Previously, it has been shown that the Dsg-specific autoantibodies in patients with pemphigus with active disease tend to be IgG4,12–15
although to our knowledge no previous studies have reported elevation of total serum IgG4 in patients with pemphigus. In the current study, we have shown that serum IgG4 is enriched in patients with pemphigus compared with unaffected individuals (). Dsg-specific autoantibodies comprise a median of 7·1% and 4·2% of total serum IgG4 in patients with PV and PF, respectively (). Overall, an eightfold enrichment of anti-Dsg3 antibodies and a fourfold enrichment of anti-Dsg1 antibodies were observed in the serum IgG4 vs. the serum IgG1 fractions in patients with PV and PF.
In some patients, antigen-specific antibodies comprised the majority of serum IgG4, which could account for the elevation of total serum IgG4. However, in other patients with elevated serum IgG4, Dsg-specific antibodies did not comprise the majority. It is possible that in these patients antibodies against other keratinocyte autoantigens (such as Dsg1 in mucocutaneous PV, or other keratinocyte cell surface antigens) may contribute to elevation of serum IgG4. Alternatively, chronic cutaneous antigen stimulation may generally skew the immune response towards a hyper-IgG4 state (discussed further below).
Why enrichment of autoantibodies in the IgG4 fraction occurs in pemphigus has not been well established. The variable region of an antibody is formed in developing B cells, before exposure to antigen, through a process of germline DNA rearrangement known as VDJ recombination. After the variable regions have recombined, the constant region of the antibody is joined to the variable region through further germline DNA rearrangement. Initially all B cells produce IgM, but subsequently they may undergo class switch recombination to a new isotype (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgE). Due to the order of constant region genes in chromosomal DNA, if a variable region recombines initially with a proximal constant region, such as IgG1, it can later recombine again to a distal constant region, such as IgG4. However, because DNA recombination involves irreversible excision of intervening DNA, once a variable region recombines with a more distal constant region, such as IgG4, that variable region can never switch back to a more proximal isotype such as IgG1. IgG1 is the most abundant serum IgG subclass and is associated with an interleukin (IL)-4/T-helper2 (Th2) immune response.23
Repeated antigenic exposure can encourage subsequent isotype switching. IL-4 and IL-13 promote isotype switching first to IgG4 and subsequently to IgE.24
IL-10 may potentiate IL-4-induced switching to IgG4 over IgE.25
Upregulation of the Th2 cytokines IL-4, IL-10 and IL-13 has been described in patients with pemphigus.26,27
These cytokines would promote an IgG4 > IgG1 serum autoantibody profile, coinciding with the observed autoantibody IgG subclasses in patients with active disease.
IgG4 autoantibodies are predominant in other chronic autoimmune conditions, such as bullous pemphigoid, cicatricial pemphigoid, epidermolysis bullosa acquisita, thrombotic thrombocytopenic purpura, autoimmune sclerosing pancreatitis and Mikulicz’s disease.28–33
Elevated serum IgG4 is also observed in immune conditions characterized by repetitive antigen exposure, such as in beekeepers and individuals undergoing allergic desensitization therapy.19
These observations support the hypothesis that IgG4 is potentially a specific marker of antibodies resulting from chronic immune reaction against antigen, or autoantigen in the case of autoantibody-mediated diseases.
The enrichment of anti-Dsg antibodies in the IgG4 fraction raises the question of whether IgG4 could serve as a therapeutic target in pemphigus, for example through subclass-specific immunoadsorption or by selective depletion of surface-IgG4 positive memory B cells. Currently there are no therapies approved by the Food and Drug Administration for pemphigus, which is life-threatening if left untreated.34
Most therapies for pemphigus rely on general immune suppression, which is associated with significant side-effects including fatal infection and secondary cancers. Thus, a major frontier for autoimmunity research is to identify and target only the disease- causing antibodies. IgG4 is a potentially attractive therapeutic target, as it is the least abundant IgG subclass, and unlike other IgG subclasses it does not activate complement and therefore is thought to be relatively unimportant for fighting infection. Although selective IgG4 deficiency has been associated with recurrent upper respiratory tract infections in some patients, other studies have estimated that up to 50% of children have no detectable serum IgG4 and are otherwise healthy.35,36
Our finding that patients with PV and PF have lower overall total IgG levels compared with unaffected individuals () suggests that current immunosuppressive therapy generally targets all subclasses, but does not correct the IgG4 enrichment in pemphigus. Therefore, selective depletion of IgG4 or the cells that produce it could represent a novel and safer strategy for the treatment of pemphigus.
Demonstrating the feasibility of this approach, we have found that IgG4 depletion reduces the pathogenic activity of pemphigus sera. IgG4 depletion reduces keratinocyte dissociation by PV IgG by a mean of 81%, indicating that pathogenic antibodies are preferentially enriched in the serum IgG4 fraction (). One remaining question is whether remaining Dsg-specific IgG1 could perpetuate active disease in patients who are depleted of IgG4. Our studies suggest that IgG4 is the major pathogenic IgG subclass in patients with PV relative to the other subclasses, as PV IgG depleted of IgG4 (which would contain the IgG1, IgG2 and IgG3 subclasses) demonstrated pathogenicity similar to a negative control (). Prior studies have shown that patients in clinical remission, and even unaffected relatives of patients with pemphigus, can express Dsg-specific IgG1 without evidence of clinical disease. 12–15
Additionally, if IgG1 autoantibodies subsequently switch to IgG4 with chronic active disease, IgG4 depletion strategies will ultimately capture these pathogenic autoantibody populations. We are currently pursuing studies to investigate the genetic relationships of pathogenic and nonpathogenic antibodies among the different IgG subclasses in patients with pemphigus at the mAbs level. Even if some pathogenic antibodies reside in the IgG1 subclass, sparing of the IgG1 population could be considered a prudent compromise in designing treatment strategies for chronic autoimmune diseases that are currently without a cure. If IgG4 depletion is safe, then chronic IgG4 depletion may be preferable to pan-B-cell depletion therapies that risk fatal infection and often require repeated infusions for maintenance of disease control.