Identification of Autoreactive B Cell Subpopulations in Peripheral Blood of Autoimmune Patients With Pemphigus Vulgaris

Abstract

Pemphigus vulgaris (PV) is a rare blistering disease caused by IgG autoantibodies against the epidermal adhesion molecules desmoglein (Dsg)3 and Dsg1 providing a well-characterized paradigm of an antibody-mediated organ-specific autoimmune disease. In PV patients who have achieved clinical remission after B cell-depleting therapy, relapses often coincide with a reoccurrence of B cells and Dsg-specific autoantibodies. Here, we analyzed Dsg3-specific B cell subpopulations (i.e., total CD19⁺ B cells, CD19⁺CD27^-B cells, CD19⁺CD27⁺ memory B cells, and CD19⁺CD27^hiCD38^hi plasmablasts) in peripheral blood of both PV patients (n = 14) at different stages of disease and healthy individuals (n = 14) by flow cytometry using fluorescently labeled recombinant human Dsg3 protein. Applying this approach, Dsg3-specific B cells could be detected at low frequencies (0.11-0.53% of CD19⁺ B cells) and numbers of Dsg3-specific memory B cells were significantly increased in PV patients in clinical remission receiving minimal immunosuppressive therapy. Finally, we confirmed in vitro that Dsg3-reactive memory B cells were able to produce anti-Dsg3 IgG autoantibodies upon ex vivo activation. Thus, monitoring of Dsg3-specific B cells in PV is of particular interest to further characterize the immunopathogenesis of PV.

Keywords: B cells; autoimmunity; desmoglein 3; flow cytometry; pemphigus vulgaris.

Figure 1

Detection of Dsg3-specific B cell hybridoma (BCH) using fluorescently labeled Dsg3. (A) Schematic drawing: the recombinant human extracellular domain (EC1-EC5) of Dsg3 was labeled with the fluorescent dye Alexa Fluor 647 (Dsg3-AF647) and was used for staining of Dsg3-specifc B cell receptors (BCR). (B) Binding of Dsg3-AF647 to Dsg3 ± addition of the monoclonal Dsg3-specific antibody AK23 was evaluated with atomic force microscopy. Cumulative data from 3 individual measurements with five replicates for each condition are presented as mean + SD. Statistical analysis was performed by multiple t-tests followed by Šidák correction. Differences between groups were considered statistically significant at p-values of <0.05 indicated as ^*. (C) Binding efficacy of Dsg3-AF647 to Dsg3-specific BCR was determined by staining of a Dsg3-specific BCH (2C10) and an unrelated BCH (1F12) together with anti-IgG antibody. Binding of Dsg3-AF647 was blocked by preincubation with unlabeled Dsg3. FACS plots shown are representative of three individual experiments. (D) Specificity of monoclonal BCH cells for Dsg3 was tested by ELISA. Anti-E-Tag served as positive and culture medium as negative control. (E) Correlation of mean fluorescence intensity (MFI) of Dsg3-AF647 with surface IgG. (F) Titration of Dsg3-specific BCH (2C10) cells in unrelated 1F12 cells in a calculated ratio of 1:16 (6.25%), 1:32 (3.13%), 1:64 (1.56%), and 1:128 (0.78%) representative of three individual experiments.

Figure 2

Dsg3-specific memory B cells are increased in remitting PV patients on minimal therapy. (A) Representative FACS plot showing staining for CD19⁺Dsg3-AF647⁺ B cells in one PV patient. (B) On the upper row: Dsg3-specific B cell populations (total CD19⁺, CD19⁺CD27⁻ mature naïve, CD19⁺CD27⁺ memory, and CD19⁺CD27^hiCD38^hi plasmablasts) were analyzed in PV patients (n = 14) and healthy controls (HC; n = 14). On the lower row: PV patients were further subdivided based on their systemic treatment into minimal to no (n = 6) and moderate therapy (n = 8) showing highest numbers of Dsg3-specific CD19⁺CD27⁺ memory B cells on minimal therapy. ■: after Rituximab treatment; □: no Rituximab treatment. (C) CD19⁺CD27⁺Dsg3-AF647⁺ cells were isolated from peripheral blood of a PV patient and a healthy control by FACS sorting and stimulated with R848 and IL-2 to induce plasma cell differentiation. Dsg3-IgG-secreting and total IgG-secreting B cells were subsequently detected using ELISpot by seeding 250 cells per well on ELISpot plates coated either with recombinant human Dsg3 or human collagen VII as control protein. Statistical analysis was performed with two-tailed nonparametric Mann-Whitney-U-Test. Differences between groups were considered statistically significant at p-values of <0.05 indicated as ^*.