Identification of regulatory T (Treg) cells and Treg-cell subsets using multicolor flow cytometry. A, CD4/CD25 coexpression on peripheral blood mononuclear cells and gating of CD4+ cells (R1). PE represents phycoerythrin. B, CD45RO/FOXP3 expression of CD4+ cells identifies FOXP3+CD45RO−naive Treg cells (R2), FOXP3+CD45RO+ memory Treg cells (R3), FOXP3−CD45RO−naive helper T (TH) cells (R4), and FOXP3-CD45RO+ memory TH cells (R5). APC indicates antigen-presenting cells. C, CD31 expression on naive Treg and naive TH cells identifies RTE-Treg cells (M1) and RTE-TH cells (M2) cells. FITC indicates fluorescein isothiocyanate.
Prevalence of recent thymic emigrant (RTE)–helper T cells (TH) and regulatory RTE-T cells (Treg) increases with interferon beta (IFN-β) therapy. Prevalence of TH (A) and Treg-cell (B) subsets in samples of peripheral blood mononuclear cells (PBMC) obtained from 20 patients with relapsing-remitting multiple sclerosis (RRMS) before treatment initiation and after 3 and 6 months of therapy with IFN-β and from 18 healthy control individuals (HC). Values represent the median. *P < .05 and †P < .01 compared with the indicated study cohorts.
Enhanced suppressive capacity of regulatory T cells (Treg) during therapy with interferon beta (IFN-β). A, Suppression rates of regulatory Treg cells obtained from 20 patients with relapsing-remitting multiple sclerosis (RRMS) and 18 healthy control subjects (HC) before treatment and after 3 and 6 months of therapy with IFN-B. Values represent the median.*P < .01 compared with the indicated study cohorts. B, Time courses of Treg cell–mediated suppression and recent thymic emigrant (RTE) Treg-cell prevalence during therapy from 6 random RRMS samples.
Korporal M, Haas J, Balint B, Fritzsching B, Schwarz A, Moeller S, Fritz B, Suri-Payer E, Wildemann B. Interferon Beta–Induced Restoration of Regulatory T-Cell Function in Multiple Sclerosis Is Prompted by an Increase in Newly Generated Naive Regulatory T Cells. Arch Neurol. 2008;65(11):1434-1439. doi:10.1001/archneur.65.11.1434
Naturally occurring regulatory T (Treg) cells are functionally impaired in patients with relapsing-remitting multiple sclerosis. We recently showed that prevalences of newly generated CD31-coexpressing naive Treg cells (recent thymic emigrant–Treg cells) are critical for suppressive function of circulating Treg cells, and a shift in the homeostatic composition of Treg-cell subsets related to a reduced de novo generation of recent thymic emigrant–Treg cells may contribute to the multiple sclerosis (MS)–related Treg-cell dysfunction. Interferon beta, an immunomodulatory agent with established efficacy in MS, lowers relapse rates and slows disease progression. Emerging evidence suggests that Treg-cell suppressive capacity may increase in patients with MS undergoing treatment with interferon beta, although the mechanisms mediating this effect are uncertain.
To evaluate the effect of interferon beta treatment on the suppressive activity and the homeostasis of circulating Treg cells in patients with MS.
Twenty patients with relapsing-remitting MS and 18 healthy control subjects.
Administration of interferon beta.
Main Outcome Measures
Effect of interferon beta on Treg-cell homeostasis and suppressive capacity.
Suppressive capacities of Treg cells were consistently upregulated at 3 and 6 months after treatment with interferon beta. The restoration of Treg-cell function was paralleled by increased naive recent thymic emigrant–Treg cells and a coincidental reduction in memory Treg cells.
The increase in Treg-cell inhibitory capacity mediated by interferon beta treatment can be explained by its effect on the homeostatic balance within the Treg cell compartment.
Reduced prevalence or suppressive capacity of CD4+CD25+FOXP3+ regulatory T (Treg) cells have been described in various autoimmune diseases in human beings.1- 11 We and others have shown that Treg cells derived from patients with relapsing-remitting multiple sclerosis (RRMS) demonstrate functional impairment as their potential to inhibit myelin-specific and antigen nonspecific T-cell proliferation is diminished compared with that in healthy individuals.8- 11 Moreover, Treg cells delay the onset and progression of experimental autoimmune encephalomyelitis and promote recovery from active disease.1,12 Hence, Treg-cell dysfunction in multiple sclerosis (MS) may be causally related to facilitated activation and migration of autoreactive effector T (Teff) cells into the central nervous system. This implies that pharmacologic modulation of Treg-cell prevalence or function has the potential to beneficially influence the severity and long-term course of MS. In accord with this assumption, recent observations of experimental autoimmune encephalomyelitis indicate that enhancement of Treg-cell effector functions by either adoptive transfer of preactivated Treg cells or by direct in vivo stimulation of Treg cells provides protection from disease.12- 14
Interferon beta is an immunomodulatory drug with established therapeutic efficacy in MS. It decreases relapse rates and slows disease progression as measured clinically and by using magnetic resonance imaging.15,16 The mechanisms behind these beneficial effects are not fully understood. Numerous studies have demonstrated anti-inflammatory properties of interferon beta treatment such as downregulation of activated T cells, apoptotic elimination of T lymphocytes, and amelioration of blood-brain barrier dysfunction.15,16 There is emerging evidence that interferon beta may also affect the Treg-cell compartment.17,18 We recently showed that the prevalence of CD31-coexpressing naive helper T cells (TH) and naive Treg cells that have just entered the circulation as recent thymic emigrants (RTE-Treg cells) (CD4+CD45RA+CD45RO−CD31+ RTE-TH and CD4+CD45RA+CD45RO−CD31+FOXP3+ RTE-Treg cells) are decreased in patients with RRMS.19 We further demonstrated that the prevalence of RTE-Treg cells is critical for suppressive function of circulating Treg cells, which suggests that a shift in the homeostatic composition of Treg- cell subsets related to a reduced thymic-dependent de novo generation of RTE-Treg cells with compensatory expansion of memory Treg cells contributes to the MS-related functional Treg cell impairment.
We assessed the effects of interferon beta treatment on the Treg-cell compartment in 20 patients with RRMS undergoing immunomodulatory therapy. We determined prevalence, phenotype, and suppressive function of Treg cells before treatment and after 3 and 6 months of therapy.
We demonstrate that exposure to interferon beta increases Treg cell inhibitory capacity in most patients with RRMS. Whereas restoration of Treg-cell function coincided with only slightly enhanced prevalence of Treg cells in the peripheral T-cell compartment, we observed a marked increase in the RTE-Treg cell subsets and a parallel decrease in CD4+CD45RA−CD45RO+FOXP3+ memory Treg cells. Thus, the increase of Treg cell inhibitory capacity mediated by interferon beta can be explained by its effect on the homeostatic balance within the Treg-cell compartment.
Blood specimens were obtained from 20 patients with RRMS (mean age, 36.8 years; age range, 21-53 years) and 18 healthy control individuals (31.1 years; 19-54 years). All patients had definite RRMS according to the criteria of McDonald et al20 or Poser et al21 and had experienced, on average, 2 relapses (range, 1-4). Mean duration of disease was 3 years (range, 0-8 years). The mean Expanded Disability Status Scale was 1.9 (range, 1-3). Eighteen patients received interferon beta-1a drugs (11 patients received Rebif [Serono, Inc, Randolph, Massachusetts], and 7 patients received Avonex [Biogen, Inc, Cambridge, Massachusetts]). Two patients received interferon beta-1b (Betaferon; Bayer Schering Pharma AG, Berlin, Germany). Rebif was given at a dose of 22 μg 3 times per week initially; 4 patients continued to receive Rebif, 44 μg/wk, 3 times per week after 4 weeks. Blood samples for analysis of Treg-cell function were obtained at 3 and 6 months after treatment. During the observation period, clinical status in all patients remained stable.
The Treg cells from 2 healthy control subjects and 2 patients with MS were tested in parallel for their capacity to inhibit both syngeneic and allogeneic responder T cells ex vivo. The protocol was approved by the University of Heidelberg Ethics Committee, and all study participants gave written informed consent.
Monoclonal antibodies (mAbs) were obtained from BD Pharmingen (Heidelberg, Germany; CD4-APC.Cy7, CD45RO-PE.Cy7, CD45RA-ECD, CD31-FITC), Miltenyi Biotech Inc (Auburn, California; CD25-PE), and eBioscience, Inc (San Diego, California; FOXP3-APC). FOXP3 staining was performed according to the manufacturer's protocol. For 6-color flow cytometric analysis, cells were first stained with surface mAb specific for CD4, CD25, CD45RO, CD45RA, and CD31, followed by FOXP3 intracellular staining. Flow cytometry was performed immediately with a cytometer (FACSCanto; BD Biosciences, San Jose, California) and analyzed with commercially available software (FACSDiva; BD Biosciences). Quantification of Treg-cells and Treg-cell subsets was performed as previously described.19
In short, stained peripheral blood mononuclear cells were gated on CD4+ cells and analyzed for expression of FOXP3 and CD25. Inasmuch as only CD4+ T cells expressing the FOXP3+ gene product scurf in exhibit suppressive function,22 CD4+CD25+FOXP3+ cells were defined as Treg cells and CD4+FOXP3- cells as TH. Treg and TH cells were further analyzed for their CD45RA/CD45RO surface expression to identify CD45RA−CD45RO+ memory and CD45RA+CD45RO−naive subsets. RTE-Treg and RTE-TH were identified by coexpression of the CD31 molecule on naive Treg and TH cells (Figure 1).
Peripheral blood mononuclear cells were isolated from 50 mL of peripheral blood using density gradient centrifugation with Ficoll-Hypaque (Biochrom AG, Berlin, Germany). CD4+ T cells were isolated from peripheral blood mononuclear cells using a negative CD4+ T-cell isolation kit (Dynal Biotech GmbH, Hamburg, Germany). CD25high Treg and CD25low/CD4+CD25int Teff were isolated from the pure untouched CD4+ T cells using CD25 magnetic beads (Dynal Biotech GmbH). Immune magnetic separation constantly yielded highly pure Treg cells with more than 90% CD4+CD25high cells, as previously described.9 Purity of random samples was further tested using intracellular FOXP3 staining, which revealed more than 93% FOXP3+ cells in preparations of total Treg cells.19 Furthermore, flow cytometry analysis revealed constant purities of both Treg- and Teff-cell preparations obtained from serial blood specimens during the observation period.
Cell proliferation assays were performed as described previously.9,19 In brief, 105 freshly isolated Teff cells were incubated in 96-well plates (Nunc GmbH & Co, Wiesbaden, Germany) in 200-μL culture medium alone or in 4:1 coculture with 2.5 × 104 total Treg cells. For T-cell receptor stimulation, soluble anti-CD3 (1 μg/mL) and anti-CD28 mAbs (1 μg/mL) were added to the culture medium. After 4 days at 37°C in 5% carbon dioxide, 1 μCi of tritium-labeled thymidine per well was added for an additional 16 hours. Proliferation was measured using a scintillation counter. Inhibition rate (percent) of Treg cells in coculture experiments was defined as follows:
[1-3(H) Thymidine Uptake (in Counts per Minute [cpm]) in Treg/Teff Coculture / cpm of Teff Alone] × 100.
Each experiment was performed in triplicate. The CD3 and CD28 mAbs used for cell culture experiments were purified from hybridoma supernatants using protein A affinity purification as described previously.23 Cell culture media and mAbs were endotoxin-free (<10 pg/mL) as assessed by the Limulus assay (Sigma-Aldrich, Taufkirchen, Germany).
We used the nonparametric 2-tailed Mann-Whitney test to analyze for differences in cell counts and ex vivo Treg cell–mediated suppressions at various times. P < .05 was considered significant. Statistical analyses were performed using commercially available software (SPSS version 14; SPSS, Inc, Chicago, Illinois).
As described by Haas et al,19 mean (SD) RTE-TH cells were significantly reduced in patients with MS compared with healthy controls (11.5% [4.2%]; median, 12.2%; vs 25.2% [6.1%]; median, 26.3%; P < .01; Figure 2A). Similarly, total naive TH cells were decreased (28.6% [12.3%]; median, 26.5%; vs 45.3 [12.0%]; median, 43.6%; P = .03), whereas memory cells among CD4+ T cells were increased (54.0% [14.2%]; median, 54.5%; vs 42.0% [4.8%]; median, 41.0%; P < .01).
The mean (SD) peripheral CD4+ T-cell population in patients with MS contained slightly lower numbers of FOXP3+ Treg cells compared with healthy controls (4.6% [1.5%]; median, 4.9%; vs 5.6% [1.1%]; median, 5.2%; P = .08; Figure 2B). Patient-derived Treg cells harbored reduced prevalence of naive Treg cells (24.2% [6.4%]; median, 26.0%; vs 30.7% [9.5%]; median, 29.8%; P = .09) and RTE-Treg cells (3.5% [1.2%]; median, 3.5%; vs 8.2% [4.4%]; median, 8.4%; P < .01) but moderately higher prevalence of memory Treg cells (62.6% [6.7%]; median, 60.0%; vs 56.3% [11.0%]; median, 53.7%; P = .12), as previously reported.19
Treatment with interferon beta was associated with decreased prevalence of leukocytes and lymphocytes (data not shown), whereas the prevalence of CD4+ T cells was unchanged (Figure 2A). Mean (SD) naive TH and RTE-TH cells had markedly increased at 3 and 6 months after treatment (naive TH cells, 3 months: 37.7% [9.3%]; median, 39.1%; P = .06; and 6 months: 49.2% [14.6%]; median, 45.5%; P < .01; RTE-TH cells, 3 months: 20.8% [8.6%]; median, 22.5%; P = .01; and 6 months: 25.3% [10.9%]; median, 23.7%; P = .04). Conversely, memory TH cells had decreased (3 months: 50.9% [8.8%]; median, 50.5%; P = .03; and 6 months: 38.6% [13.1%]; median, 40.4%; P = .02).
The prevalence of FOXP3+ Treg in CD4+ T cells was slightly elevated at 3 and 6 months after therapy (5.3% [1.6%]; median, 6.0%; P = .15; and 5.6% [1.8%]; median, 5.9%; P = .11), though the changes were not statistically significant (Figure 2B). Naive Treg and RTE-Treg cells had increased at 3 and 6 months (3 months: 28.3% [8.5%]; median, 28.7%; P = .08; and 6.8% [3.1%]; median, 7.4%; P = .03; and 6 months: 34.7% [11.2%]; median, 35.0%; P = .11; and 9.0% [3.3%]; median, 8.5%; P < .01). Coincidental with the increases in RTE-Treg cells, memory-Treg cells had decreased (3 months: 54.2% [12.4%]; median, 52.6%; P = .09; and 6 months: 48.7% [10.9%]; median, 46.9%; P = .05).
Similar to recent reports,8,9,11,18 mean (SD) patient-derived Treg cells exhibited markedly reduced in vitro inhibitory activity compared with healthy control-derived Treg cells (32.3% [13.0%]; median, 27.4%; vs 64.8% [15.3%]; median, 66.8%; P = .01; Figure 3A). Low suppression rates (<30%) were noted in 16 of 20 patients.
After 3 and 6 months of interferon beta treatment, mean (SD) inhibition rates had increased significantly to 65.5% (15.4%; median, 63.3%; P < .01) and 58.3% (12.9%; median, 59.7%; P < .01). A clear increase in Treg-cell inhibition rates (>10%) was present in 15 patients, whereas 5 individuals exhibited only small increases in Treg-mediated suppression (0%-10%). At 6 months after therapy, low suppression rates (<30%) were present in only 1 patient. In contrast, the functional impairment was unchanged when Treg cells were serially derived from patients without immunomodulatory treatment, as shown previously.9
The treatment-associated increase in Treg-cell suppressive activities was linked to increased RTE-Treg cell prevalence in 14 of 15 patients, whereas in 4 of 5 individuals without clear improvement in Treg cell suppressive activities, prevalence of RTE-Treg cells had not changed at follow-up (Figure 3B).
The increase of Treg cell–mediated suppression and the increased RTE-Treg-cell prevalence in patients with MS receiving interferon beta therapy occurred independently of the interferon beta preparation used for treatment and of clinical parameters including Expanded Disability Status Scale score, disease duration, and number of relapses.
We reciprocally mixed patient Treg cells before and 4 weeks after initiation of interferon beta treatment with anti-CD3/CD28-stimulated syngeneic Teff and in parallel with healthy control Teff, and vice versa, in 2 independent experiments. Patient Treg cells exhibited weak antiproliferative effects (mean [SD]) on both autologous (29.5% [9.8%]) and allogeneic (21.0% [10.5%]) Teff, whereas healthy control Treg cells mediated a stronger reduction of proliferation of both Teff-cell subsets (autologous Teff, 54.8% [6.5%]; allogeneic Teff, 49.9% [13.1%]). After 4 weeks of treatment, Treg cells from the same patients exhibited markedly improved suppression of both healthy control Teff (53.0% [10.6%]) and patient Teff cells (55.4% [13.5%]). Conversely, healthy control Treg cell–mediated inhibition remained stable (autologous Teff, 48.6% [7.7%]; and allogeneic Teff cells, 49.0% [9.2%]; data not shown). This observation confirms that direct targeting of Treg cells contributes to enhanced Treg-cell suppressive capacity prompted by interferon beta therapy.
Mean (SD) proliferative response of 1 × 105 Teff cells from 20 treatment-naive patients was 45.2 (35.5) × 103 cpm at day 5 after polyclonal stimulation in vitro. During therapy, Teff-cell immune responses remained stable (3 months: 38.4 [27.2] × 103 cpm; and 6 months: 39.1 [16.0] × 103 cpm; data not shown). Likewise, Treg cells remained anergic in response to polyclonal stimuli during the observation period. Proliferative responses of 2.5 × 104 Treg cells did not change either, and ranged from 0.1 to 2.2 × 103 cpm at baseline and from 0.1 to 1.9 × 103 cpm after 6 months of treatment.
The functional Treg-cell defect detectable in MS may imbalance the interaction between responder T cells and Treg cells and compromise peripheral immune regulation.8,9,11 Herein, we show that Treg-cell dysfunction is counteracted by pharmacologic modulation because interferon beta treatment markedly augments the Treg-cell suppressive capacity independent of the interferon beta preparation used for treatment (data not shown). In 16 of 20 patients, Treg cells produced low suppression of Teff cell-proliferation at baseline and became a more potent Teff cell-suppressor after treatment. These observations are in accord with a previous study that reported an augmentive effect of interferon beta on Treg-cell function in 10 patients with RRMS after 3 and 6 months of treatment.17 In this study, Treg cells exposed to interferon beta in vivo for 4 weeks showed an improved suppressive effector function ex vivo toward allogeneic IFN-β–naive Teff cells compared with baseline suppression, which suggests a direct effect of interferon beta on Treg cells.
We have recently shown that Treg-cell dysfunction in patients with RRMS is linked to an imbalanced homeostatic composition of circulating Treg cells, because prevalence of newly generated naive Treg cells is significantly decreased along with a coincidental increase in Treg cells exhibiting a memory-phenotype.19 Moreover, previous findings consistently demonstrate contraction of the entire naive T-cell compartment in MS.24,25 The altered Treg-cell homeostasis effects Treg-function as prevalences of RTE-Treg cells correlate with Treg-cell inhibitory properties and depletion of RTE-Treg-cell prompts decreased suppression rates.19 In an attempt to establish whether interferon beta mediates changes in the composition of peripheral Treg cells, we demonstrated that restoration of Treg-cell suppressive function induced by interferon beta therapy is associated with a shift in Treg-cell homeostasis, resulting in an increase in RTE-Treg-cell prevalence to levels found in healthy controls and a coincidental decrease in memory cells. This effect was consistently detectable during treatment with all 3 interferon beta preparations (data not shown). The treatment-associated increase in RTE-Treg cells correlated with upregulation of Treg-cell inhibitory function in 14 of 15 patients, whereas RTE-Treg-cell prevalence remained unaltered in 4 of 5 patients with stable low levels of Treg cell suppressive activity, which suggests a causal link between Treg-cell homeostasis and function. Therapy with interferon beta also promoted an increase in naive TH and RTE-TH cells, corroborating earlier observations with other immunomodulatory agents used for treatment of MS. Upregulation of naive T cells together with downregulation of memory T cells was reported during therapy with linomide.26 Moreover, in a recent longitudinal study, glatiramer acetate prompted a significant increase in the percentage of CD4+CD45RA+ T cells.27
In this study, interferon beta therapy was accompanied by a mild decrease in total leukocytes and lymphocytes, as previously reported.28 The relative percentages of CD4+ T cells remained unaltered, and we observed a mild though nonsignificant increase in the percentage of FOXP3+ Treg cells (approximately 1%). In contrast, FOXP3+ Treg cells were moderately increased (<2%) in 15 patients with RRMS treated with interferon beta compared with 40 patients who received no treatment.18 However, the study by Venken et al18 used a nonprospective cross-sectional design to evaluate FOXP3+ Treg cells in patients with heterogeneous treatment durations and did not include follow-up assessments. In line with our data, CD25high Treg-cell prevalence did not differ in patients with untreated MS compared with patients undergoing therapy with disease-modifying drugs including interferon beta.29
In conclusion, IFN-β markedly upregulates the Treg-cell inhibitory capacity and reverses Treg cell dysfunction associated with RRMS. This effect seems to be conferred by drug-induced normalization of peripheral Treg-cell homeostasis. The extent to which this effect contributes to stabilization of clinical disease activity remains to be determined in long-term follow-up studies. These findings, however, clearly demonstrate that Treg cells are responsive to pharmacologic modulation, and efforts to selectively enhance Treg-cell expansion or Treg-cell function are a highly promising new treatment option in MS.
Correspondence: Brigitte Wildemann, MD, Division of Molecular Neuroimmunology, Department of Neurology, University of Heidelberg, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany (firstname.lastname@example.org).
Accepted for Publication: February 22, 2008.
Author Contributions: Dr Korporal had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Drs Korporal and Haas contributed equally to this study. Study concept and design: Korporal, Haas, Suri-Payer, and Wildemann. Acquisition of data: Korporal, Haas, Balint, Fritzsching, Moeller, and Fritz. Analysis and interpretation of data: Haas, Schwarz, and Wildemann. Drafting of the manuscript: Korporal, Haas, and Wildemann. Critical revision of the manuscript for important intellectual content: Korporal, Haas, Balint, Fritzsching, Schwarz, Moeller, Fritz, Suri-Payer, and Wildemann. Statistical analysis: Haas and Schwarz. Obtained funding: Suri-Payer and Wildemann. Administrative, technical, and material support: Korporal, Haas, Balint, Fritzsching, Moeller, Fritz, and Wildemann. Study supervision: Korporal, Haas, and Wildemann.
Financial Disclosure: None reported.
Funding/Support: This study was supported by grants 1.319.110/01/11 and 1.01.1/04/003 from the Gemeinnützige Hertie-Stiftung; grants SFB 405, 5H, and SFB 571, B7 from Deutsche Forschungsgemeinschaft; and a Young Investigator Award from the Faculty of Medicine, University of Heidelberg (B.F.)
Role of the Sponsor: None of the funders had a role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, or approval of the manuscript.
Additional Contributions: We thank the patients with RRMS and the healthy controls for their participation in this study.