Some immature B cells bearing an autoreactive B cell receptor become deleted by central tolerance mechanisms operative in the bone marrow. The immigration of immature B cells into the spleen occurs through the terminal branches of the central arteriole that drains into sinusoids of the MZ (21). After migration to the splenic follicles, non-self-reactive IgM+/IgD− immature B cells penetrate the MZ sinus to migrate through the interface between the periarteriolar lymphoid sheaths and B cell follicles, and eventually become IgM+/IgD+ naive mature B-2 or MZ B cells. This process is strongly dependent on high concentrations of the cytokine BAFF, which is expressed in splenic follicles. Most self-reactive immature B cells that passed central tolerance induction are energized or deleted in the spleen before they reach the BAFF-rich follicles. This peripheral tolerance mediating process relies on the activation of self-reactive B cells by autoantigens, which results in arrest of migration before these cells can enter the splenic follicle, and as a consequence they die as a result of BAFFdeprivation (21). Despite this, autoantibodies are also found in healthy individuals (22, 23), suggesting that some autoreactive B cells escape both the central and peripheral tolerance inducing mechanisms.

Mature B cells recirculate through the blood and accumulate in the follicles of secondary lymphoid tissues (24). Interestingly enough, mature B cells express a certain degree of migrational diversity. While all mature B cells are found in systemic and mucosal secondary lymphoid tissues, only a subpopulation of mature B cells recirculate through the bone marrow, a process of unknown biological significance, but which is clearly well controlled. In mice, these B cells express CD22, an Ig superfamily member serving as an adhesion receptor for sialic acid-bearing ligands. CD22 binds to CD22-ligands specifically present on sinusoidal endothelial cells in the bone marrow, but not on endothelial cells in other lymphoid tissues (25). A heterogeneous population of B cells also recirculates through the skin, as has been shown in animal models (26). These B cells express typical skin-homing receptors, e.g., ligands for E-selectin and α-4 and β-1 integrins, and exhibit a different immunophenotype than lymph node B cells. It is thought that these cells activate T cells at the site of inflammation and can increase local antibody production (26). However, the possibility that skin-homing B cells contribute to autoimmune skin diseases remains to be established.

After stimulation with their cognate (self-) antigen, mature B cells eventually may form short-lived plasma cells, memory B cells, or long-lived plasma cells (27, 28). As discussed below, these cell types markedly contribute to the pathogenesis of autoimmune diseases and exhibit a specific response to distinct therapeutic approaches.

# Memory B Cells

Memory B cells are formed within germinal centers and differ from naive B cells with respect to several features. While many human and murine memory B cells still express IgM, a significant proportion has already switched to the production of downstream antibody subclasses, mainly IgA and IgGs (29–31). Antigenmediated crosslinking of membrane IgG provides a much stronger activation signal than IgM (32), which contributes to the reduced activation threshold observed for memory B cells and their capacity to quickly give rise to antibody-secreting plasma cells (33–36). Independent from their subclass, memory B cells have down-regulated the expression of genes that negatively control BCR signaling and have up-regulated the expression of their counterparts (37, 38). Moreover, memory B cells also express higher affinity B cell receptors, which not only strengthens the effector functions of the antibodies secreted by their plasma cell progeny, but also allows memory B cells to sense very low antigen doses. Consequently, memory B cells resemble very powerful antigen-presenting cells (APCs) (39, 40). While dendritic cells definitively represent the most important APC during the initiation of the immune response, memory B cells may take over later. Hence, antigen-presentation by memory B cells might be of particular importance during the later phases of chronic immune reactions, such as autoimmune diseases. Human, but not murine, memory B cells show increased expression of CD27. The interaction of CD27 with its ligand on T cells, CD70, promotes the differentiation of activated memory B cells into plasma cells (41). Compared with naive cells, human memory B cells also exhibit a distinct expression profile of homing molecules; these molecules help B cells to interact with T cells and present their cognate antigen in the context of MHC class II in an optimal manner (42–47). The capacity to efficiently interact with T cells is crucial to quickly boosting both the formation of plasma cells and antibody secretion, but likely also increases the capacity of memory B cells to modulate T cell responses.

In conclusion, memory B cells are optimized to interact with T cells and to yield strong antibody responses even in response to relatively little stimulation. Autoreactive memory antibodies are likely to contribute to the chronic and progressive course typically observed for autoimmune disorders (48). High frequencies of memory B cells are associated with poor clinical responses to RTX treatment (49). RTX treatment results in the efficient depletion of memory B cells in peripheral blood, and relapse after this treatment is thought to be associated with the repopulation of IgD-CD27− and IgD-CD27+ memory B cells (50). However, RTX may not deplete the whole B cell memory compartment. RTXtreated patients can generate robust recall responses to repeated influenza vaccination, as indicated by the increase in serum antibody levels and peripheral blood plasmablast frequencies (51). This memory response is comparable to that observed in healthy controls. Hence, a significant fraction of memory B cells seems to be resistant to RTX treatment and is most likely localized in lymphoid or inflamed tissues (51). In mice, distinct layers of memory B cells have been identified, suggesting that the memory compartment is much more complex and diverse than expected (52, 53). Similarly, in human donors, distinct subpopulations of memory B cells that distinctly express homing receptors, such as CXCR3, have been observed in blood (46, 54). In response to IFN-γ, activated B cells upregulate CXCR3, which thereafter is stably expressed on their memory B cell progeny (46, 54). These findings may indicate that a subpopulation of memory B cells is formed under inflammatory conditions, such as during an autoimmune disease flare-up, which then might be able to migrate into inflammatory tissues where they are relatively protected from therapeutic intervention.

# Short-Lived and Long-Lived Plasma Cells

Successful activation of naive B cells leads to a massive clonal expansion and eventually the formation of short-lived plasma cells in the extra-follicular areas of secondary lymphoid tissues (55). In mice, these cells exhibit lifetimes of less than a week (56). Cytostatic drugs, such as cyclophosphamide, prevent B proliferation and the formation of new antibody-secreting cells, hence eliminating the short-lived plasma cell compartment (57). Simultaneous to the formation of short-lived plasma cells, germinal centers may give rise to both memory B cells and long-lived plasma cells. For a brief period following vaccination, precursors of long-lived plasma cells (plasmablasts) are found in human peripheral blood (54); these cells then give home to deposit tissues, such as bone marrow, mucosal tissue, or sites of inflammation. The tissue homing of these cells is tightly regulated by adhesion molecules, chemokines, and their receptors (58–60). In peripheral blood, plasma cell subpopulations are found that exhibit different expression profiles of homing receptors. For example, migratory plasmablasts, induced by intracutaneous vaccination, express L-selectin, which are associated with homing to peripheral lymph nodes (61, 62). Similar to the return of locally activated plasma cell precursors to mucosal sites, cells that are activated in the context of a pro-inflammatory response seem to be programmed to relocate to inflamed tissues. Here, the high levels of inflammatory cytokines can support plasma cell survival, likely enabling these cells survive for the period of inflammation (60). These data suggest that plasma cells formed in the course of an inflammation-associated immune response can maintain the production of antibodies but will disappear when the cause of inflammation has resolved (46, 63).

In human tissues, various plasma cell subpopulations exist which exhibit different phenotypes and stages of differentiation (46, 64, 65). Some plasma cell stages still express CD20, indicating that they might be susceptible to RTX treatment. However, mature human plasma cells have lost CD20 expression (66–68). Accordingly, even B cell depletion has been found to have no impact on the production of human memory antibodies, e.g., specific for tetanus toxoid (63). In general, serum antibodies of different specificities show a great variety of responses following B cell depletion, ranging from no response to depletion below the detection limit (69), possibly suggesting that antibodies are maintained by various mechanisms, including short-lived plasma cell populations that are replenished by various memory B cell subsets and by long-lived plasma cells. The notion that long-lived plasma cells contribute to the production of autoantibodies (57), but are not affected by conventional immunosuppressive drugs such as steroids or cyclophosphamide, or by B cell depletion, has identified them as a novel target cell requiring specific therapeutic approaches (70).

# B Lineage Cells Exert Multiple Roles That Drive Pathogenesis but Also Control the Severity of Inflammation

B cells play multiple functions. Following differentiation into plasma cells, they secrete huge amounts of antibodies into the body fluids. Autoantibodies can contribute to the pathogenesis of autoimmune diseases in multiple ways (71). They can initiate immune-complex-mediated inflammation, deplete specific cell types or modulate important signaling pathways, relevant in SLE, antibody-mediated hemolytic anemia or Hashimoto's thyroiditis and Graves' disease, respectively (72–75). However, more recent studies indicate that antibodies can also exert significant anti-inflammatory effects, which limit or even inhibit autoimmune pathogenesis. The pro- and anti-inflammatory effects of antibodies depend on their isotype (76) and on their Fc *N*-linked glycosylation patterns (77, 78). While IgGs with low levels of galactosylation promote inflammation, sialylated IgGs have a strong anti-inflammatory capacity (79, 80). Highly glycosylated IgG antibodies have been shown to inhibit autoimmune inflammation in mouse models (76, 81, 82). Changes in autoantibody glycosylation have been observed during the course of human autoimmune diseases, possibly providing an interesting novel diagnostic tool, but also suggesting that the antibody glycosylation pattern can alter the clinical course of autoimmune disorders (83).

B lineage cells can also present antigens in the context of MHC II and secrete immunomodulatory cytokines, thereby playing a prominent role for the modulation of antigen-specific T cell responses (84–86). B cells probably only play a minor role in T cell priming. However, during secondary or chronic immune reactions B cells resemble very potent APCs that drive the expansion of activated T helper cells (87–90). Hence, B cells are likely to be both important antigen-presenting and T cell promoting cells during chronic and repeated immune reactions, such as occurs during the course of autoimmune diseases.

In addition, various B lineage cells, including those with a CD138+ plasmablast/plasma cell phenotype, have been shown to express a variety of pro-inflammatory cytokines that can stimulate innate effector cells and significantly contribute to inflammation and immune protection in murine models (91–93). Moreover, while some B lineage cells promote inflammation, others exhibit profound immunosuppressive capacities (94). As shown in many models, B cells and plasma cells can also suppress autoimmune inflammation through the production of cytokines, such as IL-10, TGF-beta, or IL-35 (86, 95–98). Interestingly, IL-10 and IL-35 are produced by B cells that have different phenotypes (98). As discussed below, the therapeutic induction of immunosuppressive B lineage cells may be an interesting direction for the development of future therapies.

# LESSONS FROM RTX TREATMENT OF AUTOIMMUNE DISEASES

Regarding the clinical use of B cell targeting therapies, the majority of the information comes from studies using RTX. This chimeric mouse/human monoclonal antibody targets the pan B cell marker CD20, a transmembrane protein expressed on all B lineage cells, from early pre-B to mature B and memory B cells. CD20 has been shown to mediate Ca2+ influx across plasma membranes and is important in maintaining intracellular Ca2+ concentration and activation of B cells (99). RTX was the first anti-CD20 antibody approved by the U.S. Food and Drug Administration for medical use in 1997 as Rituxan®, originally to treat B cell non-Hodgkin lymphomas (100). Later it was also approved for use in RA, granulomatosis with polyangiitis, and microscopic polyangiitis and there is growing clinical use of RTX in other autoimmune diseases, such as MS, SLE, and autoimmune blistering skin diseases (101–105).

# Mode of Action of RTX

After binding of RTX to membrane bound CD20 it mediates strong complement-dependent cytotoxicity directed to its target cell due to the enhanced clustering of antibody Fc regions (106, 107). Based on its ability to redistribute CD20 into lipid rafts, which provides the molecular basis for RTXs engagement of complement factors, RTX is classified as a type I anti-CD20 antibody. By contrast, type II antibodies cannot cause this redistribution of CD20 (108–111), do not induce complement-dependent cytotoxicity to the same extent (111), but appear to induce a greater degree of directly induced, non-apoptotic cell death, upon binding to target cells (112).

CD20 expression is lost during differentiation into mature antibody-secreting plasma cells (66–68). This lack of CD20, particularly on long-lived plasma cells, explains why RTX treatment does not interfere with the production of memory antibodies, such as anti-tetanus/-measles/-mumps/-rubella (63, 113). Depending on the stage of plasma cell differentiation and tissue localization, early plasma cells (plasmablasts) exhibit various levels of CD20 expression (114). While mature long-lived plasma cells are apparently not depleted by RTX, it is not known to which extent earlier plasma cell stages are affected.

Independent of disease, RTX leads to the depletion of peripheral B cells from approximately 90% to close to 100% (**Table 1**). Despite this fact, the clinical efficacy of RTX varies broadly among different autoimmune diseases, and also among individual patients. Following withdrawal of RTX treatment, B cell levels recover within 6–20 months, with the rate of recovery greatly varying between individual patients (115).

# Use of RTX in Pemphigus Vulgaris (PV)

RTX has shown promising results in the treatment of PV, an autoantibody-driven blistering skin disease. In three independent clinical studies, comprising a total of 43 patients with this rare autoimmune disorder, RTX treatment achieved a complete remission in over 80–95% of patients (103, 116, 117) who, importantly, were refractory to steroid therapy. This impressive clinical outcome was achieved in parallel with B cell depletion close to undetectable levels in almost all patients. One study showed that in most, but not all patients, the levels of anti-keratinocyte cell-surface IgG4 autoantibodies dropped to undetectable levels (116). In another study, anti-desmoglein 1 and 3 (Dsg1/3) autoantibodies were measured and found to be reduced on average by 65–80% (103). Generally, the response to RTX treatment seems to correlate with the extent of B cell depletion. However, two treated patients showed clinical remission despite persistent high levels of anti-Dsg1/3 autoantibodies, although remission was delayed compared with that in patients who showed remarkably reduced autoantibody levels (103). Hence, a reduction in the levels of autoantibodies seems to be a major factor in the success of RTX in treating PV. Nevertheless, the finding that RTX can improve clinical symptoms in patients that still exhibit high levels of anti-Dsg1/3 autoantibodies, suggests that RTX can also act *vi*a additional mechanisms. It would be interesting to consider this concept while designing future clinical studies.

# Use of RTX in RA

Studies using RTX in RA have provided additional evidence that its therapeutic efficacy is not merely based on the reduction of autoantibody levels. In this disease, RTX is recommended for use in patients refractory to standard therapy (118). Although the B cell depletion rate is almost 100%, a clinical response was observed in approximately 60–70% of patients (11, 105, 119). Considering that these patients did not respond to other therapies, this was deemed to be a significant success. Results from more recent studies have suggested that RTX has better long-term efficacy when used in patients with fewer previous treatments and lower disease activity (120). Interestingly, the clinical response to RTX in RA positively correlates with the presence of anti-CCP autoantibodies, but is inversely correlated with the IgG-levels present before treatment (121–124). Autoantibodies and IgG-levels together with serum IL-33 have also been reported to predict the clinical response to RTX (125).

Based on the correlation between serum autoantibodies and response to RTX, on first view it seems possible that suppression

#### Table 1 | Efficacy of RTX treatment varies.

*RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; PV, Pemphigus vulgaris; MS, multiple sclerosis; ACR, American College of Rheumatology Criteria, standard criteria to measure the effectiveness of arthritis treatments in clinical trials for rheumatoid arthritis, 20/50/70 refers to the improvement in tender or swollen joint counts as a percentage; HACA, human anti-chimeric antibodies; CCP, cyclic citrullinated peptides; RF, rheumatoid factor; MTX, methotrexate; MMF, methyl mofetil; Std, standard therapy; BILAG, British Isles Lupus Activity Group, organ-specific 86-question assessment based on the principle of the clinical intent to treat; SLEDAI, SLE Disease Activity Index, a list of 24 items (16 clinical items including seizure, psychosis, organic brain syndrome, visual disturbance, other neurological problems, hair loss, new rash, muscle weakness, arthritis, blood vessel inflammation, mouth sores, chest pain that worsens with deep breathing, and manifestations of pleurisy and/or pericarditis and fever, and eight laboratory results, including urinalysis testing, blood complement levels, increased anti-DNA antibody levels, low platelet count, and low white blood cell count); EDSS, Expanded Disability Status Scale is a method of quantifying disability in multiple sclerosis and monitoring changes in the level of disability; CDP, delayed time to confirmed disease progression; Dsg, desmoglein; cANCA, anti-neutrophil cytoplasmic antibodies; MOG, myelin oligodendrocyte glycoprotein; aab, autoantibodies; dsDNA, double-stranded DNA.*

Hofmann et al. Targeting B Cells

of autoantibodies is a relevant mechanism by which RTX affects the clinical outcome of RA. The presence of autoantibodies such as rheumatoid factor (RF) and anti-CCP correlate well with disease activity in RA patients and indicate the presence and activation of autoreactive B cells. Moreover, anti-CCP and IgM-RF can enhance pro-inflammatory macrophage functions *in vitro* (126), supporting the idea that these autoantibodies may also contribute to RA pathogenesis *in vivo.* However, the reduction of serum autoantibody levels in RTX-treated responders is only very moderate, i.e., approximately 50 and 30%, for anti-CCP and RF, respectively, with a high degree of variation being observed (69). It seems questionable that such minor reductions in autoantibody levels cause the clinical response. Moreover, the absolute autoantibody levels persisting in responders after treatment were still two to fivefold higher than in non-responders (69). If autoantibodies in responders and non-responders exhibit similar pathogenicity, the moderate depletion of autoantibodies in responders to levels above of non-responders would not explain the success of the therapy. Other studies have also shown very moderate RTX effects on IgA and IgG anti-CCP autoantibodies, suggesting that a significant proportion is produced by long-lived plasma cells (127). The question of whether these antibodies contribute to RA pathogenicity remains to be addressed.

# RTX Effects on CD4 T Cells

Alternative explanations for the anti-inflammatory effects of RTX in RA patients include the suppression of inflammatory T cells in favor of regulatory T cells subsets, possibly in combination with an induction of high galactosylated anti-inflammatory antibodies. There is good evidence that CD4 T cells can activate myeloid and synovial cells, which in turn activate and recruit macrophages to synovial tissue, eventually leading to joint inflammation and cartilage destruction, whereas regulatory T cells are protective (128–131).

The production of pro- and anti-inflammatory cytokines such IFN-γ, IL-17, or IL-10 seems to be key for T cell mediated control of RA pathogenesis. There is evidence that the clinical response to RTX therapy in RA is associated with lower IFN-γ levels (132). Moreover, a study including 52 patients showed that RTX often induces a reduction in the number of peripheral blood CD4 T cells, an effect that was strongly associated with the clinical response (133). It is possible that the depletion of a small subpopulation of CD20+ T cells contributes to this effect (134).

However, there is good evidence that B cells can efficiently promote CD4 T cell functions through antigen-presentation and cytokine release. As discussed above, while B cells may play only a minor role in T cell priming, during secondary or chronic immune reactions, B cells can act as very potent APCs that drive the expansion of activated T helper cells (87–89, 135). Hence, B cell depletion may impair the formation, clonal expansion, and function of T memory cells.

Accordingly, T cell activation in the synovium of RA patients has been reported to be dependent on B cells (136). Hence, it is highly likely that RTX mediates its beneficial effects in RA at least partly through the depletion of T cell stimulating B lineage cells, although this remains to be studied further.

# Effects of RTX Mediated B Cell Depletion on Cytokines

Of note, under certain conditions, B cells can also directly promote innate inflammatory effector cells through the production of cytokines such as IFN-γ, IL-6, granulocyte-macrophage colony-stimulating factor, and IL-17 (91, 93, 137, 138). In mice, B cell ablation has been shown to ameliorate autoimmune diseases by depleting IL-6-producing B cells (93). Moreover, a recent study has provided evidence that B cells from RA patients show abnormal IL-6 signaling and altered cytokine production, and that this may contribute to disease (139). Hence, B cells resemble an underestimated source of inflammatory cytokines and the depletion of such pro-inflammatory cytokine producing B cells is likely to contribute to the therapeutic outcome of RTX treatment.

# RTX Effects on Antibody Glycosylation

T cell help in germinal centers has been found to be important for the induction of inflammatory low-glycosylated IgG antibodies (82). In particular, the T cell-derived cytokines such as IFN-γ and IL-17 are capable of synergistically promoting the production of low-sialylated, and potentially pathogenic, antibodies (82, 139). Based on the notion that the clinical response to RTX in RA is associated with lower IFN-γ levels (132), it is possible that a reduction in IFN-γ levels, in turn, leads to alterations in antibody glycosylation.

In accordance with the idea that these processes are of pathophysiological relevance, reduced antibody glycosylation has been reported to precede disease onset and to correlate with disease activity in RA patients (140). This phenotype is highly prevalent in, but not restricted to, autoantibodies (141). Hence, it is possible that acting through a reduction in IFN-γ levels, RTX corrects the shift toward the production of pro-inflammatory antibodies that is observed in RA. Of note, high-glycosylated antibodies can mediate anti-inflammatory effects independent of their specificity, as indicated by the therapeutic effects of intravenously administered immunoglobulins (142), although antigen-specific effects might be considerably stronger and require lower antibody concentrations (79, 81, 143). Accordingly, highly glycosylated IgGs have been reported to induce antigen-specific tolerance (78). Hence, RTX may partly mediate its beneficial effects through changes in antibody glycosylation, either *via* a reduction in autoantibody pathogenicity, through the generation of antiinflammatory autoantibodies, or by the induction of persistent high levels of anti-inflammatory total IgGs that mimic continuous IVIG treatment.

# Effects of RTX in MS and SLE

Similar to what has been observed in RA, the therapeutic effect of RTX in SLE and MS is variable. Its impact on total antibody levels as well as on autoantibody levels shows a high degree of diversity (**Table 1**). In a recent study, only 11 out of 32 SLE patients with IgG hypergammaglobulinemia before treatment showed reduced IgG-levels after 12 months of treatment (144). Likewise, a reduction in anti-double-stranded DNA levels was incomplete, with high inter-individual variety and differences between antibody subclasses (145–148). Despite homogenous B cell depletion rates in MS of over 90 and 95% in spinal fluid and in the periphery, respectively, the disease outcome showed great variation (104, 149–151). Interestingly, RTX has even been found to worsen the clinical outcome of MS (104).

These variable results might be not be surprising in the light of the finding that B lineage cells play multiple pro-and anti-inflammatory roles in experimental autoimmune encephalomyelitis (EAE), a murine model of MS. B cell-derived IL-6 has been shown to be crucial for the initiation of EAE, suggesting that B cells can promote MS pathogenesis through the production of this pro-inflammatory cytokine (93). However, there is an abundance of evidence that anti-inflammatory B cell subsets can also efficiently suppress CD4 T cells mediating neuroinflammation, and that these effects are mediated by B lineage-derived IL-10, TGF-β, and IL-35 (98, 152). These findings led to the concept of regulatory B cells (Bregs), which, however, have never been clearly defined. Recent results indicate that these IL-10+ B lineage cells have a plasmablast phenotype (98, 153). Similarly, investigations conducted by our group have identified plasmablasts/plasma cells as an important source of IL-10, capable of suppressing skin inflammation in a murine model of epidermolysis bullosa acquisita (EBA) (85). In EAE, B lineage-derived IL-6 and IL-10 were shown to have an impact on the induction and resolution of inflammation, respectively (93, 98, 153). These findings may partly explain the heterogeneity of the clinical response to RTX observed in MS. Depending on the major role of B lineage cells as drivers or inhibitors of inflammation in individual patients, and possibly related to timing, RTX may be either beneficial or worse for the clinical course of MS.

# ALTERNATIVE B CELL TARGETING APPROACHES

# Second Generation Anti-CD20 Antibodies

The great clinical success of the chimeric antibody, RTX, has stimulated the development of the second generation anti-CD20 antibodies, ocrelizumab, obinutuzumab, veltuzumab, and ofatumumab (154). These second generation anti-CD20 antibodies are humanized or even fully human, exhibit improved effector functions, and compared with rituximab show greater potential *in vitro*. Due to these properties, they are expected to be more effective, to exhibit lower immunogenicity, and to be better tolerated. However, these expectations, which may be validated by head-to-head trials of these second generation anti-CD20 antibodies and RTX, have not been confirmed till date.

Ocrelizumab has recently been approved for the treatment of relapsing-remitting MS, and is the first approved treatment for primary progressive MS. In RA, however, ocrelizumab seems to have no benefit over current treatments, leading to a halt in the development of ocrelizumab for the treatment of RA (155).

Clinical trials exploring the use of obinutuzumab, veltuzumab, and ofatumumab in autoimmune disorders are at various stages of development and have been extensively reviewed by Du and colleagues (154). In general, it appears that the new anti-CD20 antibodies are effective against autoimmune diseases, against which RTX is also beneficial, but more studies are needed to evaluate their efficiency and long-term safety profiles in greater detail.

# Antibodies Targeting CD19

Other B cell depleting reagents that promise a great potential for the treatment of autoimmune diseases are a series of reagents targeting CD19, which are currently under development. These include humanized antibodies, "bi-specific T cell engagers," and antibody-drug conjugates, such as inebilizumab, blinatumomab, and SAR3419, among several others (156, 157).

CD19 targeting therapeutics generally exhibit a broader target spectrum than anti-CD20 based reagents. CD19 is expressed very early in B cell development, being evident already on pro-B cells and on all later B cell stages, whereas CD20 is expressed later, starting at the immature B cell stage. Possibly of greater importance for the treatment of autoimmune diseases, in addition to all mature B cell subsets, CD19 is expressed on a significant proportion of plasmablasts and plasma cells, particularly outside the bone marrow (64, 114). Accordingly, CD19 targeting reagents have been reported to deplete pre-existing peripheral antibodysecreting cells, at least in humanized mouse models (158). Hence, CD19 targeting reagents might be not only suitable for treating B cell malignancies, but also exhibit great potential for therapeutic use in autoimmune diseases, particularly for those diseases with a strong involvement of pathogenic antibodies, e.g., SLE, pemphigus, and neuromyelitis optica.

# BAFF/APRIL Antagonists

An alternative therapeutic strategy to target B cells is the blockade of B lineage survival factors, such as BAFF and its homolog APRIL and their receptors (159). Together, BAFF and APRIL along with their receptors form a complex system, which is very important for the survival of mature B cells and plasma cells. There are three receptors that bind BAFF and APRIL with different affinities. BAFF binds to BAFF-R, transmembrane activator and calcium modulator and cyclophilin ligand interactor, and B cell maturation protein. These three receptors are differentially expressed at various times during B cell ontogeny (160). Most BAFF circulates as a soluble active homo-trimer (161) that binds to BAFF-R and this interaction is required for survival of late transitional, MZ, and mature naive B cells, all of which are depleted by BAFF-blockade (162, 163).

Several BAFF/APRIL targeting drugs are currently under development. The humanized anti-BAFF antibody belimumab has already been approved in the EU and the USA for the treatment of adult patients with active, autoantibody-positive, and SLE despite standard therapy. It is generally well tolerated with low rates of immunogenicity. Belimumab in combination with standard therapy reduces the overall disease activity and the incidence and severity of flares, has steroid-sparing effects, and can maintain disease control for at least 10 years (164). Interestingly, despite its clinical efficiency, belimumab only partly inhibits the production of IgG-autoantibodies. While some studies found no reduction of IgG-autoantibody levels following treatment with belimumab, others have reported a decline of 40–60% within 2–7 years of treatment (165–167). This is in accordance with the finding that belimumab depletes both naive and activated B cells, but not memory B cells (165, 167).

Similar to what has been observed with RTX, B cell targeting by blockade of BAFF and APRIL using belimumab, tabalumab, or atacicept also shows greatly variable effects on the levels of autoantibodies and the clinical outcome of autoimmune diseases (118, 168–181), and of note, seem to have only limited effects on the production of (auto)antibodies (**Table 2**).

Generally, B cell targeting represents a powerful strategy for the treatment of autoimmune diseases. Its mechanism of action seems to be diverse and complex, and needs further elucidation.

# DIRECT TARGETING OF PLASMA CELLS BY BORTEZOMIB

As originally shown by us and others, in mice, memory antibodies are secreted by long-lived plasma cells (182, 183). The notion that these cells can contribute to the production of autoantibodies but do not respond to current therapeutic approaches (57), has led to the search for novel plasma cell targeting agents.

The small molecule proteasome inhibitor bortezomib promotes plasma cell apoptosis and is approved for the treatment of multiple myeloma. Nearly a decade ago, Voll and colleagues reported that this drug could also be useful for the treatment of antibody-mediated autoimmune diseases. They demonstrated that bortezomib efficiently depletes both short-lived and long-lived plasma cells and protects mice with lupus-like disease from nephritis (70). Its efficacy was later proven in various models of antibody-mediated autoimmune diseases (184–186). There is now increasing evidence that bortezomib can also efficiently deplete autoantibodies in patients, resulting in the improvement of clinical symptoms, as has been described for refractory primary Sjögren's syndrome, refractory SLE, thrombotic thrombocytopenic purpura, and among others (9, 187–197). The potential development of severe side effects, such as peripheral neuropathies may limit the use of bortezomib in autoimmune diseases. However, its unique capacity to deplete antibody-producing plasma cells suggests that the safety and efficacy of bortezomib should be evaluated in clinical trials including more patients who are refractory to standard therapeutic approaches.

# INDUCTION OF ANTI-INFLAMMATORY B LINEAGE CELLS: A PROMISING THERAPEUTIC TREATMENT OPTION?

IL-10+ B lineage cells have been known as potent suppressors of autoimmune inflammation for decades (198). Over the last decade, the expansion of IL-10+ B cells using various approaches has been shown to efficiently suppress both autoimmune and allergic inflammation in numerous models (85, 199, 200). The first report that IL-10+ B cells exert a suppressive function was in 2002 by Fillatreau and colleagues (86), who showed that chimeric mice with a B cell specific IL-10 deficiency do not recover from EAE. Later, IL-10+ B cells were termed as Bregs or B10 cells. However, no surface marker or transcription factor unique to these cells has been identified to date. These cells are only functionally defined by their production of anti-inflammatory cytokines such as IL-10, and more recently IL-35, and the resulting suppression of inflammation and autoimmune diseases (201, 202).

# Phenotype and Origin of Human IL-10-Producing B Cells

The combination of markers used to describe "regulatory B cells" in human and mice is controversial. The phenotypic identification of these B cells and their possible origin and development have been excellently reviewed elsewhere (198, 203). In humans, the ability to produce anti-inflammatory IL-10 has been reported in B cells at various stages of development: immature/ transitional B cells (CD19+ CD38hi CD24hi) (204), plasmablasts (CD27int CD38hi) (153, 205), and memory B cells (CD19+ CD27+) (206, 207). It is likely that IL-10+ B cells represent a transient stage with a functional program rather than a terminally differentiated stage, and that any B cell can acquire suppressive properties within a certain environment. Nevertheless, it is debatable if these cells arise from a single shared progenitor, from individual progenitors, or are induced under certain environmental stimuli (198, 203). Interestingly, in this context autocrine IL-10 can promote human IL-10+ B cells to differentiate into IgG- and IgM-secreting plasma cells (208).

# IL-10-Producing B Cells in Patients Suffering from Autoimmune Diseases

The first report describing human IL-10-producing B lineage cells in autoimmune diseases was in 2010 by Iwata et al. (207), who described abnormally high frequencies of peripheral IL-10+ B cells in various autoimmune diseases, such as SLE, RA, MS, Sjögren's syndrome, and blistering skin diseases. An increase in the number of blood IL-10+ CD19+ CD24hi CD38hi cells was also found in PV patients, but these B cells were functionally unable to suppress Th1 immune responses (209). By contrast, several studies on RA (206, 210–212), SLE (204), systemic sclerosis (SSc) (213, 214), and MS (215–217) patients have shown a reduced number of peripheral IL-10-producing B cells compared with that in the controls. This was often accompanied by an impaired suppressive capacity of CD4 T cells. An overview describing the modulation of human IL-10-producing B lineage cells in different autoimmune diseases has been provided by Miyagaki et al. (218).

# IL-10-Producing B Cell Dynamics Following B Cell Targeting Therapy

In myasthenia gravis, depletion of B cells with RTX showed that IL-10+ B cells can be found to have repopulated in the periphery after several months (219). Immunosuppressive treatments, TNF-therapy, and BAFF-blockade in RA (206), SSc (213), and experimental diabetes mellitus type 1 (220), respectively, have shown that IL-10-producing B lineage cells enrich after treatment and that their frequency is even higher than before treatment. In relapsing-remitting MS, the frequencies of IL-10+ CD19+ B cells were significantly reduced in patients experiencing a relapse compared with that in patients in remission (217), indicating that the clinical outcome of the disease also depends on the availability of IL-10-producing B cells.

Moreover, a "good responder" to RTX in myasthenia gravis showed a rapid repopulation of CD19+ IL-10+ B cells after

Targeting B Cells

# Frontiers in Immunology | www.frontiersin.org

#### Table 2 | Efficacy of belimumab, tabalumab, and atacicept.

*RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; SS, Sjörgen's syndrome; MS, multiple sclerosis; ACR, American College of Rheumatology Criteria, standard criteria to measure the effectiveness of arthritis treatments in clinical trials for rheumatoid arthritis, 20/50/70 refers to the improvement in tender or swollen joint counts as a percentage; CCP, cyclic citrullinated peptides; RF, rheumatoid factor; MMF, methyl mofetil; std, standard therapy; BILAG, British Isles Lupus Activity Group, organ-specific 86-question assessment based on the principle of the clinical intent to treat; SELENA, Safety of Estrogens in Systemic Lupus Erythematosus National Assessment; SLEDAI, SLE Disease Activity Index, a list of 24 items (16 clinical items, including seizure, psychosis, organic brain syndrome, visual disturbance, other neurological problems, hair loss, new rash, muscle weakness, arthritis, blood vessel inflammation, mouth sores, chest pain that worsens with deep breathing, and manifestations of pleurisy and/or pericarditis and fever, and eight laboratory results, including urinalysis testing, blood complement levels, increased anti-DNA antibody levels, low platelet count, and low white blood cell count); EDSS, Expanded Disability Status Scale is a method of quantifying disability in multiple sclerosis and monitoring changes in the level of disability; aab, autoantibodies; C3* + *4, Complement factors 3 and 4; ACPA, Anti-citrullinated peptide/protein antibodies; CRP, C-reactive protein; SRI-4, Systemic Lupus Erythematosus Responder Index for 4-point reduction due to SLEDAI; EULAR SS, The European League Against Rheumatism for Sjörgen's Syndrome; Ig, immunoglobulins; dsDNA, double-stranded DNA.*

from 8 to 9 months compared with a "non-responder," where repopulation was delayed (219). This shows that the kinetics of the IL-10+ B cell repopulation is related to the responsiveness to RTX. Similarly, Colliou et al. (221) have shown that RTX-treated PV patients in complete remission had fourfold higher numbers of IL-10+ CD19+ B cells compared with patients in incomplete remission. In SLE patients responding well to RTX treatment, IL-10+ CD24hi CD38hi B cells were found to repopulate and exhibited a restored suppressive function compared to nonresponders (222).

Differences in IL-10-producing B cells in individual patients and types of autoimmune diseases could explain the differential outcome and benefit of B cell specific therapies. For example, in SLE and lupus nephritis, pan B cell therapies, such as RTX, show only moderate or even no benefit, despite the significant role of B cells in this disease (145, 223). The depletion of antiinflammatory B cells could contribute to this unexpected result. Very few studies to date have included an analysis of the kinetics and function of IL-10-producing B cells after B cell depleting therapy. To better understand the individual clinical outcome of the patients and the differences between certain autoimmune diseases treated with the same B cell targeting agent, it would be of great benefit to include an analysis of IL-10+ B lineage cells in further studies.

# Challenges Hampering the Development of IL-10**+** B Cell-Based Therapies

Restoring the regulatory capacity and the number of IL-10+ B cells is a promising therapeutic goal for the treatment of autoimmune diseases. However, currently two unsolved problems hamper the development of a therapy based on IL-10+ B cells. First, the methods used to generate IL-10+ B cells for therapeutic approaches are not suitable for a clinical setting. Second, the identity and phenotype of IL-10+ B cells remain uncertain. Nevertheless, recent progress has been made with respect to both issues. Giacomini et al. (224) have shown that stimulation of peripheral blood mononuclear cells (PBMCs) from MS patients with thymosin-α1 (Tα1) increases IL-10 and IL-35 secretion and expands transitional- and plasmablast-like B cell populations. Upon exposure to pro-inflammatory cytokines, such as IL-21, IL-6, and IL-1β, an expansion of IL-10+ B cell population has been observed. In RA patients, IL-21 increases the number of IL-10-producing B cells in the memory compartment and induces IL-10+ plasmablasts (206), whereas in mice, gut microbiota-derived IL-1β and IL-6 promote the formation of various IL-10+ B lineage cells in the spleen and lymph nodes (225). By contrast, the anti-inflammatory cytokine IL-35 can also induce human B cells to produce IL-10 and IL-35 (226). Nevertheless, inducing anti-inflammatory B cells *in vivo via* inflammatory cytokines bears the risk of undesirable pathogenic side effects by also activating other effector cell types. If not expanded *in vivo*, IL-10+ B cells could be also induced from patient PBMCs *in vitro* and transferred back. Here, the questions of the amount of B cells required to improve clinical symptoms and the stability of the IL-10+ phenotype and function arise. The difficulties and potential of these therapies were recently discussed by Mauri and Menon (227).

# Induction of IL-10-Producing Plasma Cells/Plasmablasts: Potential as a Novel Treatment Option

Progress has been made in defining the identity of IL-10+ B cells that could be used to develop a novel therapeutic strategy. During the last decade, several phenotypically distinct murine B cell subsets have been described that produce IL-10 upon *in vitro* stimulation, which was able to limit autoimmune diseases (198). These cells include B cells with a CD5+ CD1dhi phenotype (B10) (228), CD5+ B cells (B1-a) (229), transitional type 2-MZ precursors (230), and MZ B cells (231).

Of note, the surface markers used to characterize the identity of the IL-10+ B cells change following activation and might be not suitable to define a specific B cell subtype under inflammatory conditions. Interestingly in this context, it has been shown that "B10" cells upregulate the expression of the transcription factors Blimp1 and IRF4 while downregulating that of Pax5, suggesting that these cells undergo plasma cell differentiation. Moreover, upon transfer into recipient mice, "B10" cells become antibody-secreting cells (232). More recently, CD138hi plasmablasts in murine spleen (98) or lymph nodes (153) were described as the major producer of antiinflammatory IL-10 and IL-35 *in vivo* with the ability to limit EAE. In accordance with these findings, we found that IL-10+ plasma cells exhibit profound anti-inflammatory activities in a model of EBA, a rare autoimmune skin disease (85). These cells induce IL-10 expression but reduce IFN-γ production in CD4 T cells, promote IL-10 production by CD4+/Foxp3+ Tregs and suppress neutrophil functions. Hence, IL-10+ plasmablasts/plasma cells represent an important anti-inflammatory B cell subtype.

Identification of the identity of IL-10+ B lineage cells may help to develop a novel method to induce these cells in a therapeutic setting. Stimulation of B cells with CpG-oligonucleotides induces both plasma cell differentiation and IL-10 expression. Accordingly, experimental induction of IL-10+ B lineage cells by adaptive transfer of CpG-stimulated B cells has recently been shown to suppress ongoing EAE inflammation in a therapeutic setting (200). This approach may open a novel perspective for the treatment of inflammatory autoimmune diseases.

# CONCLUDING REMARKS

The success of current B cell targeting therapies emphasizes the important roles B cells play in the pathogenesis of autoimmune diseases. There is overwhelming evidence from animal models indicating that B lineage cells exhibit multiple powerful pro- and anti-inflammatory capacities. The current experience with B cell targeting therapies suggests that these findings also hold true in the clinic. Hence, therapies that specifically deplete pathogenic B cells and plasma cells, or generate immunosuppressive B cells/plasma cells could hold great potential for the treatment of autoimmune diseases. In an optimal setting, the therapy would be tailored to the individual patient based on his/her predicted needs, benefits, and risks.

# AUTHOR CONTRIBUTIONS

KH, A-KC, and RM contributed to reviewing the current literature and writing of the manuscript.

# FUNDING

KH was supported by the Research Training Group 1727 "Modulation of Autoimmunity" (GRK 1727). A-KC was founded

# REFERENCES

by the Clinical Research Unit 303 "Pemphigoid Diseases – Molecular Pathways and their Therapeutic Potential" (CRU303). RM was supported by the Excellence Cluster "Inflammation at Interfaces" (EXC 306/2).

autoimmune and infectious diseases. *Nature* (2014) 507:366–70. doi:10.1038/ nature12979

chemokines, and hematopoietic growth factors. *J Clin Immunol* (2011) 31:89–98. doi:10.1007/s10875-010-9456-8

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Hofmann, Clauder and Manz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Autoantibody Signaling in Pemphigus vulgaris: Development of an integrated Model

*Thomas Sajda and Animesh A. Sinha\**

*Department of Dermatology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, United States*

Pemphigus vulgaris (PV) is an autoimmune skin blistering disease effecting both cutaneous and mucosal epithelia. Blister formation in PV is known to result from the binding of autoantibodies (autoAbs) to keratinocyte antigens. The primary antigenic targets of pathogenic autoAbs are known to be desmoglein 3, and to a lesser extent, desmoglein 1, cadherin family proteins that partially comprise the desmosome, a protein structure responsible for maintaining cell adhesion, although additional autoAbs, whose role in blister formation is still unclear, are also known to be present in PV patients. Nevertheless, there remain large gaps in knowledge concerning the precise mechanisms through which autoAb binding induces blister formation. Consequently, the primary therapeutic interventions for PV focus on systemic immunosuppression, whose side effects represent a significant health risk to patients. In an effort to identify novel, disease-specific therapeutic targets, a multitude of studies attempting to elucidate the pathogenic mechanisms downstream of autoAb binding, have led to significant advancements in the understanding of autoAb-mediated blister formation. Despite this enhanced characterization of disease processes, a satisfactory explanation of autoAb-induced acantholysis still does not exist. Here, we carefully review the literature investigating the pathogenic disease mechanisms in PV and, taking into account the full scope of results from these studies, provide a novel, comprehensive theory of blister formation in PV.

Keywords: pemphigus vulgaris, autoantibodies, signaling pathways, p38MAPK, calcium, epidermal growth factor receptor, Rho GTPases

# INTRODUCTION

Pemphigus vulgaris (PV) is an autoimmune skin blistering disease characterized by the presence of autoantibodies (autoAbs) directed against keratinocyte surface antigens. It has been well established that autoAbs alone are capable of driving blister formation in PV. Early studies identified the primary target of pathogenic autoAbs as desmoglein 3 (Dsg3) and to a lesser extent, desmoglein 1 (Dsg1). More recently, additional autoAbs specificities have been identified in PV patients that could potentially also contribute to disease pathogenicity (1, 2). Despite extensive research, the exact mechanisms through which autoAbs induce a loss of cell–cell adhesion (also termed acantholysis) are not well understood.

Since the primary target of PV autoAbs was shown to be a desmosomal protein, most of the earliest theories of acantholysis suggested that the loss of cell adhesion was simply the result of

*Edited by:*

*Ralf J. Ludwig, Universität zu Lübeck, Germany*

#### *Reviewed by:*

*Robert Gniadecki, University of Alberta, Canada Sergei Grando, University of California, Irvine, United States*

> *\*Correspondence: Animesh A. Sinha<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Immunological Tolerance and Regulation, a section of the journal Frontiers in Immunology*

*Received: 01 February 2018 Accepted: 21 March 2018 Published: 19 April 2018*

#### *Citation:*

*Sajda T and Sinha AA (2018) Autoantibody Signaling in Pemphigus Vulgaris: Development of an Integrated Model. Front. Immunol. 9:692. doi: 10.3389/fimmu.2018.00692*

autoAbs sterically hindering the homo- and heterophillic binding of desmosomal proteins between neighboring cells (3, 4). Studies showing that staphylococcal exfoliative toxins, which cleave Dsg1, could produce blisters similar to those seen in PF, indicated that disturbance of desmosomal proteins was capable of causing a loss of cell–cell adhesion (5, 6). The relationship between desmoglein expression and cell adhesion was further supported by the observation that Dsg3-deficient mice develop mucosal lesions similar to those seen in PV patients (7). Together, these data demonstrated that interference with desmoglein interactions was sufficient to drive a loss of cell–cell adhesion.

The observation by multiple groups that PVIgG was seen only to bind at desmosomal areas also supported the idea that autoAb interference of desmosomal interaction drives blister formation (8–10). In addition, in areas of acantholysis, PVIgG was seen to bind to both desmosomal plaques and split desmosomes (11), indicating that PVIgG could access desmosome-associated desmogleins.

Further support for the steric hindrance theory came from later studies attempting to characterize the fine-epitope specificity of anti-Dsg3 autoAbs. It was shown that autoAbs primarily target the amino terminal of Dsg3 which, based on the crystal structure of other classical cadherins, was predicted to facilitate trans-interaction (12–17). Furthermore, only anti-Dsg3 auto-Abs targeting the amino terminal EC1-2 of Dsg3 could illicit blister formation when passively transferred to neonatal mice, not anti-Dsg3 autoAbs targeting the more carboxyl domains EC3-5. Another study demonstrated that anti-Dsg3 autoAbs preferentially recognize the mature form of Dsg3, but not an immature form, which requires additional proteolytic processing to participate in adhesion (18, 19). It was also shown that autoAb binding was dependent on the proper calcium stabilized formation of Dsg3, which is known to be required for proper adhesive functioning (13, 20, 21). Later experiments using atomic force microscopy (AFM) were able to demonstrate that PVIgG could directly interfere with Dsg3 trans-interaction (22).

One potential liability of the steric hindrance theory was the possibility that blistering effects were mediated by the constant regions of the Abs. However, experiments showing that Fab and F(ab2) fragments, as well as single-chain variable fragments which lack the Fc domain, were able to induce blister formation *in vitro* proved that Fc-dependent mechanisms were not necessary for blister formation (23–26). Additional experiments demonstrating the pathogenicity of PVIgG in C5a-deficient mice indicated that compliment activation was not required for acantholysis (23).

Over time, evidence has accumulated suggesting steric hindrance may not be the primary or sole pathogenic mechanism operative in PV. One of the earliest indications that alternative mechanisms may drive pathogenesis was the observation that IgG from PF patients could induce disease in mice without interfering with trans-adhesion of Dsg1 (27). It was noted in multiple studies that PVIgG was seen to bind extra-desmosomal spaces on the surface of keratinocytes, allowing for the possibility that binding of autoAbs outside of desmosomes may affect disease (3, 28). It was also shown that PVIgG binding induced cytoskeletal changes and the retraction of keratin intermediate filaments before any visible changes in desmosomes (29–33). It was also noted that in early PV lesions keratinocytes first separate at inter-desmosomal areas and desmosomes are still intact and interacting with neighboring desmosomes (29, 34–36). Together, these findings suggested that desmosomal separation may be downstream of other processes induced by the binding of autoAbs. Recently, one research group used AFM to demonstrate that the loss of Dsg3 binding alone was not sufficient to cause a loss of cell adhesion, strongly indicating that steric hindrance by itself cannot sufficiently explain acantholysis in PV (37).

An early alternative to the steric hindrance theory was suggested by results showing that the binding of autoAbs initiated the activation of proteases which in turn degraded Dsg3 and inhibited cell–cell adhesion. Specifically, plasminogen activator was thought to play a role in disease (38). PVIgG was shown to induce signaling that led to increased production of plasminogen activator (39, 40). Furthermore, PVIgG induced keratinocyte expression of plasminogen activator receptor (38, 41). However, inhibition of plasminogen *via* dexamethasone did not prevent PVIgG-induced acantholysis (42). The role of other proteases was also shown not to be essential in disease by the failure of protease inhibitors and gene ablation to prevent blister formation (43, 44).

One of the earliest studies that indicated that autoAbs may exert their pathogenic effect through the activation of intracellular cascades demonstrated that plakoglobin (Pkg)-deficient mice were protected from PVIgG-induced blister formation (45). Pkg, an armadillo family protein, is well established as a major signaling molecule involved in the regulation of cell adhesion (46, 47). The inability of PVIgG to induce blisters in the absence of Pkg strongly suggests that alteration of Pkg signaling is a primary pathogenic mechanism of PVIgG. In addition, keratinocytes incubated at 4°C did not show any effects of PVIgG on cell adhesion, suggesting that the mechanisms underlying blister formation are energy dependent (48).

Identification and characterization of the precise signaling pathways driving autoAb-induced acantholysis has been a significant focus for PV research. As a result, large amounts of (often conflicting) information concerning the signaling alterations downstream of anti-Dsg and PVIgG binding have been characterized. Moreover, studies showing that autoAbs in PV sera directed at non-desmoglein antigens can also elicit intracellular signaling have further complicated efforts to elicit the precise mechanisms driving disease (49, 50). The primary signaling pathways and the evidence that supports their role in PV pathogenesis are reviewed below (see **Table 1** for evidence supporting steric hindrance vs. intracellular signaling).

# SIGNALING PATHWAYS IMPLICATED IN PV

# p38MAPK

p38 is one of the three major families of mitogen-activated protein kinases (MAPK), which are known to play a prominent role in a wide range of cellular pathways (51). In general, p38MAPK proteins can be activated by environmental stress and regulate the transcription of inflammatory cytokines (52). All MAPKs require dual phosphorylation for enzymatic activity, and each contains a

#### TABLE 1 | Evidence supporting steric hindrance vs. intracellular signaling.

#### Evidence supporting the steric hindrance theory

#### Evidence indicating a role for intracellular signaling

characteristic dual phosphorylation sequence which affect both the substrate specificity and ability to auto-phosphorylate (53, 54). There are four types of p38MAPK: α, β, γ, and δ, each displaying unique patterns of tissue expression (52). Only p38MAPK α, β, and δ are known to be expressed in keratinocytes (52, 54–58) and have been primarily associated with differentiation and apoptosis (59).

The significance of p38MAPK signaling in PV pathogenesis was first suggested by a study which observed that PVIgG induced significant increases in the phosphorylation of p38MAPK, MAPKAP2 (MK2), and heat-shock protein (Hsp)27 (60). The degree of phosphorylation was shown to increase when cells were treated with higher concentrations of PVIgG. In addition, treatment of cells with p38MAPK inhibitors was able to prevent PVIgG-induced acantholysis as well as changes in the actin cytoskeleton and the retraction of KIFs from desmosomal attachments, both of which are hallmarks of acantholysis in PV. Inhibition of p38MAPK also prevented PVIgG-induced phosphorylation of MK2, Hsp27, and p38MAPK (60).

Other studies assessing the role of PVIgG on p38MAPK activation identified that PVIgG causes keratin retraction and p38MAPK activation within 30 min, and another peak at 6–10 h in cultured human keratinocytes. Only inhibition at the earlier time point was associated with prevention of blister formation and keratin retraction (61). Another study showed that AK23, a mouse-derived monoclonal anti-Dsg3 antibody, could also activate p38MAPK, demonstrating that autoAb binding to Dsg3 specifically can lead to p38MAPK activation (62).

The relevance of p38MAPK in disease was emphasized by findings that both p38MAPK and Hsp27 are phosphorylated in the lesional skin of PV patients (63). Further studies demonstrated that p38MAPK inhibitor blocks acantholysis *in vivo*, as well as p38MAPK activation, suggesting auto-phosphorylation of p38MAPK (64). p38MAPK activation was also shown to cause Dsg3 internalization, and p38MAPK inhibition can prevent this phenomenon *in vivo* (65). A more detailed assessment of the effects of p38MAPK showed that p38 depletes extra-desmosomal Dsg3 early as 30 min, and also is responsible for later depletion (2–24 h) of other desmosomal cadherins as well as DP (66–70).

The regulation of cytoskeletal changes by p38MAPK is especially relevant in understanding how p38MAPK plays a role in PV pathogenesis. In epithelial cells, cell detachment has been shown to induce p38MAPK activation (71), indicating a close relationship between p38MAPK and cellular adhesion. Furthermore, p38MAPK activation can lead to phosphorylation, and subsequent destabilization of keratin intermediate filaments (72), which could be one explanation for the characteristic retraction of KIFs seen in PV. p38MAPK is known to regulate actin filaments as well (73). Since extra-desmosomal Dsg3 complexes with actin cytoskeleton and is required to bring DP to desmosomal plaques (74, 75), it is possible that PVIgG-induced dysregulation of p38MAPK could interfere with proper desmosome assembly.

MAPKAP2 is phosphorylated and activated by p38MAPK (76, 77). The activation of MK2 has been associated with cell cycle control, cytokine production, and regulation of the keratin and actin cytoskeletons (78–80). The inhibition of MK2 has been shown to prevent PVIgG-induced spontaneous blister formation in mice, but not blistering solicited *via* the application of mechanical stress (81). This suggests that while MK2 may mediate some of the pathogenic effects of PVIgG, additional pathways downstream of p38MAK are likely also contributing to acantholysis.

Heat-shock protein 27 is another signaling molecule activated by MK2 (82). Hsp27 regulates both actin (83–85) and keratin cytoskeleton (86, 87). Both p38MAPK and MK2 regulate the effect of Hsp27 on the cytoskeleton *via* phosphorylation (88–90). Taken together, these findings demonstrate that one possible pathogenic mechanism of PVIgG-induced p38MAPK activation could be the perversion of typical cytoskeletal regulation, resulting in impaired cell adhesion.

The degree of evidence supporting a role for p38MAPK activation in the pathogenesis of PV has led researchers to investigate the utility of p38MAPK inhibition for the clinical treatment of PV. In a small clinical trial, 15 PV patients were treated with KC-706, a small molecule allosteric inhibitor of p38MAPK. Unfortunately, the trial was terminated before completion due to severe side effects of the drug. At the time of cessation, half of the patients were seen to exhibit at least a partial response to treatment, whereas the other half showed no improvement or a worsening of symptoms (91). Hopefully, the development of newer, more specific inhibitors of p38MAPK and other downstream targets will allow for effective pathway inhibition while avoiding serious side effects (92).

In addition to the effects listed above, p38MAPK can affect epidermal growth factor receptor (EGFR) signaling (93), RhoA activation (60, 68, 94, 95), and various apoptotic pathways (96). All of these pathways have been implicated in PV pathogenesis and are discussed in greater detail below.

# Calcium/Protein Kinase C (PKC)/ Phospholipase C (PLC)

The role of calcium signaling in keratinocyte differentiation and adhesion is well established. Increasing the Ca2+ concentration in keratinocyte culture medium increases intracellular calcium which in turn induces cell–cell contact (mainly adherens junctions) within 5 min, and formation of desmosomes within 2 h (97–104). PLC, an isoenzyme that is responsible for the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), plays a significant role in calcium-induced keratinocyte differentiation (103, 105–108). PKC is a downstream target of PLC and is activated by calcium and DAG. Of the five isoforms known to be expressed in keratinocytes, PKC-alpha has been shown to play a major role in epidermal differentiation and proliferation (109–112).

Early studies showing that PVIgG leads to a rapid increase in intracellular calcium in keratinocytes were the first to implicate calcium signaling as a pathogenic signaling pathway in PV (113, 114). Additional studies demonstrated that PKC was activated within 30 s of treatment of PVIgG (115). A significant role for calcium signaling in PV pathogenesis was strengthened by studies which showed inhibition of PKC could prevent acantholysis both *in vitro* and *in vivo* (116, 117). It was also shown that inhibition of PLC prevented PVIgG-induced acantholysis, as well as increases in intracellular calcium and PKC activation (118). These results suggest that PVIgGs may exert their pathogenic effect by eliciting an increase in intracellular calcium, which leads to the activation of downstream signaling pathways.

The identification of PKC as a potential driver of PVIgGinduced pathogenesis is especially interesting due to the wellestablished role of PKC in cell adhesion. It has been shown that PKC activation leads to weakened cell–cell adhesion, whereas PKC inhibition results in increased adhesion (119, 120). In addition, PKC is known to be required for desmosome assembly and disassembly (41, 97, 121–126). The association of PKC with cell adhesion and desmosomal regulation may provide insight to help identify exactly how PVIgG induced PKC activation results in a loss of cell adhesion. One study showed that PVIgG-induced activation of PKC leads to its dissociation from KIFs and subsequent phosphorylation of DP, which resulted in desmosomal instability (127, 128). Another mechanism through which PKC activation may contribute to disease pathogenesis is by its effect on KIF turnover. It has been shown that PKC directly phosphorylates keratin molecules, leading to a turnover in KIFs (129, 130). This phenomenon may also explain the mechanistic background for the detachment of KIFs which is the hallmark of acantholysis.

# Epidermal Growth Factor Receptor

Epidermal growth factor receptor is a well-studied signaling pathway that impacts a multitude of cellular processes either through the direct binding of its ligand, epidermal growth factor, or by cross-activation from a number of other signaling pathways (131–134). EGFR signaling has been shown to impact cell adhesion *via* both adherens junctions and desmosomes (135–137). Specifically, association with Dsg1 has been shown to suppress EGFR extracellular signal-regulated kinase 1/2 signaling in skin (138). In addition, it has been shown that EGFR activation can induce the phosphorylation of Pkg and decrease the association of desmoplakin with the desmosome, resulting in weakened cellular adhesion (139). In general, these studies associate an activation of EGFR signaling with destabilization of desmosomal adhesion.

The association between EGFR signaling and cell adhesion provided a rationale for researchers to investigate if EGFR signaling played a role in acantholysis, and it was eventually shown that PVIgG led to the activation of EGFR in keratinocytes (49, 93). Further studies determined that the activation of EGFR could be detected as early as 30 min after exposure to PVIgG, but the activation occurred downstream of p38MAPK activation (49, 66, 67, 140). Another group was able to show that anti-Dsg3 autoAbs could also lead to EGFR activation (14). Multiple studies then showed that the inhibition of EGFR could prevent PVIgGinduced skin blistering both *in vivo* and in human skin explants (66, 93, 141).

In addition, EGFR is implicated in PV pathogenesis *via* a second signaling axis independent of p38MAPK. PVIgG binding induces secretion of EGF and related mediators from basal keratinocytes, which in turn activate Src, focal adhesion kinase, and mammalian target of rapamycin (mTOR) *via* EGFR and nitric oxide synthase. The end result is the activation of caspases 3 and 9, which have been proposed to contribute to bister formation (142–145). The role for traditional apoptosis in PV pathogenesis is currently unclear; our group showed that the induction of apoptosis-related mechanisms after anti-Dsg antibody binding is reversible and independent of the Fas/FasL axis (146). However, studies showing that inhibition of the low level caspase-3 induction caused by PVIgG prevented acantholysis *in vitro* and *in vivo* suggest that caspase-3 activation does indeed play a role in disease (147).

# Rho Family GTPases

Rho and Rac are both members of the Rho small GTPases family, which are known to play a role cytoskeletal reorganization, cell polarity, morphogenesis, and cell migration (61). These Rho family GTPases are known to affect the turnover of adherens junctions through multiple pathways (148). Both Rho and Rac are required for the establishment of adherens junctions (149, 150), and the activation of Rac has also been shown inhibit desmosomal adhesion in human keratinocytes (151).

The first association of Rho GTPases with PV pathogenesis was seen in experiments which demonstrated that the activation of Rho GTPases could prevent PVIgG-induced blister formation in human skin. Additional studies were able to show that RhoA activation was able to block the PVIgG-induced retraction of KIFs as well as loss of cell adhesion in HaCaT cells (41). Also, it was observed that HaCaT cells treated with PVIgG demonstrated a reduction in RhoA activity. In addition, p38MAPK inhibitors were shown to block the PVIgG-induced reduction of RhoA activity (94). These results suggest that PVIgG-induced activation of p38MAPK may induce blister formation, at least in part, by inhibiting the activity of RhoA. Given that the formation of adherens junctions has been shown to be necessary for proper assembly and disassembly of desmosomes (47, 152, 153), the loss of desmosomal adhesion seen in PV may be secondary to the inhibition of adherens junctions caused by RhoA inhibition.

For a summary of evidence in support of PV-associated signaling pathways, see **Table 2**.

# INTEGRATED MODEL OF PV autoAb-INDUCED SIGNALING

The pathogenic processes following the binding of autoAbs that eventually drive the loss of cell adhesion are diverse and complex. Taken together, the above data suggest that the disease mechanisms underlying PV are the result of both the direct steric interference of adhesion molecule interaction by autoAb binding *and* the activation of intracellular signaling pathways elicited *via* autoAb binding. A viewpoint that could tie both these mechanisms together is to see the desmosome/keratin intermediate filament complex as a signaling complex which participates in mechanosensing in addition to providing structural stability. Although desmosomal proteins have not yet been shown to function in this manner, a wealth of data exists which describes a similar function in adherens junctions (154). If this was shown to be the case with desmosomal proteins as well, it would be reasonable to assume that, in addition to physically interfering with desmosomal adhesion, the binding of autoAbs in PV may alter signaling pathways associated with the desmosomal complex, such as p38MAPK, EGFR, and PKC that ultimately interfere with the structural stability of keratinocytes.

Taking into consideration the breadth of experimental data detailing autoAb-induced activation of intracellular signaling pathways, we propose that blister formation in PV may result from the following mechanism: (1) binding of autoAbs to target desmosomal antigens (either desmosomal or extra-desmosomal) induces the activation of PLC, leading to the activation of PKC *via* Ca2+ and DAG, which in turn activates p38MAPK *via* MAP3ks [such as Ask1 (155, 156)]; (2) activated PKC (either through direct phosphorylation of keratin filaments or DP) and p38MAPK (*via* MK2 and Hsp27 phosphorylation) then induce the retraction of the KIFs as well as the turnover of the actin cytoskeleton; (3) the retraction of KIFs from the desmosomal plaques, as well as cytoskeletal rearrangements, then cause a destabilization of desmosomes, and a weakening of cell adhesion; (4) finally, the weakening of cell adhesion, coupled with the mechanical stress induced by cytoskeletal rearrangements, induces a cellular stress response, resulting in the activation of Src, EGFR, and Rac1 (as well as other pathways), and re-initiation/perpetuation of the pathological cycle (**Figure 1**).

A primary advantage of this model is the ability to provide a mechanistic framework for understanding how the widely variegated set of factors that have been implicated across multiple studies may be contributing to PV pathogenesis. For example, multiple studies have linked increased levels of reactive oxygen species (ROS) to PV (157–161), but a potential mechanistic contribution to disease has not been well defined. Using our model, however, the known ability of ROS to affect KIF and actin cytoskeletons as well as PKC, Src, p38MAPK, and RhoA signaling (162–167) potentially demonstrates how ROS may contribute to blister formation in a number of ways. As a result, our model can also explain how anti-mitochondrial autoAbs, which have been suggested to play a role in PV and shown to increase ROS production, can directly mediate disease pathogenesis (50, 168–172). Furthermore, the activation of pro-apoptotic pathways by autoAb binding has been shown to modulate p38MAPK (50) and may further contribute to the signaling pathologies that drive acantholysis.

Although this model lays a comprehensive framework for the mechanism of blister formation incorporating all of the major signaling pathways implicated in pathogenesis by the literature, future experiments are required to test the validity of this model and more precisely define the series of signaling events which occur downstream of autoAb binding. Determining the degree to which both desmosomal and extra-desmosomal Dsg proteins associate with signaling molecules and whether or not the binding of autoAbs to these proteins is sufficient to elicit intracellular signaling is a high priority to ascertain if these molecules could function as signal transducers and if so, through which pathways. Immunofluorescent studies of keratinocyte monolayers before and after PVIgG treatment may be one way to determine which molecules are physically associated with desmosomal proteins and determine if autoAb

FIGURE 1 | Proposed comprehensive model of signaling mechanisms underlying blister formation in pemphigus vulgaris. (1) Binding of autoantibodies (autoAbs) to target keratinocyte antigens (either desmosomal or extra-desmosomal) induces the activation of phospholipase C (PLC), and subsequently the activation of protein kinase C (PKC) *via* Ca2+ and diacylglycerol (DAG), which then activates p38MAPK *via* MAP3ks (such as Ask1); (2) activated PKC {either through direct phosphorylation of keratin filaments or DP} and p38MAPK {*via* MAPKAP2 (MK2) and heat-shock protein (Hsp)27 phosphorylation} then induce the retraction of the KIFs as well as the turnover of the actin cytoskeleton; (3) the retraction of KIFs from the desmosomal plaques, as well as cytoskeletal rearrangements, then cause a destabilization of desmosomes, and a weakening of cell adhesion; (4) finally, the weakening of cell adhesion, coupled with the mechanical stress induced by cytoskeletal rearrangements, induces a cellular stress response, resulting in the activation of Src, epidermal growth factor receptor (EGFR), and Rac1 (as well as other pathways), and re-initiation/perpetuation of the pathological cycle. RhoA is inhibited by multiple pathways (such as PKC, EGFR/Src, and Rac1). As RhoA activation has been shown to inhibit PVIgG-induced acantholysis, an inhibition of RhoA would promote cell dissociation. Blue arrow-headed lines indicate activation, while red bar-headed lines indicate inhibition of selected molecules/pathways.

binding leads to their activation/translocation. Another important step would be to more precisely define the upstream MAPK cascade that leads to p38MAPK activation. There are multiple upstream signaling molecules which are known to activate p38MAPK and the characterization of the molecules involved in this process could allow for a better understanding of how autoAbs, or additional factors such as hormones or cytokines (which are known to utilize specific cascades) could activate p38MAPK (173–175). Protein microarray analysis, which allows for rapid, highly specific, multiplexed analysis of the signal transduction pathways (176, 177), would be well suited for this task.

Desmosomal proteins as a mechanosensing signaling complex would provide a logical intersection between the two primary theories of autoAb-induced blister formation in PV. Although future studies are still needed, there exists a great potential to significantly and directly affect the development of future treatments in PV. Identification of the signals transduced by autoAb binding could lead to the identification of potentially novel drug targets, or, at the very least allow researchers to focus on disease-specific signaling pathologies, resulting in small molecule inhibitors with a lower chance of harmful side effects and the ability to minimize systemic suppression of the immune system. In addition, large scale profiling of the signal transduction pathways upstream of MAPK may represent an especially beneficial endeavor as such studies could identify cytokines or hormones which may be contributing to disease process. In addition to facilitating the use of disease specific anti-cytokine or hormonal therapies which may already be in use for other disease modalities, such information could be individualized to the specific pathologies driving disease in a given patient, informing more tailored and effective treatment strategies and greatly enhancing clinical management of disease.

# REFERENCES

# AUTHOR CONTRIBUTIONS

AS and TS contributed to the conceptualization and writing of the manuscript.

# ACKNOWLEDGMENTS

We thank K. Seiffert-Sinha for critical review of the manuscript. We thank B.K.S. and M.S. for their continued guidance and support.

disease pathophysiology. *J Biol Chem* (2010) 285:3695–704. doi:10.1074/jbc. M109.081570

Rous sarcoma, mammalian target of rapamycin and focal adhesion kinase. *Exp Dermatol* (2013) 22:125–30. doi:10.1111/exd.12088

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Sajda and Sinha. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Ultra-Low Dosage Regimen of Rituximab in Autoimmune Blistering Skin Conditions

#### *Mauro Alaibac\**

*Unit of Dermatology, Department of Medicine, University of Padua, Padua, Italy*

#### Keywords: rituximab, autoimmune blistering conditions, skin, pemphigus, B-cells

Rituximab is a chimeric human-mouse monoclonal anti-CD20 antibody initially developed to treat B cell lymphoproliferative disorders (1). It is now increasingly being used for the treatment of B cell-mediated skin autoimmune disorders, including autoimmune blistering skin diseases (2, 3). Rituximab targets all CD20 expressing B-cells, including precursor B cells and mature and memory B cells, whereas it does not interact with CD20-negative early pre-B cells and terminally differentiated plasma cells. Consequently, it completely depletes peripheral memory B cells, but it does not affect preexisting titers of serum antibody produced by long-lived antibody-secreting plasma cells (4). Rituximab mainly acts through antibody-dependent cell-mediated cytotoxicity, although alternative mechanisms may also be involved in complement-dependent cytotoxicity and in direct effect of the drug leading to apoptosis (5). Peripheral B cell depletion by rituximab treatment is usually observed for 6–9 months. After 1 year, many patients show complete mature B cell recovery, whereas the population of circulating memory B cells may be slow to recover after treatment (6). On the other hand, it is plausible that rituximab treatment does not completely eliminate memory B-cells which could be responsible for subsequent disease relapse.

#### *Edited by:*

*Randy Q. Cron, University of Alabama at Birmingham, United States*

#### *Reviewed by:*

*Pui Y. Lee, Harvard University, United States Matthew Stoll, University of Alabama at Birmingham, United States*

#### *\*Correspondence:*

*Mauro Alaibac<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology*

*Received: 05 March 2018 Accepted: 03 April 2018 Published: 18 April 2018*

#### *Citation:*

*Alaibac M (2018) Ultra-Low Dosage Regimen of Rituximab in Autoimmune Blistering Skin Conditions. Front. Immunol. 9:810. doi: 10.3389/fimmu.2018.00810*

Autoimmune blistering skin diseases are a group of heterogeneous skin conditions characterized by the presence of serum autoantibodies targeting desmosomal structural proteins (pemphigus group) or the hemidesmosomal anchoring complex (pemphigoides group) (7, 8). These disorders are generally treated with systemic corticosteroids which are often combined with other immunosuppressive and/or immunomodulatory approaches, notably azathioprine, mycophenolate mofetil, dapsone, tetracyclines, plasmapheresis, immunoadsorption, and high-dose intravenous immunoglobulins (9). These treatments may not be effective for either induction or maintenance of remission or, alternatively, need to be discontinued because of unmanageable adverse effects. B-cells play a central role in the pathogenesis of autoimmune blistering disorders and rituximab, which selectively targets B cells, has proven to be an effective and safe therapeutic agent in patients with autoimmune blistering skin disorders refractory to conventional treatments (9). To this regard, the recent published randomized controlled trial of rituximab and short-course prednisone versus standard-dose prednisone in newonset pemphigus has clearly demonstrated that rituximab with short-course prednisone was a more effective treatment than the high-dose prednisone regimen used in this trial, which has been the mainstay of therapy for pemphigus over the years (10). Furthermore, rituximab associated with short-course prednisone showed lower rates of grade 3 or 4 adverse events compared with standarddose prednisone (10). These results indicate that rituximab associated with short-course prednisone should be considered the first-line therapy for new-onset pemphigus.

The optimal dosing of rituximab in autoimmune blistering skin conditions is currently poorly defined. Initially, rituximab was used for the treatment of pemphigus following the lymphoma dosing regimen (375 mg/m2 weekly for 4 weeks) (11). However, given the fact that B-cell burden in autoimmune blistering skin diseases is much lower than that in lymphoproliferative disorders, several studies have investigated lower dosage. Horwath et al. treated patients with pemphigus with a single course of two infusions of rituximab (500 mg each) at an interval of 2 weeks with satisfactory responses and relapses generally occurring at the end of the second year (12). Several low-dose protocols for the treatment of pemphigus have been reviewed in a recent metaanalysis, such as the rheumatoid arthritis protocol (2 × 1,000 mg doses at a 2-week interval) and the standard low-dose rituximab (2 × 500 mg doses at a 2-week interval) with the concomitant use of immunoadsorption or high-dose intravenous immunoglobulins (13). Moreover, a very recent investigation has shown impressive results in healthy volunteers treated with ultra-low dosage of rituximab. In this study, the authors demonstrated that <1% of the conventional rituximab doses induced a nearly complete depletion of circulating B lymphocytes (1 mg/m2 rituximab depleted 97% of all B-cells and doses of 0.3 mg and 0.1 mg/m2 depleted, respectively 75 and 66% of circulating B-lymphocytes) (14). After 4 weeks from the infusion of rituximab circulating B-cells returned to approximately 60% of normal levels in the 1 mg/m2 dose group, whereas recovery was completed by 9 months after infusion in the 1 mg/m2 dose group and about

# REFERENCES

1 month after infusion in the 0.3 mg and 0.1 mg/m2 dose groups. From the data of the study, the authors extrapolated that 100 mg rituximab may be sufficient to induce a depletion of B-cells for 3 months and, consequently, two doses of 100 mg every 3 months could deplete the B-cell population for 6 months. Thus, alternative ultra-low dosing schedules for rituximab could be proposed for antibody-dependent autoimmune diseases, including autoimmune blistering skin disorders, as they may provide a considerable improvement of the cost effectiveness. Moreover, well-designed clinical trials are warranted to determine the efficacy of ultra-low dosage rituximab and to assess whether there are other potential advantages over conventional low-dose protocols, in particular lower risk for infection and lower risk for infusion reaction.

# AUTHOR CONTRIBUTIONS

MA conceived and wrote the manuscript.

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer MS and handling Editor declared their shared affiliation.

*Copyright © 2018 Alaibac. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Humoral epitope Spreading in Autoimmune Bullous Diseases

*Dario Didona1 and Giovanni Di Zenzo2 \**

*1Clinic for Dermatology and Allergology, University Hospital Marburg, University of Marburg, Marburg, Germany, 2Molecular and Cell Biology Laboratory, Istituto Dermopatico dell'Immacolata (IDI)-IRCCS, Rome, Italy*

Autoimmune blistering diseases are characterized by autoantibodies against structural adhesion proteins of the skin and mucous membranes. Extensive characterization of their autoantibody targets has improved understanding of pathogenesis and laid the basis for the study of antigens/epitopes diversification, a process termed epitope spreading (ES). In this review, we have reported and discussed ES phenomena in autoimmune bullous diseases and underlined their functional role in disease pathogenesis. A functional ES has been proposed: (1) in bullous pemphigoid patients and correlates with the initial phase of the disease, (2) in pemphigus vulgaris patients with mucosal involvement during the clinical transition to a mucocutaneous form, (3) in endemic pemphigus foliaceus, underlining its role in disease pathogenesis, and (4) in numerous cases of disease transition associated with an intermolecular diversification of immune response. All these findings could give useful information to better understand autoimmune disease pathogenesis and to design antigen/epitope specific therapeutic approaches.

#### *Edited by:*

*Ralf J. Ludwig, Universität zu Lübeck, Germany*

#### *Reviewed by:*

*Abhigyan Satyam, Harvard Medical School, United States Jens Waschke, Ludwig-Maximilians-Universität München, Germany*

> *\*Correspondence: Giovanni Di Zenzo<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Immunological Tolerance and Regulation, a section of the journal Frontiers in Immunology*

*Received: 25 January 2018 Accepted: 28 March 2018 Published: 17 April 2018*

#### *Citation:*

*Didona D and Di Zenzo G (2018) Humoral Epitope Spreading in Autoimmune Bullous Diseases. Front. Immunol. 9:779. doi: 10.3389/fimmu.2018.00779*

Keywords: autoantibody, antigen, epitope, epitope spreading, pathogenesis, specific therapy, desmoglein, BP180

# INTRODUCTION

Autoimmune diseases are caused by dysregulation of the immune system or arise as a consequence of microbial infection (1–3). In human, many antigens have been identified in patients and several studies have demonstrated that autoantigens, autoantibody, or related autoreactive lymphocytes can transfer autoimmune disease in animal models. Autoantigens are often clustered on the basis of tissue-specific expression or their structural organization. Several studies conducted in animal models and patients with autoimmune disease suggest that epitope spreading (ES) is the probable explanation for this clustering of specificities in human autoimmune diseases. The ES is an important component of the protective immune responses that acts to enhance its efficiency.

The dynamics of autoimmune response has been investigated both in patients and in animal models. However, although several animal models of autoimmune disease have demonstrated the functional value of ES, in patients limited data are available. A possible explanation could be the early occurrence of ES before the diagnosis and the inhibitory effect of therapy on the autoimmune response.

Autoimmune bullous diseases are organ-specific diseases of the skin and mucous membranes characterized by circulating and tissue bound autoantibodies to structural proteins that maintain cell–cell and cell–matrix adhesions. Studies on prospective cohort of patients have allowed to characterize the dynamics of humoral response and have provided in some studies and reports evidence of the functional role of ES.

**Abbreviations:** ES, epitope spreading; IIF, indirect immunofluorescence; DIF, direct immunofluorescence; IB, immunoblotting; BMZ, basement membrane zone; Dsg, desmoglein; Coll VII, collagen VII; PV, pemphigus vulgaris; PF, pemphigus foliaceus; EPF, endemic pemphigus foliaceus; MMP, mucous membrane pemphigoid; PNP, paraneoplastic pemphigus; EBA, epidermolysis bullosa acquisita; BP, bullous pemphigoid.

To study ES can give a better insight into the autoimmune response elucidating: (i) the initiation and progression of the disease and (ii) the meaning of the autoantigen clustering and specific reactivity profile in patients. Furthermore, to understand the possible influence of ES in autoimmune progression and to gain knowledge of relevant autoantigen and epitopes could be crucial for diagnosis and for designing antigen-specific treatments.

Here, we summarize the significance and mechanism of ES and review current literature on humoral ES in the autoimmune bullous diseases that are paradigmatic autoantibody mediated disorders.

## ES: DEFINITION AND SIGNIFICANCE

Epitope spreading represents the process of diversification of B and/or T-cell response from the initial dominant epitope to a secondary epitope over time. To expand the antigenic epitopes corresponds to optimizing the Ag recognition, to enhancing the neutralization function of antibodies and, in general, to contributing to the efficiency of the immune response.

Epitope spreading that occurs within a single antigen or involves different antigens is termed intramolecular and intermolecular ES, respectively. The intramolecular ES consists of the diversification of immune response in the same autoantigen; the intermolecular ES commonly involves different antigens of a single macromolecular complex or that colocalize in the same anatomical site.

The reactivity that spreads from a single autoantigen to multiple antigens through cross-reactivity is not authentic ES. However, the cross-reactivity could be an initial step to ES. In systemic lupus erythematosus (SLE), autoreactivity to three ribosomal P protein (P1, P2, and P3) is due to a cross-reactive C-terminal epitope present in all three molecules (4). Nevertheless, additional ES phenomenon has been described (5). In this context, the cross-reactivity at the base of molecular mimicry phenomenon can initiate the spreading of an autoimmune response in genetically susceptible hosts. In multiple sclerosis (MS), the initial inflammatory response to a virus infecting the central nervous system can be the triggering factor that induces ES toward autoantigens (6). Similarly, patients with infectious mononucleosis possess circulating IgM to p542, a hematopoietic cell antigen that cross-reacts with Epstein–Barr virus (EBV) nuclear antigen. Afterward, they develop also IgM autoantibodies to p554 that do not cross-react with EBV antigens and that probably arise through ES (7, 8).

In autoimmune response against molecular targets, ES can vary between patients and sometimes reveals a predictable hierarchy. The hierarchy of dominant and secondary epitopes is due to a differential protein processing and presentation, MHC restriction, and to the availability of epitope-specific T and B cells, taking into account central and peripheral tolerance mechanisms.

The predictable sequential ES cascade can present a clinical value. In a subset of patients with mild SLE and relevant skin involvement, autoantibody responses are directed only to the 60 kDa RoAg. In contrast, many patients with primary Sjogren's syndrome and SLE possess IgG autoantibodies for both Ro and La Ag (9, 10).

The functional value of ES in disease development and progression has been clearly demonstrated in immune response in animal models with autoimmune disease. In relapsing experimental autoimmune encephalomyelitis (EAE), an experimental animal model for MS, the contribution to the pathogenesis and specifically to the relapsing clinical episodes of T cell response against epitopes released as a result of tissue damage *via* ES is evident. Specifically, emerging responses to myelin epitopes are clearly able to mediate disease relapse in this mouse model (11, 12). The measles virus infections in Lewis rats are a further example of the causative role of ES in disease induction. This infection induces inflammatory demyelinating lesions in central nervous system. T cells from rats have been reported to proliferate in response to myelin basic protein (MBP) and no cross-reactivity between MBP and virus was demonstrated (13, 14). T cells activated with MBP and adoptively transferred in recipient rats induce lesions resembling those of EAE model (15). Interesting data on functional ES are also provided in non-obese diabetic (NOD) mice, a model of type 1 diabetes where destruction of pancreatic β cells is mediated by CD4 and CD8 T cells specific for numerous epitopes expressed on insulin (Ins) and other autoantigens. Using NOD splenocytes coupled with intact Ins and several additional diabetogenic epitopes Prasad et al. have demonstrated that Ins B9–23 is a dominant initiating epitope, but autoimmune responses to Ins epitope(s) distinct from Ins B9–23 are driven by ES (16). In this context, the progression to overt disease is associated with responses to epitopes distinct from the initiating B9–23 region (16).

However, a pathological role for ES was not always demonstrated in human diseases. Limited data on the functional relation between ES, clinical severity, and disease pathogenesis have been reported. One reason may be that published studies were small in scale (17–19) or that at the time of the diagnosis the ES phenomenon might already have occurred (20, 21). In addition, since treatments are based on inhibition of autoimmune response ES, could be negatively modulated. Indeed, some interesting data on functional value of ES are reported in patients affected by autoimmune blistering diseases. In bullous pemphigoid (BP), the ES phenomenon seems to be associated with disease severity at diagnosis (22). Furthermore, several reports describe a transition from a certain bullous disease to another one due to an intermolecular ES phenomenon that involves a different disease specific antigen. Maeda et al. described a patient who developed BP 12 years after pemphigus foliaceus (PF) being diagnosed (23). Another example of ES occurred during the progression to SLE. Patient autoantibody profiles showed a target diversification over time. Specifically, anti-nRNP-A antibodies bind to the N-terminus of the protein more frequently in later stages when compared to the diagnosis suggesting a role of this diversification in the progression of autoimmune disease (24).

Finally, further evidence of a functional ES in humans is associated with transplantation of allografts. In several cases, ES of the host response to the allograft and organ allograft rejection are clearly correlated (25, 26).

## MECHANISM OF ES

Immune responses are characterized by the immunodominance of epitopes within antigens and a great diversity of T- and B-cell epitope specificity. The broadening of the immune response in autoimmune diseases is induced by tissue damage and inflammation, endocytic processing, antigen presentation, and somatic hypermutation (SHM).

There are two major mechanisms at the base of B cell ES in autoimmune diseases: the first is independent of a physical association of antigens while the second is dependent (27). In the "independent" mechanism, tissue damage inflammation and cytokines induce T cells to recognize cryptic epitopes and activate B cells. On the other hand, a "dependent" response relies on the activation of T and B cells by processing and presentation of physical associated antigens.

# Mechanism Independent of Physical Association of Antigens

The development of secondary epitopes in the initial autoantigen or in different autoantigens can depend on the release of antigens or the disclosure of part of antigens during a chronic autoimmune or inflammatory response. In this context, a chronic tissue damage can induce the activation and recruitment of autoreactive lymphocytes specific for epitopes, which are distinct from and non-cross-reactive with the disease-inducing epitope.

An example is the spreading from viral to self epitopes that is shown to play a pathological role in several virus-induced autoimmune disease models (28). A persistent infection can cause the activation of microorganism-specific T cells which mediates tissue damage and release of self peptides (29). Moreover, the induced inflammation can also result in an increased infiltration of T cells at the site of infection and a non-specific activation of self-reactive T cells. Another possible scenario during microbial infection is driven by the IFN-γ secreted by both activated T cells and infected tissue cells. This cytokine can activate antigen presenting cells (APC) and lead to the engulfment of self-antigens. Increased protease production and different processing of captured self-antigens can result in presentation of cryptic selfepitopes that possibly activate autoimmune T and B cells (29). One example of molecular mimicry involves the relation between EBV infection and the development of SLE. The EBV nuclear Ag-1 (EBNA-1) contains a peptide sequence that closely resembles a region on the Smith Ag (Sm) targeted by autoantibodies in SLE patients. Immunization of rabbits with EBNA-1 peptide leads to development of cross-reactivity to Sm antigen that spreads toward an immune response to Sm and nRNP complexes (30). A prospective study on BP patients showed that the autoimmune response starts with autoantibodies specific for an extracellular antigen and then spreads to an intracellular one, suggesting that tissue damage and inflammation play a role in the dynamics of the response (22).

# Mechanism Dependent of Physical Association of Antigens

Autoantigens are frequently part of multiantigen complexes. T cells specific for one epitope of an antigen can activate B cells that are specific for other different antigens of the complex, allowing production of autoantibodies even against antigens/ epitopes not originally targeted by the immune response (31). In this case, B cells directed against disease-initiating epitopes mediate antigen-specific uptake of large protein complexes leading to the efficient presentation of self-epitopes from distinct proteins. Immune responses are initiated by presentation of immunodominant epitopes to CD4 T cells by APC such as dendritic cells. The primed CD4 helper T cells then activate antigenspecific B cells. The APC function of B cells induce an increased uptake of antigen that leads to a stimulation of CD4 T cell also by peptides that weakly bind MHC class II molecules. In this context, the processing of antigens is directed by the specificity of the immunoglobulin receptor on B cells. In fact, the epitope bound by the immunoglobulin is protected from proteolysis, and this alters the array of peptides presented to naive CD4 T cells (31). An example of ES dependent of physical association of antigens is the development of autoantibodies to multiple components of the La/Ro ribonucleoprotein complex in SLE and SS. The initiation of an immune response on a single component is at the base of the appearance of mixed autoantibody patterns in these systemic autoimmune diseases (32). The splicesome consists of multiple proteins and nucleic acids associated with each other. Thus, immunization with various SmD peptides leads to the development of autoantibodies not only against regions of the SmD protein but also against the U1-associated ribonucleoprotein (A-RNP) and induces lupus-like symptoms in a mouse model (33).

The mechanism at the base of inter- and intramolecular ES dependent on physical association of antigens can be associated with endocytic processing and SHM. After binding to B cell receptors, the antigen is endocytosed, cleaved and loaded into a compatible MHC class II complex on the cell surface (34). This process can allow the presentation of previously unrecognized epitopes or, in case of endocytosis of multi-antigen complex, the presentation of novel antigen (27, 35). On the other hand, after B cells activation in response to Ag, they undergo SHM in germinal centers. IgV gene experiences single nucleotide substitutions at a frequency of about 10−3 per base pair in each B lymphocyte generation (36). Thereafter an affinity maturation process allows a selection of cells with a higher affinity for the Ag. In the context of cell selection, intramolecular ES can occur during the selection of B cells with higher affinity for a different epitope of the Ag or also different Ag (37). Autoantibodies specific for dsDNA can be generated through SHM in developing SLE (38).

Finally, several factors that could influence the ES in the physically linked antigens have been described. In SLE, the strength of non-covalent interactions between Ku antigen and the DNA protein kinase catalytic subunit (p350) is stabilized by autoantibodies that may enhance ES to the p350 antigen (39). In addition, antibody binding of autoantigens may mask some epitopes during antigen presentation or also facilitate presentation of poorly tolerized epitopes (40, 41). In organ-specific autoimmunity, the anatomical isolation of antigens is presumably associated with a less efficient central or thymic T-cell tolerance requiring intrathymic antigen presentation during development of the T-cell repertoire. This partial central tolerance to tissue specific antigens could facilitate the ES. Finally, ES may also be determined by MHC genes, as observed in the experimental model of La/Ro autoimmunity (27).

# HUMORAL ES IN AUTOIMMUNE BULLOUS DISEASES

Autoimmune bullous diseases of the skin and mucosae constitute a large group of diseases, including BP, pemphigus vulgaris (PV), PF, paraneoplastic pemphigus (PNP), epidermolysis bullosa acquisita (EBA), linear IgA bullous disease, dermatitis herpetiformis (DH), mucous membrane pemphigoid (MMP), lichen planus pemphigoid (LPP), and others. Their clinical presentation is polymorphic and the pathogenesis is mainly associated with autoantibodies targeting distinct components of the basement membrane zone (BMZ) and desmosome of stratified epithelia. These autoantigens represent structural proteins important for maintenance of epidermal and dermoepidermal integrity. Over the past few decades, identification of autoantigens and relative autoantibodies has improved understanding of the pathogenesis laying the foundation for the study of the dynamics of humoral ES.

# Bullous Pemphigoid

Bullous pemphigoid, the most frequent autoimmune bullous disease, typically affects the elderly and is associated with a significant morbidity and mortality. The cutaneous manifestations of BP are protean. In the prodromal, non-bullous phase, patients complain of severe itch sometimes accompanied by eczematiform, papular, and or urticarial lesions. In the bullous stage, vesicles and bullae develop on apparently normal or erythematous skin. Involvement of the oral cavity is observed in 10–30% of cases (42). BP is associated with autoantibody to BP antigen 1 and 2 (BPAG 1 and BPAG2) also known as BP230 and BP180, two components of junctional adhesion complexes in human skin. The diagnosis is based on clinical evaluation and detection by direct immunofluorescence (DIF) of IgG and/or complement deposition along the BMZ in a linear pattern. Serological tests such as indirect immunofluorescence (IIF) and antigen-based ELISAs have a confirmatory value (42). The vast majority of BP sera react with an immunodominant extracellular domain of BP180, termed non-collagenous 16A (NC16A) domain (43, 44). The pathogenic relevance of autoantibodies to NC16A is supported by several experimental evidences (43, 45–47). Although *in vitro* and *in vivo* animal models have shown that IgG autoantibodies to BP180 are pathogenic, the role of anti-BP230 antibodies is only partially clear (48, 49). Several studies in BP patients demonstrated that, in addition to the NC16A domain, other epitopes of BP180 and BP230 are targeted by both autoaggressive B and T cells (50–57). To study the dynamics of IgG reactivity against different BP180 epitopes, Di Zenzo et al. have employed a mouse model in which skin obtained from transgenic mice expressing human BP180 was grafted on the back of wild-type mice. The grafting induced an immune response to BP180 perfectly located in its natural molecular context (58). Interestingly, the IgG reactivity with extracellular epitopes preceded IgG recognition of intracellular domain. Indeed, the spread of humoral immune responses in this graft model was exclusively target specific and the kinetics of graft loss is completely different from alloantigen-related graft rejection previously described in this model (59). A possible interpretation of these data is that antibodies directed against extracellular epitopes of BP180 may have induced tissue damage with consequent exposure of intracellular epitopes (**Figure 1**). These findings were in accord with a previous study by Di Zenzo et al. on the analysis of the humoral response in a large cohort of BP patients. Reactivity results obtained on a vast array of BP230 and BP180 epitopes demonstrated that the IgG recognition of intracellular epitopes was already present at an early stage of the disease (52). Data from mouse model and BP patients raise the possibility that the development of IgG against intracellular epitopes or antigens may correlate with the initial phase of the disease when the tissue damage starts occurring (**Figure 1**).

In order to assess the evolution of IgG autoantibodies, a multicenter prospective study in 35 BP patients over a 12-month observation period was performed (22). ELISA and immunoblotting (IB) assays were utilized to assess circulating autoantibodies to BP180 and BP230 peptides. In particular, ES events were detected in 17 of 35 patients and preferentially occurred at an early stage of the disease. The functional role of ES was supported by its significant relation to disease severity at diagnosis. Moreover, in line with data obtained by Di Zenzo et al. in mice grafted with human BP180, in three patients the spreading of IgG reactivity to intracellular epitopes of BP180 and BP230 (an intracellular antigen) was preceded by recognition of the BP180 ectodomain (**Figure 1**).

All these findings suggest that IgG recognition of the BP180 ectodomain is an early event, followed by intra- and intermolecular ES events, which shape the individual course of BP. In addition, these data could give useful information for design therapeutical approaches. In particular, to deplete pathogenic autoantibodies, other pathogenic region of BP180 (in addition to NC16A) should be considered as possible therapy using decoy peptides to block autoantibody binding *in vivo.* However, this approach may require early and massive administration of several different peptides.

Several cases of automimmune bullous disease patients indicate that an ES phenomenon often leads to a disease transition. Recently, Sardy et al. reported an interesting case of an 18-yearold Caucasian woman, affected by BP, who showed clinical and laboratory transition to MMP 5 years after the BP diagnosis (60). The patient developed a severe esophageal stenosis, that was not present at the time of the BP diagnosis. DIF detected linear IgG and C3 fluorescence along the mucosal side of the BMZ. Furthermore, IgG autoantibodies directed against the immunodominan epitope of BP180 (NC16A domain) and the BP230 COOH-terminal were identified. Laminin 332 ELISA was positive. Immunoblot studies showed presence of IgG4 autoantibodies to laminin 332, and both IgA and IgG against desmoglein 3 (Dsg3). Collagen VII (Coll VII) autoantibodies were detected neither by ELISA nor by immunoblot. At the time of BP diagnosis, no evidence of MMP have been detected, and anti-laminin 332, autoantibodies were not tested. In addition, retrospective analysis of the serum samples showed no reactivity. After the BP diagnosis, the patient was started on topical steroid therapy and a short course of oral prednisolone obtaining a partial BP remission. The authors postulated that insufficient immunosuppressive treatment of BP might facilitate ES, and lead to a more complex clinical course (60). Another interesting

case was reported by Kasperkiewicz et al. In a 70-year-old man with hemorrhagic blisters, widespread crusted erosions, and the immunopathological characteristics of anti-p200 (γ1 laminin subunit) pemphigoid, treatment with doxycycline, topical corticosteroids and immunoadsorption led to rapid clinical remission. Nineteen weeks later, a relapse occurred with an altered clinical phenotype together with autoantibodies against both p200 and NC16A suggested a functional role of ES in the disease transition (61). Autoimmune bullous diseases often develop in patients with psoriasis. BP was the most prevalent followed by antilaminin γ1 pemphigoid (62). A possible explanation of the development of BP in psoriatic patients is provided by previous studies that have shown that laminin 1 and laminin α1 within BMZ are disrupted in both involved and uninvolved psoriatic lesions (63, 64). In general, the damage to BMZ in patients with psoriasis may induce the development of several antibodies such as anti-laminin γ1 and -BP180. However, although a Taiwanese population study disclosed that psoriasis occurred significantly more in patients with BP than that in the control group, large population studies in each country are necessary to support this association (65).

and intermolecular ES events (orange lines), which likely shape the individual course of BP (22, 58).

# Pemfigus Vulgaris

Pemphigus includes a group of potential life-threatening autoimmune blistering diseases of the skin and mucous membranes. These diseases are characterized by autoimmune responses mainly directed against two transmembrane components of desmosomes, Dsg3 and Dsg1 (66). Similar to other cadherins, Dsg3 and Dsg1 comprise five extracellular subdomains of approximately equal size (EC1–EC5). Several distinct forms of pemphigus have been reported (PV, PF, PNP, and others); the most common is PV. In PV, the critical role of autoantibodies to Dsg3 is largely demonstrated (67–71). The diagnosis clinical and histopathological findings are supported by demonstration of the IgG deposition on the surface of the epithelial cells by DIF. Detection of circulating autoantibodies against the cell surface by IIF and/or to recombinant Dsgs by ELISA can confirm the diagnosis (69). Usually, the oral mucosa is first involved and then blisters arise on the whole body. In the skin, Dsg3 is mainly expressed in the basal and suprabasal layers, while Dsg1 is predominantly expressed in the upper epidermal layers. However, in oral mucosa Dsg3 is highly expressed throughout the epithelium, while Dsg1 is less expressed. According with "Dsg compensation" theory, the Dsg3/Dsg1 autoantibody profile is at the base of different clinical variants of pemphigus depending on differential expression pattern of Dsg1 and Dsg3 (72). More specifically, in PV patients antibodies to Dsg3 cause mucosal disease due to lack of compensation by Dsg1, while they don't induce cutaneous disease because of compensation by Dsg1. In PV, patients who acquire antibodies to Dsg1, compensation is no longer possible, resulting in cutaneous as well as mucosal disease (67, 73, 74). On the other hand, in PF antibodies to Dsg1 cause cutaneous disease, while they cannot cause mucosal disease, because Dsg3 is present at high levels throughout the epithelium (75). In accord with this theory are the results obtained in a mouse model by Ding et al. The authors showed that the passive transfer of PV anti-Dsg1 and -Dsg3 antibodies were able to generate cutaneous lesion while anti-Dsg3 antibodies alone were not (73).

To investigate the ES phenomenon in PV and to identify the basis of clinical transition from mucosal to mucocutaneous involvement, Salato et al. analyzed sera from PV patients taken at various times during the course of disease (76). A subset of PV patients transitioned from mucosal PV to mucocutaneous PV and their autoantibodies profile were evaluated. One representative patient, with only mucosal involvement at an early PV stage, had autoantibodies specific for C-terminal region of Dsg3 ectodomain in the EC5 subdomain. At this time the circulating autoantibodies were not able to bind human skin by IIF (76) (**Figure 2**). Several years later, the patient produced autoantibodies directed to EC1 subdomain of Dsg3 demonstrating an intramolecular ES phenomenon. Interestingly, the autoantibodies started to bind the human skin by IIF and an intermolecular ES toward Dsg1 occurred (**Figure 2**). At this stage, the patient exhibited non-cross-reactive autoimmunity to both Dsg3 and Dsg1 and developed cutaneous as well as mucosal blisters (**Figure 2**). In this context, it is important to underline that the pathogenic activity of anti-Dsg1 autoantibodies affinity purified from a PV patient serum was previously demonstrated in a mouse model

the human skin and an intermolecular ES toward Dsg1 occurred with development of cutaneous as well as mucosal blisters (76).

based on antibody passive transfer (77). Previous cases with mucosal involvement and anti-Dsg3 autoantibodies that spread to Dsg1 with cutaneous involvement in addition to mucosal one were also reported (73, 78). A major limitation of study from Salato et al. was the use of Dsg1/Dsg3 swapping domains approach for the epitope mapping of autoantibody reactivity. In fact, although the majority of anti-Dsg3 IgG autoantibodies in PV did not cross-react with Dsg1, in mucocutaneous-type PV, with both anti-Dsg1 and anti-Dsg3, autoantibodies mapping results from this approach, may be less reliable, because of the sequence homology of two cadherins. In addition, the exclusive presence of autoantibody to the C-terminal domain of Dsg3 in PV patients with mucosal lesions appeared in contrast with several studies that showed that the vast majority of PV sera react with the N-terminal portion of Dsg3 and rarely bind the C-terminal one (79, 80). On the other hand, the fact that an intermolecular ES from Dsg3 to Dsg1 and not the presence of cross-reactive antibodies is at the base of clinical phenotype transition in PV was demonstrated by an inhibition assay in PV patients with mucocutaneous lesions (67). However, it could be hypothesized the presence of few cross-reactive not detectable antibodies that shift the reactivity from Dsg3 to Dsg1 possibly followed by an intramolecular ES event in the Dsg1 molecule. Indeed, the isolation from PV patients with Dsg1/Dsg3 cross-reactive monoclonal autoantibodies could corroborate this hypothesis (42, 81).

In contrast with data from Salato et al., another study using the same approach has reported a stable epitope profile during disease course in four PV patients (80). A further study analyzed ES events in a larger cohort of PV patients (53 patients) at multiple disease stages (20). In this case, a different system based on Dsg3 (or Dsg1)/Dsg2 domain-swapped molecules was employed. In this approach using Dsg2 cadherin as the backbone of the domain-swapped molecules, the epitopes recognized by anti-Dsg3 IgG could be analyzed without the influence of anti-Dsg1 IgG autoantibodies and vice versa, because PV sera showed no reactivity with Dsg2. As previously reported, the major epitopes recognized by PV sera were calcium-dependent and mapped at the N-terminal region of Dsgs (**Figure 3A**). Interestingly, the recognized region appeared to be unchanged over the course of disease in both anti-Dsg3 mucosal dominant-type PV and anti-Dsg3/Dsg1 mucocutaneous-type PV (20). Intramolecular epitope shift was not evident in the vast majority of PV cases and only two patients of 53 showed an intramolecular ES event in the Dsg3 during the disease course. Specifically, a PV patient (PV1) in active stage reacted to EC1/4 and shifted to EC2/3 during remission, strongly reducing the anti-EC1 reactivity, and another one (PV2) showed a transition from active to moderate disease characterized by a stable anti-EC1 reactivity and the appearance of anti-EC2/4 antibodies (**Figure 3A**) (20). Independent of disease stage, in most PV patients the major Dsg3 epitopes are localized in the EC1–2 subdomains, while in PV with cutaneous involvement dominant epitopes of Dsg1 are in addition present in EC1 but not in the EC2–5 subdomains (**Figure 3A**) (20). In contrast to the results of Salato et al., no mucosal dominant-type PV sera reacted only with the EC4 or EC5 subdomain of Dsg3. Further studies with larger cohort of well characterized patients with only mucosal and mucocutaneous phenotype are needed to clarify the relation between recognized epitope and clinical phenotype and to better understand the role of ES in this phenotype transition. All these studies show three major limitations (i) it was impossible to evaluate ES before the onset of disease, (ii) treatment could influence the ES during the course of the disease, and (iii) a possible intradomain ES was not investigated.

In addition to Dsg3 and Dsg1, several other organ-specific non-Dsg autoantibodies in pemphigus patient sera could be involved in an intermolecular ES phenomenon. For example, antibodies against an acetylcholine receptor and pemphaxin that are not pathogenic alone, but may act synergistically with pathogenic anti-Dsg3 antibodies (82–85). Another example are antibodies targeting keratinocyte mitochondria that contribute to the process of acantholysis and could be also involved in ES phenomena associated with the tissue damage induced by pathogenic autoantibodies (85, 86). Other autoantibodies specific for intracellular domains of Dsgs, a calcium pump encoded by ATP2C1, desmocollin 1, BP230, periplakin, E-cadherin, Dsg4, desmoplakin 1, and desmoplakin 2 have been detected in pemphigus patients (69). However, since their pathogenic role has not been demonstrated they could be considered a result of an intermolecular ES phenomenon without any functional role in the disease. According with this possible interpretation are findings from our study on the presence of other non-disease specific autoantibodies in autoimmune bullous disease such as anti-BP180, BP230 and Coll VII autoantibodies in PV patients or anti-Dsg3 autoantibodies in BP. Interestingly, the reactivity against additional non disease-specific antigens was rare and remained stable during disease course, while in the same patients reactivity against disease-specific antigens fluctuated in parallel with disease severity. This atypical reactivity possibly represented an epiphenomenon unrelated to the evolution and progression of the autoimmune disease (87). Further studies are needed to understand the possible role of ES in the generation of these autoantibodies and their role in disease pathogenesis.

In a recent study, Cho et al. have postulated a functional role of ES in the pathogenesis of PV. In particular, they have isolated cross-reactive rotavirus/Dsg3 antibodies from PV patients. A subset of these antibodies had the potential to confer protection against rotavirus infection but also to cause pathogenic effects on skin, suggesting a role of molecular mimicry in the disease pathogenesis (88). In this context, the authors speculate that even in the presence of non-pathogenic and cross-reactive antibodies, the activation of a cross-reactive B cell could stimulate Dsg3-reactive T cells to trigger a broader anti-Dsg3 B cell response, that react to other and possibly pathogenic epitopes leading to PV (88).

## Pemfigus Foliaceus

Pemphigus foliaceus is an autoimmune blistering skin disease characterized by superficial cutaneous erosions. Tissue bound and circulating autoantibodies that react to Dsg1 have shown a pathogenic role in the disease (75, 89) and provide important diagnostic clues in addition to clinical evaluation. The most common subtype is sporadic PF occurring all over the world, whereas endemic PF (EPF) is found in rural areas of Brazil, where the disease is known as fogo selvagem (90–92).

FIGURE 3 | Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) patients present Dsg3 and Dsg1 epitope profiles stable over the disease course. A schematic representation of the antigenic regions recognized by autoantibodies of a PV (A) and PF (B) patients at diagnosis, with relative percentage of reactivity, and during the disease course (20, 21). Only two patients (for PV and PF) in whom epitope spreading (ES) phenomenon occurred have been shown. In most PV and PF patients, the major Dsg3 and Dsg1 epitopes are localized in the EC1–2 subdomains. In the vast majority of PV and PF patients, the epitope profile remained stable during the course of the disease. Of note, two patients (PV patient 1 and PF patient 2) in active stage reacted to EC1 and shifted with an intramolecular ES to other subdomains (EC2/EC3 and EC2) (20, 21).

Didona and Di Zenzo ES in Bullous Disease

In 2010, Chan et al. have characterized the dynamics of immunoreactivity to various Dsg1 extracellular subdomains in non-endemic PF patients during the course of the disease by using the Dsg1/Dsg2 domain-swapped molecules approach. According to data obtained on PV patients, most of the anti-Dsg1 antibodies bind to the N-terminus region of Dsg1 (**Figure 3B**), and this reactivity prevails across various activity stages. Only two PF patients lost their EC1 reactivity upon remission and in one case shifting to EC2 (**Figure 3B**) (21). Thus, in PV such as in PF patients, the Dsg3 and Dsg1 epitope profiles remained stable over the disease course and, because of the rarity of ES, treatments based on the depletion or abrogation of autoantibodies to the N-terminal domains of Dsg3/Dsg1 should be promising (**Figure 3B**). Another possible involvement of ES is described in a previous study from Ishii et al. that have isolated several pathogenic and non-pathogenic monoclonal antibodies from PF patients (93). Some non-pathogenic antibodies bound to the proprotein of Dsg1, which are thought to be synthesized as intracellular inactive precursor proteins with prosequences that are cleaved to yield mature and extracellular adhesive molecules. Similarly, non-pathogenic autoantibodies from PV patients were also able to bind the precursor of Dsg3 (94, 95). Interestingly, these findings could suggest that PV and PF patients at first develop non-pathogenic antibodies against the intracellular Dsg precursor to which they would not be expected to have tolerance, and in some susceptible patients the antibody response might shift through ES to pathogenic autoantibodies specific for the mature molecule (93–95). Further studies are needed to confirm this hypothesis.