Bone Marrow Transplantation (2014) 49, 349–354 & 2014 Macmillan Publishers Limited All rights reserved 0268-3369/14 www.nature.com/bmt
ORIGINAL ARTICLE
Peripheral blood regulatory T cells in patients with diffuse systemic sclerosis (SSc) before and after autologous hematopoietic SCT: a pilot study J Baraut1, EI Grigore1, F Jean-Louis1, SH Khelifa2, C Durand3, F Verrecchia4, D Farge1,2 and L Michel1 The present pilot study aims to evaluate the frequency and the function of regulatory T (Treg) cells in patients with diffuse cutaneous SSc (dcSSc) before and after autologous hematopoietic SCT (aHSCT). Peripheral blood lymphocytes from seven dcSSc patients were analyzed before and 24 months after aHSCT and were compared with those from seven healthy donors (controls). Immunophenotyping of CD4 þ CD25highFoxP3 þ natural Treg (nTreg), CD4 þ CD25 þ TGF-b þ and CD4 þ CD25 þ IL-10 þ adaptive Treg (aTreg) cell subsets was performed using four-color flow cytometry. Treg-suppressive capability was measured after coculture with autologous T effector cells by evaluation of T-cell proliferation using 3H-thymidine incorporation. Peripheral CD4 þ CD25highFoxP3 þ (2±0.5 vs 4.2±1.1, Po0.01), CD4 þ CD25 þ TGF-b þ (6.9±1.8 vs 14.6±5.0, Po0.05) and CD4 þ CD25 þ IL-10 þ (10.7±0.5 vs 16.1±3.2, Po0.01) Tregs as well as CD4 þ CD25highCD127low Tregs suppressive capacity (Po0.05) were decreased in dcSSc patients vs controls. After aHSCT (n ¼ 7), the percentages of CD4 þ CD25highFoxP3 þ (4.1±1.8) and CD4 þ CD25 þ IL-10 þ (15.7±2.2) Treg cells and the suppressive activity of CD4 þ CD25highCD127low were restored to the levels in controls. The decreased frequency and the functional defect of peripheral Treg cells from patients with dcSSc are reversed following aHSCT to reach those observed in controls. This pilot study brings evidence of an effective restoration of nTreg and aTreg subsets, and recovery of nTreg suppressive function following aHSCT. Bone Marrow Transplantation (2014) 49, 349–354; doi:10.1038/bmt.2013.202; published online 23 December 2013 Keywords: systemic sclerosis; regulatory T cells; hematopoietic stem cell transplantation; T-cell proliferation
INTRODUCTION Systemic sclerosis (SSc) is a chronic autoimmune disease with a heterogeneous clinical presentation. Although its exact pathogenesis is still unknown, it is characterized by alterations of the microvasculature, disturbances of the immune system with high production of autoantibodies and progressive fibrosis of the skin and internal organs.1 Whatever the original stimulus, abnormal immune stimulation, production of autoantibodies and cytokine release may be,2 all contribute to increased collagen synthesis and deposition, fibroblast activation and microvascular damage. Several studies have highlighted the key role of the adaptive immune response in SSc patients,3 as evidenced by infiltration of activated CD4 þ and CD8 þ T cells in skin tissues, lung interstitium and bronchio-alveolar fluids and in peripheral blood.4 T regulatory (Treg) cells represent 5–10% of the peripheral CD4 þ T cells in humans5 and can be characterized by the expression of CD4, CD25 and Foxp3, a transcription factor critical to the development of natural Treg (nTreg) cells,6 namely CD4 þ CD25highFoxP3 þ . Two major subsets of Treg cells have been described: a nTreg population developing in the thymus and an adaptive Treg (aTreg) subset developing in the periphery under particular conditions of suboptimal antigen exposure and/or costimulation. The aTreg cells, which include CD4 þ CD25highFoxp3 þ
and CD4 þ CD25highFoxp3 T cells, are generated from mature T-cell populations (classical T-cell subsets or nTreg cells) after antigenic stimulation.7 The aTreg cells mediate their inhibitory activities by producing immunosuppressive cytokines, such as TGF-b and IL-10.2,7 The various Treg cells have key roles in maintaining self-tolerance and modulating the allergic and autoimmune responses, while controlling autoreactive T cells.8–11 Several studies have reported a deficiency in the number of Treg and/or function in autoimmune disease patients,12,13 namely in systemic lupus erythematous,14 rheumatoid arthritis,15 multiple sclerosis,16 autoimmune polyglandular syndrome II,17 myasthenia gravis18 and type I diabetes.19 However, results concerning the number of circulating Treg cells and their suppressive function have been contradictory. In SSc patients, few studies investigated the various Treg subsets and their suppressive function, these also with conflicting results, showing either reduced,20–22 increased23,24 or the same25–27 number of circulating Treg cells in peripheral blood as compared with controls. These discrepancies might be mostly due to variations in study techniques, with setting analysis of either CD4 þ CD25 þ ,21,23 CD4 þ CD25high,12,25,26 CD4 þ CD25high Foxp3 þ ,20,24,26 CD25 þ Foxp3 þ CD127–23 or CD4 þ CD25high CD127low subsets.22 Despite these controversies, in most of these studies of SSc patients’ samples,22,23,26 impaired Treg-suppressive function was associated with SSc disease severity. Of interest, Klein
1 INSERM U976 and Universite´ Paris Diderot Sorbonne Paris Cite´, Hoˆpital Saint-Louis, Paris, France; 2Service de Me´decine Interne, Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Saint-Louis, Paris, France; 3Service de Me´decine Interne, Hoˆpital Hoˆtel Dieu, Nantes, France and 4INSERM U957 and Universite´ Nantes Atlantique, Nantes, France. Correspondence: Dr L Michel, Institut de Recherche sur la Peau, Inserm U976, Pavillon Bazin, Universite´ Paris Diderot Sorbonne Paris Cite´, Hoˆpital Saint-Louis – 1, Avenue Claude Vellefaux, 75010 Paris, France. E-mail: [email protected] Received 13 August 2012; revised 26 September 2013; accepted 10 October 2013; published online 23 December 2013
Treg in SSc patients after autologous HSCT J Baraut et al
350 et al.26 reported that there was a reduction in Treg within the skin, but not in the peripheral blood of SSc patients. Use of autologous hematopoietic SCT (aHSCT) has recently been shown to induce long-term remission in severe or rapidly progressive diffuse cutaneous SSc (dcSSc), with rapid and sustained regression of skin and lung fibrosis, and improved functional status.27,28 After aHSCT, the clinical regression of skin fibrosis has been correlated with reappearance of functional B cells, thymic and extrathymic T-cell development, reconstitution of effector cells and efficient antigen presentation to reconstitute the pretransplantation immune repertoire.29,30 However, further analysis of the immune reconstitution of the various Treg subsets and their function after aHSCT in SSc patients is warranted. To address this issue, the present pilot study aimed to analyze the respective levels of Treg subsets in SSc patients before and after aHSCT, and the suppressive functions of these cells from SSc patients as compared with those from healthy donors.
CD4 þ CD25– and CD4 þ CD25 þ T cells were isolated from PBMCs using a Miltenyi Regulatory T Cell Isolation Kit with LD and MS columns according to the manufacturer’s instructions (Miltenyi Biotech, Bergisch Gladbach, Germany), and were stained with FITC-conjugated anti-CD4, PE-conjugated anti-CD127 and APC-conjugated anti-CD25. CD4 þ CD25highCD127low (Treg cells) and CD4 þ CD25lowCD127high (T effector cells: Teff) were isolated using BD Biosciences FACSAria III cell sorter. Highly purified Treg and Teff cells were immediately cocultured with T-feeder cells (irradiated CD4 T cells) in a 96-well plate previously coated with anti-CD3 and antiCD28 antibodies. Cocultures were assessed with Teff to Treg ratio ranging from 1:1 to 3:1. After 5 days of culture, cell proliferation was measured by incorporation of 3H-thymidine radioactive (37 mBcq/mL/well) during the last 18 h. The percentage of cell proliferation inhibition was calculated as (1 (% of proliferating target T cells in coculture with Treg cells)/(% of proliferating target T cells cultured alone)) 100.
Statistical analysis All results are expressed as mean±s.d. Statistical significance for all experiments was assessed using the paired Student’s t-test. Differences were considered as significant at Po0.05.
PATIENTS AND METHODS Patients Seven patients (mean age 46±18 years) with early rapidly progressive dcSSc), defined according to the American Rheumatism Association criteria,31 were enrolled for receiving aHSCT treatment (Department of Internal Medicine, St Louis Hospital). The transplant procedure and followup were performed as previously described.28 Written informed consent was obtained and each protocol was approved by the local ethics committee of the St Louis Hospital institute. Table 1 provides an overview of the patients’ clinical characteristics. As controls, venous blood was collected from seven healthy donors (four males, three females) of the same age range as that of SSc patients (39±16 years), recruited by the Etablissement Franc¸ais du Sang after informed consent. Peripheral blood cells were isolated by Ficoll–Hypaque centrifugation followed by elimination of monocytes by adhesion for 2 h. Treg subpopulations from SSc patients and their suppressive capacity were analyzed before and 24 months after aHSCT and results were compared with controls.
Flow cytometry analysis Peripheral blood lymphocyte (PBL) samples were stimulated for 5 days with anti-CD3 antibody, anti-CD28 antibody and IL2, and during the last 5 h, PMA (0.2 mg/mL), Ionomycin (1 mg/mL) and GolgiStop (BD Biosciences, San Jose`, CA, USA) were added to the cultures. Monoclonal antibodies conjugated with either fluorescein isothiocyanate (FITC), phyco-erythrin (PE) or allophycocyanin (APC) and directed to CD4, CD25, IL-10, TGF-b (BD Biosciences), Foxp3 (eBiosciences, San Diego, CA, USA) or matched isotype controls were used for immunophenotyping and intracellular staining, according to the manufacturer’s protocols. Evaluation of Treg subpopulations by flow cytometry was determined as CD25brightFoxP3 þ percentage expression within the CD3 þ CD4 þ compartment. aTreg percentages were CD4 þ TGF-b- or IL-10-producing aTregs. Data from patients (n ¼ 7) and controls (n ¼ 7) were acquired and analyzed on a four-parameter flow cytometer FACSCalibur using ProCellQuest software (BD Biosciences).
Table 1.
Suppressor assays of Treg cells
Characteristics of patients with SSc
Patients
Age
Gender
Total mRSS (M0)a
Total mRSS (M24)a
1 2 3 4 5 6 7
63 60 46 58 62 41 50
F F M M M F F
29 25 32 37 35 20 33
8 4 10 24 11 2 13
Abbreviations: F ¼ female; M ¼ male; mRSS ¼ modified Rodnan skin score; SSc ¼ systemic sclerosis. aTotal mRSS was measured before (M0) and 24 months after (M24) HSCT.
Bone Marrow Transplantation (2014) 349 – 354
RESULTS Frequency of peripheral CD4 þ CD25highFoxP3 þ nTreg cells in SSc patients before and after aHSCT As detected by flow cytometry analysis, we observed a significant decrease in the percentage of CD4 þ CD25highFoxP3 þ nTreg cells in SSc patients evaluated before aHSCT (2±0.5, n ¼ 7) as compared with the respective percentage in healthy donors (4.2±1.1, n ¼ 7, Po0.01; Figure 1a). Figure 1b represents the fluorescence-activated cell sorting (FACS) dotplots for one representative healthy donor (left panel) and one SSc patient before any treatment (middle panel). To investigate whether aHSCT induced reconstitution of the various Treg subsets in SSc patients, we compared the percentage of nTreg cells from SSc patients before and after aHSCT versus those from healthy donors. As shown in Figure 1a, the percentage of CD4 þ CD25highFoxP3 þ T cells increased in SSc patients 24 months after aHSCT (4.1±1.8, n ¼ 7) as compared with their values before transplant. Figure 1b (left panel) exhibits the FACS dotplots for one representative SSc patient treated with aHSCT. These data indicate that aHSCT allowed to induce an increase in the percentage of nTreg in SSc patients up to the levels detected in healthy donors. aHSCT restored the reduced percentage of TGF-b- and IL-10-producing CD4 þ CD25 þ aTreg cells from SSc patients The mean percentage of CD4 þ CD25 þ IL-10 þ T cells was significantly decreased in SSc patients before aHSCT (10.7±0.5, n ¼ 3) as compared with those in healthy donors (16.1±3.2, n ¼ 7, Po0.01l; Figure 2a). Conversely, an increase in the mean percentage of CD4 þ CD25 þ IL-10 þ T cells was detected at 24 months after treatment by aHSCT, up to 15.7±2.2 (n ¼ 3), and back to control values. Figure 2b represents the FACS dotplots for one representative healthy donor (left panel), one SSc patient before (middle panel) and 24 months after (right panel) aHSCT. The mean percentage of CD4 þ CD25 þ TGF-b þ T cells was significantly lower in SSc patients (6.9±1.8, n ¼ 3) compared with healthy donors (14.6±5.0, n ¼ 7, Po0.05), as shown in Figure 2c. Twenty-four months after aHSCT, the mean percentage of CD4 þ CD25 þ TGF-b þ T cells remained significantly lower than in controls, although it was increased (17.2±4.5, data not shown) in two patients studied at 48 months after aHSCT. In Figure 2d, FACS dotplots are presented for one representative healthy donor (left panel), as well as in one SSc patient before aHSCT (middle panel) and 24 months after (right panel) aHSCT. Taken together, & 2014 Macmillan Publishers Limited
Treg in SSc patients after autologous HSCT J Baraut et al
351 the Teff:Treg cell ratio. In contrast, Treg cells from SSc patients exhibited a significantly reduced suppressive capacity compared with those from healthy donors (Po0.05), suppressing the proliferation of CD4 þ target T cells by a mean percentage of 59.8±16.1 (n ¼ 3) at ratio 1:1 (Figure 3b, red dots). This low inhibitory effect was maintained up to a dilution of 3:1 Teff:Treg. The suppressive activity of Treg cells from SSc patients 24 months after aHSCT (83.4±11.7 at ratio 1:1, n ¼ 3, Figure 3b, green dots) was significantly higher compared with their mean suppressive capacity before aHSCT (Po0.05). These results clearly indicated that aHSCT can restore the suppressive activity of Treg cells in SSc patients, in parallel with regression of skin fibrosis as shown by a fall in Rodnan skin score for each patient at 24 months (Table 1).
our results demonstrated that SSc patients have a markedly decreased frequency of both IL-10- and TGF-b-producing CD4 þ CD25 þ adaptive T cells. aHSCT restored suppressive activity in CD4 þ CD25highCD127low T cells from SSc patients To further standardize our FACS analysis, the sorting gate was adjusted to detect Treg CD4 þ CD25highCD127low and Teff CD4 þ CD25lowCD127high cells from SSc patients and healthy donors (Figure 3a). The percentage of isolated CD4 þ CD25highCD127low T cells obtained using these cell sorting parameters reached 4.4% (±2.2, n ¼ 4) in SSc patients before aHSCT as compared with 2.6% (±0.2, n ¼ 4) in normal donors, although with no significance. In the functional suppression assay, CD4 þ CD25highCD127low Treg cells were cocultured with autologous target CD4 þ CD25lowCD127high Teff cells at three ratios (Teff:Treg 1:1, 2:1 and 3:1). As expected, Treg cells from healthy controls efficiently suppressed the proliferation of Teff cells with a mean percentage of inhibition of 70.8±13.4 (n ¼ 3) at ratio 1:1 (Figure 3b, blue dots). At higher dilutions, the percentage of suppression correlated with
DISCUSSION The present pilot study illustrates, for the first time to our knowledge, that aHSCT allowed restoration of the levels of both nTreg and aTreg subsets in this small group (n ¼ 7) of SSc patients. In addition, our results confirmed that dcSSc patients have significantly decreased levels of circulating CD4 þ CD25highFoxP3 þ
% CD4+ CD25highFoxp3+ T cells after HSCT
% CD4+ CD25high Foxp3+ T cells
NS 8 **
6
4
2
Control
SSc M0
SSc M24 post graft SSc patient M24 HSCT
SSc patient M0
Healthy control 104
104
CD25+ Foxp3+ 4.3%
103
104
CD25+ Foxp3+ 0.7%
103
102
102
102
101
101
101
100 100
CD25
101
102
103
104
100 100
101
102
103
104
CD25+ Foxp3+ 7.8%
103
100 100
101
102
103
104
Foxp3
Figure 1. Flow cytometry analysis of CD4 þ CD25highFoxP3 þ T cells. (a) Percentage of CD4 þ CD25highFoxP3 þ T cells are presented for healthy donors (control, n ¼ 7), SSc patients (n ¼ 7) before HSCT (SSc M0) and 24 months (SSc M24) post transplantation (n ¼ 6). (b) One representative individual FACS dotplot from healthy control cells (left panel), SSc patient before any HSCT treatment (middle panel) and 24 months after HSCT (right panel). **Po0.01. & 2014 Macmillan Publishers Limited
Bone Marrow Transplantation (2014) 349 – 354
Treg in SSc patients after autologous HSCT J Baraut et al
352 % CD4+ CD25+ IL-10+ T cells after HSCT
104
104
CD25+ IL-10+ 103
**
SSc patient M24 HSCT
SSc patient M0
104
NS 25
CD25+ IL-10+ 103
17.3%
CD25+ IL-10+ 103
10.7%
20
102
102
102
15
101
101
101
10
100 100
101
CD25
% CD4+CD25+IL10+
Healthy control
5
102
103
100 104 100
101
102
103
100 104 100
16.5%
101
102
103
104
IL-10
0 Control
SSc M0 SSc M24 post graft
% CD4+ CD25high TGF-β T cells after HSCT
104
CD25+ TGF-β+ 103
20
19.6%
SSc patient M24 HSCT
SSc patient M0 104 CD25+ TGF-β+ 103
6.9%
CD25+ TFG-β+ 103
102
102
102
101
101
101
100
100 104
4.3%
15 10
* *
5
100
101
CD25
% CD4+CD25+TGF-β
25
Healthy control 104
0 Control
102
103
100
101
102
103
100 104 100
101
102
103
104
TGF-β
SSc M0 SSc M24 post graft
Figure 2. Flow cytometry analysis of IL-10- and TGF-b- producing CD4 þ CD25 þ T cells. (a) Percentage of CD4 þ CD25 þ IL-10 þ T cells and (c) CD4 þ CD25 þ TGF-b þ T cells were detected by intracellular staining in lymphocytes from healthy donors (Control, n ¼ 7), SSc patients before (SSc M0, n ¼ 3) and 24 months after HSCT (SSc M24, n ¼ 3). (b and d) Representative dotplots from one normal volunteer, one SSc patient before and 24 months after HSCT. *Po0.05. **Po0.01.
natural T cells, as recently reported by Antiga et al.20 in 15 patients. We also show a decrease of IL-10- and TGF-b-producing CD4 þ CD25 þ adaptive T cells in SSc patients, as reported in two previous studies that investigated CD4 þ IL-10 þ and CD4 þ TGF-b þ T cells.20,21 In contrast to our study, Klein et al.26 recently showed no significant difference in the frequency of CD4 þ CD25highFoxP3 þ from SSc patients as compared with healthy donors. This discrepancy might be due to systemic treatment with corticosteroids and/or cyclophosphamide, which might have restored the frequency of Treg in the studied SSc patients, as described in human lupus erythematous.32 Nevertheless, the authors reported a decrease in cutaneous Treg content that might be essential during the early phase of cutaneous disease. To determine the regulatory potential of the natural Treg cells in SSc patients, we performed functional suppressor assays using a new biomarker as an alternative to Foxp3. Indeed, most studies have identified CD127 as a viable surface marker that could distinguish highly suppressive Treg (CD127low) cells from Teff (CD127high) cells in humans.33 These new sorting parameters for Treg avoid contamination with activated T cells that expressed intermediate levels of CD25. Our results demonstrate that CD4 þ CD25highCD127low Treg cells functions were impaired in SSc patients as compared with normal healthy controls, and also that suppressive functions were effectively restored 24 months after aHSCT. This clearly indicates that aHSCT can restore immunological selftolerance in dcSSc patients who could recover suppressive function of Treg cells similar to normal, as previously found by Zhang et al.34 in 15 HSCT lupus patients studied before and after aHSCT. Few studies with different methods, each of them in a small number of patients, have analyzed the immune reconstitution Bone Marrow Transplantation (2014) 349 – 354
process after aHSCT for various types of autoimmune disease, including multiple sclerosis,35 SSc,29,30 juvenile arthritis36 and SLE.37 All studies reported that long-term remission in patients who received aHSCT was associated with a de novo regeneration of the T-cell compartment that can be delayed for years after T-cell depleting conditioning for aHSCT. In particular, they demonstrated a drastic reactivation of thymic activity reflected by the increased T-cell receptor excision circle (TREC) levels, as well as an increased thymic output as evidenced by the increased diversity of TCRB CDR3 repertoire. The immune reconstitution process after aHSCT is associated with the reemergence of naive T cells, the depletion of autoreactive memory T cells, the regeneration of thymic-derived FoxP3 þ regulatory T cells and the recovery of the naive B-cell compartment.29,30,35,38 Two important observations for SSc patients treated by aHSCT emerged from the present pilot study: first, both nTreg and aTreg cells regenerated to frequencies comparable with those in normal controls; second, the renewal of Treg subsets was associated with a restoration of nTreg suppressive capacity, as reported by Zhang et al.34 in SLE patients treated by aHSCT. Altogether, our data support the ‘resetting’ of the adaptive immune system induced by aHSCT, as previously reported for other types of autoimmune diseases treated by aHSCT and always based on the analysis of a small number of patients.29,34,36,37 To further prove the specific relationship to SSc, these results have to be compared with aHSCT effects on a population of patients receiving transplantation for another indication. Therefore, aHSCT could be used as a therapeutic intervention in several autoimmune diseases in which Treg functions are impaired, such as SLE, lupus or type I diabetes. Future studies are needed to evaluate the mechanisms by which Tregsuppressive functions could be altered in SSc and restored by & 2014 Macmillan Publishers Limited
Treg in SSc patients after autologous HSCT J Baraut et al
353 SSc patient
105
105
104
104 CD25 APC-A
CD25 APC-A
Healthy control
103
103
102 0 -102
102 0 -102
-235
-265 -105
102
0
103 CD127 PE-A
104
105
-92
0
102
104 103 CD127 PE-A
CD127 PE...
CD25 AP...
#Events
#Parent
Mean
Mean
P4
22,392
95.8
439
195
P4
23,221
P6
4,442
19.8
793
178
P6
7,620
P5
545
2.4
94
1,937
P5
1,712
5.0
Population
Population
#Events
105
CD127 PE...
CD25 AP...
Mean
Mean
98.7
518
358
32.8
800
169
148
2,755
#Parent
Suppression assay of CD4+ CD25highCD127low T cells from SSc patients compared with healthy donors 100 Healthy donors 90
SSc patients M0 SSc patients M24
80
% of inhibition
70 60 50 40 30 20 10 0 1:1
2:1
3:1
1:0
T effector cells:Tregcells þ
Figure 3. Isolation of CD4 CD25 CD127 T cells for suppression activity analysis. (a) FACS analysis of CD4 þ CD25highCD127low Treg cells was performed in healthy controls (n ¼ 3) and SSc patients before and 24 months after treatment by HSCT (n ¼ 3). Representative cell sorter dotplots from one healthy donor (left panel) and from one SSc patient before HSCT (right panel) are shown. The gate was set in such a way that Treg cells (P5) expressed the highest CD25 expression and the lowest CD127 expression, while Teff cells (P6) expressed the highest CD127 and the lowest CD25 expressions. (b) Suppression assays of CD4 þ CD25highCD127low Treg cells from healthy donors (n ¼ 3) (blue dots), SSc patients (n ¼ 3) before any treatment (red dots) and 24 months after HSCT (green dots). The percentage of inhibition was determined by comparing proliferation of Teff cells alone to the proliferation of Teff cells in coculture with Treg cells at different ratios. high
low
aHSCT. Alteration might be attributed to either an impaired suppressor function of the Treg cells from SSc patients or a resistance to suppression by SSc Teff cells, as previously demonstrated in SLE.39 This resistance has been attributed to pro-inflammatory cytokines such as tumor necrosis factor-a, interleukin (IL)-6, IL-7 and IL-1b.40,41 Further investigation in a larger cohort of patients with dcSSc treated by aHSCT as compared with standard therapy, such as those included ASTIS or SCOTT trials, the still ongoing large phase III randomized trials,42 will help to answer these questions and to correlate encouraging & 2014 Macmillan Publishers Limited
results from the present pilot study with patient clinical parameters and remission. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We acknowledge the Association des Scle´rodermiques de France (ASF) and the Groupe Francophone de Recherche sur la Scle´rodermie (GFRS). This work was supported by grants from the Groupe Franc¸ais de Recherche sur la Scle´rodermie (GFRS).
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Treg in SSc patients after autologous HSCT J Baraut et al
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sclerosis (SSc) is related to a diminished CD69 and TGFbeta expression. PLoS One 2009; 4: e5981. Giovannetti A, Rosato E, Renzi C, Maselli A, Gambardella L, Giammarioli AM et al. Analyses of T cell phenotype and function reveal an altered T cell homeostasis in systemic sclerosis. Correlations with disease severity and phenotypes. Clin Immunol 2010; 137: 122–133. Slobodin G, Ahmad MS, Rosner I, Peri R, Rozenbaum M, Kessel A et al. Regulatory T cells (CD4( þ )CD25(bright)FoxP3( þ )) expansion in systemic sclerosis correlates with disease activity and severity. Cell Immunol 2010; 261: 77–80. Klein S, Kretz CC, Ruland V, Stumpf C, Haust M, Hartschuh W et al. Reduction of regulatory T cells in skin lesions but not in peripheral blood of patients with systemic scleroderma. Ann Rheum Dis 2011; 70: 1475–1481. Burt RK, Shah SJ, Dill K, Grant T, Gheorghiade M, Schroeder J et al. Autologous non-myeloablative haemopoietic stem-cell transplantation compared with pulse cyclophosphamide once per month for systemic sclerosis (ASSIST): an open-label, randomised phase 2 trial. Lancet 2011; 378: 498–506. Vonk MC, Marjanovic Z, van den Hoogen FHJ, Zohar S, Schattenberg AVMB, Fibbe WE et al. Long-term follow-up results after autologous haematopoietic stem cell transplantation for severe systemic sclerosis. Ann Rheum Dis 2008; 67: 98–104. Farge D, Henegar C, Carmagnat M, Daneshpouy M, Marjanovic Z, Rabian C et al. Analysis of immune reconstitution after autologous bone marrow transplantation in systemic sclerosis. Arthritis Rheum 2005; 52: 1555–1563. Bohgaki T, Atsumi T, Bohgaki M, Furusaki A, Kondo M, Sato-Matsumura KC et al. Immunological reconstitution after autologous hematopoietic stem cell transplantation in patients with systemic sclerosis: relationship between clinical benefits and intensity of immunosuppression. J Rheumatol 2009; 36: 1240–1248. LeRoy EC, Medsger Jr TA. Criteria for the classification of early systemic sclerosis. J Rheumatol 2001; 28: 1573–1576. Kuhn A, Beissert S, Krammer PH. CD4( þ )CD25 ( þ ) regulatory T cells in human lupus erythematosus. Arch Dermatol Res 2009; 301: 71–81. Liu W, Putnam AL, Xu-Yu Z, Szot GL, Lee MR, Zhu S et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4 þ T reg cells. J Exp Med 2006; 203: 1701–1711. Zhang L, Bertucci AM, Ramsey-Goldman R, Burt RK, Datta SK. Regulatory T cell (Treg) subsets return in patients with refractory lupus following stem cell transplantation, and TGF-beta-producing CD8 þ Treg cells are associated with immunological remission of lupus. J Immunol 2009; 183: 6346–6358. Muraro PA, Douek DC, Packer A, Chung K, Guenaga FJ, Cassiani-Ingoni R et al. Thymic output generates a new and diverse TCR repertoire after autologous stem cell transplantation in multiple sclerosis patients. J Exp Med 2005; 201: 805–816. de Kleer I, Vastert B, Klein M, Teklenburg G, Arkesteijn G, Yung GP et al. Autologous stem cell transplantation for autoimmunity induces immunologic self-tolerance by reprogramming autoreactive T cells and restoring the CD4 þ CD25 þ immune regulatory network. Blood 2006; 107: 1696–1702. Alexander T, Thiel A, Rosen O, Massenkeil G, Sattler A, Kohler S et al. Depletion of autoreactive immunologic memory followed by autologous hematopoietic stem cell transplantation in patients with refractory SLE induces long-term remission through de novo generation of a juvenile and tolerant immune system. Blood 2009; 113: 214–223. Radbruch A, Thiel A. Cell therapy for autoimmune diseases: does it have a future? Ann Rheum Dis 2004; 63: ii96–ii101. Venigalla RKC, Tretter T, Krienke S, Max R, Eckstein V, Blank N et al. Reduced CD4 þ ,CD25 T cell sensitivity to the suppressive function of CD4 þ , CD25high,CD127 -/low regulatory T cells in patients with active systemic lupus erythematosus. Arthritis Rheum 2008; 58: 2120–2130. van Amelsfort JMR, van Roon JAG, Noordegraaf M, Jacobs KMG, Bijlsma JWJ, Lafeber FPJG et al. Proinflammatory mediator-induced reversal of CD4 þ ,CD25 þ regulatory T cell-mediated suppression in rheumatoid arthritis. Arthritis Rheum 2007; 56: 732–742. O’Sullivan BJ, Thomas HE, Pai S, Santamaria P, Iwakura Y, Steptoe RJ et al. IL-1 beta breaks tolerance through expansion of CD25 þ effector T cells. J Immunol 2006; 176: 7278–7287. Farge D, Gluckman E. Autologous HSCT in systemic sclerosis: a step forward. Lancet 2011; 378: 460–462.
& 2014 Macmillan Publishers Limited
ORIGINAL ARTICLE
Peripheral blood regulatory T cells in patients with diffuse systemic sclerosis (SSc) before and after autologous hematopoietic SCT: a pilot study J Baraut1, EI Grigore1, F Jean-Louis1, SH Khelifa2, C Durand3, F Verrecchia4, D Farge1,2 and L Michel1 The present pilot study aims to evaluate the frequency and the function of regulatory T (Treg) cells in patients with diffuse cutaneous SSc (dcSSc) before and after autologous hematopoietic SCT (aHSCT). Peripheral blood lymphocytes from seven dcSSc patients were analyzed before and 24 months after aHSCT and were compared with those from seven healthy donors (controls). Immunophenotyping of CD4 þ CD25highFoxP3 þ natural Treg (nTreg), CD4 þ CD25 þ TGF-b þ and CD4 þ CD25 þ IL-10 þ adaptive Treg (aTreg) cell subsets was performed using four-color flow cytometry. Treg-suppressive capability was measured after coculture with autologous T effector cells by evaluation of T-cell proliferation using 3H-thymidine incorporation. Peripheral CD4 þ CD25highFoxP3 þ (2±0.5 vs 4.2±1.1, Po0.01), CD4 þ CD25 þ TGF-b þ (6.9±1.8 vs 14.6±5.0, Po0.05) and CD4 þ CD25 þ IL-10 þ (10.7±0.5 vs 16.1±3.2, Po0.01) Tregs as well as CD4 þ CD25highCD127low Tregs suppressive capacity (Po0.05) were decreased in dcSSc patients vs controls. After aHSCT (n ¼ 7), the percentages of CD4 þ CD25highFoxP3 þ (4.1±1.8) and CD4 þ CD25 þ IL-10 þ (15.7±2.2) Treg cells and the suppressive activity of CD4 þ CD25highCD127low were restored to the levels in controls. The decreased frequency and the functional defect of peripheral Treg cells from patients with dcSSc are reversed following aHSCT to reach those observed in controls. This pilot study brings evidence of an effective restoration of nTreg and aTreg subsets, and recovery of nTreg suppressive function following aHSCT. Bone Marrow Transplantation (2014) 49, 349–354; doi:10.1038/bmt.2013.202; published online 23 December 2013 Keywords: systemic sclerosis; regulatory T cells; hematopoietic stem cell transplantation; T-cell proliferation
INTRODUCTION Systemic sclerosis (SSc) is a chronic autoimmune disease with a heterogeneous clinical presentation. Although its exact pathogenesis is still unknown, it is characterized by alterations of the microvasculature, disturbances of the immune system with high production of autoantibodies and progressive fibrosis of the skin and internal organs.1 Whatever the original stimulus, abnormal immune stimulation, production of autoantibodies and cytokine release may be,2 all contribute to increased collagen synthesis and deposition, fibroblast activation and microvascular damage. Several studies have highlighted the key role of the adaptive immune response in SSc patients,3 as evidenced by infiltration of activated CD4 þ and CD8 þ T cells in skin tissues, lung interstitium and bronchio-alveolar fluids and in peripheral blood.4 T regulatory (Treg) cells represent 5–10% of the peripheral CD4 þ T cells in humans5 and can be characterized by the expression of CD4, CD25 and Foxp3, a transcription factor critical to the development of natural Treg (nTreg) cells,6 namely CD4 þ CD25highFoxP3 þ . Two major subsets of Treg cells have been described: a nTreg population developing in the thymus and an adaptive Treg (aTreg) subset developing in the periphery under particular conditions of suboptimal antigen exposure and/or costimulation. The aTreg cells, which include CD4 þ CD25highFoxp3 þ
and CD4 þ CD25highFoxp3 T cells, are generated from mature T-cell populations (classical T-cell subsets or nTreg cells) after antigenic stimulation.7 The aTreg cells mediate their inhibitory activities by producing immunosuppressive cytokines, such as TGF-b and IL-10.2,7 The various Treg cells have key roles in maintaining self-tolerance and modulating the allergic and autoimmune responses, while controlling autoreactive T cells.8–11 Several studies have reported a deficiency in the number of Treg and/or function in autoimmune disease patients,12,13 namely in systemic lupus erythematous,14 rheumatoid arthritis,15 multiple sclerosis,16 autoimmune polyglandular syndrome II,17 myasthenia gravis18 and type I diabetes.19 However, results concerning the number of circulating Treg cells and their suppressive function have been contradictory. In SSc patients, few studies investigated the various Treg subsets and their suppressive function, these also with conflicting results, showing either reduced,20–22 increased23,24 or the same25–27 number of circulating Treg cells in peripheral blood as compared with controls. These discrepancies might be mostly due to variations in study techniques, with setting analysis of either CD4 þ CD25 þ ,21,23 CD4 þ CD25high,12,25,26 CD4 þ CD25high Foxp3 þ ,20,24,26 CD25 þ Foxp3 þ CD127–23 or CD4 þ CD25high CD127low subsets.22 Despite these controversies, in most of these studies of SSc patients’ samples,22,23,26 impaired Treg-suppressive function was associated with SSc disease severity. Of interest, Klein
1 INSERM U976 and Universite´ Paris Diderot Sorbonne Paris Cite´, Hoˆpital Saint-Louis, Paris, France; 2Service de Me´decine Interne, Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Saint-Louis, Paris, France; 3Service de Me´decine Interne, Hoˆpital Hoˆtel Dieu, Nantes, France and 4INSERM U957 and Universite´ Nantes Atlantique, Nantes, France. Correspondence: Dr L Michel, Institut de Recherche sur la Peau, Inserm U976, Pavillon Bazin, Universite´ Paris Diderot Sorbonne Paris Cite´, Hoˆpital Saint-Louis – 1, Avenue Claude Vellefaux, 75010 Paris, France. E-mail: [email protected] Received 13 August 2012; revised 26 September 2013; accepted 10 October 2013; published online 23 December 2013
Treg in SSc patients after autologous HSCT J Baraut et al
350 et al.26 reported that there was a reduction in Treg within the skin, but not in the peripheral blood of SSc patients. Use of autologous hematopoietic SCT (aHSCT) has recently been shown to induce long-term remission in severe or rapidly progressive diffuse cutaneous SSc (dcSSc), with rapid and sustained regression of skin and lung fibrosis, and improved functional status.27,28 After aHSCT, the clinical regression of skin fibrosis has been correlated with reappearance of functional B cells, thymic and extrathymic T-cell development, reconstitution of effector cells and efficient antigen presentation to reconstitute the pretransplantation immune repertoire.29,30 However, further analysis of the immune reconstitution of the various Treg subsets and their function after aHSCT in SSc patients is warranted. To address this issue, the present pilot study aimed to analyze the respective levels of Treg subsets in SSc patients before and after aHSCT, and the suppressive functions of these cells from SSc patients as compared with those from healthy donors.
CD4 þ CD25– and CD4 þ CD25 þ T cells were isolated from PBMCs using a Miltenyi Regulatory T Cell Isolation Kit with LD and MS columns according to the manufacturer’s instructions (Miltenyi Biotech, Bergisch Gladbach, Germany), and were stained with FITC-conjugated anti-CD4, PE-conjugated anti-CD127 and APC-conjugated anti-CD25. CD4 þ CD25highCD127low (Treg cells) and CD4 þ CD25lowCD127high (T effector cells: Teff) were isolated using BD Biosciences FACSAria III cell sorter. Highly purified Treg and Teff cells were immediately cocultured with T-feeder cells (irradiated CD4 T cells) in a 96-well plate previously coated with anti-CD3 and antiCD28 antibodies. Cocultures were assessed with Teff to Treg ratio ranging from 1:1 to 3:1. After 5 days of culture, cell proliferation was measured by incorporation of 3H-thymidine radioactive (37 mBcq/mL/well) during the last 18 h. The percentage of cell proliferation inhibition was calculated as (1 (% of proliferating target T cells in coculture with Treg cells)/(% of proliferating target T cells cultured alone)) 100.
Statistical analysis All results are expressed as mean±s.d. Statistical significance for all experiments was assessed using the paired Student’s t-test. Differences were considered as significant at Po0.05.
PATIENTS AND METHODS Patients Seven patients (mean age 46±18 years) with early rapidly progressive dcSSc), defined according to the American Rheumatism Association criteria,31 were enrolled for receiving aHSCT treatment (Department of Internal Medicine, St Louis Hospital). The transplant procedure and followup were performed as previously described.28 Written informed consent was obtained and each protocol was approved by the local ethics committee of the St Louis Hospital institute. Table 1 provides an overview of the patients’ clinical characteristics. As controls, venous blood was collected from seven healthy donors (four males, three females) of the same age range as that of SSc patients (39±16 years), recruited by the Etablissement Franc¸ais du Sang after informed consent. Peripheral blood cells were isolated by Ficoll–Hypaque centrifugation followed by elimination of monocytes by adhesion for 2 h. Treg subpopulations from SSc patients and their suppressive capacity were analyzed before and 24 months after aHSCT and results were compared with controls.
Flow cytometry analysis Peripheral blood lymphocyte (PBL) samples were stimulated for 5 days with anti-CD3 antibody, anti-CD28 antibody and IL2, and during the last 5 h, PMA (0.2 mg/mL), Ionomycin (1 mg/mL) and GolgiStop (BD Biosciences, San Jose`, CA, USA) were added to the cultures. Monoclonal antibodies conjugated with either fluorescein isothiocyanate (FITC), phyco-erythrin (PE) or allophycocyanin (APC) and directed to CD4, CD25, IL-10, TGF-b (BD Biosciences), Foxp3 (eBiosciences, San Diego, CA, USA) or matched isotype controls were used for immunophenotyping and intracellular staining, according to the manufacturer’s protocols. Evaluation of Treg subpopulations by flow cytometry was determined as CD25brightFoxP3 þ percentage expression within the CD3 þ CD4 þ compartment. aTreg percentages were CD4 þ TGF-b- or IL-10-producing aTregs. Data from patients (n ¼ 7) and controls (n ¼ 7) were acquired and analyzed on a four-parameter flow cytometer FACSCalibur using ProCellQuest software (BD Biosciences).
Table 1.
Suppressor assays of Treg cells
Characteristics of patients with SSc
Patients
Age
Gender
Total mRSS (M0)a
Total mRSS (M24)a
1 2 3 4 5 6 7
63 60 46 58 62 41 50
F F M M M F F
29 25 32 37 35 20 33
8 4 10 24 11 2 13
Abbreviations: F ¼ female; M ¼ male; mRSS ¼ modified Rodnan skin score; SSc ¼ systemic sclerosis. aTotal mRSS was measured before (M0) and 24 months after (M24) HSCT.
Bone Marrow Transplantation (2014) 349 – 354
RESULTS Frequency of peripheral CD4 þ CD25highFoxP3 þ nTreg cells in SSc patients before and after aHSCT As detected by flow cytometry analysis, we observed a significant decrease in the percentage of CD4 þ CD25highFoxP3 þ nTreg cells in SSc patients evaluated before aHSCT (2±0.5, n ¼ 7) as compared with the respective percentage in healthy donors (4.2±1.1, n ¼ 7, Po0.01; Figure 1a). Figure 1b represents the fluorescence-activated cell sorting (FACS) dotplots for one representative healthy donor (left panel) and one SSc patient before any treatment (middle panel). To investigate whether aHSCT induced reconstitution of the various Treg subsets in SSc patients, we compared the percentage of nTreg cells from SSc patients before and after aHSCT versus those from healthy donors. As shown in Figure 1a, the percentage of CD4 þ CD25highFoxP3 þ T cells increased in SSc patients 24 months after aHSCT (4.1±1.8, n ¼ 7) as compared with their values before transplant. Figure 1b (left panel) exhibits the FACS dotplots for one representative SSc patient treated with aHSCT. These data indicate that aHSCT allowed to induce an increase in the percentage of nTreg in SSc patients up to the levels detected in healthy donors. aHSCT restored the reduced percentage of TGF-b- and IL-10-producing CD4 þ CD25 þ aTreg cells from SSc patients The mean percentage of CD4 þ CD25 þ IL-10 þ T cells was significantly decreased in SSc patients before aHSCT (10.7±0.5, n ¼ 3) as compared with those in healthy donors (16.1±3.2, n ¼ 7, Po0.01l; Figure 2a). Conversely, an increase in the mean percentage of CD4 þ CD25 þ IL-10 þ T cells was detected at 24 months after treatment by aHSCT, up to 15.7±2.2 (n ¼ 3), and back to control values. Figure 2b represents the FACS dotplots for one representative healthy donor (left panel), one SSc patient before (middle panel) and 24 months after (right panel) aHSCT. The mean percentage of CD4 þ CD25 þ TGF-b þ T cells was significantly lower in SSc patients (6.9±1.8, n ¼ 3) compared with healthy donors (14.6±5.0, n ¼ 7, Po0.05), as shown in Figure 2c. Twenty-four months after aHSCT, the mean percentage of CD4 þ CD25 þ TGF-b þ T cells remained significantly lower than in controls, although it was increased (17.2±4.5, data not shown) in two patients studied at 48 months after aHSCT. In Figure 2d, FACS dotplots are presented for one representative healthy donor (left panel), as well as in one SSc patient before aHSCT (middle panel) and 24 months after (right panel) aHSCT. Taken together, & 2014 Macmillan Publishers Limited
Treg in SSc patients after autologous HSCT J Baraut et al
351 the Teff:Treg cell ratio. In contrast, Treg cells from SSc patients exhibited a significantly reduced suppressive capacity compared with those from healthy donors (Po0.05), suppressing the proliferation of CD4 þ target T cells by a mean percentage of 59.8±16.1 (n ¼ 3) at ratio 1:1 (Figure 3b, red dots). This low inhibitory effect was maintained up to a dilution of 3:1 Teff:Treg. The suppressive activity of Treg cells from SSc patients 24 months after aHSCT (83.4±11.7 at ratio 1:1, n ¼ 3, Figure 3b, green dots) was significantly higher compared with their mean suppressive capacity before aHSCT (Po0.05). These results clearly indicated that aHSCT can restore the suppressive activity of Treg cells in SSc patients, in parallel with regression of skin fibrosis as shown by a fall in Rodnan skin score for each patient at 24 months (Table 1).
our results demonstrated that SSc patients have a markedly decreased frequency of both IL-10- and TGF-b-producing CD4 þ CD25 þ adaptive T cells. aHSCT restored suppressive activity in CD4 þ CD25highCD127low T cells from SSc patients To further standardize our FACS analysis, the sorting gate was adjusted to detect Treg CD4 þ CD25highCD127low and Teff CD4 þ CD25lowCD127high cells from SSc patients and healthy donors (Figure 3a). The percentage of isolated CD4 þ CD25highCD127low T cells obtained using these cell sorting parameters reached 4.4% (±2.2, n ¼ 4) in SSc patients before aHSCT as compared with 2.6% (±0.2, n ¼ 4) in normal donors, although with no significance. In the functional suppression assay, CD4 þ CD25highCD127low Treg cells were cocultured with autologous target CD4 þ CD25lowCD127high Teff cells at three ratios (Teff:Treg 1:1, 2:1 and 3:1). As expected, Treg cells from healthy controls efficiently suppressed the proliferation of Teff cells with a mean percentage of inhibition of 70.8±13.4 (n ¼ 3) at ratio 1:1 (Figure 3b, blue dots). At higher dilutions, the percentage of suppression correlated with
DISCUSSION The present pilot study illustrates, for the first time to our knowledge, that aHSCT allowed restoration of the levels of both nTreg and aTreg subsets in this small group (n ¼ 7) of SSc patients. In addition, our results confirmed that dcSSc patients have significantly decreased levels of circulating CD4 þ CD25highFoxP3 þ
% CD4+ CD25highFoxp3+ T cells after HSCT
% CD4+ CD25high Foxp3+ T cells
NS 8 **
6
4
2
Control
SSc M0
SSc M24 post graft SSc patient M24 HSCT
SSc patient M0
Healthy control 104
104
CD25+ Foxp3+ 4.3%
103
104
CD25+ Foxp3+ 0.7%
103
102
102
102
101
101
101
100 100
CD25
101
102
103
104
100 100
101
102
103
104
CD25+ Foxp3+ 7.8%
103
100 100
101
102
103
104
Foxp3
Figure 1. Flow cytometry analysis of CD4 þ CD25highFoxP3 þ T cells. (a) Percentage of CD4 þ CD25highFoxP3 þ T cells are presented for healthy donors (control, n ¼ 7), SSc patients (n ¼ 7) before HSCT (SSc M0) and 24 months (SSc M24) post transplantation (n ¼ 6). (b) One representative individual FACS dotplot from healthy control cells (left panel), SSc patient before any HSCT treatment (middle panel) and 24 months after HSCT (right panel). **Po0.01. & 2014 Macmillan Publishers Limited
Bone Marrow Transplantation (2014) 349 – 354
Treg in SSc patients after autologous HSCT J Baraut et al
352 % CD4+ CD25+ IL-10+ T cells after HSCT
104
104
CD25+ IL-10+ 103
**
SSc patient M24 HSCT
SSc patient M0
104
NS 25
CD25+ IL-10+ 103
17.3%
CD25+ IL-10+ 103
10.7%
20
102
102
102
15
101
101
101
10
100 100
101
CD25
% CD4+CD25+IL10+
Healthy control
5
102
103
100 104 100
101
102
103
100 104 100
16.5%
101
102
103
104
IL-10
0 Control
SSc M0 SSc M24 post graft
% CD4+ CD25high TGF-β T cells after HSCT
104
CD25+ TGF-β+ 103
20
19.6%
SSc patient M24 HSCT
SSc patient M0 104 CD25+ TGF-β+ 103
6.9%
CD25+ TFG-β+ 103
102
102
102
101
101
101
100
100 104
4.3%
15 10
* *
5
100
101
CD25
% CD4+CD25+TGF-β
25
Healthy control 104
0 Control
102
103
100
101
102
103
100 104 100
101
102
103
104
TGF-β
SSc M0 SSc M24 post graft
Figure 2. Flow cytometry analysis of IL-10- and TGF-b- producing CD4 þ CD25 þ T cells. (a) Percentage of CD4 þ CD25 þ IL-10 þ T cells and (c) CD4 þ CD25 þ TGF-b þ T cells were detected by intracellular staining in lymphocytes from healthy donors (Control, n ¼ 7), SSc patients before (SSc M0, n ¼ 3) and 24 months after HSCT (SSc M24, n ¼ 3). (b and d) Representative dotplots from one normal volunteer, one SSc patient before and 24 months after HSCT. *Po0.05. **Po0.01.
natural T cells, as recently reported by Antiga et al.20 in 15 patients. We also show a decrease of IL-10- and TGF-b-producing CD4 þ CD25 þ adaptive T cells in SSc patients, as reported in two previous studies that investigated CD4 þ IL-10 þ and CD4 þ TGF-b þ T cells.20,21 In contrast to our study, Klein et al.26 recently showed no significant difference in the frequency of CD4 þ CD25highFoxP3 þ from SSc patients as compared with healthy donors. This discrepancy might be due to systemic treatment with corticosteroids and/or cyclophosphamide, which might have restored the frequency of Treg in the studied SSc patients, as described in human lupus erythematous.32 Nevertheless, the authors reported a decrease in cutaneous Treg content that might be essential during the early phase of cutaneous disease. To determine the regulatory potential of the natural Treg cells in SSc patients, we performed functional suppressor assays using a new biomarker as an alternative to Foxp3. Indeed, most studies have identified CD127 as a viable surface marker that could distinguish highly suppressive Treg (CD127low) cells from Teff (CD127high) cells in humans.33 These new sorting parameters for Treg avoid contamination with activated T cells that expressed intermediate levels of CD25. Our results demonstrate that CD4 þ CD25highCD127low Treg cells functions were impaired in SSc patients as compared with normal healthy controls, and also that suppressive functions were effectively restored 24 months after aHSCT. This clearly indicates that aHSCT can restore immunological selftolerance in dcSSc patients who could recover suppressive function of Treg cells similar to normal, as previously found by Zhang et al.34 in 15 HSCT lupus patients studied before and after aHSCT. Few studies with different methods, each of them in a small number of patients, have analyzed the immune reconstitution Bone Marrow Transplantation (2014) 349 – 354
process after aHSCT for various types of autoimmune disease, including multiple sclerosis,35 SSc,29,30 juvenile arthritis36 and SLE.37 All studies reported that long-term remission in patients who received aHSCT was associated with a de novo regeneration of the T-cell compartment that can be delayed for years after T-cell depleting conditioning for aHSCT. In particular, they demonstrated a drastic reactivation of thymic activity reflected by the increased T-cell receptor excision circle (TREC) levels, as well as an increased thymic output as evidenced by the increased diversity of TCRB CDR3 repertoire. The immune reconstitution process after aHSCT is associated with the reemergence of naive T cells, the depletion of autoreactive memory T cells, the regeneration of thymic-derived FoxP3 þ regulatory T cells and the recovery of the naive B-cell compartment.29,30,35,38 Two important observations for SSc patients treated by aHSCT emerged from the present pilot study: first, both nTreg and aTreg cells regenerated to frequencies comparable with those in normal controls; second, the renewal of Treg subsets was associated with a restoration of nTreg suppressive capacity, as reported by Zhang et al.34 in SLE patients treated by aHSCT. Altogether, our data support the ‘resetting’ of the adaptive immune system induced by aHSCT, as previously reported for other types of autoimmune diseases treated by aHSCT and always based on the analysis of a small number of patients.29,34,36,37 To further prove the specific relationship to SSc, these results have to be compared with aHSCT effects on a population of patients receiving transplantation for another indication. Therefore, aHSCT could be used as a therapeutic intervention in several autoimmune diseases in which Treg functions are impaired, such as SLE, lupus or type I diabetes. Future studies are needed to evaluate the mechanisms by which Tregsuppressive functions could be altered in SSc and restored by & 2014 Macmillan Publishers Limited
Treg in SSc patients after autologous HSCT J Baraut et al
353 SSc patient
105
105
104
104 CD25 APC-A
CD25 APC-A
Healthy control
103
103
102 0 -102
102 0 -102
-235
-265 -105
102
0
103 CD127 PE-A
104
105
-92
0
102
104 103 CD127 PE-A
CD127 PE...
CD25 AP...
#Events
#Parent
Mean
Mean
P4
22,392
95.8
439
195
P4
23,221
P6
4,442
19.8
793
178
P6
7,620
P5
545
2.4
94
1,937
P5
1,712
5.0
Population
Population
#Events
105
CD127 PE...
CD25 AP...
Mean
Mean
98.7
518
358
32.8
800
169
148
2,755
#Parent
Suppression assay of CD4+ CD25highCD127low T cells from SSc patients compared with healthy donors 100 Healthy donors 90
SSc patients M0 SSc patients M24
80
% of inhibition
70 60 50 40 30 20 10 0 1:1
2:1
3:1
1:0
T effector cells:Tregcells þ
Figure 3. Isolation of CD4 CD25 CD127 T cells for suppression activity analysis. (a) FACS analysis of CD4 þ CD25highCD127low Treg cells was performed in healthy controls (n ¼ 3) and SSc patients before and 24 months after treatment by HSCT (n ¼ 3). Representative cell sorter dotplots from one healthy donor (left panel) and from one SSc patient before HSCT (right panel) are shown. The gate was set in such a way that Treg cells (P5) expressed the highest CD25 expression and the lowest CD127 expression, while Teff cells (P6) expressed the highest CD127 and the lowest CD25 expressions. (b) Suppression assays of CD4 þ CD25highCD127low Treg cells from healthy donors (n ¼ 3) (blue dots), SSc patients (n ¼ 3) before any treatment (red dots) and 24 months after HSCT (green dots). The percentage of inhibition was determined by comparing proliferation of Teff cells alone to the proliferation of Teff cells in coculture with Treg cells at different ratios. high
low
aHSCT. Alteration might be attributed to either an impaired suppressor function of the Treg cells from SSc patients or a resistance to suppression by SSc Teff cells, as previously demonstrated in SLE.39 This resistance has been attributed to pro-inflammatory cytokines such as tumor necrosis factor-a, interleukin (IL)-6, IL-7 and IL-1b.40,41 Further investigation in a larger cohort of patients with dcSSc treated by aHSCT as compared with standard therapy, such as those included ASTIS or SCOTT trials, the still ongoing large phase III randomized trials,42 will help to answer these questions and to correlate encouraging & 2014 Macmillan Publishers Limited
results from the present pilot study with patient clinical parameters and remission. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We acknowledge the Association des Scle´rodermiques de France (ASF) and the Groupe Francophone de Recherche sur la Scle´rodermie (GFRS). This work was supported by grants from the Groupe Franc¸ais de Recherche sur la Scle´rodermie (GFRS).
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Treg in SSc patients after autologous HSCT J Baraut et al
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