Blocking of CTLA-4 on lymphocytes improves the sensitivity of lymphocyte transformation tests in a patient with nickel allergy

ARTICLE

ejd.2012.1641

Auteur(s) : Kazunari Sugita¹ k-sugita@med.uoeh-u.ac.jp, Kenji Kabashima², Yu Sawada¹, Sanehito Haruyama¹, Manabu Yoshioka¹, Tomoko Mori¹, Miwa Kobayashi¹, Kouetsu Ogasawara³, Yoshiki Tokura⁴

¹ Department of Dermatology, University of Occupational and Environmental Health, Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

² Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan

³ Department of Immunobiology, Institute of Development, Aging and Cancer, Tohoku University Sendai, Japan

⁴ Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan

Allergic contact dermatitis is a T cell-mediated cutaneous inflammatory response typically induced by contact with low-molecular weight chemicals. The patch test remains the gold standard method of confirming allergic contact dermatitis and to identify the specific causative allergen, although it is time-consuming, subjective, and not completely safe since it can induce sensitization to allergens during the test. The frequency and morbidity of allergic contact dermatitis and the problems related to the patch test necessitate the development of an alternative test.

Several in vitro alternative tests have been described in the literature, and the lymphocyte transformation test (LTT) is one of the most promising possibilities [1]. The low sensitivity of the LTT limits its usefulness as a diagnostic tool, however. We describe a patient with nickel allergy in whom the LTT was initially negative but turned positive after the addition of a neutralizing monoclonal antibody (mAb) to cytotoxic T lymphocyte antigen 4 (CTLA-4).

A 56-year-old woman with a past history of metal-induced allergic contact dermatitis following ear piercing was referred to our department. Patch testing was performed for various metal allergens, including 19 ready-made patch test reagents (Torii Pharmaceutical Corporation, Tokyo, Japan) [2]. The patch test was positive for nickel (2+, according to the International Contact Dermatitis Research Group scale), confirming that the patient had a metal allergy to nickel.

To evaluate the LTT as a diagnostic tool for contact dermatitis, peripheral blood mononuclear cells (PBMCs) of the patient and a healthy control donor were freshly isolated and incubated with 50 μM of nickel sulfate (Ni) and 50 μM of cobalt (Co) as previously reported [3]. Briefly, PBMCs were incubated in RPMI1640 medium supplemented with 10% heat-inactivated fetal calf serum. Cells were stimulated with 3 μg/ml of concanavalin A as a positive control. Cultures were performed in triplicate in 96-well flat bottomed plates for six days. Eighteen hours before harvesting, 1 μCi of ³H-thymidine was added to each culture, and T cell proliferation was measured based on ³H-thymidine incorporation.

It has been demonstrated that IL-10-, TGF-b-, and CTLA-4-dependent mechanisms may contribute to the suppression of T cell proliferation by regulatory T cells (Tregs) [4]. Therefore, we hypothesized that lymphocyte proliferation was likewise suppressed by Tregs, despite the presence of effector T cells for Ni in the culture. To address this issue, we added neutralizing mAb to IL-10 (10 μg/ml) (eBioscience, San Diego, CA) and CTLA-4 (10 μg/ml) (BD Biosciences, San Jose, CA) and a selective inhibitor of TGF-β (1 μM) (Sigma-Aldrich, St.-Louis, MO, USA). Intriguingly, when anti-CTLA-4 mAb was added to the PBMCs, lymphocytes proliferated well in response to Ni (figure 1A). In contrast, no such enhanced proliferation was observed in response to Co in the patient's PBMCs (figure 1A). Cavani et al. reported that CD4⁺ T cells purified from the peripheral blood of healthy subjects proliferate to Ni in vitro [5]. In our study, however, no responses to Ni or Co were found in three healthy controls, as represented by an individual shown in figure 1B.

It is possible that the CTLA-4-mediated suppressive mechanism depends on contact allergens, but our results suggest that CTLA-4 is involved in the mechanism responsible for suppression by Tregs, at least in the LTT assay for certain antigenic molecules. Since CTLA-4 is important in Ni-specific T cell proliferation, Treg-antigen presenting cell interaction could be a possible mechanism in our case. Although the number of cases is limited in this study, our method represents a modified LTT with improved sensitivity for Ni, thus warranting further investigation.

Disclosure

Financial support: none. Conflict of interest: none.

References

1. Merk HF. Lymphocyte transformation test as a diagnostic test in allergic contact dermatitis. Contact Dermatitis 2005; 53: 246.

2. Hosoki M, Bando E, Asaoka K et al. Assessment of allergic hypersensitivity to dental materials. Biomed Mater Eng 2009; 19: 53-61.

3. Minang JT, Arestrom I, Troye-Blomberg M et al. Nickel, cobalt, chromium, palladium and gold induce a mixed Th1- and Th2-type cytokine response in vitro in subjects with contact allergy to the respective metals. Clin Exp Immunol 2006; 146: 417-26.

4. Nouri-Aria KT. Foxp3 expressing regulatory T-cells in allergic disease. Adv Exp Med Biol 2009; 665: 180-94.

5. Cavani A, Mei D, Guerra E et al. Patients with allergic contact dermatitis to nickel and nonallergic individuals display different nickel-specific T cell responses. Evidence for the presence of effector CD8+ and regulatory CD4+ T cells. J Invest Dermatol 1998; 111: 621-8.