Frequency Analysis of Autoreactive T-helper 1 and 2 Cells in Bullous Pemphigoid and Pemphigus Vulgaris by Enzyme-linked Immunospot Assay

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)-gamma 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-gamma- or IL-5-producing spots per </= 5 x 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.