Association Alopecia Areata | Evaluation de l’efficacité et de la tolérance du traitement par interleukine 2 dans la pelade sévère résistante aux traitements systémiques conventionnels

Evaluation de l’efficacité et de la tolérance du traitement par interleukine 2 dans la pelade sévère résistante aux traitements systémiques conventionnels

Auteur : Pr Thierry Passeron – Service de Dermatologie & INSERM U1065 équipe 12, C3M CHU de Nice 150 route de Ginestière 06200 Nice FRANCE

Travail subventionné par l’AAA (subvention 2012) à hauteur de 4350 €.

Subvention débloquée le 27 mars 2013.

Compte rendu prévu en novembre 2013. Fourni le 30 mai 2014.

 

ABSTRACT

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

Alopecia areata (AA) is one of the most frequent autoimmunediseases, with a prevalence of 1.7%in the general population.1 It leads to various extentsof 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 ofCD4+ 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 immunetolerance 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 cellswasreported to playakey role inAA.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 contactdermatitiswith 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.

Report of Cases

We conducted amonocentric prospectiveopenpilot study from August 2012 to November 2013. The study was approvedby 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 1previous 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 3million 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 scalpwere performed at day 0, the last day of the treatment, and 2 months after the end of the treatment. Immunohistochemical studieswere performed on 5-μm-thick sections of formalin-fixed, paraffin-embedded tissue, using the standardtechniques involving heat-induced epitope retrieval buffer, and primary antibodies againstCD3(2GV6, prediluted; VentanaMedical Systems Inc), CD4 (SP 35, prediluted; Ventana Medical Systems Inc), CD8 (SP57, prediluted; VentanaMedical Systems Inc), FoxP3 (236A-E7, dilution 1:200; eBioscience), CD25 (4C9, prediluted; Ventana Medical Systems Inc). Appropriate positive and negative controlswere included. Intensity of staining was scored as 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong).

Serumsampleswere taken at day 0, day 5, day 25, day 45, and M2. Immunophenotypical analysis was performed using 8-color flowcytometry (FACSCanto II, BDBiosciences). The following antibodieswere used in this study : fluorescein isothiocyanate–conjugated CD127; phycoerythrin-antiFoxP3 (FoxP3-PE); peridinin chlorophyll protein–Cy5.5–conjugated CD8; PECy7–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 EuroFlowStandard Operating Procedures. Identification of lymphocytes was done using a combination of side-scattered light/forwardscattered 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.

Results

All the subjectswerewomen,with

Results

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 6months after the end of the treatmentwere 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 assessedwith a 10-point visual analog scale 2months 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 in4of the5patients at the endof the treatment compared with baseline (Table and eFigure in Supplement). Aconcomitant decrease of the CD8+ infiltratewas observed. The Treg cells were still present in the skin biopsy specimens 2months 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).

Discussion

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.

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