0
We're unable to sign you in at this time. Please try again in a few minutes.
Retry
We were able to sign you in, but your subscription(s) could not be found. Please try again in a few minutes.
Retry
There may be a problem with your account. Please contact the AMA Service Center to resolve this issue.
Contact the AMA Service Center:
Telephone: 1 (800) 262-2350 or 1 (312) 670-7827  *   Email: subscriptions@jamanetwork.com
Error Message ......
Case Report/Case Series |

Effects of Low-Dose Recombinant Interleukin 2 to Promote T-Regulatory Cells in Alopecia Areata FREE

Emeline Castela, MD1; Florence Le Duff, MD1,2; Catherine Butori, MD3; Michel Ticchioni, MD, PhD4; Paul Hofman, MD, PhD3; Philippe Bahadoran, MD, PhD1,2; Jean-Philippe Lacour, MD1; Thierry Passeron, MD, PhD1,5
[+] Author Affiliations
1Department of Dermatology, University Hospital of Nice, Nice, France
2Centre de Recherche Clinique (CRC), University Hospital of Nice, Nice, France
3Laboratory of Clinical and Experimental Pathology, University Hospital of Nice, Nice, France
4Laboratory of Immunology, University Hospital of Nice, Nice, France
5Institut National de la Santé et de la Récherche Médicale (INSERM) U1065, Team 12, Mediterranean Centre of Molecular Medicine, Nice, France
JAMA Dermatol. 2014;150(7):748-751. doi:10.1001/jamadermatol.2014.504.
Text Size: A A A
Published online

Importance  An impaired inhibitory function of circulating CD4+CD25+ regulatory T (Treg) cells was reported to play a key role in alopecia areata (AA). We report the first use to our knowledge of low-dose interleukin 2 for treating severe AA by promoting the recruitment of Treg cells.

Observations  We conducted a prospective open pilot study in 5 patients with severe AA resistant to previous systemic treatments. Subcutaneous interleukin 2 (1.5 million IU/d) was administered during 5 days, followed by three 5-day courses of 3 million IU/d at weeks 3, 6, and 9. The primary outcome was the evolution of the Severity of Alopecia Tool (SALT) score, evaluated by 2 independent investigators on standardized photographs. Lesional skin biopsy specimens and peripheral blood lymphocyte phenotype were analyzed. The median SALT score went from 82 (range, 63-100) at baseline to 69 (range, 28-100) at 6 months. Immunochemical analysis revealed the appearance or a notable increase in Treg cell count in 4 of 5 patients at the end of the treatment compared with baseline. No serious adverse event was reported.

Conclusions and Relevance  The partial regrowth achieved in 4 of 5 patients and the recruitment of Treg cells in lesional skin support the interest of promoting Treg cells for treating AA. Further investigations are now required to confirm and to optimize the design in order to enhance the Treg cell response.

Trial Registration  clinicaltrials.gov Identifier: NCT01840046

Figures in this Article

Alopecia areata (AA) is one of the most frequent autoimmune diseases, with a prevalence of 1.7% in the general population.1 It leads to various extents of hair loss of the scalp and other areas of the body. The pathobiological determinants of AA remain unclear.2 Several observations indicate environmental factors and genetic predisposition. It is now well established that AA is an autoimmune disease related to the infiltration of CD4+ and CD8+ T lymphocytes around hair follicles. Depletion experiments in mouse models have showed that these lymphocytes are essential to the development of AA.3 Enhanced T-cell–mediated immunity and breakdown of immune tolerance due to deficiency in T-regulatory (Treg) cells may facilitate its occurrence. It has been showed that transfer of CD8+ cells induces localized hair loss, whereas CD4+/CD25 cells promote systemic AA and CD4+/CD25+ cells prevent disease onset in a mouse model.4 Recently, an impaired inhibitory function of circulating CD4+/CD25+ Treg cells was reported to play a key role in AA.5 Quality of life is deeply altered in most affected patients.6 The treatment of severe AA remains highly challenging. Many local and systemic treatments have been proposed, but none have been validated by randomized clinical trials.7 Topical immunotherapy is considered to be the most efficient, with a response rate ranging from 40% to 60%.8 It consists in inducing contact dermatitis with a chemical sensitizer leading to a decrease in the perifollicular inflammation due to AA. The pathomechanisms are not clearly understood, but they may involve an induction of tolerance by the Treg cells recruited at the end of the contact dermatitis reaction.9 However, owing to its numerous adverse effects, the use of topical immunotherapy is limited. Low-dose interleukin 2 (IL-2) is essential for homeostasis of Treg cells.10 In a mouse model of dermatitis, treatment with a fusion protein IL-2–IgG activates the Treg lymphocyte population and inhibits the dermatitis.11 Two open trials showed that low-dose IL-2 led to Treg cell recovery and concomitant clinical improvement in patients with hepatitis C virus–induced vasculitis or graft-vs-host disease.12,13 We hypothesized that low-dose IL-2 could be effective in the treatment of severe AA by promoting the recruitment of Treg cells.

We conducted a monocentric prospective open pilot study from August 2012 to November 2013. The study was approved by the ethics committee of Nice Medical University and was conducted in accordance with the Declaration of Helsinki Principles. Five patients with severe AA (>50% of the scalp surface affected) resistant to at least 1 previous systemic treatment (pulse steroid therapy and/or methotrexate) were included after written informed consent was obtained. The main objective of the study was to assess the rate of complete response under low-dose recombinant IL-2 treatment 2 and 6 months after the end of the injections (M2 and M6, respectively). The secondary objectives were to evaluate the rate of partial response to the treatment, the safety of this treatment, and its effects on the Treg lymphocytes in the blood and lesional skin.

All the patients received 1 course of subcutaneous IL-2 (1.5 million IU/d) during 5 days, followed by three 5-day courses of 3 million IU/d at weeks 3, 6, and 9.12,13 Adverse events, vital signs, and laboratory parameters were evaluated throughout the study.

The primary outcome was achieving a Severity of Alopecia Tool (SALT) score of 90. The SALT was performed as previously described14 by 2 independent investigators on standardized pictures at baseline as compared with M2 and M6. Secondary outcomes were achieving a SALT score of 50 at M2, effects of the treatment on nails and body hair, physician global assessment (0, no regrowth; 1, <25% of regrowth; 2, 25%-49% of regrowth; 3, 50%-74% of regrowth; 4, 75%-99% of regrowth; 5, 100% of regrowth), Dermatology Life Quality Index, patient global assessment (0, no regrowth; 1, <25% of regrowth; 2, 25%-49% of regrowth; 3, 50%-74% of regrowth; 4, 75%-99% of regrowth; 5, 100% of regrowth).

Skin biopsies of lesional scalp were performed at day 0, the last day of the treatment, and 2 months after the end of the treatment. Immunohistochemical studies were performed on 5-μm-thick sections of formalin-fixed, paraffin-embedded tissue, using the standard techniques involving heat-induced epitope retrieval buffer, and primary antibodies against CD3 (2GV6, prediluted; Ventana Medical Systems Inc), CD4 (SP 35, prediluted; Ventana Medical Systems Inc), CD8 (SP57, prediluted; Ventana Medical Systems Inc), FoxP3 (236A-E7, dilution 1:200; eBioscience), CD25 (4C9, prediluted; Ventana Medical Systems Inc). Appropriate positive and negative controls were included. Intensity of staining was scored as 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong).

Serum samples were taken at day 0, day 5, day 25, day 45, and M2. Immunophenotypical analysis was performed using 8-color flow cytometry (FACSCanto II, BD Biosciences). The following antibodies were used in this study: fluorescein isothiocyanate–conjugated CD127; phycoerythrin-antiFoxP3 (FoxP3-PE); peridinin chlorophyll protein–Cy5.5–conjugated CD8; PE-Cy7–conjugated CD19; allophycocyanin (APC)-conjugated CD25; APC-H7 conjugated CD3; V450-conjugated CD4; and V500-conjugated CD45, all purchased from BD Bioscience except for FoxP3-PE, which was obtained from eBiocience. The fixation and permeabilization steps were performed according to the instructions of the manufacturer (eBioscience). Instrument setup was performed according to the EuroFlow Standard Operating Procedures. Identification of lymphocytes was done using a combination of side-scattered light/forward-scattered light properties and CD45 expression. The gating strategy was next based on the gating of CD3+CD4+ and CD3+CD8+ lymphocytes, with the gate FoxP3+CD127CD25+ being set on CD3+CD4+ T cells, using the CD3+CD8+ T cells as a negative control as previously described.15 Kendall rank correlation tau (τ) was used to analyze the interrater reliability of the scoring. The 2-tailed t test was used to analyze differences for values obtained with cytometry. P < .05 was considered significant.

All the subjects were women, with a median (range) age of 34.6 (21-42) years and a median (range) baseline SALT score of 82 (63-100). The mean (range) duration of AA was 10 (5-20) years. All the patients completed the study. No serious adverse event was reported. Treatment adverse events were mild to moderate with the following symptoms: asthenia, arthralgia, urticaria, and local reactions at injection sites. All the patients had at least some regrowth of the scalp and/or body hairs. Of 5 patients, 4 had a regrowth of scalp hair with a continuation of the improvement at 6 months compared with 2 months after treatment. The median (range) SALT scores 2 and 6 months after the end of the treatment were 76 (37-100) and 69 (28-100), respectively. The interrater reliability of the scoring between the 2 physicians was good (τ: 0.9623; P = .001). The Physician Global Assessment score 6 months after the end of the treatment was 0 for 1 patient, 1 for 3 patients, and 2 for 1 patient (Figure). The median (range) DLQI score decreased from 6 (1-10) to 2.5 (2-14). The mean (range) satisfaction of the patient assessed with a 10-point visual analog scale 2 months after the end of the treatment was 6.3 (3.2-9.2). Findings from immunochemical studies showed the appearance or a notable increase in Treg cell count in 4 of the 5 patients at the end of the treatment compared with baseline (Table and eFigure in Supplement). A concomitant decrease of the CD8+ infiltrate was observed. The Treg cells were still present in the skin biopsy specimens 2 months after the end of treatment. Interestingly, the patient without recruitment of Treg cells in the skin sample was the one who did not have hair regrowth. A slight but not statistically significant increase in circulating blood Treg cell count was noted at the end of the treatment (median value, 6.64% [beginning of treatment]; 8.22% [end of treatment]; P = .08).

Place holder to copy figure label and caption
Figure.
Clinical Evolution Under Low-Dose Recombinant Interleukin 2 Treatment

A, Patient 3 at baseline (Severity of Alopecia Tool [SALT] score, 98); B, clinical evolution 6 months after the end of the treatment (SALT score, 50).

Graphic Jump Location
Table Graphic Jump LocationTable.  Histological Results of Skin Biopsies Specimens of Lesional Scalp Obtained at Baseline, the Last Day of the Treatment, and 2 Months After the End of Treatment

These results show that low-dose IL-2 can have a positive effect on the recruitment of CD4+CD25+FoxP3+ Treg cells in lesional skin of AA. None of the patient achieved a complete regrowth of the hair that was the main objective of the study. However, all the patients had severe AA with a mean duration of 10 years and were resistant to all previous systemic treatments. Some hair regrowth was observed in all the patients, and a partial regrowth of hair scalp was achieved in 4 of the 5 patients. Interestingly, the results were maintained at 6 months and the regrowth was higher at 6 months compared with 2 months after treatment. The low-dose IL-2 treatment allowed recruitment of Treg cells within the lesional scalp skin, whereas a slight, although not statistically significant, increase in circulating blood Treg cell count was noted at the end of the treatment. This difference, compared with the significant increase in circulating Treg cell count reported in hepatitis C virus–induced vasculitis or graft-vs-host disease achieved with the same protocol, is probably explained by the fact that the immune reaction is restricted in the hair follicle in AA and is systemic in hepatitis C virus–induced vasculitis or graft-vs-host disease. Interestingly, the only patient who did not show hair regrowth on the scalp was the one without recruitment of Treg cells in the affected skin. This correlation between the clinical response and the recruitment of Treg cells within the affected skin supports the responsibility of the Treg cell response in the regrowth observed in those patients. However, the clinical results obtained with the present protocol are not fully satisfactory, and further investigations are now required to confirm and to optimize the protocol of treatment to enhance the Treg cell response. These results open a new research avenue in the treatment of AA.

Accepted for Publication: February 27, 2014.

Corresponding Author: Thierry Passeron, MD, PhD, Department of Dermatology, Archet 2 Hospital, 150, Route de Ginestière, 06200 Nice, France (passeron@unice.fr).

Published Online: May 28, 2014. doi:10.1001/jamadermatol.2014.504.

Author Contributions: Dr Passeron 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.

Study concept and design: Castela, Lacour, Passeron.

Acquisition, analysis, or interpretation of data: Castela, Le Duff, Butori, Ticchioni, Hofman, Bahadoran, Passeron.

Drafting of the manuscript: Castela, Passeron.

Critical revision of the manuscript for important intellectual content: All authors.

Statistical analysis: Castela, Ticchioni.

Obtained funding: Castela.

Administrative, technical, or material support: Le Duff, Ticchioni, Bahadoran, Lacour.

Study supervision: Butori, Lacour, Passeron.

Conflict of Interest Disclosures: None reported.

Funding/Support: This study was performed with grant support from Laboratoires Phytosolba, Association Alopecia Areata, and Association Bien Vivre la Pelade.

Role of the Sponsors: The sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Previous Presentation: The abstract of an earlier version of this article was presented as a poster at the 2013 Annual Meeting of the French Society of Dermatology; December 11-14, 2013; Paris, France.

Safavi  KH, Muller  SA, Suman  VJ, Moshell  AN, Melton  LJ  III.  Incidence of alopecia areata in Olmsted County, Minnesota, 1975 through 1989. Mayo Clin Proc. 1995;70(7):628-633.
PubMed   |  Link to Article
Gilhar  A, Etzioni  A, Paus  R.  Alopecia areata. N Engl J Med. 2012;366(16):1515-1525.
PubMed   |  Link to Article
Gilhar  A, Ullmann  Y, Berkutzki  T, Assy  B, Kalish  RS.  Autoimmune hair loss (alopecia areata) transferred by T lymphocytes to human scalp explants on SCID mice. J Clin Invest. 1998;101(1):62-67.
PubMed   |  Link to Article
McElwee  KJ, Freyschmidt-Paul  P, Hoffmann  R,  et al.  Transfer of CD8(+) cells induces localized hair loss whereas CD4(+)/CD25(-) cells promote systemic alopecia areata and CD4(+)/CD25(+) cells blockade disease onset in the C3H/HeJ mouse model. J Invest Dermatol. 2005;124(5):947-957.
PubMed   |  Link to Article
Shin  BS, Furuhashi  T, Nakamura  M, Torii  K, Morita  A.  Impaired inhibitory function of circulating CD4+CD25+ regulatory T cells in alopecia areata. J Dermatol Sci. 2013;70(2):141-143.
PubMed   |  Link to Article
Chu  SY, Chen  YJ, Tseng  WC,  et al.  Psychiatric comorbidities in patients with alopecia areata in Taiwan: a case-control study. Br J Dermatol. 2012;166(3):525-531.
PubMed   |  Link to Article
Delamere  FM, Sladden  MM, Dobbins  HM, Leonardi-Bee  J.  Interventions for alopecia areata. Cochrane Database Syst Rev. 2008;(2):CD004413.
PubMed
Rokhsar  CK, Shupack  JL, Vafai  JJ, Washenik  K.  Efficacy of topical sensitizers in the treatment of alopecia areata. J Am Acad Dermatol. 1998;39(5, pt 1):751-761.
PubMed   |  Link to Article
Vocanson  M, Hennino  A, Rozières  A, Poyet  G, Nicolas  JF.  Effector and regulatory mechanisms in allergic contact dermatitis. Allergy. 2009;64(12):1699-1714.
PubMed   |  Link to Article
Zorn  E, Nelson  EA, Mohseni  M,  et al.  IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo. Blood. 2006;108(5):1571-1579.
PubMed   |  Link to Article
Rückert  R, Brandt  K, Hofmann  U, Bulfone-Paus  S, Paus  R.  IL-2-IgG2b fusion protein suppresses murine contact hypersensitivity in vivo. J Invest Dermatol. 2002;119(2):370-376.
PubMed   |  Link to Article
Saadoun  D, Rosenzwajg  M, Joly  F,  et al.  Regulatory T-cell responses to low-dose interleukin 2 in HCV-induced vasculitis. N Engl J Med. 2011;365(22):2067-2077.
PubMed   |  Link to Article
Koreth  J, Matsuoka  K, Kim  HT,  et al.  Interleukin 2 and regulatory T cells in graft-versus-host disease. N Engl J Med. 2011;365(22):2055-2066.
PubMed   |  Link to Article
Olsen  EA, Hordinsky  MK, Price  VH,  et al; National Alopecia Areata Foundation.  Alopecia areata investigational assessment guidelines—part II. J Am Acad Dermatol. 2004;51(3):440-447.
PubMed   |  Link to Article
Presicce  P, Moreno-Fernandez  ME, Lages  CS, Orsborn  KI, Chougnet  CA.  Association of two clones allows for optimal detection of human FOXP3. Cytometry A. 2010;77(6):571-579.
PubMed   |  Link to Article

Figures

Place holder to copy figure label and caption
Figure.
Clinical Evolution Under Low-Dose Recombinant Interleukin 2 Treatment

A, Patient 3 at baseline (Severity of Alopecia Tool [SALT] score, 98); B, clinical evolution 6 months after the end of the treatment (SALT score, 50).

Graphic Jump Location

Tables

Table Graphic Jump LocationTable.  Histological Results of Skin Biopsies Specimens of Lesional Scalp Obtained at Baseline, the Last Day of the Treatment, and 2 Months After the End of Treatment

References

Safavi  KH, Muller  SA, Suman  VJ, Moshell  AN, Melton  LJ  III.  Incidence of alopecia areata in Olmsted County, Minnesota, 1975 through 1989. Mayo Clin Proc. 1995;70(7):628-633.
PubMed   |  Link to Article
Gilhar  A, Etzioni  A, Paus  R.  Alopecia areata. N Engl J Med. 2012;366(16):1515-1525.
PubMed   |  Link to Article
Gilhar  A, Ullmann  Y, Berkutzki  T, Assy  B, Kalish  RS.  Autoimmune hair loss (alopecia areata) transferred by T lymphocytes to human scalp explants on SCID mice. J Clin Invest. 1998;101(1):62-67.
PubMed   |  Link to Article
McElwee  KJ, Freyschmidt-Paul  P, Hoffmann  R,  et al.  Transfer of CD8(+) cells induces localized hair loss whereas CD4(+)/CD25(-) cells promote systemic alopecia areata and CD4(+)/CD25(+) cells blockade disease onset in the C3H/HeJ mouse model. J Invest Dermatol. 2005;124(5):947-957.
PubMed   |  Link to Article
Shin  BS, Furuhashi  T, Nakamura  M, Torii  K, Morita  A.  Impaired inhibitory function of circulating CD4+CD25+ regulatory T cells in alopecia areata. J Dermatol Sci. 2013;70(2):141-143.
PubMed   |  Link to Article
Chu  SY, Chen  YJ, Tseng  WC,  et al.  Psychiatric comorbidities in patients with alopecia areata in Taiwan: a case-control study. Br J Dermatol. 2012;166(3):525-531.
PubMed   |  Link to Article
Delamere  FM, Sladden  MM, Dobbins  HM, Leonardi-Bee  J.  Interventions for alopecia areata. Cochrane Database Syst Rev. 2008;(2):CD004413.
PubMed
Rokhsar  CK, Shupack  JL, Vafai  JJ, Washenik  K.  Efficacy of topical sensitizers in the treatment of alopecia areata. J Am Acad Dermatol. 1998;39(5, pt 1):751-761.
PubMed   |  Link to Article
Vocanson  M, Hennino  A, Rozières  A, Poyet  G, Nicolas  JF.  Effector and regulatory mechanisms in allergic contact dermatitis. Allergy. 2009;64(12):1699-1714.
PubMed   |  Link to Article
Zorn  E, Nelson  EA, Mohseni  M,  et al.  IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo. Blood. 2006;108(5):1571-1579.
PubMed   |  Link to Article
Rückert  R, Brandt  K, Hofmann  U, Bulfone-Paus  S, Paus  R.  IL-2-IgG2b fusion protein suppresses murine contact hypersensitivity in vivo. J Invest Dermatol. 2002;119(2):370-376.
PubMed   |  Link to Article
Saadoun  D, Rosenzwajg  M, Joly  F,  et al.  Regulatory T-cell responses to low-dose interleukin 2 in HCV-induced vasculitis. N Engl J Med. 2011;365(22):2067-2077.
PubMed   |  Link to Article
Koreth  J, Matsuoka  K, Kim  HT,  et al.  Interleukin 2 and regulatory T cells in graft-versus-host disease. N Engl J Med. 2011;365(22):2055-2066.
PubMed   |  Link to Article
Olsen  EA, Hordinsky  MK, Price  VH,  et al; National Alopecia Areata Foundation.  Alopecia areata investigational assessment guidelines—part II. J Am Acad Dermatol. 2004;51(3):440-447.
PubMed   |  Link to Article
Presicce  P, Moreno-Fernandez  ME, Lages  CS, Orsborn  KI, Chougnet  CA.  Association of two clones allows for optimal detection of human FOXP3. Cytometry A. 2010;77(6):571-579.
PubMed   |  Link to Article

Correspondence

CME
Also Meets CME requirements for:
Browse CME for all U.S. States
Accreditation Information
The American Medical Association is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The AMA designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 CreditTM per course. Physicians should claim only the credit commensurate with the extent of their participation in the activity. Physicians who complete the CME course and score at least 80% correct on the quiz are eligible for AMA PRA Category 1 CreditTM.
Note: You must get at least of the answers correct to pass this quiz.
Please click the checkbox indicating that you have read the full article in order to submit your answers.
Your answers have been saved for later.
You have not filled in all the answers to complete this quiz
The following questions were not answered:
Sorry, you have unsuccessfully completed this CME quiz with a score of
The following questions were not answered correctly:
Commitment to Change (optional):
Indicate what change(s) you will implement in your practice, if any, based on this CME course.
Your quiz results:
The filled radio buttons indicate your responses. The preferred responses are highlighted
For CME Course: A Proposed Model for Initial Assessment and Management of Acute Heart Failure Syndromes
Indicate what changes(s) you will implement in your practice, if any, based on this CME course.
Submit a Comment

Multimedia

Supplement.

eFigure. Patient 2: Immunostaining With CD25, FoxP3, and CD8 Antibodies

Supplemental Content

Some tools below are only available to our subscribers or users with an online account.

1,453 Views
8 Citations

Related Content

Customize your page view by dragging & repositioning the boxes below.

See Also...
Articles Related By Topic
Related Collections
Jobs
×