Multiple sclerosis patients have reduced resting and increased activated CD4+CD25+FOXP3+T regulatory cells

Resting and activated subpopulations of CD4+CD25+CD127loT regulatory cells (Treg) and CD4+CD25+CD127+ effector T cells in MS patients and in healthy individuals were compared. Peripheral blood mononuclear cells isolated using Ficoll Hypaque were stained with monoclonal antibodies and analysed by flow cytometer. CD45RA and Foxp3 expression within CD4+ cells and in CD4+CD25+CD127loT cells identified Population I; CD45RA+Foxp3+, Population II; CD45RA−Foxp3hi and Population III; CD45RA−Foxp3+ cells. Effector CD4+CD127+ T cells were subdivided into Population IV; memory /effector CD45RA− CD25−Foxp3− and Population V; effector naïve CD45RA+CD25−Foxp3−CCR7+ and terminally differentiated RA+ (TEMRA) effector memory cells. Chemokine receptor staining identified CXCR3+Th1-like Treg, CCR6+Th17-like Treg and CCR7+ resting Treg. Resting Treg (Population I) were reduced in MS patients, both in untreated and treated MS compared to healthy donors. Activated/memory Treg (Population II) were significantly increased in MS patients compared to healthy donors. Activated effector CD4+ (Population IV) were increased and the naïve/ TEMRA CD4+ (Population V) were decreased in MS compared to HD. Expression of CCR7 was mainly in Population I, whereas expression of CCR6 and CXCR3 was greatest in Populations II and intermediate in Population III. In MS, CCR6+Treg were lower in Population III. This study found MS is associated with significant shifts in CD4+T cells subpopulations. MS patients had lower resting CD4+CD25+CD45RA+CCR7+ Treg than healthy donors while activated CD4+CD25hiCD45RA−Foxp3hiTreg were increased in MS patients even before treatment. Some MS patients had reduced CCR6+Th17-like Treg, which may contribute to the activity of MS.

. (A) Gating Strategy for analysis of CD4 + and Treg subpopulations: PBMC isolated from a MS patient were stained for multicolour flow cytometry. Cells were first gated on SSC-A vs. SSC-H to exclude doublets, then lymphocytes were gated based on forward scatter (FSC-A) and side-scatter (SSC-A). CD4 + cells within the lymphocyte population were gated. Staining of CD25 vs. CD127 identified CD4 + CD25 + CD127 lo Treg within CD4 gate. Subpopulations were then identified based on Foxp3 vs. CD45RA expression either from CD4 + gate or from CD4 + CD25 + CD127 lo Treg gate. Within CD4 + cells five different populations were studied as defined by Miyara et al. 43 . Treg are thought to be located within populations I, II and III, with activated Th-like Treg thought to lie within population II. Populations IV and V are Foxp3 − and CD25 − and are respectively defined as activated/memory (CD45RA − ) and effector CD4 + cells (CD45RA + ). Populations I, II and III were then analysed for chemokine receptor expression (CXCR3, CCR6 or CCR7) to examine frequencies of specific Th-like Treg subsets, showing CXCR3 Th1-like Treg as an example. (B): Comparison of percentage of lymphocyte subsets in HD and MS patients. PBMC from MS patients (n = 36) were compared to PBMC from HD (n = 20) for lymphocyte count (a), CD4 + cell count (b) and their proportion within lymphocytes (d), and CD4 + CD25 + CD127 lo Foxp3 + Treg count (c) and their proportion within CD4 + cells (e). Regression analysis for Treg proportion within CD4 + cells vs. CD4 + cell numbers did not change with change in CD4 + cell numbers (f).

Human ethics and subjects
This study was approved by the Human Ethics Committee of the South Western Sydney Local Health District at Liverpool Hospital, Liverpool, NSW Australia, and complied with the Declaration of Helsinki-Ethical Principles for Medical Research involving Human Subjects. All subjects voluntarily gave informed written consent. HD were recruited by an internal email invitation within the hospital. All volunteers were screened to confirm the absence of diseases such as autoimmune conditions, HIV, other infections and anaemia. 20 healthy donors (HD) and 36 MS patients were recruited, and their characteristics are detailed in Table 1.
All MS patients had a clinical diagnosis of MS made according to the McDonald Criteria 2017 59 . Total 36 MS patients were recruited including recently diagnosed MS patients and patients who had received prior treatment with immunomodulatory drugs. Patients were assigned to three groups: (i) a treatment naïve group of 12 patients who had never received treatment with immune modulating therapies, including interferon β (Treatment Naïve), (ii) 8 MS patients who were clinically stable and had not received immune modulating therapy in the last three months, (iii) 15 MS patients who had received immune modulating therapy in the last three months (on therapy). For some analysis, Group I and II were combined as one group of patients not currently on immune modulating therapy and clinically stable (off therapy, n = 20). MS patients were defined to have active disease if they had a relapse or deterioration of symptoms within the last 3 months of sampling.
Immune modulating therapy included 15 patients given interferon-β, all had stopped this therapy 18 months to 10 years prior. Four patients had received glatiramer acetate (Copaxone, Teva Pharma Australia), which had been stopped 3-6 years ago. Eight patients had received natalizumab (Tysabri, Biogen Australia): two stopped 4 weeks prior sampling, the others 8 months to 6 years prior. 14 patients had been treated with fingolimod (Gilenya, Novartis Australia), nine had stopped this therapy in the previous 3 months, the other five stopped 2-4 years prior sampling. Five patients had received dimethyl fumarate (Tecfidera, Biogen Idec Australia), two were currently on the drug and three stopped over a year ago. Five patients had been treated with Rituximab (Mabthera, Roche, Australia) 7-12 months prior sampling. Four patients were treated with alemtuzumab (Lemtrada, Sanofi Aventis Australia), two a year prior and the other two 7-8 years ago. Two patients had been treated with cladribine (Mavenclad, Merck Australia), one remained on the drug, and the other had it a year prior sampling. One patient received IvIg (CSL, Melbourne Australia) 4 months prior. One patient received teriflunomide (Aubagio, Sanofi Aventis, Australia) 3 years prior. One patient had received methotrexate and another an unsuccessful stem cell transplant in Mexico, both several years prior. No patient had received anti-CD25 monoclonal antibody therapy.

Materials and methods
Cell isolation. Peripheral blood lymphocyte counts (PBL) were assessed by a clinical haematological laboratory. Peripheral blood mononuclear cells (PBMC) were isolated from 30ml of fresh whole blood using Ficoll-Hypaque density gradient centrifugation (Ficoll-Paque TM PLUS, GE Healthcare Bio-Sciences AB).
Fresh PBMC (1 × 10 6 ) were stained for chemokine receptors for 15 minutes at room temperature in dark before staining for CD4, CD25, CD127 and CD45RA for 30 minutes on ice. Cells were washed, fixed and permeabilized according to the manufacturer's protocol (eBioscience) then stained for intracellular Foxp3.
Phenotypic analysis was performed on stained PBMC using a FACSCanto II flow cytometer (BD Biosciences) and FACS DIVA 8.0 software. Data was analysed following the gating strategy shown in Fig. 1A using FloJo v10 software (Tree Star, Ashland, OR). Lymphocyte populations were gated after exclusion of doublets based on SSC-A vs. SSC-H.
CD4 + cells were divided into five populations based on CD45RA and Foxp3 expression as described by Miyara et al. 43 . The percentage of each population was also calculated within CD4 + CD25 + CD127 lo Treg, which mainly has cells in Population I, Population II and Population III (Fig. 1A). Table 1. Characteristics of healthy donors (HD) and multiple sclerosis (MS) patients. 1 Disease duration calculated from very first onset of symptoms to time of study. 2 EDSS is expanded disability status scale. 3 Active MS defined as relapse or clinical progression of MS in 3 months before study. 4 Non active MS, no progression or relapse in last 6 months. 5 No recent treatment-no immunomodulatory therapy received in 3 months before study. *Some clinical data not available for this post-hoc analysis. www.nature.com/scientificreports/ Each population was further examined for the Th-like Treg phenotype using expression of chemokine receptors; CXCR3 for Th1-like Treg, CCR6 for Th17-like Treg and CCR7 for circulating naïve Treg. The gating for chemokine receptor expressing cells was based on a Flourescence Minus One (FMO) control. Statistical analysis. Statistical analysis was performed using Graphpad Prism 8.0.2 and IBM SPSS Statistics 25. For comparison of unpaired data, the Mann-Whitney U test was used. The Kruskal-Wallis Test was used in cases where multiple independent groups were assessed. Linear regression analysis was performed to compare the effect of age and disease duration with Treg parameters. Results were expressed as mean ± SEM unless otherwise specified and significance was p < 0.05.

Results
Comparison of peripheral lymphocyte counts (PBL) in HD and MS patients. The demographic characteristics of HD and MS patients are summarised in Table 1. HD had higher PBL counts than MS patients, 2.19 ± 0.60 (mean ± SEM) vs. 1.77 ± 0.15 × 10 9 /L (p = 0.035) (Fig. 1Ba). MS patients with no previous therapy had PBL counts of 2.45 ± 0.25 × 10 9 /L that was not significantly different to HD. Compared to HD, PBL counts were lower in MS patients whose disease was inactive (p = 0.004) or had treatment in last three months (p = 0.012), but not in those not treated in last three months (p = 0.054). Regression analysis of PBL counts showed no significant effect of age, disease duration or time since last flare of MS (R 2 = 0.053, p = 0.316).
Patients with active MS had higher numbers and proportions of CD4 + cells compared to those with inactive disease (p = 0.02). CD4 + cell counts or proportion of CD4 + cells (p = 0.184) were not different in MS patients that received therapy in last three months compared to MS patients who received no therapy in the last three months. Regression analysis found neither the number nor the proportion of CD4 + cells in MS patients correlated with age, disease duration or time since last flare of MS (R 2 = 0.109, p = 0.087).
Regression analysis of CD4 + cell numbers with proportion of Treg in CD4 + cells in MS patients showed no correlation (R 2 = 0.001) (Fig. 1Bf). This demonstrated that the ratio of Treg as a proportion of CD4 + cells did not relate to total CD4 + cell number. Thus, all results were expressed either as proportions of either CD4 + or of CD4 + CD25 + CD127 lo Treg.
Comparison of the three Treg populations based on CD45RA and Foxp3 expression in HD and MS patients. We next examined if there were changes in the subpopulations of CD4 + cells and CD4 + CD25 + CD127 lo Treg as described by Miyara et al 43 for CD4 + cells identifying resting and activated Treg by staining for CD45RA and Foxp3/CD25. A representative plot of five subpopulations within CD4 + cells and CD4 + CD25 + CD127 lo Treg population is shown as Fig. 2A. To identify if changes demonstrated in studies of all MS patients ( Fig. 2B-D), were a marker of immune changes intrinsic to MS or a consequence of immune modifying therapy, we compared CD4 + and CD4 + CD25 + CD127 lo Treg from treatment naïve MS patients (n = 12) and HD ( Fig. 2E-G).
Within CD4 + CD25 + CD127 lo Treg, Population I was also significantly smaller in MS than HD (p = 0.004) (Fig. 2C). Population I was significantly lower in MS patients who were off treatment in last three months compared to HD (p = 0.008), but not for those who were on treatment in the last three months (data not shown).
There was no significant difference of Population III in either CD4 + cells (Fig. 2B,E) or CD4 + CD25 + CD127 lo Treg (Fig. 2C,F), when comparing all MS or treatment naïve MS to HD. Population III includes activated effector CD4 + that are activated and transiently express Foxp3 and CD25 41,60 .
The ratio of Population I to Population V was not different between HD and MS (p = 0.867) (Fig. 3B). The ratio of activated Treg Population II to activated effector Population IV also showed no difference between HD and MS (p = 0.188) (Fig. 3A).
MS patients who were off treatment for three months had significantly higher Population II than HD (p = 0.026) (Fig. 3C), as did MS patients who were on treatment (p = 0.04).
Treg Population II increased significantly in patients with inactive MS compared to HD (p = 0.012) but not with active MS (Fig. 3D).

Changes in populations with age: comparison of HD and MS patients. Previous studies showed
CD45RA + CD25 + Foxp3 + naïve Treg decrease with age. Within the CD4 + gate there was a significant decrease in the Population I (R 2 = 0.571, p < 0.001) with age in HD but not MS (data not shown). www.nature.com/scientificreports/ Within the CD4 + CD25 + CD127 lo Treg gate, Population I decreased with age in HD (R 2 = 0.542, p < 0.001) and to a lesser degree in MS (R 2 = 0.116, p = 0.042) (Fig. 4A,B). The decline with age was less apparent in MS patients (p = 0.042) compared to HD (p < 0.001).
Population III, in both CD4 + and CD4 + CD25 + CD127 lo Treg gates had no significant relationship with age in HD or MS patients (data not shown).

Changes in populations with disease duration in MS patients. Population I significantly decreased
with MS duration within both, the CD4 + (R 2 = 0.151, p = 0.026) and CD4 + CD25 + CD127 lo Treg gates (R 2 = 0.16, p = 0.021) (data not shown). No significant changes in Population II with disease duration were identified, however.
Linear regression analysis of Treg with time since last flare of MS showed no correlation for Population I (data not shown), but Population II increased with length of clinical remission (R 2 = 0.143, p = 0.047) (Fig. 4E). www.nature.com/scientificreports/ Population III in the CD4 + CD25 + CD127 lo Treg population increased with time since last clinical relapse (R 2 = 0.151, p = 0.041). Fig. 5A,B. CCR7 was expressed by 90% of Population I but only a small portion of Population II and III (Fig. 5C). This is consistent with CCR7 + naïve Treg recirculating from blood to lymphoid tissues. In Population I, lack of expression of CCR7 identifies TemRA Treg, which in MS were 5.55 + 9.74% (n=33), similar to 6.43 + 9.23% (n=19) in HD.

Fig. 5A,B show chemokine receptor positive cells in Populations I-V of CD4 + cells and in Treg Population 1-III respectively. The gate for CCR + cells is set based on chemokine receptor FMO control in respective subpopulations, top row in
CCR7 was significantly lower in Population II (26.52±1.60%) compared to Population I (94.45±1.70%, p < 0.0001) and Population III (53.63±2.37%, p < 0.0001). Population III also had lower expression of CCR7 than Population I (p < 0.0001) (Fig. 5C).
Majority of CD4 + T cells in Population V express CCR7, which promotes their migration into secondary lymphoid tissues.Within Population V, there are CD4 TemRA cells that express CD45RA but not CCR7 47,61 . These are not naïve T cells. In this study MS patients had 12.25 + 23.3% of Population V that were CCR7 − compared to 5.16 + 8.46% in HD.
Activated Treg express chemokine receptors that promote their migration to sites of inflammation. CXCR3 promotes migration to sites of Th1 inflammation and CCR6 to sites of Th17 inflammation 53 . In both HD and MS patients, within CD4 + CD25 + CD127 lo Treg gate, the highest expression of CXCR3 was in Population II (50.49±2.28%) compared to Population I (12.63±1.72%, p < 0.0001) and Population III (33.8±1.73%, p < 0.0001) (Fig. 5C). Population III had higher expression than Population I (p < 0.0001).
The staining methods used in this study did not allow identification of CXCR3/CCR6 double positive cells. In our ongoing studies, we examined a group of MS patients with clinically active disease (n=10) to HD (n=10). In Population II, CXCR3 + CCR6 + cells were 20.2 + 20.1% in MS compared to 13.1 + 7.9% in HD (NSD, p = 0.31). In Population III, CXCR3/CCR6 double positive cells were 6.2 + 4.8% in MS and 8.0 + 9.1% in HD. Thus, a small population of CXCR3 + CCR6 + cells can be identified in HD and MS.

CCR6 expression is decreased in Treg of MS patients.
A significant decrease in the number of CCR6 + cells in MS patients was observed within Population III of CD4 + CD25 + CD127 lo Treg compared to HD (p = 0.05) (Fig. 5C). Some MS patients had low expression of CCR6 in Population II. Reduced frequency of CCR6 + Treg did not associate with activity of MS, treatment or length of MS (data not shown). There were a few cases of low CCR6 expression in untreated, in any clinical groups. A larger cohort is required to resolve the meaning of this observation.
CXCR3 or CCR7 expression in subpopulations of CD4 + cells or CD4 + CD25 + CD127 lo Treg did not show any significant difference between MS and HD (Fig. 5C).

Discussion
This study identified trends in subpopulations of CD4 + cells that may have pathogenic significance in MS. As Treg function on ratio basis to effector CD4 + cells, we focused on the proportion of Treg subpopulations within whole CD4 + cells gate or within CD4 + CD25 + CD127 lo Treg gate and not on the absolute number of Treg. CD45RA expression was used to distinguish resting from activated/memory cells. Subtypes of activated Treg were analysed by expression of Th17 and Th1 associated chemokine receptors. Both activated/memory Treg (Population II in CD4 + CD25 + CD127 lo Treg) and activated effector T cells (Population IV in CD4 + ) were increased in MS. Both CD4 + CD45RA + CD25 − Foxp3 − cells (Population V in CD4 + ) and CD4 + CD25 + Foxp3 + CD127 lo Treg (Population I in Treg) were reduced in MS.
Our main findings were differences in subpopulations of Treg identified by expression of CD45RA or chemokine receptors. We found a shift from resting to activated/ memory Treg in MS patients. The most consistent finding is reduced numbers of resting CD4 + CD25 + CD45RA + Foxp3 + Treg in MS relative to age-matched controls. Resting Treg decrease with age in HD, accompanied by an increase in activated/memory Treg 62 , likely due to natural exposure to various antigens throughout life activating resting Treg. The reduction in CD45RA + cells in both Population I (resting Treg) and V (CD4 + effectors) may be due to activation of resting cells by the autoimmune response. It was not due to loss of TemRA effector or regulatory cells. We observed a decline in resting Treg with age in MS patients as well, as occurs in HD.
Resting Treg have been reported as lower than in HD in newly diagnosed MS in both children 14 and in adults 29 . In adults, there is no change in total Treg, but reduced CD31 + Treg and increased memory Treg in adults 29 . In paediatric MS, resting Treg and CD31 + RTE Treg were reduced while memory Treg increased 14 . Our study confirmed reduced resting Treg in MS patients with no prior immune modifying therapy, as well as in established MS. Whether depletion of resting Treg is a contributor to induction of MS or a consequence of the auto immune responses, needs to be resolved. Depletion of resting Treg did not appear to be a consequence of therapy, however. A minority of Population I Treg did not express CCR7, and are effector memory Treg. Their proportion in Population I was not different in MS compared to HD, and these cells were thus also relatively depleted.
We found nine studies identifying memory/activated Treg in MS patients by expression of CD45RO or lack of CD45RA. Five reported an increase in activated/memory Treg 14,29,31,34,37 , two found no difference 24,27 , and two reported reduced memory Treg 10 In this study, activated/memory CD45RA − Treg were increased, particularly in the CD25 hi and Foxp3 hi cells. Population II was greater in MS patients without clinical activity and increased the longer they were not clinically active. This is consistent with the increased activated Treg pool controlling MS, as reported 29,37 .
The CD45RA + and CD45RO + subsets of Treg, the naïve and memory Treg respectively, are affected differently in MS. In the acute phase of MS, suppressive function is impaired in both subsets, but activated/memory Treg recover in chronic MS 11 . This is accompanied by an increased activated/ memory Treg frequency in chronic patients, potentially explaining the observed recovery of Treg in secondary progressive MS 5,63 and the positive correlation between MS duration and Treg frequency 11,15 . However, in chronic disease the number of resting Treg remains low. Higher proportions of activated/memory Treg are also found in CSF of MS patients compared to their peripheral blood 10,12,24 .
Previous studies on activated Treg in MS assayed other markers, but none found a consistent increase in Treg. CD39, a rate-limiting enzyme in ATP/ADP-AMP-adenosine pathway, produces adenosine, inhibits activated T effector cells and thereby limits immune inflammation. CD39 promotes a major pathway for Treg inhibition of autoimmunity. In eight studies of CD39 expression by Treg in MS, five reported reduced CD39 + Treg 7,16,22,28,30 , one no difference 37 and two increased numbers 32,36 . Most studies examined CD39 expression by the whole Treg population. Dalla Libera et al. 7 showed CD4 + CD25 + Foxp3 + or CD4 + CD25 + CD39 + or CD4 + CD39 + Foxp3 + are reduced in stable RRMS but restored to normal in acute relapse in RRMS. Alvarez-Sanchez et al. 36 showed increased CD39 + cells in CD4 + CD25 + CD127 lo Foxp3 + in RRMS compared to HD. CD39 + Treg have been found to be associated with relapsing-remitting MS and are increased in relapsing patients and is significantly correlated with EDSS score 36 . Patients have reduced CD39 + Treg in a stable phase but in acute relapse had comparable CD39 + Treg to HD 28 . Of the two studies examining CD39 + cell numbers in conjunction with CD45RA/RO, one reported reduced CD39 + cells in memory/activated Treg 22 . The other study found no difference in CD39 expression in MS and HD within total Treg, secreting Treg (Population III) or activated Treg (Population II) 37 . CD39 + Treg were reduced in stable RRMS patients but their suppressive ability was not compromised 7 . Others reported that CD39 + Treg have reduced capacity to suppress IL-17 production 30 . HD have marked variation ranging from 2% to 60% CD39 + cells within the CD4 + CD25 hi Treg population 28 .
Another marker of activated Treg is expression of class II MHC 64,65 . In four studies in MS 27,33,35,37 , two monitored HLA-DR + Treg in response to therapy and made no comparison to HD 33,35 and one found no difference in HLA-DR + Treg 27 . Only one study reported an increase in HLA-DR + Treg within total and activated/memory The proportion of cells expressing CXCR3, CCR6 and CCR7 was examined within CD4 + CD25 + CD127 lo Treg populations I-III that were identified based on Foxp3 vs. CD45RA expression as outlined in Fig. 2A. Values shown as the percentage of cells expressing respective CCR within each population. Both in HD (n = 19) and in MS patients (n = 35), expression of CXCR3 and CCR6 was higher in Treg Population II compared to Population I (p < 0.0001) or in Population III (p < 0.0001). Population III also had higher expression of CXCR3 and CCR6 compared to Population I (p < 0.0001). CCR7 expression in Treg Population II in both HD (n = 19) and MS (n = 33) was significantly lower compared to Treg Population I (p < 0.0001) or Population III (p < 0.0001). Population III also had significantly lower CCR7 expression compared to Treg Population I in both MS and HD. Approximately half of the MS patients have low CXCR3 and CCR6 expression in Population II. CCR6 expression was lower in Population III in MS compared to HD (p = 0.05). www.nature.com/scientificreports/ Treg in MS compared to HD 37 . There is one study showing increased PD-1 expression on Treg in MS compared to in HD 22 . Overall the published studies do not show an increase in these activated Treg in MS patients. In MS patients, CCR6 expression was lower in Population III, and for a fraction of patients in Population II when gating was on CD4 + CD25 + CD127 lo cells, but not when gating was on all CD4 + cells. CCR6 is the chemokine receptor of Th17 cell responses. Th17 cells contribute to auto-inflammation in MS 48 , and infiltration of CCR6 + CXCR3 + Th1 and Th17 effector memory cells into the CNS occurs in early disease activity in MS 66 . Given the role of CCR6 in chemotaxis 67 with homeostatic accumulation of CCR6 + Treg into the CNS following EAE induction in mice 68 , the decreased expression of CCR6 by CD4 + CD25 + CD45RA − Treg may lead to the reduced migration of these Treg to inflammatory lesions.
Jones et al. found an increase in Th17-like Treg in CIS patients 34 . They defined Treg as CD4 + Foxp3 + CXCR5 − and hence their analysis would have excluded cells that may co-express both CXCR5 and CCR6. They also studied another marker of Th17 cells, CD161, in CIS 34 , These Th17-like Treg may control the Th17 effector response in CIS and prevent progression to MS. We found no differences in expression of CXCR3 the Th1 CCR. In a pilot study, we found a minority of cells in Population II and III express both CXCR3 and CCR6, but this was similar in HD and MS.
Our study identified several differences between MS patients and HD that may prove useful in monitoring MS. The proportion of activated/memory CD45RA − Foxp3 hi Treg was increased in MS and appeared to be greatest in patients with longer clinical remissions. Increased activated/memory Treg may prove to be a marker of stable MS, with a reduced risk of relapse. CCR6 expression was also low in activated Treg and a relative deficiency of these cells may allow progression of MS.
This report is based on a limited number of patients and requires confirmation in a larger longitudinal study examining the subsets of activated/ memory Treg, identified in this study. The effects of therapy on the Treg populations and disease activity were not assessed.
However, this study including a large proportion of patients who never had immune modulating therapy, or who had no immune modulating therapy within three months prior sampling, confirmed that resting Treg are depleted in MS without immune modulating therapy, at a rate greater than natural attrition of these cells with age. Whether this makes patients more prone to develop MS or is a consequence of accelerated activation of Treg by the autoimmune response remains to be resolved.
Our findings identify an increased proportion of CD25 hi Foxp3 hi Treg in many MS patients a finding supported by other smaller studies 31,37 . Similar changes were reported in sarcoidosis, a Th1 mediated disease, but not in SLE 43 . This suggests activated/memory Treg, which should include autoantigen specific Treg are generated to control immune inflammation in MS.
The decreased frequency of CCR6 + cells in Population III and low numbers of CCR6 + activated/memory Treg in Population II in some MS patients, suggested that failure to produce Th17-like Treg may contribute to lack of control of inflammation in MS.
The methodology will allow more detailed studies of Treg in MS especially with the highly activated Treg in Population II, which can be further divided into Th1 and Th17-like Treg, and other markers of activated Treg such as CD39, Class II MHC and PD1. Whether the increased proportion of activated/memory Treg leads to inhibition of immune inflammation and disease activity requires resolution. It may potentially guide therapy.