoxP3+PD1+) in the peripheral blood of the same patients and controls. The boxes stretch from the 25th to the 75th percentile; the lines across the boxes indicate the median values; the lines stretching from the boxes indicate interquartile ranges. Statistical significance is shown.

Antigen stimulation induces PD1 expression: MBP-stimulated CD4+ CD25bright FoxP3+PD1+ Treg cells in MS patients
PBMCs of all patients were stimulated in vitro with MBP peptide pool. As previously reported, antigenic stimulation induced the surface expression of intracellular PD1, and, as a consequence, PD1– Treg lymphocytes diminished, whereas PD1– expressing Treg cells increased in all groups of individuals considered. CD4+CD25bright FoxP3+PD1+ MBP-stimulated Treg lymphocytes quantified after a 24 h antigenic incubation were reduced in AMS patients compared to COPA-responsive individuals and HCs. These differences, nevertheless, did not reach statistical significance (data not shown).

Suppression of MBP peptides and anti-CD3-stimulated proliferation by Treg cells
The ability of Treg cells to suppress the proliferative ability of antigen- and anti-CD3-stimulated proliferation was analyzed in all groups of patients enrolled in the study. CD4+CD25bright Treg cells were isolated from freshly drawn peripheral blood, and Treg-depleted cells were stimulated with either MBP or with anti-CD3. Treg cells were then added back in a 1:1 ratio of Tresp/Treg lymphocytes. Results obtained in MBP-stimulated cultures indicated that the suppressive ability of CD4+CD25bright Treg cells on activated cells was similar (mean percentage 75.5, 82, 83%, respectively) in SMS, COPA-responsive, and IFNβ-treated patients. This activity was significantly higher as compared to what was observed in AMS and in COPA-unresponsive individuals (9.5 and 14.5%, respectively; P<0.01) (Fig. 6 ). Results obtained in anti-CD3-stimulated cell cultures confirmed those seen in the MBP stimulation. Thus, a strong suppression of proliferation by CD4+CD25bright Treg cells on activated cells was seen in SMS, COPA-responsive, and IFNβ-treated patients (mean percentage 62, 53, and 43%, respectively); this activity was significantly augmented compared to what was observed in AMS (4%; P=0.01 vs. SMS; P<0.01 vs. COPA-responsive and IFNβ) and in COPA-unresponsive individuals (13%; P=0.03 vs. SMS and IFNβ; P=0.01 vs. COPA-responsive) (Fig. 6) .


Figure 6
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  		Figure 6. Inhibition of the proliferative response by CD4+ 25bright (Treg) in RR-MS patients and in HCs. Mean percentage inhibition of the proliferative response caused by CD4+25bright (Treg) in PBMCs of patients with either SMS or AMS. Results obtained in patients responding (COPA) or nonresponding (NRCOPA) to therapy with glatiramer acetate and of individuals responding to therapy with IFNβ are also presented. MBP-stimulated proliferation (A) and plate-bound anti-CD3-stimulated proliferation (B). In all experiments a 1:1 Treg:Tresp ratio was used.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Treg lymphocytes modulate immune responses against autologous antigens, thus playing a pivotal role in preventing autoimmune diseases. Although a specific surface marker for this T cell subset has not yet been identified, Treg cells can be characterized on the basis of the high expression of CD25, the memory phenotype (CD45RO+), and the intracellular expression of the forkhead transcription factor 3 (FoxP3) (31 , 32) . Other proteins, such as CTLA-4, glucocorticoid-induced TNFR, OX40 (CD134), CD103, CD39, and lymphocyte activation gene-3, can also be expressed by Treg lymphocytes (33 , 34) . This rapidly expanding set of data therefore seems to indicate that the definition of Treg is in reality an oversimplified way to describe a number of subpopulations that are phenotypically distinct and have different abilities to mediate immune suppression against autologous antigens. In particular, recent results have suggested that the intra- or extracellular localization of PD1 distinguishes Treg cells into 2 subpopulations: naive and activated Treg, respectively (25) .

Quantitative and qualitative alterations affecting Treg cells play a role in the ethiopathogenesis of MS, a chronic, degenerative, autoimmune disease of the central nervous system; contrasting results have nevertheless been reported. Thus, whereas some authors showed that the percentage of CD4+/CD25++ Treg cells was comparable in peripheral blood of MS patients and HCs (35 , 36) , other investigators reported a diminished expression of FoxP3 mRNA or expression at the single-cell level in circulating Treg cells obtained from MS patients (37 , 38) . Finally, Treg lymphocytes were shown to augment in the CSF of patients (39) . Most investigators agree on the observation that the functional ability of mitogen-stimulated Treg cells to suppress myelin-specific and Ag-nonspecific T cell proliferation is diminished in MS individuals (35 36 37 38 39 40) . These findings are supported by results obtained in the experimental autoimmune encephalomyelitis (EAE) model: Treg cells migrate to the lesional sites in mice with EAE, but they are ineffectual in suppressing disease (41) . Taken together, these results seem to indicate a disease model characterized by a dichotomy between the number of circulating Treg lymphocytes and their function: Treg cells might not be diminished in MS, but their ability to suppress autoimmune reactions is impaired. It is interesting to observe that this disease model, a discrepancy between T lymphocyte numbers and functions, would not be unique to MS because it had been described early in HIV research (42) .

Based on the assumption that recent thymic emigrants (RTEs) express CD31, a recent study analyzed CD31-expressing (CD4+CD25+CD45RA+CD45RO–FOXP3+CD31+) Treg lymphocytes in MS patients (43) . Results showed that RTE Treg cells within peripheral blood are significantly reduced in MS patients. We decided to address this issue differently and focused our attention on characterizing naive Treg cells by analyzing their intracellular expression of PD1. Results herewith indicate that, whereas the percentage of circulating PD1– Treg cells is similar in MS patients and HCs, a clinical subdivision of patients reveals that these cells are significantly reduced in patients undergoing disease relapse compared to those in quiescent phases of disease. Our results also indicate that PD1– Treg cells augment in patients successfully treated with COPA, but not in individuals responding to IFNβ. The observation that the significant differences seen when these lymphocytes were compared between different groups of patients all but disappeared in MBP-stimulated cells; thus, when activated, "classic" CD4+CD25+FoxP3+PD1+ Treg cells were quantified, reconciles our results with those of authors who did not report a decrease of classic Treg cells in MS. Finally, because PD1– Treg cells have strong immunomodulatory properties, their decrease in AMS individuals justifies previous data indicating a functional impairment of the suppressive ability of Treg lymphocytes in these patients. It is nevertheless necessary to underline that the observations that 1) PD1– naive Treg cells are augmented in the CSF of MS patients compared to patients with noninflammatory diseases and 2) the percentage of these cells is significantly reduced in peripheral blood of HCs compared to SMS patients indicate that an "autoimmune status" characterizes MS patients, in whom disease control is possible only in the presence of a greatly augmented quantity of Treg cells. In this model, PD1– Treg cells would not be responsible for the prevention of the initial events of MS, but rather would be associated with control over disease progression achieved either spontaneously (SMS individuals) or secondarily to therapy.

To measure suppression, we isolated CD4+CD25bright cells from freshly drawn blood (PD1–-based sorting was not technically possible); as a consequence we did not formally measure the suppressive capacity of PD1– Treg cells. Nevertheless, the observation that the percentages of PD1– Treg cells are significantly different in diverse patient populations allows the speculation that the different suppressive abilities we observed are secondary to the diverse percentages of PD1– Treg cells detected in each one of these subpopulations. Interestingly, whereas PD1– Treg cells were increased in COPA-responsive MS patients, their number was not affected by therapy with IFNβ. The suppressive effect of PD1+ Treg lymphocytes on proliferative responses was nevertheless comparable between the two groups of individuals. These results suggest that different pathways mediate the biological effects of COPA and IFNβ: COPA could be beneficial because it increases PD1– Treg cells; IFNβ would up-regulate the functionality of these cells. On a speculative basis, it could be hypothesized that the suppressive activity of COPA-associated PD1– Treg cells would be mainly mediated by cell-cell contact, whereas IFNβ would induce a higher production of IL-10 and TGFβ by these lymphocytes. In this regard, preliminary results indicate that addition of IL-10- and TGFβ-neutralizing antibodies diminishes by >50% the suppressive ability of PD1– Treg cells obtained in IFNβ-treated patients, but affect only marginally the suppression mediated by these cells in COPA-responsive patients (unpublished results).

IFNβ was recently suggested to have a direct effect on the induction of IFNβ-responsive genes in the microglia, resulting in a beneficial immunomodulatory effect. The exact mechanism of action of this drug nevertheless still needs to be fully elucidated. The effect of COPA is a little clearer. Thus, COPA exerts its effect via a bystander effect mediated through the generation of TH2-like cytokines in response to the cross-recognition of MBP proteins and could compete with binding sites for MBP peptides on major histocompatability complex class II molecules (44 45 46 47) .

The use of COPA has also been associated with the development of both CD8+ (48 , 49) and CD4+ Treg cells (50) Additional, recent data have also shown that COPA increases the expression of FoxP3 in Treg cells in a model of myelin oligodendrocyte glycoprotein-induced EAE in mice (51) . Our results confirm these findings and suggest that the beneficial effect of COPA is at least partially mediated by the ability of this compound to increase PD1– Treg lymphocytes.

The data herein indicate that PD1– Treg cells might play a pivotal role in the control of disease progression in MS patients; these cells are up-regulated in patients responding to COPA. Increases in PD1– Treg cells could be monitored in MS patients starting COPA-based therapy in the attempt to prune away patients who will not respond to this regimen. Additionally, given the high ability of these cells to suppress inflammatory processes, these results could encourage trials aiming at up-regulating PD1– Treg cells in other autoimmune diseases by the use of glatiramer acetate (52 , 53) .


Figure 1
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  		Figure 1. Representative results obtained by staining PBMCs with mAbs specific for CD4, CD25, PD1, intracellular PD1, and FoxP3. Data observed in a representative MS patient are shown. Expression of CD25bright T lymphocytes on CD4+ T cells (A); intracellular FoxP3, cells gated on CD4+CD25bright (B); surface expression of PD1 (PD1+ cells), cells gated on CD4+CD25bright (C); intracellular PD1 (PD1– cells), cells gated on CD4+CD25bright (D). Dotted line in B–D represents the isotype control.


   ACKNOWLEDGMENTS
 
This study was supported by grants from the Istituto Superiore di Sanitá "Programma Nazionale di Ricerca sull’ AIDS," the EMPRO and AVIP EC WP6 projects, the Japan Health Science Foundation, 2007 Ricerca Finalizzata (Italian Ministry of Health), 2007 Ricerca Corrente (Italian Ministry of Health), and project FIRB RETI, Rete Italiana Chimica Farmaceutica, CHEM-PROFARMA-NET (RBPR05NWWC). M.C. designed research and wrote the paper; M.S designed and performed research; I.M. analyzed results and drew figures; and R.L., F.L., D.T., L.M., and D.C. performed research. The authors declare no competing financial interests.

Received for publication March 25, 2008. Accepted for publication May 29, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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