Before the procedure, a 1250 IE heparin i.v. bolus was given. Access to circulation was provided *via* the cubital vein. Peripheral blood was drawn, anti-coagulated, and separated into plasma and blood cells. Extracorporal anti-coagulation was achieved by sodium citrate (ACD-A, 2.2%; 1:15–1:20 v/v to blood). The plasma was pumped through an adsorber column (Globaffin®) regulated by the Adasorb® device. The separated plasma was perfused through the columns at a flow rate between 11 and 28 ml/min; one cycle contained 250 ml plasma. The treated plasma was subsequently remixed with the other blood components and reinfused. During one cycle of treatment, the twofold volume of the individually calculated plasma volume was cleared by the adsorber.

## Statistical Analysis

Student's *t*-test was used to compare variables between the groups. Values of *P* < 0.05 are significant. Continuous data are presented as medians (with ranges). We performed comparisons between groups with use of Fisher's exact test for categorical values and the Mann–Whitney *U* test for continuous variables. In addition, statistical analysis was done using four-field analysis.

# RESULTS

# Sera of Patients with POAG and OHT Contain AAb Stimulating Rat Cardiomyocytes

Immunoglobulin-enriched fractions of sera of patients with POAG, OHT, cataract, and of healthy donors (NHD) were analyzed for AAbs against GPCRs. We calculated the cutoff as mean of the beating rate in normals added to three times of the SD. As shown in **Figure 1**, the beat rate of cardiomyocytes was increased by the immunoglobulin fractions in POAG, OHT, yet not in cataract and NHD. 73% of the POAG patients and 78% of the OHT patients showed increased beat rates of the cardiomyocytes, whereas 0% of the cataract and NHD yielded an increased beat rate. This indicates the presence of agonistic AAb in patients with POAG and OHT. To identify the specific target(s) and IgG fraction of these AAb receptor-specific analyses were performed.

# Immunoglobulins from POAG and OHT *Patients Interact* with the **β**2AR

The positive chronotropic responses of the AAb from patients with POAG and OHT were analyzed for their receptor specificity using respective antagonists. The action of AAb on the cardiomyocytes was blunted by ICI118.551 and the β1/β2 adrenergic antagonist propranolol (**Figure 2**), but not by bisoprolol (β1AR

Figure 1 | Antibodies stimulating rat cardiomyocytes in sera of patients with POAG and OHT. Immunoglobulin-enriched serum fractions were added to cardiomyocyte cultures in a 1:20 dilution and incubated for 60 min at 37°C. The data are displayed as the changes of the beats/min. Each point represents 6–10 separate single-cell check points in a culture flask. Repeated measurements of the effects with different cell cultures prepared on different days resulted in almost identical data. The differences between patients with POAG or OHT versus healthy controls were highly significant. Primary open-angle glaucoma (POAG, *n* = 37); ocular hypertension (OHT, *n* = 9); cataract (*n* = 10); healthy donors (NHD, *n* = 10).

Figure 2 | β2-adrenergic receptor (β2AR) agonistic autoantibodies (AAbs) in patients with primary open-angle glaucoma (POAG) or ocular hypertension (OHT). The neonatal cardiomyocytes were incubated with the immunoglobulin fractions from POAG (*n* = 33) and OHT (*n* = 14) patients as described in Figure 1. The agonistic effect of the AAbs was blocked by the β2AR antagonist ICI118551 (ICI; 0.1 µM). ICI (0.1 µM) did not affect the basal beating rate of the cardiomyocytes.

antagonist) or losartan (AT1 antagonist; not shown). The β2ARspecific agonist clenbuterol enhanced the cardiomyocyte beat rate of all samples, indicating functionality of the detection system (not shown).

Reportedly, agonistic AAb against the β1 adrenoceptor or monoclonal antibodies against the second extracellular loop of the β2 adrenoceptor did not desensitize the corresponding receptors within 4 to 6 h (49). Therefore, we analyzed the long term effect of the β2 AAb prepared from patients with POAG. The AAb did not induce desensitization of the β2AR within 5 h. ICI118.551 normalized the beating rate of the cardiomyocytes. A successive washing procedure removed ICI118.551 and the AAbs. When the washed cells were stimulated with the β2 agonist clenbuterol, a maximal response to this agonist was to be observed (**Figure 3A**). On the other hand, clenbuterol desensitized the receptor mediated signal cascade and decreased the beating rate of the cardiomyocytes. After washing out the agonist and a further stimulation with clenbuterol, we observed an increase of the beating rate by only 30% of the response obtained in the first stimulation with clenbuterol. We conclude that, in contrast to clenbuterol (**Figure 3B**), AAb did not desensitized the β2AR mediated signal cascade.

# The **β**2 AAb Recognize Two Peptide Epitopes of the Second Extracellular Loop II of the **β**2AR

The binding of the AAb to distinct extracellular loops of β2AR was characterized in the presence of loop-specific synthetic peptides. As presented in **Figure 4** only the loop II peptide neutralized the agonistic activity. This indicates that the second extracellular loop of β2AR is the target of the AAb in the sera of patients with glaucoma.

To identify the epitopes of the AAbs, we pretreated the AAb with short overlapping peptides corresponding to the second extracellular loop of the β2AR. For these studies five synthetic

oligopeptides were prepared covering the loop II amino acid 172–197. Only the loop II-borne peptides AINCYAN (181–187) and ANETCCD (186–192) abolished the agonistic activity of the AAb from five representative patients with POAG (**Figure 5**) and, thus, represent the dominant epitope of the β2AR.

# Affinity Purified AAb of POAG and OHT Patients Induce Agonistic Effects in a Dose-Dependent Manner

Affinity purified AAb against the β2AR exert a dose-dependent positive chronotropic effect on cultured spontaneously beating

Figure 4 | The agonistic anti-β2AR autoantibodies (AAbs) of patients with primary open-angle glaucoma (POAG) recognized the second extracellular loop of the β2-adrenergic receptor. The antibody preparations from patients with POAG (*n* = 5) were pretreated with loop-specific peptides corresponding to the extracellular loops I–III. The antibody–peptide complexes were added to the cardiomyocytes to measure remaining agonistic capabilities. The final AAb dilution was 1:40. The data are presented as the increase in beats/min versus baseline beating rates.

ANETCCD of loop II. The procedure to detect epitope-specific responses of immunoglobulins isolated from POAG patients is similar to the one used in the studies for loop analyses. The final AAb dilution was 1:40. Data are displayed as individual measurements of five independent patients with POAG.

rat cardiomyocytes. The maximal response for the POAG and OHT was observed at a dilution of the antibodies of 1:200 and 1:100, respectively.

# Surface Plasmon Resonance Analysis Confirmed the Receptor Specificity of the Agonistic AAb

Surface plasmon resonance allows to determine direct physicochemical interactions between macromolecules. Analyzing the IgG fractions of sera from patients with glaucoma we observed a significant binding activity to a loop II peptide from the β2AR (**Table 6**). We subjected the values to a statistical four-field analysis. The Fisher-test revealed a significance of *p* = 0.01282, a relative risk (CI) of 0.37721 (0.37093–0.84993), a sensitivity of 0.35 and a specificity of 0.0066. Therefore, the AAb from the sera of patients with glaucoma display a higher affinity for the loop II of the β2AR than those of controls.

# The *Agonistic* **β**2AR AAb in the *Sera* of *Patients* with POAG Are of the IgG3 *Isotype*

To define the nature of the molecules in immunoglobulinenriched fractions, which increased beating rates of cardiomyocytes, we used antibodies against immunoglobulins IgG and IgM (**Figure 6**). Only anti-IgG significantly prevented the agonistic activity. We next analyzed the IgG subclass of the agonistlike β2AR AAb with antibodies specifically depleting certain IgG subclasses (**Figure 7**). Only IgG3-precipitating antibodies eliminated the agonistic activity. Therefore, most of the agonistic AAb are of the IgG3 isotype.

# Clinical Parameters in Correlation to AAbs

We analyzed the clinical parameters of the patients with respect to treatment for glaucoma, IOP, and general diseases including

Table 6 | Surface plasmon resonance analysis of binding of IgG fractions to the biotinylated H19C peptide (biotinyl-LC-HWYRATHQEAINCYANETC) derived from loop II of the β2AR.

Figure 6 | Agonistic anti-β2AR IgG autoantibodies in patients with primary open-angle glaucoma. The neutralization of IgG from the immunoglobulinenriched fraction lead to the loss of the stimulatory potential. Antibodies against IgM had no effect. The β2 receptor agonist clenbuterol served as positive control (not shown).

cardiovascular disease and diabetes (**Tables 3** and **4**). We could not detect any correlation of these parameters between AAb positive POAG and OHT patients with NHD or AAb negative patients (data not shown).

# Pilot Proof-of-Principal Study on IA for Patients with POAG

The IA was well tolerated in all patients—no adverse events were to be observed. Specifically, no change in visual acuity, visual field, and intraocular flare measured by laser flare cell meter was detected (not shown). The levels of the AAb before and after IA are shown in **Table 7**. The IOP course is presented in **Table 8** for each patient. Time course of IOP and levels of AAbs during IA are shown in **Figure 8**.

The individual IOP course of each patient was as follows.

## Patient 1 (Male, 71 Years Old)

Mean IOP before immune adsorption of the first patient showed a reduction only during the first days of IA (**Figure 8A**. The IOP dropped from 15.6 ± 1.8 mmHg to 15.1 ± 03 mmHg (3.2%) during the first day and raised to 23.9 ± 1.9 mmHg during the last day of IA. Because of the advanced stage of glaucoma, trabeculectomy was performed 4 weeks after IA.

### Patient 2 (Female, 52 Years Old)

In the second patient largest reduction of IOP was found 2 months after IA (visit 4) (**Figure 8B**). IOP dropped about 27.8% at the right eye (OD) and 32.1% at the left eye (OS) if compared to IOP at visit 1. At visit 7 (12 months after IA) mean IOP was still decreased by about 21.9% at the right eye and 12.5% at the left eye in comparison to visit 1.

### Patient 3 (Female, 56 Years Old)

Largest drop of IOP was found in the third patient (**Figure 8C**). At visit 4, the reduction of IOP was 11% for the right eye and 46.3% for the left eye with additional two topical medications. Without topical medications, IOP reduction was found to be 12.5% at the left eye, yet IOP increase of about 11% at the right eye (visit 2, 2 weeks after IA) compared to IOP at visit 1.

### Patient 4 (Male, 57 Years Old)

The fourth patient showed the lowest IOP measurement (without topical medication) at the end of the observation period (without medication visit 7) (**Figure 8D**). One month after IA, IOP was equal to IOP before IA (without medication, visit 1) with an increase of mean IOP up to 23.5 ± 4.6 mmHg at the right eye and 23.5 ± 4.0 mmHg at the left eye (6 months after IA, visit 6). In comparison to visit 0 (three medications), no topical medication was necessary at the end of the observation period.

Taking together, in all patients, AAb and other IgGs were washed out almost completely by IA, resulting in autoantibody concentrations below the detection limit at the end of treatment. In two patients (1 and 3) AAb remained undetectable during the 6 months follow-up period. The two other patients showed detectable AAb-levels 4 months after IA. The reduction of total IgG and IgG3 levels ranged from 88 to 95 and from 64 to 80%, respectively. Four weeks after IA total IgG and IgG3 concentrations reached normal values.

# DISCUSSION

In the present study, we describe formerly unknown circulating AAb in patients with glaucoma (50). We screened patient sera for antibodies that activate a GPCR expressed by cardiomyocytes and detected AAb against the β2AR employing specific inhibitory peptides. These agonistic AAb induced a dose-dependent stimulation and their effects were blocked by the selective antagonist

Table 7 | AABs, IgG (mg/dl), and IgG3 (mg/dl) before and after immunoadsorption (IA).

ICI118.551, and neutralized by peptides corresponding to the second extracellular loop of the β2AR (AS 181–192).

Autoantibody can be detected by several methods: (1) enzymelinked immunosorbent assay detects the patient's antigen bound AAb by a specific (to human IgG) animal sourced and enzyme (peroxidase or alkalic phosphatase) labeled antibody; (2) fluorescence (microscopy) detects AAb as (1), but the detection of the AAb is fluorescence labeled (e.g., fluorescein isothiocyanate, FITC); (3) radioimmunoassay detects AAb as (1), however, the detection of the AAb is radio labeled (e.g., iodine); (4) bioassay (with spontaneous beating neonatal rat cardiomyocytes) measures the increase in the beating rate, induced by added IgG (fraction from human serum) due to the binding of AAb to the corresponding receptor (here: β2-AR). The detection methods (1)–(4) measure the AAb indirectly, however method (4) additionally directly. As the detected signal extent of the bioassay is proportional to the AAb bound to the surface fixed target, we decided to choose the cardiomyocyte bioassay for the detection of the AAb. Next to the detection of the AAbs, this method enables functional analyses.

The AAb found in glaucoma patients are of the IgG3 subclass. The antibodies that trigger effector functions, and that are most likely to be involved in immunoregulatory activity, are IgG3 and IgG1 (51). In addition, it is known that IgG3 is involved in antibody-dependent cellular cytotoxicity (52). Complement activation is most effective with IgG3 and, to a lesser extent, with IgG1, IgG2, and IgG4. Usually, complement binding and activation leads to destruction of the target structure. The plasma concentration of IgG3 is low, and its half-life is shorter than that of any of the other IgG-subclasses. Further investigations are necessary to elucidate whether complement activation and cell death are involved in the pathogenesis of glaucomatous diseases.

Recent studies support the hypothesis that glaucomaassociated neurodegenerative processes are partially caused by immune-mediated mechanisms (53–56). These studies report complex AAb repertoires in patients with glaucoma which were directed against retinal and optical nerve antigens. Such autoantibody reactivities were shown in optical nerve extracts from bovine eyes. The later findings are of interest in respect to ocular pressure-dependent and independent autoimmune responses. With regard to AAb-mediated immunoreactions, these pathomechanisms seem to be driven by inflammation.

The β2AR is one of the best investigated GPCR. The modulation of cardiac contractility is also mediated by β2AR, and its activation might have chronic effects on cellular metabolism, on cell growth, or on excitability (57). This receptor seems to be involved in allergic asthma, several cardiomyopathies, such as Chagas' disease, and cardiac electrical disturbances (58–60). However, whereas the AAb in the case of Chagas disease were functionally active in cardiomyocytes, β2-AAb from asthmatic patients react in the opposite way. Respective loop analyses explained these findings; in the Chagas-related cardiac disorders the loop II was identified to be the target of AAbs, whereas in the asthmatic disease the loop III was recognized. Thus, it was concluded that the various extracellular loops of the β-AR are functionally linked to distinct antibody-mediated responses.

Several years ago, the effect of monoclonal antibodies directed against the second extracellular loop of the beta2 adrenoceptor was investigated. Interestingly, similar results were detected as seen with the AAbs prepared from the sera of glaucoma patients (61). The agonist-like effect of this mAB (6H8) was blocked by the specific β2-adrenergic antagonist ICI118.551 and a peptide HWYRATHQEAINCYANETC corresponding to the second extracellular loop of the β2-adrenoceptor. The β1-adrenoceptor antagonist bisoprolol was without influence. The AAb were directed against loop II and recognized the overlapping epitopes AINCYAN and ANETCCD. Considering that the peptide corresponding to the second extracellular loop and the short overlapping peptides cannot form the correct conformation of the extracellular loop, the anti-β2 receptor antibodies were pretreated with the peptides corresponding to the first, second or third extracellular loop for 1 h. Under these conditions, only Table 8 | Intraocular pressure (IOP) before and after immunoadsorption (IA)—Visit 0: less than 3 months before IA under maximal combination therapy.

After IA

Patient

Before IA

Visit 0

IOP

1

2

 OD

 20.0 ± 2.0

3

 23.3 ± 3.7

0

 22.4 ± 3.9

2

 23.8 ± 4.5

2

 16.8 ± 1.9

3

 18.2 ± 2.3

3

 23.8 ± 4.5

3

 18.2 ± 2.3

3

(15–22, 7)

(19–30, 11)

(15–22, 7)

(15–20, 5)

(19–30, 11)

(17–29, 12)

(17–33, 16)

(17–23, 6)

OS

 26.0 ± 4.5

3

 29.7 ± 4.6

0

 23.0 ± 4.4

2

 21.2 ± 3.8

2

 20.2 ± 2.3

3

 26.0 ± 3.0

3

 21.2 ± 3.8

3

 26.0 ± 3.0

3

(22–31, 9)

(16–26, 10)

(22–31, 9)

(18–24, 6)

(16–26, 10)

(19–30, 11)

(22–40, 18)

(21–33, 12)

3

 OD

 17.4 ± 3.7

2

 15.5 ± 5.2

0

 17.2 ± 6.9

0

 14.3 ± 3.5

2

 13.8 ± 0.4

2

 13.2 ± 1.6

2

 14.8 ± 3.4

2

 13.8 ± 2.4

2

(12–16, 4)

(12–19, 7)

(12–16, 4)

(12–14, 1)

(10–19, 9)

(12–31, 19)

(10–23, 13)

(12–24, 12)

OS

 23.9 ± 7.9

2

 25.5 ± 8.4

0

 22.3 ± 14.6

0

 14.7 ± 3.6

2

 13.7 ± 0.8

2

 15.2 ± 1.8

2

 22.5 ± 6.8

2

 15.5 ± 2.4

2

(12–19, 7)

(16–32, 16)

(12–17, 5)

(12–16, 2)

(10–19, 9)

(14–52, 38)

(16–42, 26)

(16–40, 24)

4

 OD

 17.6 ± 3.3

4

 20.4 ± 2.8

0

 21.5 ± 5.4

0

 20.7 ± 5.3

0

 21.5 ± 5.4

0

 23.5 ± 4.6

0

 23.5 ± 4.6

0

 19 ± 1.8

0

(17–22, 5)

(17–28, 11)

(17–28, 11)

(14–29, 15)

(15–26, 11)

(14–30, 16)

(18–24, 6)

(11–25, 14)

OS

 17.4 ± 2.9

4

 21.0 ± 2.5

0

 22.4 ± 4.3

0

 21.2 ± 4.7

0

 22.8 ± 4.5

0

 23.7 ± 4.1

0

 23.5 ± 4.0

0

 19.7 ± 4.0

0

(15–26, 11)

(20–28, 8)

(20–28, 8)

(16–29, 13)

(16–26, 10)

(17–30, 13)

*Visit 1: two days before IA with systemic medications (acetazolamide), yet without topical medications. Mean* ± *SD (minimum–maximum, range) of 24 h IOP curve are presented.*

(18–24, 6)

(12–23, 11)

 OD

21.7 ± 4.0

3

18.4 ± 2.8

0

27.0 ± 6.5

2

 TE

(20–37, 17)

(13–21, 8)

(17–26, 9)

 Med

IOP

 Med

IOP

 Med

IOP

 Med

IOP

 Med

IOP

 Med

IOP

 Med

IOP

 Med

Visit 1

2 weeks

1 month

2 months

4 months

6 months

12 months

the peptide that corresponds to the second extracellular loop was able to neutralize the AAb activity. The peptides of the first and third extracellular loops were without effect. Similar results we observed also for the short overlapping peptides with an amino acid sequence of 5–6 amino acids. In these experiments, the sequence (AINCYANETCCD) was able to neutralize the AAb action. Therefore, it is assumed that this sequence represents the epitope of the AAb on the second extracellular loop. Plasmon resonance and affinity purification confirmed the direct interaction of the AAb with loop II of the β2AR. The agonistic β2AR antibodies described here are good mechanistic candidates that might induce and maintain an elevated IOP as a critical step in the development of glaucomatous diseases.

In this study, we present first *in vivo* data of four patients with POAG that underwent IA in a pilot proof-of- principal study: glaucoma therapy is considered to be effective when IOP reduction is 20% from wash out baseline (62). Even not all patients in a study group must show a substantial IOP decrease of 20%, in cases when representative parts of the patients display even more than 20% IOP reduction. Thus, an IOP decrease could be observed in 3 of 4 patients after IA: patients 2 and 3 showed an IOP decrease of 27.8% (OD)/32.1% (OS) and 11% (OD)/46.3% (OS) using equal number of topical antiglaucomatous eye drops. An IOP decrease of 2.9% (OD) and 6.2% (OS) could be observed 12 months after IA in patient 4. As it is known that each single millimeter of mercury increases glaucoma progression rate about 12–13% (18) even the IOP decrease in this patient is important and enables winning sighted lifetime. The IOP of patient 4 is considered to be even lower after IA as the number of local antiglaucomatous eye drops decreased from 4 to 0, thus, measured IOP values under no local therapy can be seen as a lowered IOP after IA with even higher IOP under quadruple therapy before IA. Summarizing, immunoadsorption seemed to lower IOP or even number of antiglaucomatous therapy. Possibly, the decrease of IOP could even be larger, if β2AR AAbs are blocked specifically. Because IA is a non-specific method for adsorbing AAbs and other IgGs, several other AAb were removed in addition to the agonistic β2AR AAb. As known for other autoimmune diseases, AAb relapse. This effect was also seen in our patients. Detection of β2AR AAbs went along with an IOP increase (patients 2 and 4). The delay in the decrease of IOP might be possibly due to a re-sensitization of the β2AR in the absence of stimulating AAbs. In addition, the therapy was well tolerated and no adverse effects were observed.

The most important target of glaucoma treatment is elevated IOP. A large body of evidence has established the importance of the reduction of the IOP in the medical management of glaucomatous diseases (13–17, 63). The pathogenic damage of the optic nerve in POAG is not well understood. It is supposed to be associated with increased IOP, glutamate toxicity (64), interrupted transport of neutrophins (8), apoptosis (4, 65), extracellular matrix changes (5, 6), and hypoxia due to ocular and systemic vascular dysregulation (66).

The aqueous humor dynamic plays the major role in the regulation of IOP. Four factors maintain the steady-state IOP of the healthy eye. These are flow of aqueous humor, resistance to outflow, episcleral venous pressure (67), and uveoscleral outflow (68).

Figure 8 | Time course of IOP, levels of autoantibodies (AAbs) and number of local antiglaucomatous eye drops during immunoadsorption (IA) for the patient with primary open-angle glaucoma (POAG). Patient 1/2/3/4 (A)/(B)/(C)/(D): a decrease in IOP under reduced number of local antiglaucomatous therapy was to be observed in all patients, going along with a decrease of the AAb. Intraocular pressure (IOP) increased, when AAbs relapsed after the IA; OD, right eye (black); OS, left eye (red); grayish area, data before IA; visit 1, IOP graph: less than 3 months before immunoadsorption under maximal combination therapy, AAB against β2AR graphs: before immunoadsorption; visit 2 – IOP graph: two days before immunoadsorption with system medications (acetazolamide), yet without topical medications, AAB against β2AR graphs: at the end of immunoadsorption; IOP/AAB against β2AR graphs: visit 3–2 weeks after immunoadsorption; visit 4–1 month after immunoadsorption; visit 5–2 months after immunoadsorption; visit 6–4 months after immunoadsorption; visit 7–6 months after immunoadsorption; visit 8–12 months after immunoadsorption.

The IOP reflects the balance between in- and outflow of aqueous humor. A major strategy in medical treatment of glaucoma is the reduction of inflow and, thereby, normalization of IOP. Understanding the mechanisms and regulation of inflow is, thus, of undoubted clinical relevance. Several mechanisms underlying increased inflow have been identified, however, the integration and regulation of these mechanisms is still elusive (69). The balance of in- and outflow of aqueous humor is modulated by the local β2AR activity and may be chronically influenced by agonistic AAbs.

Usually, night-time aqueous flows are approximately half of those observed at daytime in active humans (70–72). The ability of aqueous flow stimulation by epinephrine during night-time shows a low basal β-adrenergic stimulation during the night (23). Patients with POAG form in average 15% more aqueous humor/24 h than control subjects of the same age. This is due to a significantly higher aqueous flow in the night in comparison to control subjects (73). Thus, a β-adrenergic overdrive precludes the downregulation of the aqueous flow during the night. This observation is underlined by the lack of receptor desensitization by agonistic AAb displayed in **Figure 3**. Usually, β2AR agonists such as clenbuterol desensitize the receptor within 1–2 h (**Figure 3B**). By contrast, the β2AR AAb do not desensitize the β2AR *in vitro* for at least 5 h. This chronic β2-adrenergic stimulation may explain the higher aqueous flow and the elevation of the IOP in patients with POAG. Alteration of the circadian rhythm of IOP in POAG can be explained by the presence of AAb. The lack of tachyphylaxia was also observed for agonistic antibodies against anti-β1AR-, anti-α1AR-, anti-AT1-, and anti-muscarinic M2 receptor in several cardiovascular diseases (74, 75).

The AAb might also play a modulatory role in the aqueous outflow. Putney et al. (76) found an increase of cell volume of trabecular meshwork cells *via* Na+-K+-Cl− cotransporters in glaucoma leading to a reduced outflow. As β-adrenergic stimulation results in an activation of Na<sup>+</sup>-K<sup>+</sup>-Cl<sup>−</sup> cotransporters, agonistic AAb might also contribute to reduced outflow facility.

An "organ specificity" for the AAb against the β2AR can be assumed as patients of glaucoma suffered not of other diseases, which can be caused by these AAb (e.g., allergic asthma). It can be hypothesized that the chemical surrounding has to be changed before the autoantibodies can act specifically. Previous data showed that the βAR is not accessible for the AAb under adequate oxygen conditions. However, the βAR seemed to be unmasked, if ischemia occurred (77, 78). This could be a potential reason as glaucoma is hypothesized to be a disorder with local ischemia (79).

The data presented here offer another insight into eye disorders associated with high IOP. The newly identified AAb against the β2AR were exclusively found in the sera of patients with POAG and OHT but neither in those with cataract, nor in controls. Based on these observations, we suggest that these AAbs are not only immunological markers with a high incidence in POAG and OHT, but might also be pathomechanistically relevant β2AR activating molecules. The common, broadly successful therapy with β2AR antagonists further supports the idea that AAbmediated mechanisms might be of pathogenic importance. In addition to their effect on IOP, the AAb might also have a direct effect on retinal ganglion cells as β-adrenoreceptor antagonists showed neuroprotective properties (80).

# CONCLUSION

In patients with POAG and OHT, however, not in controls, agonistic AAb directed to β2AR were detected. These AAb interacted with the second extracellular loop of β2AR (peptides 181–187 and 186–192), thus functionally different links to distinct antibodymediated responses are assumed. Data of our clinical pilot proofof-principal study might suggest that a reduction of total IgG, including AABs against β2AR (IgG3 isotype) by IA, transiently decreases IOP. These findings might indicate a possible role of these AAb in the dynamics of aqueous humor and might support a contribution of adaptive autoimmunity in the etiopathogenesis of POAG and OHT. A specific blocking of AABs against β2AR might be of interest for further studies.

# ETHICS STATEMENT

The study followed the tenets of the declaration of Helsinki for research. Written informed consent to use serum samples for research purposes was obtained from each participants of the study. The institutional review board of the University Hospital Erlangen approved the protocols (3483; ClinicalTrials.gov Identifier NCT00494923).

# AUTHOR CONTRIBUTIONS

RK, MH, and GW had the idea and planned the study; GW, QF, and SB performed the laboratory work; AJ, JR, and RV planned and performed the clinical trial; HK did the synthetic work; JH did laboratory work; AS and BH performed data acquisition and statistical analysis; RR was involved in the analysis and revised the manuscript, FH was involved in the analysis, BH was responsible for the draft of the manuscript. US-S interpreted results and edited the manuscript. All authors have no financial interest in any topic regarding the present study and approved the final version of the manuscript.

# ACKNOWLEDGMENTS

We thank Karin Karczewski (MDC, Berlin) for excellent technical assistance and Dr. med. Sabine Froese-Duill (Celle) for helpful discussions about the pathomechanism of glaucoma.

# FUNDING

Globaffin was kindly provided by Fresenius Medical Care Deutschland GmbH, Bad Homburg, Germany. This work was

# REFERENCES

supported by "Freundeskreis of the MDC, Berlin," "Deutsche Forschungsgemeinschaft" SFB 643 (project B5), SFB 539 ("glaucoma including Pseudoexfoliation syndrome"), by the Interdisciplinary Center for Clinical Research (IZKF) (project number A4 and N2) at the University Hospital of the Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), by the European Commissions [E.U. (QLK3-CT-2002-02017\_APOCLEAR)], by the Lupus Erythemathodes Selbsthilfegemeinschaft e.V., by the responsif GmbH Erlangen, and the University of Erlangen's ELAN program, and by the Doktor Robert Pfleger Foundation, Bamberg.

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**Conflict of Interest Statement:** (1) Patent: EP 1832600 A1 (2) RK is working for Fresenius Medical Care GmbH.

The reviewer RK declared a shared affiliation, with no collaboration, with one of the authors, HK, to the handling editor.

*Copyright © 2018 Jünemann, Hohberger, Rech, Sheriff, Fu, Schlötzer-Schrehardt, Voll, Bartel, Kalbacher, Hoebeke, Rejdak, Horn, Wallukat, Kunze and Herrmann. 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.*

*Michael Kasperkiewicz1 \*, Sophie-Charlotte Mook1 , Diana Knuth-Rehr <sup>1</sup> , Artem Vorobyev1,2, Ralf J. Ludwig1,2, Detlef Zillikens1,2, Philip Muck3 and Enno Schmidt1,2*

*1Department of Dermatology, University of Lübeck, Lübeck, Germany, 2 Lübeck Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany, 3Department of Internal Medicine, University of Lübeck, Lübeck, Germany*

#### *Edited by:*

*Mette Søndergaard Deleuran, Aarhus University Hospital, Denmark*

#### *Reviewed by:*

*Rene De Waal Malefyt, Merck, United States Giampiero Girolomoni, University of Verona, Italy*

#### *\*Correspondence:*

*Michael Kasperkiewicz<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Dermatology, a section of the journal Frontiers in Medicine*

*Received: 24 October 2017 Accepted: 26 January 2018 Published: 12 February 2018*

#### *Citation:*

*Kasperkiewicz M, Mook S-C, Knuth-Rehr D, Vorobyev A, Ludwig RJ, Zillikens D, Muck P and Schmidt E (2018) IgE-Selective Immunoadsorption for Severe Atopic Dermatitis. Front. Med. 5:27. doi: 10.3389/fmed.2018.00027*

Keywords: atopic dermatitis, IgE, immunoadsorption, immunoglobulin, SCORAD

# INTRODUCTION

Atopic dermatitis (AD) is characterized by pruritic, eczematous skin lesions, and is commonly associated with elevated serum IgE levels. AD can considerably impact the patient's quality of life and is one of the most frequent chronic inflammatory cutaneous disorders. For patients who are severely affected by the disease, conventional immunosuppressive treatments may not show uniform efficacy and can be limited by severe side effects (1).

Although the role of IgE in the pathophysiology of AD is not fully understood, sequestering free IgE by the anti-IgE monoclonal antibody omalizumab showed some clinical benefit in AD patients with poor response to traditional therapy (2). However, studies of this treatment have yielded controversial results. A recent systematic review and meta-analysis of the efficacy of omalizumab in 103 AD patients from 13 studies revealed that serum IgE concentrations of >700 kU/L were associated with less-favorable clinical responses compared with lower levels (2). The limited effect of omalizumab in AD patients with high total IgE serum levels may be related to insufficient IgE neutralization by omalizumab. The recommended dosing table for omalizumab is limited to 150–1,200 mg/month, according to total IgE concentrations within a range between 30 and 1,500 kU/L (3).

While adjuvant immunoadsorption (IA) has been introduced in dermatology by its successful application in patients with severe and/or refractory pemphigus (4), its use in AD is still limited to individual centers. We and others, however, have recently demonstrated that IA may represent an alternative IgE depletion method in patients with very high IgE concentrations leading to normalization of cutaneous inflammation parameters and clinical improvement of AD (5–9). While panimmunoglobulin IA was initially employed, an IgE-specific adsorber column has more recently become available (6, 9). Since experience with this novel treatment approach in AD is limited and recommendation for its use in clinical practice will require more clinical data, we employed IgE-selective IA in a further series of patients with severe AD and considerably elevated serum IgE levels. For this group of patients, there still remains a large unmet medical need for powerful new treatment options.

# MATERIALS AND METHODS

# Patients

In this study, 10 patients (seven males and three females, mean age 40.3 ± 18.7, range 18–70 years) with severe AD [mean SCORAD (SCORing AD index) 67.5 ± 5.4, range 61.5–81] and greatly elevated serum IgE levels (mean 5,377.7 ± 6,775.3 kU/L, range 931–21,510 kU/L) were enrolled at the Department of Dermatology of the University of Lübeck. The inclusion criteria were as follows: (1) severe AD, i.e., SCORAD ≥ 60, requiring systemic immunosuppressive treatment, (2) total serum IgE level ≥750 kU/L, and (3) ≥18 years of age. The exclusion criteria were as follows: (1) known hypersensitivity or allergy to materials used in the adsorber columns, (2) no possibility of an adequate anticoagulation (e.g., multiple allergies to various anticoagulants), (3) bleeding disorders including hypo- and hypercoagulabilities, (4) severe cardiovascular disease (cardiac failure, NYHA III and IV), (5) severe systemic infections extending beyond skin, (6) serum IgG level <250 mg/dL, (7) severe immunodeficiency (e.g., AIDS), (8) treatment with an angiotensin-converting enzyme inhibitor that could not be omitted 72 h before IA, and (9) pregnancy. The majority of patients had previously not adequately responded to or had side effects from treatment with systemic corticosteroids and/or cyclosporine A. Written informed consent was obtained from all patients before participation in this study, which was approved by the ethics committee of the University of Lübeck and followed the Declaration of Helsinki.

# Treatment

Two cycles of five IA sessions on days 1 to 5 (week 1) and days 29 to 33 (week 5) were performed as described previously (5, 6), except for one patient in whom the second cycle was split into two parts (2 × IA and 3 × IA 2 weeks apart) because of an adverse event. In each IA, plasma was separated using a blood cell separation technique (Life-18 Apheresis Unit; Miltenyi Biotec, Bergisch Glattbach, Germany) followed by alternate application of two patient plasma volumes to two adsorption columns (30–35 cycles of approximately 8,000 mL separated plasma) containing monoclonal mouse anti-human IgE (TheraSorb®-IgE; Miltenyi Biotec).

In half of the patients, a peripheral venous catheter was used, and in the other half, central venous access was required due to difficult peripheral veins. Patients receiving a central venous catheter were instructed to prophylactically apply topical fusidic acid to the neck area for 3 days before each IA cycle. Previous topical [corticosteroids (*n* = 9 patients in total; class I: *n* = 1, class II: *n* = 1, class III: *n* = 1, class IV: *n* = 3, class V: *n* = 2, class VII: *n* = 3), calcineurin inhibitors (*n* = 1; tacrolimus)] and/ or systemic treatments [antihistamines (*n* = 5 patients in total; cetirizine 10–20 mg/day: *n* = 3, hydroxyzine 25 mg/day: *n* = 3, desloratadine 20 mg/day: *n* = 1, loratadine 20 mg/day: *n* = 1), corticosteroids (*n* = 1; prednisolone 10 mg/day), cyclosporine A (*n* = 0)] were initially continued without change of frequency and/or dosage and were subsequently allowed to be modified as needed.

# Clinical and Laboratory Examinations

Patients were prospectively assessed by dermatologists both as inpatients (on the days when IA was performed) and outpatients at the Department of Dermatology of the University of Lübeck. During a follow-up period of up to 6 months following IA (weeks 1, 3, 5, 9, 13, 17, and 25), the SCORAD index was applied to evaluate the clinical course of the patients. Additionally, the use of concomitant therapy for AD and adverse events were documented. Total levels of serum IgE, IgG, IgM, and IgA were measured before and after IA using the UniCAP system (Phadia; IgE) and the BN Prospec Nephelometer system (Siemens; IgG, IgM, and IgA) (5).

# Statistical Analyses

Data are presented as means ± SD (SCORAD) and box-andwhisker plots (serum immunoglobulins). Repeated measurements of SCORAD were compared with baseline values using one-way ANOVA, with *P* < 0.05 considered statistically significant.

# RESULTS

# Clinical Course

Of the 10 patients enrolled, two patients withdrew from the study due to an adverse event (central venous catheter-associated *Staphylococcus aureus* septicemia and pain related to intravenous catheter insertion, respectively) during the first IA cycle. Two other patients dropped out because of a coping/compliance problem and absence from follow-up examinations in week 5, but were included in the analysis. Other adverse events included fatigue in one patient necessitating to split the second IA cycle into two parts and edema formation of the hands and feet in another patient. Otherwise, IA was relatively well tolerated.

The mean initial SCORAD (68.3 ± 5.7) improved significantly by 19% at week 3 (to a mean score of 54.5 ± 9.9; *p* < 0.05), by 29% at week 5 (to a mean score of 48.0 ± 10.7; *p* < 0.001), by 43% at week 9 (to a mean score of 38.9 ± 18.5; *p* < 0.001), by 21% at week 13 (to a mean score of 54.0 ± 8.0 *p* < 0.05), by 25% at week 17 (to a mean score of 50.6 ± 14.2; *p* < 0.01), and by 29% at week 25 (to a mean score of 48.2 ± 13.1; *p* < 0.01), with individual maximal SCORAD reductions ranging from 15 to 80% and a SCORAD50 response (i.e., an at least 50% reduction of the baseline SCORAD) in 50% of patients. A slight increase in the mean SCORAD was observed in week 13, although by this time and later, only 3–4 patients could be analyzed as the remainder had dropped out or was additionally lost to follow-up (**Table 1**; **Figures 1** and **2**).

There were generally no major changes in the use of the previously prescribed topical and systemic medications during the study, except for one patient in whom cyclosporine A (250 mg/day) was reinitiated because of worsening of the disease in week 17.

# Serum Immunoglobulin Levels

Each IA cycle induced an average decrement of total IgE levels by 92%. Less substantial reductions in IgM, IgA, and IgG were

*IA, immunoadsorption.*

found, with means of 43, 38, and 35%, respectively. Levels of all immunoglobulins were lowered only transiently and reached similar precycle values again (**Figure 3**).

# DISCUSSION

We recently reported a successful response of a series of treatment-refractory AD patients with excessively high-serum IgE levels to panimmunoglobulin IA and two such patients to IgE-selective IA (5, 6). Using the same treatment protocol of two cycles of five apheresis sessions as in these previous case series, this new study with a further cohort of patients with severe AD and greatly elevated IgE levels extends our knowledge regarding the efficacy of IgE-specific IA. Although somewhat lower mean SCORAD reductions were observed compared with our two preceding reports (e.g., by 19% and 33–38% at week 3 and by 21% and 53–59% at week 13, respectively) (5, 6), the observed clinical improvements were still constantly significant at all examination time points including the last follow-up week 25 compared with baseline.

Recently, an independent head-to-head comparison trial of three cycles of a total of 10 IA sessions of either panimmunoglobulin IA or IgE-selective IA applied over a 2-month period in patients with severe AD with excessive IgE levels revealed that there are no major differences in the positive clinical response between the two different IA treatment groups (9).

Like in our two previous studies (5, 6), serum IgE levels were effectively but transiently reduced by a mean of more than 90% with each IA cycle. In contrast to panimmunoglobulin IA which decreased all other immunoglobulin isotypes to a similar degree (5), serum concentrations of IgM, IgA, and IgG were found to be reduced by only less than half with the IgE-selective adsorber.

This latter finding is considered to be nonspecific and has been attributed to IA-related elution and dilution procedures as reported before (6, 9).

Figure 2 | Time-dependent mean SCORAD changes following immunoadsorption (IA). *n* = 3–8 patients (8, 5, 8, 5, 3, 3, and 4 patients at week 1, 3, 5, 9, 13, 17, and 25, respectively). \**P* < 0.05, \*\**P* < 0.01, and \*\*\**P* < 0.001.

Correlation between serum IgE levels and AD severity has been suggested, but study results are partly conflicting (10, 11). A recent study combining panimmunoglobulin IA for 2–4 days with subsequent biweekly omalizumab treatment for 6 months showed that after initial reduction in total serum IgE levels by IA, free IgE levels continued to fall during omalizumab administration and began to increase again during treatmentfree follow-up. In addition, in parallel with free IgE levels, an improvement in AD was found during the treatment period, with aggravation during follow-up (8). On the other hand, we and others showed that a prolonged decrease of the SCORAD is not hampered by the commonly seen reincrease in serum IgE that occurs within a relatively short time after IA (5–7, 9). Since in our initial study, an IA-induced continuous reduction of the amount of skin-bound IgE was observed, which correlated with histoimmunological and clinical improvements (5), we speculate that this indirect effect rather than a sole impact of IA on circulating IgE contributes to amelioration of AD. In fact, omalizumab has been previously shown to interfere with antigen processing and presentation to T cells by downregulating IgE-Fcε receptor I expression on dendritic cells (12). Nevertheless, the role of circulating and tissue-bound IgE in the pathogenesis and disease activity of AD as well as the detailed mechanistic

after each IA cycle; *n* = 6–8 patients (varying by time point and analyte).

interaction of IA with IgE-associated processes in this disease remain in need of further elucidation.

It also remains to be clarified which kind of IA protocol is best suited for AD patients and how long they can ultimately benefit from this treatment. The published trials so far using different IA protocols with follow-up times ranging from 3 to 18 months uniformly revealed a satisfactory initial linear response after treatment (5–9). Results from this current study and our former two reports indicate that IA treatment results in continuous and stable clinical improvements of at least 3–6 months (5, 6), although some minor reincreases in the SCORAD were observed during the second half of the extended observation timeframe (from 3 months in the panimmunglobulin IA trial up to 6 months) in this and our previous IgE-selective IA case series (6). However, this slight worsening in the mean disease severity observed in the current investigation, which was still significantly lower than before initiation of IA, could be at least partly explained by a potential bias due to the relatively high proportion of patients who dropped out or were additionally lost to follow-up during this time period. In fact, the considerable withdrawal rate, the observation that concomitant AD treatment was basically not reduced or discontinued during the study, and the lack of an appropriate control group represent major limitations in judging the effects of adjuvant IA in this patient cohort.

In the study by Reich et al. (9), clinical effects remained stable until 6 months of follow-up in the IgE-selective IA group and slightly reincreased toward the last visit in the panimmunoglobulin arm. As mentioned before, the IA-omalizumab combinatory study revealed a steady decrease in the SCORAD throughout the 6-month treatment period, whereas a reverse trend was observed during follow-up of another 6 months (8). Finally, a study that investigated the efficacy of 1–5 series of panimmunoglobulin IA, each consisting of five consecutive treatments performed on a monthly regimen, revealed no additional benefit with regard to further SCORAD improvement when more than three series were applied. However, one of the patients of this study who completed five IA series exhibited a long lasting clinical benefit over 12 months (7).

A shortcoming of panimmunoglobulin IA is the patient's potential risk of infections because of the parallel reduction of protective immunoglobulins. In fact, in the study directly comparing unspecific with specific IA in AD, infectious adverse events were limited to the panimmunoglobulin group and comprised herpes labialis/keratitis and bacterial conjunctivitis/sinusitis (9). In contrast, however, a central venous catheter-related *S. aureus* septicemia was observed in both our previous and current study using the nonselective and selective adsorber, respectively (5). Thus, even with IgE-specific IA, a thorough risk-benefit

# REFERENCES

assessment is advised especially for patients in whom a peripheral venous access is not possible, considering that AD is strongly associated with increased susceptibility to skin infections by this bacterial pathogen.

In conclusion, our observations strengthen our and other recent results further suggesting that IgE-selective IA is an effective treatment option for patients severely affected by AD with highly elevated IgE levels. However, given the limitations of this study, including the relatively small number of patients, these results should certainly be considered suggestive rather than definite. In fact, considering that IA has only recently been introduced and that it represents a so far restrictedly used new treatment method for AD, information on efficacy and safety currently still relies on low-level evidence case series. Nevertheless, collection of available data even from these small studies, including the current one, may be important for future meta-analyses or systematic reviews to generate better estimates of the treatment outcomes and may also serve as basis for a potential randomized controlled trial. Thus, future investigations are required to better characterize, among others, the long-term effects and cost–benefit (approximately 6,000 euro for a reusable TheraSorb®-IgE adsorber column pair allowing for up to 10 treatment sessions) of IA in AD, for which more optimized treatment protocols still need to be defined.

# ETHICS STATEMENT

This study was carried out in accordance with the recommendations of the ethics committee of the University of Lübeck with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the ethics committee of the University of Lübeck.

# AUTHOR CONTRIBUTIONS

MK, RL, DZ, and ES contributed to the conception and design of study and analyzed the data. MK, S-CM, DK-R, and PM contributed to the data acquisition. MK, S-CM, DK-R, AV, RL, DZ, PM, and ES interpreted the data, drafted the manuscript, approved the final version of the manuscript, and agreed to be accountable to all aspects of this work. MK, S-CM, DK-R, AV, RL, DZ, PM, and ES approved the final version of the manuscript.

# FUNDING

This work was supported by infrastructural funding provided by Deutsche Forschungsgemeinschaft Excellence Cluster Inflammation at Interfaces (EXC 306/2).

**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 Kasperkiewicz, Mook, Knuth-Rehr, Vorobyev, Ludwig, Zillikens, Muck and Schmidt. 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.*

# A New Classification System for igG4 Autoantibodies

*Inga Koneczny\**

*Institute of Neurology, Medical University of Vienna, Vienna, Austria*

IgG4 autoimmune diseases are characterized by the presence of antigen-specific autoantibodies of the IgG4 subclass and contain well-characterized diseases such as muscle-specific kinase myasthenia gravis, pemphigus, and thrombotic thrombocytopenic purpura. In recent years, several new diseases were identified, and by now 14 antigens targeted by IgG4 autoantibodies have been described. The IgG4 subclass is considered immunologically inert and functionally monovalent due to structural differences compared to other IgG subclasses. IgG4 usually arises after chronic exposure to antigen and competes with other antibody species, thus "blocking" their pathogenic effector mechanisms. Accordingly, in the context of IgG4 autoimmunity, the pathogenicity of IgG4 is associated with blocking of enzymatic activity or protein–protein interactions of the target antigen. Pathogenicity of IgG4 autoantibodies has not yet been systematically analyzed in IgG4 autoimmune diseases. Here, we establish a modified classification system based on Witebsky's postulates to determine IgG4 pathogenicity in IgG4 autoimmune diseases, review characteristics and pathogenic mechanisms of IgG4 in these disorders, and also investigate the contribution of other antibody entities to pathophysiology by additional mechanisms. As a result, three classes of IgG4 autoimmune diseases emerge: class I where IgG4 pathogenicity is validated by the use of subclass-specific autoantibodies in animal models and/or *in vitro* models of pathogenicity; class II where IgG4 pathogenicity is highly suspected but lack validation by the use of subclass specific antibodies in *in vitro* models of pathogenicity or animal models; and class III with insufficient data or a pathogenic mechanism associated with multivalent antigen binding. Five out of the 14 IgG4 antigens were validated as class I, five as class II, and four as class III. Antibodies of other IgG subclasses or immunoglobulin classes were present in several diseases and could contribute additional pathogenic mechanisms.

### *Edited by:*

*Ralf J. Ludwig, University of Lübeck, Germany*

#### *Reviewed by:*

*Jun-ichi Kira, Kyushu University, Japan Lars Komorowski, Institute of Experimental Immunology Affiliated to Euroimmun AG, Germany*

### *\*Correspondence:*

*Inga Koneczny<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 16 November 2017 Accepted: 12 January 2018 Published: 12 February 2018*

#### *Citation:*

*Koneczny I (2018) A New Classification System for IgG4 Autoantibodies.. Front. Immunol. 9:97. doi: 10.3389/fimmu.2018.00097*

Keywords: IgG4 autoimmunity, IgG4, autoimmunity, neuronal autoantibodies, muscle-specific kinase myasthenia gravis, thrombotic thrombocytopenic purpura, pemphigus, IgG4-related disease

# INTRODUCTION

Autoantibodies are key pathogenic players in a wide range of autoimmune diseases. Immunoglobulins (Ig) of IgG or IgM class that bind either to cell surface-expressed or extracellular matrix antigens induce organ-specific damage in type II hypersensitivity diseases. More systemic autoimmune diseases are classified as type III hypersensitivity diseases and are characterized by circulating IgG and

**Abbreviations:** ADAMTS13, disintegrin and metalloproteinase with thrombospondin motifs 13; CIDP, chronic inflammatory demyelinating polyneuropathy; CNTN1, contactin 1; Caspr1,2, contactin associated protein1,2; Dsg1,3, desmoglein 1, 3; DPPX, dipeptidyl peptidase-like protein 6; FAE, Fab-arm exchange; GSH, glutathione; Iglon5, IgLON family member 5; Lgi1, leucine-rich, glioma-inactivated 1; Lrp4, low-density lipoprotein receptor-related protein 4; MBL, mannose-binding lectin; MuSK, muscle-specific kinase; MG, myasthenia gravis; PLA2R, phospholipase A2 receptor; THSD7A, thrombospondin type-1 domain-containing 7A; TTP, thrombotic thrombocytopenic purpura; vWF, von Willebrand factor.

IgM as well as IgA that binds soluble antigen and forms immune complexes. Antibody-mediated autoimmune diseases include many different pathogenic mechanisms [reviewed in detail by Ref. (1)]. Among these are complement activation, recruitment of immune cells *via* Fc receptors (leading to antibody-dependent cellular cytotoxicity, opsonization, phagocytosis, and overall immune-mediated damage and inflammation), cross-linking of antigen that leads to immune complex formation or endocytosis of transmembrane antigen, or the direct modulation of antigen function. With the exception of the latter, these mechanisms rely on antibody class and subclass, particularly on the sequence and structure of the constant regions. Single amino acids in the constant region of IgG4 are responsible for structural differences that render it unable to exert most of the known pathogenic functions of antibodies. Despite their immunological inertness, several well-described autoimmune diseases are caused by IgG4 subclass autoantibodies, such as pemphigus, muscle-specific kinase (MuSK) myasthenia gravis (MG), and thrombotic thrombocytopenic purpura (TTP), and in recent years the number of potential IgG4 autoimmune diseases has rapidly grown. By now, 14 autoantigens are known that are targeted by IgG4 subclass autoantibodies; these antigens are found throughout the body (**Table 1**), with more than half being located in the central and peripheral nervous system. Whether all of the newly described IgG4 autoantibodies are pathogenic at all, if so by what mechanism, and if they are the sole pathogenic entity in their disease are essential questions that have been addressed to variable extent in the different fields. Here, we review what is known about IgG4 autoantibodies within the scope of IgG4 autoimmunity and attempt to analyze and classify proposed IgG4 autoimmune diseases based on a set of modified Witebsky postulates to validate IgG4 pathogenicity.

# IgG4: FROM STRUCTURE TO FUNCTION

# IgG4 Structure

IgG is the predominant antibody class and one of the most abundant glycoproteins in the human plasma with concentrations of approximately 7–15 g/L. In humans, four subclasses are known, IgG1, 2, 3 and 4, which are named in descending order of frequency (136). IgG4 is the least common IgG subclass, comprising only 5% of total IgG (137, 138). It is important to keep in mind that there is a different distribution in mice which are often used as animal models for pathogenicity, here the IgG1 subclass is the non-complement fixing subclass equivalent to IgG4, and in addition there are complement fixing IgG2a, IgG2b, and IgG3 subclasses (139–141). Also, there is different capability of human IgG to bind to rodent complement or Fc receptors, which needs to be considered in passive transfer models, e.g., by additional transfer of human complement (142).

The four human IgG subclasses share a similar structure with over 90% sequence homology. IgGs are heterotetramers, consisting of two heavy (50 kDa) and two light chains (25 kDa) of either κ or λ type. The heavy chain consists of three constant domains (CH) that mediate the antibody effector functions, a hinge region between CH1 and CH2, and one N-terminal variable domain (VH), which recognizes antigen. Similarly, the light chain is comprised of one variable (VL) antigen binding and one constant (CL) region. The VH contains three complementarity-determining regions (CDR 1–3). These three CDRs, and particularly the CDR3, are hypervariable to allow for improved and highly specific binding of antigen. Increased mutation rates at this genetic locus indicate antigen-driven somatic mutations.

Disulfide bridges connect the light and heavy chains to form half-antibodies (HL), and two HL are joined together by covalent and non-covalent interactions between the heavy chains to form a whole antibody (H2L2). The heavy chains are mainly connected by disulfide bridges in the hinge region, which vary in numbers between the IgG subclasses. IgG1 and IgG4 have two disulfide bridges in the hinge region, while IgG2 has four and IgG3 11 (143–145). The disulfide bonds that connect heavy and light chains also vary between subclasses, in IgG1 they connect the n-term of the CL domain with the c-term of the CH1 domain, while in IgG2–4 the c-term of the CL domain is connected to the n-term of the CH1 region.

Upon enzymatic digestion with papain, antibodies are divided into three fragments, one Fc fragment ("Fragment crystallized"), consisting of the CH2–CH3 of both heavy chains including the hinge, and two Fab fragments ("Fragment, antigen binding"), consisting of the CH1, VH, CL, and VL. The corresponding regions in the intact antibody are thus named "Fab" or "Fc" region [**Figure 1A**, reviewed by Ref. (146)]. Particularly interesting in IgG4 is the hinge and Fc region; while there is a high degree of homology between the IgG subclasses, there are single amino acid differences that have immediate consequences for IgG4 structure and effector function.

# Functional Characteristics of IgG4: Immunological Inertness and Fab-Arm Exchange (FAE)

Single amino acid changes in the CH2 region (exchange of a proline for a serine at position 331, "P331S") prevent binding of the protein C1q (148, 149), the initial binding molecule of the classic complement cascade, and together with further changes (single amino acid replacements of L234F, P331S, and A327G) also cause a reduced binding to activating Fcγ receptors, but not to the inhibitory FcγRIIb (150–155). The exchange of a proline for a serine in the IgG4 hinge region at position 228 compared to IgG1 (P228S) leads to greater stereometric flexibility. This flexibility allows disulfide bridges in the hinge to additionally form within the same heavy chain ("intra-chain" disulfide bonds) instead connecting the two heavy chains ("inter-chain" disulfide bonds). Under reducing conditions, these states are dynamic and in equilibrium [**Figure 1A** (156–158)]. Together with reduced non-covalent interactions between the heavy chains in the CH3 region caused by an arginine instead of lysine (R409K) (159, 160), the two HL can dissociate (161–163) and recombine with other HL, in a process termed FAE [**Figure 1B** (161, 164–166)]. The resulting antibodies are bispecific and can cross-link two different

(*Continued*) Koneczny A New Classification System for IgG4 Autoantibodies

#### TABLE 1 | Continued

*ADAMTS13, disintegrin and metalloproteinase with thrombospondin motifs 13; Caspr, contactin-associated protein; CIDP, chronic inflammatory demyelinating polyradiculoneuropathy; CNTN1, contactin 1; DPPX, dipeptidyl peptidaselike protein 6; Dsg1, desmoglein 1; Dsg3, desmoglein 3; Ig, immunoglobulin; Iglon5, IgLON family member 5; Lgi1, leucine-rich, glioma-inactivated 1; Lrp4, low-density lipoprotein receptor-related protein 4; MUSK, muscle-specific kinase; PLA2R, phospholipase A2 receptor; scFv, single chain variable region fragments; THSD7A, thrombospondin type-1 domain-containing 7A; vWF, von Willebrand factor.*

February 2018

antigens instead of two antigens of the same kind [**Figure 1B** (166, 167)]. As a consequence, IgG4 is not able to activate the classic complement pathway or activate immune cells as discussed in the part of the protective role of IgG4 in immunity. The following reviews are suggested for further reading on IgG4 structure (147, 168–170).

# FAE: Regulation and Kinetics

Fab-arm exchange seems to occur spontaneously in the body, since antibodies that are bispecific for two different allergens can be found in allergy patients (166). Here, endogenous IgG4 was able to cross-link two different allergens suggesting that an exchange between two distinct allergen-specific IgG4 molecules (birch and cat allergen) had taken place. FAE also occurred between human IgG and therapeutic, humanized antibodies (natalizumab) given as therapy to patients; here, the therapeutic antibodies exchanged half-molecules *in vivo* with endogenous IgG4 (171, 172). Bi-specific IgG4 was also detected in healthy probands, as IgG4 possessing both a λ and a κ light chain simultaneously was found in their serum [**Figure 1C** (173)]. FAE is inducible *in vitro* using reducing agents, such as glutathione (GSH) at concentrations of 1 mM and less (166, 171, 174–176). It is not known if and how FAE is regulated, or where it occurs in the body. The concentrations of GSH in human serum are too low to allow for efficient FAE, but other yet unidentified compartments in the body might provide the required GSH concentrations, e.g., human red blood cells, as they contain approximately 1 mM GSH (177).

*In vitro*-induced FAE occurs rapidly, in less than an hour (167), but *in vivo* it could take hours to days. The therapeutic monoclonal anti-integrin antibody natalizumab was found to exchange Fab arms in patients with endogenous IgG4 (172), with quantitative data obtained by injection of natalizumab into rats demonstrated an exchange half-life of less than 6 h (178). Human IgG4 has a metabolic half-life of around 21 days (179). Calculations led by Peter Molenaar based on these findings using first-order kinetics suggest that up to 99% of IgG4 should be bispecific in the body (17). There is further evidence: in IgG4 the κ light chain is more commonly used with a 3:1 κ: λ ratio. After FAE, IgG4 can harbor a κ and a λ light chain simultaneously (**Figure 1C**). The chance of a κ-half molecule to exchange with another κ-half-molecule is, therefore, more likely than the exchange with a λ-half-molecule, leading to a "silent" bispecificity hidden in the κ/κ and λ/λ fractions (173). The exchange is thought to be random. Calculating recombination probabilities using a Punnet square and assuming a 100% exchange rate, we expect a ratio of 9:6:1 (κ/κ:κ/λ:λ/λ). That means 37.5% of IgG4 would be κ/λ bispecific, which is close to the values measured in one study with healthy individuals (173). Taken together, it suggests that the majority of IgG4 in the human body is bispecific under normal circumstances. Despite the reduced avidity of IgG4, autoantibody binding can be demonstrated in tissue- and cell-based assays *in vitro*, e.g (3, 72, 88, 99)., perhaps due to the high affinity of the antibody with a Kd that can be in the picomolar range (180). Bispecificity also has functional consequences, as bispecific IgG4 is unable to divalently bind and cross-link one single species of antigen.

# IgG4 Fc–Fc Interactions

The Fc region of IgG4 is also able to directly interact with IgG by Fc–Fc interactions, but only if the binding partner is immobilized on a solid phase (181–183). The relevance of this interaction is not understood, but notably occurs in rheumatoid arthritis, IgG4 related disease (IgG4-RLD), and membranous nephropathy (MN), the latter being proposed as an IgG4 autoimmune disease (184–186). It is possible that IgG4 containing immune complexes could form by this mechanism; these would not otherwise form by divalent binding and cross-linking due to the bispecificity of the antibody (163). Modifications of the Fc region of IgG4 induced the formation of artificial hexameric complexes *via* noncovalent interactions that were able to bind C1q and activate the classical complement pathway (187). Whether this is relevant for pathophysiology of IgG4 is unclear.

# IgG4 Has a Protective Role in Immunity

IgG4 and IgE are usually produced in response to chronic exposure to antigens (188). The production of IgE and IgG4 is stimulated by Th2 cytokines IL-4 and IL-13. Furthermore, the additional presence of IL-10 is thought to tip the balance toward IgG4 production and IgE inhibition (189–192). In addition, secretion of IL-10 by regulatory T cells was also found to induce IgG4 rather than IgG1 production (193) and a majority of isolated IL-10-producing regulatory B cells produced IgG4 in contrast to other B-cell subsets (194). These findings link IgG4 with anti-inflammatory tolerance mechanisms [discussed in more detail here (147)]. At present, most literature reveals that IgG4 has an anti-inflammatory function. It has been shown that its levels only rise slowly after chronic exposure to antigen (195–197) or after allergy immunotherapy (198). Thereafter, IgG4 seems to protect against antibodies of other IgG subclasses by competition for antigen without exerting an effector function, thus blocking the epitope to prevent the harmful effect of other antibody classes or subclasses [**Figure 2** (196–204)]. It has also been shown that passive transfer of IgG4 derived from hyperimmune beekeepers is clinically protective in allergy patients (205, 206) and protects mice from the effects of lethal africanized honey bee venom (207). Similarly, IgG4 subclass autoantibodies against the AChR protect against the pathogenic effects of IgG1 of the same idiotype in rhesus monkeys (166). The immunological inertness and the anti-inflammatory effect of IgG4 make it an unlikely subclass in the context of autoimmunity (**Figure 2**). In many cases [although there are exceptions (49)], antigen crosslinking and endocytosis in the context of autoimmunity rely on divalent binding [**Figure 2** (208–213)], which IgG4 cannot do (2, 3, 166). IgG4 does not form immune complexes that would stimulate antigen-presenting cells and mount an immune response nor does it activate the classical complement system (166, 170, 214). Furthermore, IgG4 inhibits immune precipitation by IgG1 antibodies (163, 200). Only pathogenic mechanisms that are independent of Fc effector functions can be exerted by IgG4, e.g., a block of protein–protein interaction or a direct activation or

inactivation of enzymes or receptors by competitive or allosteric binding (**Figure 3A**).

# DEFINITION AND VALIDATION OF IgG4 AUTOIMMUNE DISEASES

Autoantibodies are in general considered pathogenic when they bind an extracellular antigen in the target organ, e.g., the extracellular portion of a transmembrane antigen, and have a direct pathogenic effect, either directly on the target antigen (e.g., by induction of endocytosis or blocking of antigen function) or on the whole organ by recruitment and activation of complement or immune cells that attack the tissue (1, 215). Antibodies against intracellular antigens are not considered to be pathogenic as they are generally inaccessible (215), although they can be valuable biomarkers (216). IgG4 subclass autoantibodies are certainly unexpected culprits in autoimmunity. Particularly, as IgG4 can arise during an immune-dampening response to pathogenic antibodies of other classes and subclasses, candidate diseases should be carefully investigated to determine the pathogenic status of autoreactive IgG4. Using a modification of the postulates by Witebsky, Rose and Bona, and taking into account remarks by Naparstek and Plotz (215, 217, 218), the individual IgG4 autoantibodies in IgG4 autoimmunity were tested to validate the status of IgG4 autoimmunity.

Several aspects should be considered to define IgG4 autoimmunity:

	- 1. Autoantibodies with specificity for an extracellular antigen are present in the affected organ.
	- 2. A pathogenic mechanism for antibodies is demonstrated *in vitro.*
	- 3. The autoimmune disease can be reproduced in experimental animals by passive transfer of patient serum or purified abs or active immunization with antigen.
	- 4. Clinical cues for autoimmunity are present such as HLA association, genetic clustering of other autoimmune diseases in the family, and the clinical improvement of the patients after therapies that reduce autoantibody levels, such as plasma exchange or B-cell depletion therapy.
	- 1. Autoantibodies of IgG4 subclass against extracellular antigen are present.
	- 2. A pathogenic mechanism for IgG4 can be demonstrated *in vitro*. IgG4 could be purified from patient serum, and the purity of isolated IgG4 should be validated; ideally also the IgG4-depleted IgG1–3 fraction should be used as a control group to ensure the effects derived from IgG4. Alternatively, cloned patient antibodies

from IgG4+ B-cells could be used, although these may not be representative of the whole autoimmune B-cell population.

3. Reproduction of disease in animals by passive transfer of purified IgG4 from patients, monoclonal IgG or single chain variable region fragments (scFv) cloned from IgG4+ B-cells of patients.

The 14 proposed IgG4 autoantibodies and their associated diseases were evaluated according to these aspects (**Table 1**). With particular focus on the pathogenicity of IgG4 autoantibodies, three classes emerged with different levels of evidence and also with different likeliness of IgG4 pathogenicity. *Class I diseases* fulfill two or three postulates of IgG4 pathogenicity (B.1–3), here the pathogenicity of IgG4 autoantibodies could be proven by passive transfer of patient IgG4, or an IgG4 pathogenic mechanism was demonstrated *in vitro* (in most cases this is was a blocking effect). *Class II diseases* fulfill one postulate for IgG4 pathogenicity (B.1), but there exists substantial circumstantial evidence that would be in line with pathogenicity of IgG4. Examples would be if IgG4 levels correlated with disease severity or the presence of a known pathogenic mechanism that relies on functional blocking. *Class III diseases* still fulfill one postulate of IgG4 pathogenicity (B.1) but have the least supportive evidence for IgG4 pathogenicity, either because the pathogenic mechanism relies on divalent binding or is unknown. At this point, a conclusion regarding IgG4 pathogenicity cannot be drawn for class II and III diseases, and more studies are required to validate pathogenicity of their IgG4 autoantibodies.

# Class I Diseases

One of the best-studied IgG4-mediated diseases is pemphigus. Here, autoantibodies bind to transmembrane proteins of keratinocytes, which leads to loss of cell–cell adhesion and blistering of the skin (acantholysis). The antigenic targets define different subforms of disease. In pemphigus foliaceus, the antigenic target is desmoglein 1 (Dsg1), which is expressed at high concentrations in the superficial layers of the epidermis; in pemphigus vulgaris, the mucosal-dominant type antibodies target desmoglein 3 (Dsg3) in basal and parabasal layers of the skin. Patients with pemphigus vulgaris of the mucocutaneous type have antibodies against both Dsg1 and Dsg3 (38, 39). Anti-Dsg autoantibodies are predominantly IgG4 (36, 37). They bind to extracellular epitopes and disrupt cell–cell adhesion, as seen *in vitro* where they induced cell sheet dissociation of cultured human keratinocytes (57, 219) and human skin explants (51). Additionally, the disease could be reproduced by passive transfer of monoclonal patient-derived antibodies that were of IgG4 subclass (42, 43). This means that pathogenicity of IgG4 could be demonstrated in the absence of patient antibodies of other IgG subclasses. Also, depletion of IgG4 reduced pathogenicity of patient serum in one study (220). Nevertheless, this does not mean that IgG4 is the exclusive pathogenic entity, and one study demonstrated that cloned patient IgG1 against Dsg3 was as pathogenic as IgG4 (221). The role of other IgG subclasses in IgG4 autoimmunity is discussed below in more detail. There are in fact several different disease models to investigate pathophysiology of pemphigus by passive or adoptive transfer [reviewed by Ref. (52)]. Maternal antibodies can also transfer to the fetus and cause neonatal pemphigus (54). Antibodies to Dsg1/3 may also affect signal transduction pathways that affect cytoskeleton rearrangement and modulate cell adhesion in keratinocytes, and several targets have been proposed (51, 222–228). Indeed, the MAPK signaling was suggested as potential therapeutic target in pemphigus (225), which can in part modulate the disruption of cell adhesion caused by pathogenic pemphigus autoantibodies (229).

Muscle-specific kinase MG is an autoimmune disease of the neuromuscular synapse hallmarked by fatigable muscle weakness. The autoantibodies target MuSK, a protein at the neuromuscular junction. Approximately 90% of the antibodies are of IgG4 subclass (3, 16), and the pathogenic mechanism of MuSK IgG4 is known and can be demonstrated *in vitro* with purified IgG4 (2, 3). Notably, the pathogenic effect was exclusive for IgG4 subclass antibodies. Passive transfer of purified IgG4 reproduced the disease in experimental animals (4), as did active immunization of complement-deficient mice (5). There is also evidence for a genetic predisposition in these patients (14, 15). Binding of antibodies to MuSK similarly affects its function as tyrosine kinase and organizer of neuromuscular junction development and maintenance. As the binding of the agrin coreceptor low-density lipoprotein receptor-related protein 4 (Lrp4) to MuSK is blocked by MuSK-IgG4 (2, 3), MuSK autophosphorylation in response to agrin is inhibited (2), which impairs its ability not only to induce clustering of the AChR (3, 27) but also to maintain the preexisting clusters (3). This leads to reduced densities of the AChR at the synapse and reduced efficiency of neuromuscular transmission, thus causing fatigable muscle weakness. A recent study demonstrated that inhibiting MuSK dephosphorylation counteracted the effects of MuSK antibodies *in vitro*, which would be an interesting new therapeutic strategy (230). Interestingly, the MuSK antibodies also modify the cross talk between motoneuron and muscle, as presynaptic abnormalities were observed in patients and passive transfer animal models, and led to a reduced quantal release of acetylcholine neurotransmitter into the synaptic cleft (5, 21, 22, 26, 231). The affected retrograde signaling pathway is not known, but as Lrp4 may be part of a retrograde signaling pathway (232, 233), it is very likely that the Lrp4–MuSK interaction plays a role, perhaps by anchoring Lrp4 at the synapse.

Disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) is a protease in the blood circulation that cleaves von Willebrand factor (vWF). In TTP, antibodies against the spacer domain of ADAMTS13 inhibit proteolysis of vWF (60, 234), which leads to accumulation of vWF, binding to platelets and causing microthrombosis. Since the inhibitory antibodies recognize the spacer domain that is also required for binding to vWF, the functional block could be due to blocked protein–protein interaction. IgG4 (but also IgG1) antibodies cloned from TTP patients by EBV immortalization demonstrated pathogenicity *in vitro* in assays measuring ADAMTS13 activity (69). Also, several patient-derived scFv were shown to be pathogenic when expressed in mice, albeit the original subclass of the patient antibodies is unknown (59). There is also additional circumstantial evidence that pathogenic antibodies of IgG4 subclass exist in TTP as IgG4 levels are associated with relapse and some patients have exclusively IgG4 autoantibodies (235).

Contactin 1 (CNTN1), together with contactin-associated protein 1 (Caspr1), is expressed on the axonal surface and together they bind to neurofascin 155 on the surface of oligodendroglia (236–239). They are cell adhesion molecules at the axoglial junction of myelin sheaths that are important for efficient nerve impulse propagation along myelinated axons. Antibodies against CNTN1 of IgG4 and IgG1 subclass are present in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) (29, 34, 35, 124) and were proven to be pathogenic. Autoantibodies recognize CNTN1 in the paranodal axoglial junctions of motoneurons and block interaction of neurofascin 155 with CNTN1 in the CNTN1/Caspr1 complex (30). This leads to transverse band loss and paranodal loop detachment in the peripheral nerves. Pathogenicity of IgG4 was proven by passive transfer of purified IgG4 to Lewis rats (31) with loss of paranodal clusters that contain CNTN1, Caspr1, and neurofascin 155 and impaired motor nerve conduction.

# Class II Diseases

Membranous nephropathy, a cause of proteinuria and nephrotic syndrome, is hallmarked by immune deposits containing IgG [predominantly IgG4 antibodies to the phospholipase A2 receptor (PLA2R) in primary MN], autoantigens such as PLA2R, and complement beneath the basal surface of podocytes in the kidney. PLA2R-IgG4 was proposed to have a unique mechanism of complement activation *via* the lectin pathway (98, 99, 103, 108, 109). This activation is hypothesized to derive from galactose-deficient side chains in PLA2R-IgG4 (97, 98) that might recruit mannosebinding lectin (MBL), which is also seen in rheumatoid arthritis (240) and IgA nephropathy (241). MBL binding leads to the activation of the lectin pathway and formation of the membraneattack complex (103). Preliminary data by Beck and Salant from experiments with purified IgG4 from patients with primary MN showed that more of the IgG4 glycan chains lacked terminal galactose residues than did the IgG4 from control subjects and demonstrated increased binding of MBL to PLA2R-IgG4 (108). Additional evidence comes from a more recent study, where MBL deposition in glomeruli correlated with IgG4 positivity in biopsies from Japanese MN patients (109). A recent study by the group of Andreas Kistler demonstrated hypogalactosylated glycan side chains for IgG4, IgG induced complement-mediated injury to PLA2R-expressing podocytes *in vitro* and its prevention by MASP inhibitors, which adds further evidence to this unusual mechanism (poster presentation at the 2017 American Society of Nephrology meeting). However, both the Kistler and the Ma studies have not yet been published in peer-reviewed journals and need further investigation. IgG4-mediated activation of the lectin pathway may not be the only pathogenic mechanism, as one patient with characteristic PLA2R-IgG4-positive MN was deficient for MBL but revealed complement activation *via* the alternative pathway (242), and a small fraction of patients is negative for PLA2R-IgG4 (243). Additionally, a monoclonal IgG3 kappa with specificity for PLA2R was able to cause MN in the native kidney and the allograft *via* the classical pathway (244). An additional blocking mechanism was proposed since antibodies against PLA2R in MN were found to block adhesion of podocytes to collagen IV (245). In another study, PLA2R, however, was not found to bind collagen IV or I in general (106, 246). Furthermore, attempts to reproduce the disease in rodents by passive transfer have been unsuccessful, since the podocytes of rodent glomeruli do not express PLA2R [summarized by Ref. (247)]. Therefore, further studies are required to validate this finding and pinpoint the mechanism to IgG4.

Thrombospondin type-1 domain-containing 7A is expressed in glomeruli in the kidney and is another antibody targeted in MN (110, 113, 114, 118). Histologically, a thickening of the glomerular basement membrane with IgG and complement deposition can be observed. Anti-THSD7A antibodies were predominantly IgG4 (but also IgG1) (110, 116) and *in vitro* were shown to affect cytoskeletal architecture with the formation of stress fibers in cultured primary murine podocyte cells and could also induce detachment of THSD7A overexpressing HEK293 cells (111). Passive transfer of patient serum to mice led to proteinuria with late complement activation, but transient proteinuria in the absence of complement involvement was induced after passive transfer of THSD7A affinity-purified patient antibodies (111). Passive transfer of anti-THSD7A antibodies generated in rabbits could reproduce disease independent of complement activation (112). Taken together, there is evidence that THSD7A antibodies affect podocyte attachment, perhaps by a (reversible) blocking effect, but also that complement activation *via* C1q or lectinbinding pathway as a second pathogenic mechanism plays a role.

In three different neurological disorders with IgG4 subclass antibodies, targeting leucine-rich, glioma-inactivated 1 (Lgi1), contactin-associated protein 2 (Caspr2), and Neurofascin155, respectively, a pathogenic mechanism has been suggested that is based on the block of protein–protein interaction (70, 80, 87, 93, 248). The predominant mechanism of IgG4 antibodies in class 1 IgG4 autoimmune diseases was found to be a blocking mechanism, which may also be associated with a subclass switch to IgG4. It should be kept in mind that a blocking mechanism is not *per se* restricted to the IgG4 subclass [e.g., Ref. (69)], although it is more commonly associated with IgG4 autoimmunity. In detail, Lgi1 is expressed at the synaptic cleft of CNS neurons and in hippocampal neurons in culture, like CASPR2, it is part of the voltage-gated potassium channel complex, and antibodies against Lgi1 are associated with limbic encephalitis (72, 77, 93, 249). Here, serum from patients with Lgi1 encephalitis led to a block of Lgi1–ADAM22 interaction *in vitro*, which caused reduced AMPA receptors and excitation of hippocampal CA3 pyramidal cells *ex vivo* (70). These effects were not yet proven to be IgG4 specific. Caspr2 is expressed in the peripheral and central nervous system, particularly in the juxtaparanodal region of myelinated axons and in hippocampal GABAergic interneurons, and autoantibodies are associated with different clinical syndromes (88, 91, 250–252). It is suspected that the antibodies block the interaction between Caspr2 and TAG-1 and affect inhibitory interneuron function, thus leading to hyperexcitability (87). Importantly, many patients harbor not only IgG4 subclass antibodies against Caspr2 or Lgi1 but also IgG1 and IgG2 (88). Antibodies to neurofascin 155 are thought to have a similar effect to antibodies against CNTN1, with a block of CNTN1/Caspr1–neurofascin 155 interaction affecting paranodal structure and causing peripheral neuropathies (30, 79, 81, 84).

# Class III Diseases

IgLON family member 5 (Iglon5) is a CNS antigen and is targeted by autoantibodies in Iglon5 parasomnia, which is a severe autoimmune disease characterized by abnormal sleep behavior. Here, a population of pathogenic IgG1autoantibodies induced endocytosis of Iglon5 (134) (discussed in more detail below). It was demonstrated that purified Iglon5-IgG4 as well as digested Fab fragments did not induce this mechanism. It cannot be excluded that Iglon5-IgG4 antibodies have an additional pathogenic mechanism, but at this moment there is no indication that they are pathogenic at all.

Similarly, in the case of dipeptidyl peptidase-like protein 6 (DPPX) antibodies, the described pathogenic mechanism is the loss of DPPX and associated Kv4.2 from the cell surface, perhaps by antigenic modulation, which would require divalent binding and cross-linking of antigen. Unless an additional pathogenic mechanism is found for DPPX-IgG4, or alternatively, it is demonstrated that the loss of DPPX and Kv4.2 results from a mechanism that does not rely on divalent binding, it is not likely that DPPX encephalitis is an IgG4 autoimmune disease.

Only one patient has been described with IgG4 subclass antibodies against Caspr1 [a second one if antibodies against complexed CNTN1/Caspr1 are considered (35, 124)], and at this point not enough data are available to estimate pathogenicity of IgG4. There are also insufficient data available regarding pathogenicity of IgG4 subclass anti-collagen type IV (alpha3NC1 domain) antibodies in Goodpasture syndrome, the most convincing circumstantial evidence being temporal occurrence and reoccurrence of IgG4 autoantibodies and lung hemorrhage (119).

In summary, the pathogenicity of IgG4 in most IgG4 autoimmune diseases has not been proven yet. A key experiment could be the isolation of IgG4 subclass antibodies from patient serum or plasmapheresis material, followed by passive transfer to a suitable animal model, or to use purified IgG4 in already established *in vitro* models of pathogenicity.

# PROPERTIES OF IgG4 AND IgG4 AUTOIMMUNE DISEASES

One key pathogenic mechanism in antibody-mediated autoimmunity, especially in the peripheral and central nervous system, is the divalent binding, cross-linking, and endocytosis of antigen, as seen with antibodies against AChR, GlyR, NMDAR, and Iglon5 [**Figure 3B** (134, 208–210, 253)]. The effect is usually lost when using Fab fragments instead of whole IgG1, which underlines the relevance for multivalent binding. A possibility how IgG4 autoantibodies could remain pathogenic by cross-linking and endocytosis would be the loss of FAE, as then monospecific IgG4 could still cross-link and internalize antigen. An example could be the presence of point mutations or sequence variants in the IgG4 hinge or the CH3 region that would remove its ability to undergo FAE. This possibility was investigated in several IgG4 associated diseases. In IgG4-RLD, sequencing showed that the FAE abolishing variant K409 was not present (254), and a recent study demonstrated high levels of bispecific IgG4 in IgG4-RLD (255). Similarly, IgG4 autoantibodies in rheumatoid arthritis were found to be bispecific (256), and recently we were able to demonstrate the same for an example of IgG4 autoimmunity, as a large proportion of patient-derived MuSK autoantibodies were bispecific as well (17). These are only a few publications yet, and more studies are needed to validate the findings in additional IgG4-associated diseases. Until then, there is no indication that impaired FAE affects IgG4 in IgG4-associated disorders, which suggests that pathogenic mechanisms of IgG4 autoantibodies do not rely on divalent binding. Notably, the authors of the IgG4- RLD study suggest increased levels of FAE as a new biomarker. As IgG4-RLD is also associated with elevated levels of IgG4, and up to 99% of IgG4 are probably bispecific, it would be interesting to test a correlation between IgG4 concentrations and κ/λ bispecific IgG4. Enrichment of κ/λ IgG within the total IgG4 population (compared to κ/κ and λ/λ antibodies) could indicate a change in light chain usage.

# Blocking as a Main Pathogenic Mechanism of IgG4

Blocking mechanisms are independent of the Fc domain and as such are the main effector mechanisms for IgG4 autoantibodies (**Figure 3A**). The binding of antibody to antigen can cause the inappropriate activation or blocking of enzymes or receptors. As examples, blocking antibodies of IgG1–3 subclass prevent binding of the neurotransmitter acetylcholine to the acetylcholine receptor in AChR MG (257–259), blocking antibodies against thyroxin peroxidase reduce production of thyroid-stimulating hormone (TSH) thus causing hypothyroidism (260), while stimulating antibodies against the TSH receptor are associated with hyperthyroidism (261). Functional blocking as a pathogenic mechanism is found in not only the IgG4 autoimmune diseases TTP and MuSK MG but also pemphigus (2, 52, 60, 234).

Block of protein–protein interaction is overall considered a main pathogenic mechanism for IgG4 and was demonstrated in several class I and II IgG4 autoimmune diseases (**Figure 3A**). The consequence of such a block could be the block of a signal transduction pathway (MuSK, Dsg1/3) or the loss of tissue integrity (e.g., Dsg1/3, Caspr1, and CNTN1). Most IgG4 autoantibodies were found to block protein–protein interaction.

# Complement Activation

Another key mechanism of IgG1–3 autoantibodies is the recruitment of C1q protein and activation of the classical complement pathway, which leads to complement mediated damage, e.g., by antibodies against AChR or aquaporin 4 [**Figure 3B** (262–265)]. IgG4 does not bind C1q, but intriguingly, complement activation by the lectin pathway may emerge as a novel mechanism of IgG4 autoantibodies, as it is found in PLA2R-IgG4 with hypogalactosylated glycan side chains [**Figure 3A** (98, 99, 103, 108, 109)].

# IgG4 Autoimmune Diseases

IgG4 pathogenic mechanisms translate to a range of different disorders that are clinically very diverse. An excellent introduction to the different IgG4 autoimmune diseases can be found here (266). A selection of disease specific reviews is found here (9, 52, 80, 106, 235, 267–271). A good response to B-cell depletion therapy and plasmapheresis has been reported in several IgG4 autoimmune diseases (6–13, 32, 78, 272–274).

# Contribution of Other Antibody Classes and Subclasses

As IgG4 levels usually increase after repeated or strong exposure to antigen, a high level of IgG4 may coexist with (smaller quantities of) pathogenic antibody of other class or subclass (e.g., IgE or IgG1) and could have clinical implications for treatment decisions. Specifically, after the evaluation and classification of the proposed IgG4 autoimmune diseases, it emerged that in most cases several different antibody classes and/or subclasses are present in patient serum (**Table 1**), with varying degree of potential pathogenicity. Of note, IgG4 concentrations could potentially be overestimated depending on the choice of secondary antibody (16), and crossreactivity between secondary antibodies against IgG4 and IgG2 is also known to occur. A correlation between overall antibody titer and antigen-specific IgG4 titer would increase confidence in the quantification data (3).

Furthermore, pathogenic mechanisms were described for serum or IgG from patients with IgG4 autoimmunity that are likely not caused by IgG4 subclass antibodies as they depend on divalent binding or Fc effector function (**Figure 3B**, endocytosis of antigen and complement activation) or have been demonstrated using purified or cloned patient IgG1–3 [e.g., Dsg3-IgG1 (221), ADAMTS13-IgG1 (69), Lgi1-IgG3 (275), MuSK-IgG1–3 (3), or Iglon5-IgG1 (134)]. Indications for complement activation were found in several disorders (**Table 1**).

Particularly interesting are Iglon5 autoantibodies in which an irreversible internalization of Iglon5 was shown. Here, a clear pathogenic mechanism of IgG1, depending on divalent binding (134), was shown. Iglon family members are glucosylphosphatidylinositol-anchored proteins that have a role in membrane stabilization. Iglon5 parasomnia also has an unusual association with tauopathy (130, 276, 277) linking autoimmunity to neurodegenerative diseases. Combined, these findings led to the current hypothesis that Iglon5-IgG1 antibodies, by antigenic modulation of Iglon5, cause a destabilization of the cytoskeleton. This may affect microtubule stability and hyperphosphorylation of associated tau protein that could lead to the observed tauopathy (276). Whether IgG4 subclass antibodies to Iglon5 are pathogenic at all is unclear, thus making it a class III IgG4 autoimmune disease.

Similarly, DPPX antibodies cause a reversible loss of cell surface-expressed DPPX and Kv4.2, thus leading to hyperexcitation of neurons (127). This requires divalent binding and cross-linking of antigens. As IgG4 is mostly bispecific [also in the context of IgG4 autoimmune diseases (17)], it is likely that DPPX-IgG4 is not able to divalently bind and cross-link DPPX. While the precise mechanism for downregulation of cell-surfaceexpressed antigen has not yet been demonstrated, it is likely that this is a function of IgG1–3 that causes internalization by divalent binding and cross-linking of antigen. This possibility could be explored by a modification of the experiment using purified IgG subclasses and Fab fragments.

Notably, 10% of MuSK antibodies are of IgG1 and IgG2 (and very little IgG3) subclass and were pathogenic *in vitro* (3). MuSK IgG1–3 pathogenicity *in vivo* could not yet be demonstrated, but can also not yet be excluded, as passive transfer experiments were impaired by the low concentration of MuSK antibodies in the IgG1–3 fraction that were below detection limit in the experimental animals (4). The exact mechanism of pathogenicity for MuSK IgG1–3 is unknown. MuSK-IgG1–3 were able to affect MuSK function in a cellular model of the muscle and neuromuscular synapse, the C2C12 mouse myotubes. Agrin stimulation leads to MuSK activation and clustering of AChRs, which can be quantified. MuSK antibodies impaired this mechanism and led to reduced clustering of AChRs, and while it is thought that MuSK-IgG4 induce this phenomenon by blocking MuSK–Lrp4 interaction (2, 3), it is unclear how MuSK IgG1–3 affected the AChR clustering as they do not interfere with Lrp4–MuSK interaction. Interestingly, MuSK antibodies (both IgG4 and IgG1–3) also disrupted preexisting AChR clusters that were induced by overexpression of Dok7 in C2C12 myotubes [(3) Koneczny/Vincent et al. unpublished data] independent of agrin signaling, which was also found using autoantibodies against Lrp4 derived from an active immunization mouse model (278). An earlier study suggested that Lrp4 is required to make MuSK susceptible to Dok7-induced activation and induction of AChR clustering, presumably with a role for AChR prepatterning before innervation (279). MuSK IgG1–3 could also have an effect on Lrp4, though it is not clear how, or MuSK antibodies may affect Dok7-mediated MuSK activation, perhaps by altering MuSK conformation, MuSK kinase activity and/or preventing binding of Dok7 to MuSK, as observed in CMS with a MuSK mutation (280). It would be interesting to study whether MuSK-IgG1–3 antibodies affect MuSK–Dok7 interaction.

Another potential mechanism of MuSK IgG1–3 is the induction of MuSK endocytosis, which is also suggested to be part of the normal response of MuSK to agrin stimulation (281–283). One study demonstrated that serum from MuSK patients led to the internalization of MuSK in mouse muscle cells using time-lapse microscopy and confocal microscopy (28). Two different studies could not reproduce these findings using different approaches (2, 3). However, different patients were tested in these studies, and only a few patients were investigated for endocytosis in all three studies, which is important, as there could be variability in the pathogenic mechanisms in the overall MuSK MG population. In addition, there were technical limitations in the later studies [including my own, as discussed there (3)], hence it is still very possible that MuSK IgG1–3 induce MuSK endocytosis. Further studies are indicated using purified MuSK IgG1–3, MuSK expressed in muscle cells (as transient expression in non-muscle cell environment could affect MuSK endocytosis), and perhaps more appropriate methodology.

In TTP, antibodies of IgG, IgM, and IgA class are present; these may convey pathogenicity by the accelerated clearance of ADAMTS13 from the circulation (234, 284). The functional block of ADAMTS13 activity was not restricted to the IgG4 subclass and could also be induced by IgG1 (69). Therefore, the pathogenic mechanism is not linked to the antibody subclass, but rather the epitope, as only antibodies that recognize the spacer domain are associated with an inhibitory role (234).

In patients with MN with anti-PLA2R abs, a subclass switch from IgG1 to IgG4 was reported over the course of the disease, with an inverse correlation with C1q involvement (285). It is thus clear that in the initial stage of the disease, IgG1 contributes to pathogenicity by activation of the classical complement pathway (100), which may then at later stages of the disease be replenished or replaced by IgG4 that may be protective, inert, or pathogenic (99).

In pemphigus, IgG1 antibodies against Dsg1 or 3 are present and they may also contribute to the blocking mechanism (221). Additional pathogenic mechanisms in pemphigus were proposed as desmosome disassembly by clustering and endocytosis of Dsg and stimulation of signaling pathways that modulate keratinocyte cell adhesion (50, 51). It is thought that multivalent polyclonal Dsg antibodies can cross-link and endocytose Dsg, which leads to Dsg depletion from desmosomes and failed cell adhesion (44–48). However, also monovalent pathogenic anti-Dsg3 antibodies led to a depletion of Dsg3, which suggests that this could also be a mechanism of potentially bispecific (and thus monovalent) Dsg3- IgG4 (49).

Taken together, the presence of more than one pathogenic entity in IgG4 autoimmune diseases is quite possible and makes sense, if we consider that a rise of antigen-specific IgG4 in theory could have been an attempt of the immune system to dampen an inappropriate answer to the antigen by a different antibody species. This could also have clinical consequences, as monitoring of different Ig class/IgG subclass-specific autoantibodies may provide a deeper understanding of the autoimmune response, the individual antibody levels or a ratio thereof could be useful as biomarkers for disease progression or the selection of appropriate therapy.

# Presence of Multiple Antibodies in Individual Cases

Two case reports describe the co-occurrence of MuSK MG or TTP with IgG4-RLD (286, 287), which brought up the interesting question whether these diseases might be related. The term IgG4 autoimmunity is relatively new and was introduced in 2015 (266). IgG4-RLD [which is also a relatively new term (288)] is at this point considered as a separate clinical entity. IgG4-related disease is not discussed extensively here, an excellent review is suggested for further reading (289) and also a review with focus on IgG4 in IgG4-related disease (290). Due to the rareness of the IgG4 subclass and its involvement in pathology, the possibility exists that IgG4-RLD and IgG4 autoimmunity could be part of the same spectrum. Both diseases are associated with IgG4, and both have a favorable clinical response to B-cell depletion therapy with rituximab (6, 32, 78, 272–274, 289). However, IgG4-RLD is defined by the formation of tumefactive lesions in target organs, which is not normally described in IgG4 autoimmunity, and by IgG4+ plasma cell infiltrates and increased serum IgG4 levels. Normal IgG4 concentrations are variable, but are thought to comprise 5% of total IgG (137, 138). As mean IgG concentrations vary between 7 and 15 g/L with a mean around 10 g/L, IgG4 concentrations are expected to be around 0.5 g/L (but can rise up to 100-fold in immune responses). IgG4 concentrations above 1.35 g/L are considered as a diagnostic marker for IgG4-RLD (291). In IgG4 autoimmunity, few studies have looked at serum IgG4 levels, but in one study total serum IgG4 was found to be elevated in a small fraction of pemphigus patients (220), which is notable, but only occurred in a minority of patients: three of 27 pemphigus vulgaris patients and three of 16 pemphigus foliaceus patients had IgG4 concentrations above the threshold of 1.35 g/L (or 135 mg/dL). In addition, the total IgG concentrations were reduced in these patients, likely a consequence of immunosuppressive treatment [pemphigus is routinely treated with immunosuppression (292)]. Perhaps the immunosuppressive treatment in combination with an on-going IgG4 autoimmune disease also increased relative IgG4 levels, which would be interesting to study in any IgG4 autoimmune disease using patient serum before and after treatment.

A key characteristic in IgG4 autoimmunity is the presence of antigen-specific autoantibodies of IgG4 subclass with direct pathogenic function. In IgG4-RLD, the role of IgG4 is not well understood, but it is thought that IgG4 may have a blocking, antiinflammatory function. There is indirect evidence for an antigendriven pathogenicity, namely the presence of oligoclonal bands in the CSF of patients and the presence of oligoclonal expansions of somatically hypermutated IgG4+ B-cell clones, but these were not associated with any known autoantigen (293–296). Only a few antigen-specific autoantibodies have been described that are also not consistently found in the disease, and the pathogenicity of these autoantibodies is not known (297, 298). Experiments with purified antibodies from patients with a pancreatic form of IgG4-RLD showed that IgG4 blocked pathogenic effects of IgG1 in a passive transfer animal model (299), and a recent study identified Annexin A11 as an antigenic target in some patients with autoimmune pancreatitis. The study findings also suggest that the pathogenic entity may be IgG1 and that IgG4 may be upregulated as an anti-inflammatory measure that blocks pathogenic IgG1 (300). More studies are needed to validate this finding, also for other subtypes of IgG4-RLD. However, the relatively clear pathogenic role of IgG4 in IgG4 autoimmunity and the potentially protective role of IgG4 in IgG4-RLD make it as a consequence not very likely that these disorders are (closely) related.

# ETIOLOGY OF IgG4 AUTOIMMUNITY

The etiology of most autoimmune diseases and thus of most IgG4 mediated autoimmune diseases is not known, but there are a few interesting observations that could give a clue to potential factors and mechanisms that may contribute to immunopathogenesis.

# Environmental Antigens

In Europe, MuSK MG frequency correlates with geographical latitude (301), with few cases in northern countries but higher prevalence in the south. This pattern is shared by another IgG4 autoimmune disease, pemphigus (302, 303). The distribution might be coincidental, as epidemiology data of MG in the rest of the northern hemisphere do not reflect the same pattern. Or it could indicate an environmental and/or genetic factor, for example, different availability of an environmental antigen, or different climates with different exposure levels to parasites/helminths in the past could have influenced the type 1 hypersensitivity immune answer including the intensity of the IgG4 response. However, the most striking example for environmental antigen-induced autoimmunity comes from fogo selvagem, which is an endemic form of pemphigus that is found mainly in rural areas of Brazil, but also in Colombia and Tunisia (303, 304). Here, an association between insect bites and autoimmunity has been found. The bite of the sand fly (*Lutzomyia longipalpis*) exposes the patient to salivary proteins, specifically LJM11, against which most humans then develop antibodies. While LJM11 itself is a non-pathogenic environmental antigen, mounting an immune response against it is thought to be protective against parasites (specifically *Leishmania major*) that are also transmitted by the bite of the sand fly (305). Unfortunately, the responding antibodies against the non-pathogenic environmental antigen can also cross-react with self-antigen desmoglein 1 (Dsg1) in individuals with genetic predisposition (306, 307). The initial subclinical antibody response is thought to contain IgE, IgG1, and IgM (308–310), then a switch to IgG4 subclass occurs, probably associated with an intramolecular epitope spreading event (311) that is associated with disease onset (312, 313). Etiology of autoimmune diseases is rarely as well documented as in the fascinating case of fogo selvagem. It would be interesting to study if a similar mechanism could be at hand in other IgG4-mediated diseases. A test for antigen-specific IgE in IgG4-associated diseases could give first indications if this was the case. Indeed, a potential link to allergy recently emerged for a different IgG4-associated disease, IgG4-RLD. An association of IgG4-RLD with a history of allergy was already suspected (314), then a polyclonal response to multiple non-infectious environmental antigens was discovered (315) and a rise in IgE and eosinophil levels was found to predict relapse and to have potential use as diagnostic and prognostic biomarkers (316).

# Infection

Several different (potential) IgG4 autoimmune diseases may have a link to infection, generally thought to be conveyed by molecular mimicry, where antibodies against pathogens cross-react against self-antigen. The autoreactive B-cells in pemphigus showed a shared VH1–46 gene usage, and these were associated with few somatic mutations, which suggests that naïve B-cells that use VH1–46 genes are prone to react against Dsg3 (55). VH1–46 B cells are also associated with an increased reaction toward rotavirus capsid protein VP6 and may thus confer a protection toward infection, highlighting the potential for rare cross-reactive clones to trigger the onset of pemphigus autoimmunity (317, 318). In one of two known CIDP patient with Caspr1-IgG4, disease onset was 10 days after preceding virus infection (common cold) (124). Patients with DPPX encephalitis usually experience severe prodromal symptoms of diarrhea and weight loss (median 20 kg), sometimes accompanied by headache or mood disorder, then within a few months the patients develop a range of neurological symptoms (125, 127, 128). DPPX is strongly expressed in not only the myenteric plexus, a mesh of neurons in the gastrointestinal tract, but also the hippocampus, cerebellum, and striatum. The curious shift in symptoms is reminiscent of a gastrointestinal infection that leads to the mounting of an autoimmune attack against the myenteric plexus *via* molecular mimicry or bystander attack mechanisms, as seen, e.g., in *Campylobacter jejuni* infection that causes the Guillain–Barré syndrome [reviewed, e.g., by Ref. (319)]. So far, no pathogen was found to be associated with the prodromal symptoms in DPPX-encephalitis, and it is also possible that this shift may have different causality, but it is an intriguing possibility to keep in mind.

In case of PLA2R antibody-positive MN, infection and molecular mimicry were proposed to potentially contribute to immunopathogenesis, as there is partial homology between PLA2R peptides and bacterial cell wall enzymes of Clostridium species (101, 320, 321). A link between infection and Goodpasture's disease with antibodies against non-collagenous domain 1 of α3 chain of type IV collagen was also described (322, 323). One case of TTP was associated with Epstein–Barr virus reactivation (324), another case disease onset was associated with an influenza A infection (325) and another with dengue virus infection (326).

# Vaccination

There are a few cases of TTP onset or relapse after vaccination (327–329). This is interesting, as a related disease, immune thrombocytopenic purpura, which has antibodies against platelets, is also associated with vaccination (330).

# Malignancy

Overexpression of antigen by tumors can trigger an autoimmune response. THSD7A-positive MN is associated with malignancy, as THSD7A itself may also play a role in certain cancers (331) and specifically two cases were described, one with gallbladder carcinoma, one with endometrial cancer, where THSD7A was overexpressed in the tumor and taken up by follicular dendritic cells in a regional lymph node (106, 113, 115, 116).

# Summary and Conclusion

In recent years, many IgG4 autoantibodies were discovered, particularly against neuronal targets. By now, 14 different antigen targets have been described and suggested to play a causative role in "IgG4 autoimmunity" (266). Using a modified version of the Witebsky postulates to identify IgG4 autoimmune diseases with proven pathogenicity of IgG4 autoantibodies, the diseases were classified into three categories: class I with proven pathogenicity of IgG4 autoantibodies (including TTP with ADAMTS13 antibodies, pemphigus vulgaris with anti-Dsg3 antibodies, pemphigus foliaceus with anti-Dsg1 antibodies, MuSK MG and CIDP with anti-CNTN1 antibodies), class II where pathogenicity of IgG4 is highly suspected (anti-Lgi1- and Caspr2-associated encephalitis/ Morvan's syndrome, CIDP with anti-neurofascin155 antibodies, and MN with anti-PLA2R or anti-THSD7A antibodies), and finally class III with diseases where IgG4 pathogenicity has not been studied extensively yet (anti-Caspr1-associated CIDP and Goodpasture syndrome with IgG4 antibodies against Collagen IV alpha3NCI) or that have a main pathogenic mechanism that relies on divalent binding, cross-linking and endocytosis of antigen (Iglon5 parasomnia and anti-DPPX encephalitis). Class II and III diseases require further investigation to determine the pathogenicity of IgG4, ideally by passive transfer experiments with purified patient IgG4. Evaluation of the diseases suggests that IgG4 is mainly pathogenic by blocking protein–protein interaction, but that in many cases non-IgG4 autoantibodies are present that may contribute to pathogenicity by other mechanisms, in many cases activation of classical complement is suspected. This may have clinical consequences for treatment decisions. The possibility of an association between IgG4 autoimmunity and IgG4-RLD was discussed but found unlikely.

# AUTHOR CONTRIBUTIONS

The author confirms being the sole contributor of this work and approved it for publication.

# REFERENCES

# ACKNOWLEDGMENTS

I would like to express my deepest gratitude to Jan Bauer, Laurence Beck, Aimee Payne, Mårten Segelmark, Donald Siegel, and Angela Vincent (in alphabetical order) for their helpful suggestions and/ or scientific advice. Especially helpful was the thorough review of the manuscript by Jan Bauer, Laurence Beck, and Aimee Payne, which has greatly improved its quality. Furthermore, I want to thank Theo Rispens that I could base **Figure 1A** partly on one of his previously published figures. IK was funded by an Erwin-Schrödinger fellowship by the Austrian Science Fund (FWF): Project number J3545-B13.

conformationally altered IgG1 via Fc-Fc interactions. *J Immunol* (2009) 182(7):4275–81. doi:10.4049/jimmunol.0804338

repertoire of human rotavirus-specific B cells is reduced in the memory compartment through somatic mutation of nondominant clones. *J Immunol* (2008) 180(5):3279–88. doi:10.4049/jimmunol.180.5.3279

**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.

*Copyright © 2018 Koneczny. 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.*

# Pemphigus—A Disease of Desmosome Dysfunction caused by Multiple Mechanisms

#### *Volker Spindler1 \* and Jens Waschke2 \**

*1Department of Biomedicine, University of Basel, Basel, Switzerland, 2 Faculty of Medicine, Ludwig-Maximilians-Universität (LMU) Munich, Munich, Germany*

Pemphigus is a severe autoimmune-blistering disease of the skin and mucous membranes caused by autoantibodies reducing desmosomal adhesion between epithelial cells. Autoantibodies against the desmosomal cadherins desmogleins (Dsgs) 1 and 3 as well as desmocollin 3 were shown to be pathogenic, whereas the role of other antibodies is unclear. Dsg3 interactions can be directly reduced by specific autoantibodies. Autoantibodies also alter the activity of signaling pathways, some of which regulate cell cohesion under baseline conditions and alter the turnover of desmosomal components. These pathways include Ca2+, p38MAPK, PKC, Src, EGFR/Erk, and several others. In this review, we delineate the mechanisms relevant for pemphigus pathogenesis based on the histology and the ultrastructure of patients' lesions. We then dissect the mechanisms which can explain the ultrastructural hallmarks detectable in pemphigus patient skin. Finally, we reevaluate the concept that the spectrum of mechanisms, which induce desmosome dysfunction upon binding of pemphigus autoantibodies, finally defines the clinical phenotype.

#### *Edited by:*

*Ralf J. Ludwig, University of Lübeck, Germany*

#### *Reviewed by: Robert Gniadecki,*

*University of Alberta, Canada Hendri H. Pas, University Medical Center Groningen, Netherlands*

#### *\*Correspondence:*

*Volker Spindler<EMAIL_ADDRESS>Jens Waschke<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 29 November 2017 Accepted: 16 January 2018 Published: 01 February 2018*

### *Citation:*

*Spindler V and Waschke J (2018) Pemphigus—A Disease of Desmosome Dysfunction Caused by Multiple Mechanisms. Front. Immunol. 9:136. doi: 10.3389/fimmu.2018.00136*

Keywords: pemphigus, autoantibodies, cell adhesion, desmosomes, keratinocytes

# INTRODUCTION

Pemphigus is a severe autoimmune-blistering skin disease caused by autoantibodies primarily targeting the desmosomal adhesion molecules desmogleins (Dsgs) 1 and 3 (1), which are required for the firm intercellular adhesion of keratinocytes. Autoantibodies targeting Dsg3 are found during the mucosal-dominant phase in pemphigus vulgaris (mPV) which is frequently followed by a mucocutaneous phase (mcPV) with additional epidermal blistering and characterized by the presence of both anti-Dsg3 and anti-Dsg1 antibodies. By contrast, in pemphigus foliaceus (PF), flaccid blisters are found in the skin only, and their formation is associated with the occurrence of anti-Dsg1 autoantibodies. Autoantibodies against other desmosomal cadherins such as desmocollin (Dsc) 3 are rarely detectable in PV or PF but can be pathogenic (2–5). The pathogenicity of autoantibodies against a range of non-desmosomal autoantibodies that are often present in patients' sera in addition to anti-Dsg antibodies is unclear (6). We here focus on the mechanisms causing skin blistering in response to autoantibodies against Dsg1 and Dsg3. In addition to suppressing autoimmunity, which is the current basis for disease management (1), we believe that novel targeted therapeutic strategies are necessary to stabilize desmosomes in situations when autoantibodies are present. It is essential to better understand the regulation of desmosomal adhesion, and we aim to provide perspectives for future research by reevaluating the mechanisms leading to desmosome dysfunction, loss of keratinocyte cohesion, and blistering.

# HISTOLOGY AND ULTRASTRUCTURE OF PATIENT'S LESIONS HIGHLIGHT RELEVANT MECHANISMS

Cultured keratinocytes or mouse models can only partially reproduce the situation in patients. We believe that for the identification of relevant mechanisms in pemphigus pathogenesis, the careful evaluation of patients' lesions is the gold standard. On the histological level, skin blistering in PV occurs by suprabasal splitting (**Figure 1**), whereas PF is characterized by superficial lesions restricted to granular or upper spinous layers of the epidermis (1). Although exceptions occur, PF splitting is usually found in the upper half of the epidermis, whereas PV affects the lower half. At least in some parts of typical PV but not PF lesions, the histological hallmark of a "tomb-stone appearance" of keratinocytes in the blister bottom can be detected (7, 8).

By immunostaining, the clustering of Dsg3 together with autoantibodies has been detected in the mucosal and skin lesions in mcPV but also in unaffected epidermis in the mucosal-dominant type (mPV) in the absence of ultrastructural alterations of desmosomes (9, 10). By contrast, Dsg1 clustering was found in mcPV skin and PF epidermis only, i.e., when antibodies against Dsg1 were present (9, 10) (**Figure 1**). Since Dsg3 clustering was shown to be the structural correlate of Dsg3 depletion (11), these data indicate that the depletion of Dsg3 alone may be a primary mechanism in mucosal eroding but alone is not sufficient to cause epidermal blistering. Nevertheless, Dsg3 depletion may drastically worsen desmosome destabilization in the epidermis which is suggested by the fact that glucocorticoids rapidly improve the clinical phenotype (1) at least in part *via* Stat3-induced Dsg3 transcription increase (12).

On the ultrastructural level, smaller desmosomes were found only in conditions when patients presented with antibodies against Dsg1 such as in mcPV and PF but not in mPV (9, 10, 13, 14), suggesting that Dsg1 targeting is critical and may interfere with desmosome assembly or cause dismantling of existing desmosomes (**Figure 1**). Besides a reduced size, a general loss of desmosomes is present under all conditions where blistering occurred. Electron microscopy revealed the formation of doublemembrane structures in PV and PF containing desmosomes with reduced size and altered morphology which may be the correlate for the depletion of extradesmosomal Dsg molecules and the uptake of entire desmosomes (13). Similarly, interdesmosomal widening, which is the first ultrastructural sign to be detected in pemphigus lesions, may be caused by the endocytosis of extradesmosomal Dsg1 rather than of Dsg3 (13, 15). This alone appears not to be sufficient for blister formation since it was detected also in the unaffected deep epidermis and the mucosa of PF patients but not in mPV with intact Dsg1 distribution.

Split desmosomes both with and without attached keratin filaments were detected by electron microscopy and SIM on the keratinocyte surface facing blisters in PF and mcPV (13, 14). Desmosome splitting can be induced by mechanical stress (14) and may be the ultrastructural correlate for the direct inhibition of Dsg binding. Since split desmosomes in this study were of reduced size, altered desmosome structure appears to be required, suggesting an additional role of impaired desmosome assembly or the depletion of desmosomal Dsg. The final hallmark described early for both PV and PF by electron microscopy is keratin retraction (16, 17) (**Figure 1**). Recently, keratin filament retraction was observed only when desmosomes were completely absent (13). This can be interpreted in the way that keratin filaments are not the cause but rather the consequence of desmosomal loss or the changes are temporally tightly correlated.

Apoptosis is not a major mechanism because cells displaying signs of apoptotic cell death are absent or sparse in PV and PF skin lesions and therefore cannot explain acantholysis of a significant epidermal area (13, 18, 19).

# AUTOANTIBODY-TRIGGERED MECHANISMS IMPAIRING DESMOSOME TURNOVER

As outlined earlier, split desmosomes, reduced desmosome numbers and size, and keratin retraction are ultrastructural hallmarks in pemphigus skin. Reduced desmosome size or numbers cannot be explained by the direct interference of pemphigus autoantibodies with Dsg binding but rather are a consequence of the altered turnover of desmosomal proteins. These changes are likely steered by intracellular-signaling pathways, which are modulated in response to autoantibody binding and represent potential pharmacologic targets. In principal, reduced desmosome size and numbers can result either from interference with desmosome assembly or from the enhanced disassembly of desmosomes. Available data suggest that in pemphigus, both mechanisms contribute to impaired desmosome turnover, shifting the balance toward an overall reduction of desmosomal components (20).

Desmosome assembly is tightly interwoven with adherens junction formation and appears to proceed in distinct steps (21) (**Figure 2**, left panel). Desmosomal cadherins are initially transported to the cell membrane in a microtubule- and kinesindependent process (22), which, in case of Dsg2, is enhanced by its palmitoylation (23). The precise mechanisms are unclear but once membrane-localized, desmosomal cadherins appear to cluster in an intermediate junction with E-cadherin, β-catenin, and plakoglobin and probably segregate to form desmosomes clusters later on (24, 25). Plakophilins (Pkps) are essential as they are required to assemble keratin-anchored DP pools in the cortical regions of the cell (26, 27). Pkp3 was shown to participate in transferring DP clusters to the membrane and to stabilize desmosomal cadherins in a Rap1-dependent manner (28). In addition, cortical actin and actin-binding proteins such as adducins and RhoA signaling are necessary for full desmosome assembly (29–31). Desmosomal molecules localize to lipid rafts and the raft-associated proteins Flotillin-1 and -2 (32, 33). In line with this, interference with lipid raft composition prevents both desmosomal assembly and disassembly, suggesting these lipid-enriched membrane domains to be hot spots for desmosome turnover. Compared to the assembly, the disassembly of desmosomes under physiologic conditions is poorly understood, which may be related to the relative chemical inaccessibility and the stability of maturated desmosomes (34).

Pemphigus autoantibodies interfere with desmosome turnover by enhancing the internalization of desmosomal components (**Figure 2**, right panel). However, a clear separation of mechanisms

note that the distribution of Dsg1 and Dsg3 in the epidermis is indicated for all layers except the for corneal layer.

specifically affecting either the assembly or the disassembly pathway in most cases is not possible. It is conceivable that autoantibodies reach Dsgs located in extradesmosomal membrane pools more easily than those densely packed in a mature desmosome. In line with this scenario, the non-desmosomal pool of Dsg3 is the first to be depleted (35, 36). Consequently, the reduced availability of supply molecules leads to the destabilization of desmosomes and in the longer run may favor the depletion of desmosome-localized, now more easily accessible molecules. Thus, the reduced desmosome assembly route in pemphigus is at

desmosomes and promote the disassembly of existing desmosomes. The modulation of desmosome turnover is tied to changes of intracellular-signaling cascades which, for instance, lead to the phosphorylation or cleavage of desmosomal components. Linear arrays represent zones of desmosome disassembly. Split desmosomes may occur if the already-weakened desmosomes are exposed to shear stress.

least in part a result of internalized extradesmosomal molecules, summarized as "desmoglein nonassembly depletion hypothesis" (9, 37). However, the situation apparently is more complex than a simple disbalance in supply and demand. Importantly, especially the extradesmosomal molecules are considered to serve as signaling scaffolds (38, 39). As an example, it was shown that a complex of Dsg3 and Pg binds p38MAPK and suppresses its activity (40, 41). Upon the loss of Dsg3 interaction, e.g., by steric hindrance through pemphigus autoantibodies, this suppressive function is abolished and p38MAPK is activated. Indeed, p38MAPK signaling was shown to promote Dsg3 internalization (42) as well as keratin retraction (41, 43), suggesting that p38MAPK is a central signaling molecule in desmosome turnover. *Vice versa*, keratin filaments were shown to influence both desmosome stability and Dsg3-binding characteristics in a signaling-dependent manner. Under physiologic conditions, the keratin-dependent suppression of p38MAPK signaling increases Dsg3-binding strength, whereas the suppression of PKC signaling through the adapter protein Rack-1 prevents DP phosphorylation, stabilizes Dsg3 in the desmosome (44, 45), and, together with Pkp1 (46), promotes a hyperadhesive state. These functions appear to be disturbed by autoantibody-induced keratin retraction (Vielmuth et al., Frontiers Immunol, this issue). Furthermore, Pkp3 is phosphorylated in response to autoantibody binding in an Srcdependent manner (47), which may be connected to Src being present in the intermediate E-cadherin/Dsg3 complex (25). Together, these data suggest a feed-forward loop from extradesmosomal complexes to desmosomes that, at least in part through interference with keratin-dependent signaling, destabilizes desmosome composition and function. As the precise mechanisms are unclear, it is possible that keratins affect both the assembly and the disassembly pathways of desmosomes.

The occurrence of desmosome disassembly can be concluded from observations that Dsg3 together with PV-IgG is excluded from desmosomes and internalized (48). This is supported by observations that IgG autoantibodies can access Dsgs in native desmosomes (49). Clusters of desmosomal molecules localizing in arrays perpendicular to the cell–cell border may represent sites of internalization of single desmosomal components or the entire complexes (50). These may correspond to the "double-membrane structures" that are visible in patients' skin and are thought to be regions of internalization for partially dismantled desmosomes (13). Although all desmosomal proteins eventually are internalized, the degradation pathways of specific components are not uniform. Dsg3 as well as Pg colocalize with markers for endosomes and lysosomes, whereas DP and presumably other plaque proteins use different, yet unknown, routes (35, 42). Lipid rafts are thought to be important for desmosome disassembly, as the impairment of raft composition prevents the depletion at least of Dsg3 and reduces loss of cell cohesion (32) (Schlögl et al., this issue). In addition, the cleavage of Dsg3 by caspase-3 independent from apoptosis was observed and may promote internalization (51–53), the latter of which was shown to require EGFR signaling (54).