B-cell depletion induces a shift in self antigen specific B-cell repertoire and cytokine pattern in patients with bullous pemphigoid

Abstract

Bullous Pemphigoid is the most common auto-immune bullous skin disease. It is characterized by the production of auto-antibodies directed against 2 proteins of the hemi-desmosome (BP180 and BP230). We assessed the efficacy and mechanisms of action of rituximab, an anti-CD20 monoclonal antibody, in 17 patients with severe and relapsing type of bullous pemphigoid. The phenotype, cytokine gene expression, and rearrangement of BP180-specific B-cell receptor genes were performed over 2 years following treatment. At the end of the study, 5 patients had died, 3 had withdrawn from the study, and 9 patients were in complete remission. The one- and two-year relapse rates were 44.1% (95% Confidence Interval (CI): 21.0-76.0%) and 66.5%, (95% CI: 38.4-91.4%), respectively. Phenotypic analyses confirmed dramatic B-cell depletion, which lasted for 9 to 12 months. The ELISA values of serum anti-BP180 antibodies and the frequency of BP180-specific circulating B cells decreased dramatically following treatment, which paralleled the improvement of skin lesions. During B-cell reconstitution, a polyclonal IgM repertoire appeared and a shift in the rearrangement of the B-cell receptor genes of BP180-specific circulating B cells was observed. Concurrently, we observed a decrease of IL-15, IL-6 and TNFα expressing BP180-specific B cells, and the emergence of IL-10 and IL-1RA-expressing BP180-specific IgM+ B cells in patients in complete remission off therapy, suggesting the functional plasticity of BP180-specific auto-immune B cells after rituximab treatment.

Figure 1

Flow diagram of the clinical trial.

Figure 2

Evolution of anti-BP180 (panel A) and anti-BP230 (panel B) antibody ELISA values in sera from BP patients after Rituximab therapy. Blue lines correspond to patients who achieved complete remission off therapy (CRoffT). Red lines correspond to patients who were in complete remission on minimal therapy (CRMT).

Figure 3

Evolution of blood B cells in BP patients treated with rituximab. Panel A: Percentage of total CD19+ B cells (Kruskal-Wallis test Dunn’s comparison relative to day 0). Panel B: Number of BP180-specific B cell IgM+ (white bars) or IgG+ (blue bars) in BP patients at day 0, in BP patients in complete remission off therapy (CRoffT) or on minimal therapy (CRMT) at Month 24, and in healthy individuals (HI) (Mann Whitney T-Test).

Figure 4

Representation of VH family frequency usage by BP180-specific IgM (panel A) or IgG (panel B) circulating B cells from BP patients before (medium-blue column) or after (light-blue column) rituximab treatment at different time points D360, D540 and D720, compared with non-autoreactive B cells from healthy individuals (white column) (Fisher exact test).

Figure 5

Frequency of pro-inflammatory cytokines, anti-inflammatory cytokines and B cell stimulatory cytokines expressed by BP180-specific IgM+ and IgG+ B cells in CR patients before (D0) and after rituximab treatment. CR = CRMT+ CRoffT and Healthy Individuals (HI) (Fisher exact test).