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Frequency analysis of autoreactive T-helper 1 and 2 cells in bullous pemphigoid and pemphigus vulgaris by enzyme-linked immunospot assay


Dr Michael Hertl, Department of Dermatology, University of Erlangen, Hartmannstr. 14, D-91054 Erlangen, Germany. E-mail:


Background Bullous pemphigoid (BP) and pemphigus vulgaris (PV) are autoimmune bullous skin diseases mediated by autoantibodies against adhesion molecules of the skin. Previous studies have identified autoreactive T cells in patients with BP and PV, which may be critical in providing B-cell help for autoantibody production.

Objectives To evaluate the frequency of autoreactive T-helper (Th) 1 and Th2 cells in patients with BP (n = 7) or PV (n = 1) and in healthy controls (n = 11).

Methods In an enzyme-linked immunospot (ELISPOT) assay, microtitre plates were coated with antihuman interleukin (IL)-5 IgG or antihuman interferon (IFN)-γ IgG prior to culturing human peripheral blood lymphocytes (PBL) with BP180 or desmoglein (Dsg) 3 proteins for 7 days. Cytokine-producing autoreactive T cells were visualized as spot-forming units.

Results One BP patient with extensive blisters had 5·1 ± 1·5 (mean ± SD) BP180-reactive Th1 cells and 2·9 ± 1·5 Th2 cells per 105 PBL. In contrast, PBL from six BP patients in remission or on immunosuppressive therapy did not form IFN-γ- or IL-5-producing spots per ≤ 5 × 105 PBL. The patient with oral PV had 4·7 ± 2·4 Th1 cells and 3·0 ± 0·4 Th2 cells per 105 PBL and a vigorous PBL response to Dsg3. In addition, three of 10 controls had BP180-reactive Th1 (2·7–13·8 per 105 PBL) and Th2 (0·3–1·8 per 105 PBL) cells and one control had 9·0 ± 0·7 Th1 cells and 1·1 ± 0·8 Th2 cells per 105 PBL, with reactivity to Dsg3. ELISPOT reactivity correlated with 3H-thymidine incorporation in six of seven patients and controls with autoreactive T-cell responses.

Conclusions The ELISPOT assay seems to be promising for the quantitative and qualitative analysis of autoreactive T-cell responses in BP and PV.