Breaking Bad: IgG4 In Autoimmunity

The human immune system is a complex network of cells and soluble factors that can cleverly adapt to combat infection.  For example, B cells rearrange and mutate their own DNA to create a vast repertoire of antibodies capable of binding to and destroying virtually any target.

During B cell development, the antibody-coding genes are shuffled and pasted together by cellular enzymes in a coordinated, but imprecise, process known as VDJ recombination. Fortunately, humans benefit from the imprecision with which these enzymes do their job. Simply combining the various antibody genes together generates millions of different sequences, but the enzymes randomly insert or delete nucleotides at each of the gene junctions, leading to tens of billions of so-called “variable region” sequences, guaranteeing that no matter what pathogen an individual may encounter, he or she has likely made a B cell that can kill it. After the B cell leaves the bone marrow, if it meets its target, it can activate, divide, and further diversify its variable region through somatic hypermutation of its DNA, spawning clans or lineages of related but distinct B cells all descending from a single precursor, optimally evolved to ward off their preferred invading pathogen.

To add even more complexity, each variable region combines with a different constant region such as IgM, IgG, IgA, or IgE (also known as an isotype), which gives each antibody a specialized function. All B cells begin by expressing IgM, the cadets of the immune system that often nonspecifically and weakly bind to many targets. IgA antibodies act as sentries at mucosal surfaces, IgE recruit in cells specialized for allergic reactions, and IgG are general-purpose antibodies with multiple functions, divided across 4 different subclasses (IgG1-IgG4).  IgG1, IgG2, and IgG3 bind to receptors on inflammatory cells and recruit in mediators that can help to destroy targets, whereas IgG4 is relatively inert, which can be helpful for calming down inflammation by blocking other antibodies from binding and causing damage. These specializations can change through a process called class switching, in which a B cell shuffles the same variable region onto a different constant region, meaning that a single clan of B cells can express antibodies of many different isotypes and subclasses.

Unfortunately, because B cells harbor a virtually unlimited library of sequences, they have the potential to create delinquent antibodies that react against self, rather than foreign invaders. Self-reactive antibodies (autoantibodies) are usually weeded out at “tolerance checkpoints” as the B cell matures, but these self-defense tactics are far from perfect.  Autoimmune disease can attack almost any organ in the body including the skin, thyroid, brain, kidney, muscle, liver, and more, all due to autoantibodies wreaking havoc on the body’s normal functions.

IgG4: friend or foe?

Our laboratory researches a rare and debilitating autoantibody-mediated disease called pemphigus vulgaris, in which the immune system generates antibodies against desmogleins, which are proteins responsible for holding skin cells together. The result is painful, sometimes deadly, blistering of the skin and mucous membranes, requiring powerful immune suppressants such as steroids, mycophenolate, and the anti-B cell agent rituximab for disease control. Because pemphigus vulgaris has a well-characterized target, we study it as a model autoimmune disease to better understand why and how the immune system makes mistakes, so that we can develop better ways of reversing these mistakes and hence, better treat disease.

Patients with pemphigus vulgaris are known to have autoantibodies of the IgA1, IgA2, IgG1, and IgG4 subclasses, with IgG4, in particular, being a marker of active disease.  As mentioned above, IgG4 is typically considered an anti-inflammatory antibody, induced during states of chronic immune stimulation to dampen disease.

Beekeepers and individuals undergoing allergic desensitization therapy develop IgG4 antibody responses that are thought to block symptoms caused by IgG1 or IgE reacting to the same stimuli, one of the reasons why people can outgrow an allergy from childhood. However, in pemphigus vulgaris, the antibody variable region is sufficient to disrupt skin cell adhesion and cause blisters, so trying to suppress these unruly variable regions with a law-abiding constant region can’t override this pathologic effect. Thus, although the immune system is trying all its usual tricks to stop the disease, the switch of B cells to the IgG4 subclass does not allow pemphigus vulgaris patients to outgrow or remit disease but instead becomes the defining immunologic feature of their disease.

Clone Hunting

The focus of our laboratory’s research program over the last decade has been to determine the pathways by which autoantibodies arise and to identify what features of the antibody gene or protein sequence separate disease-causing autoantibodies from the normal antibodies that protect from infection. Recently, we’ve been wondering about how the isotype of anti-desmoglein B cells can point to their evolutionary pathways. At the time, no one knew how the IgG4 autoantibodies that drive the disease are related to the IgA1, IgA2, and IgG1 autoantibodies in the same patient. Because blistering often starts in the oral mucosa in pemphigus vulgaris, perhaps the pathogenic IgG4 B cells might evolve from IgA B cells, implying an initial break of tolerance within the mucosal immune system. It was also unknown whether IgG4 B cells result from a direct class switch from IgG1 B cells, which would imply that IgG4 is the final common pathway for autoreactive B cell clones in pemphigus vulgaris. Previously, these studies were impossible to pursue because the autoreactive B cells are rare and would be hard to reliably detect through traditional cloning or sequencing methods.

To determine how autoreactive B cells arise in pemphigus vulgaris patients, we mapped the B cell repertoire in patients using high-throughput, subclass-specific sequencing. In tandem, we used a technology called phage display, recognized with the Nobel Prize in Chemistry in 2018, to clone and isolate B cell sequences that can generate anti-desmoglein antibodies. Phage display has the advantage of being able to sample billions of antibody sequences at once, affording the opportunity to isolate even the rarest clones that react to a particular target. By combining high-throughput sequencing of B cell repertoires, antibody phage display for identification of desmoglein-specific B cell clones, and complex bioinformatics lineage analysis to sort the desmoglein-specific B cells into pedigrees that analyze clonal relationships across isotypes, we were able to trace the familial relationships and evolutionary history of autoreactive B cells in pemphigus vulgaris.

Many Roads to Ruin

Our results showed that autoantibodies in the IgG4 subclass tend to be “loners,” being mostly unrelated to antibodies (both normal and self-reactive) in other subclasses. In contrast, we found that autoantibodies in the IgG1, IgA1, and IgA2 subclasses have a much higher degree of interrelatedness, indicating that the dominant IgG4 subclass that is primary to disease pathogenesis in pemphigus vulgaris largely stands alone from the IgG1-IgA1-IgA2 evolutionary pathway. Thus, IgA is not the origin of IgG4 autoimmunity in pemphigus but instead is more often the offspring of IgG parental clones.

Interestingly, we found that IgG4 B cells tend to recognize specific domains of the desmoglein molecule that are associated with blister formation, whereas IgG1 and IgA B cells recognized a broader array of epitopes, including non-pathogenic domains. Although further research would be necessary to investigate mechanisms, our data support the notion that class switch to IgG4 may be induced by tissue damage, in a well-intentioned but ultimately unsuccessful effort of the immune system to dampen disease.

We also found that across patients, anti-desmoglein antibodies tend to have different nucleotide and amino acid sequences rather than common motifs, suggesting that there are many different routes to self-reactivity in pemphigus vulgaris. Studies from other groups on dengue fever infection, influenza vaccination, and other immunologic conditions have demonstrated convergent antibody sequences shared between patients, but this does not seem to be the case for pemphigus vulgaris.

Blame the Ancestors

To further understand exactly when a particular B cell becomes delinquent, we computationally inferred the sequences of the “ancestor” B cells and resurrected the antibodies they expressed. To our surprise, we found that the original ancestor and grandparents of many IgG4 B cells are themselves autoreactive, suggesting that the predisposition to autoimmunity in these rare clones is ordained shortly after the birth of the B cell due to its initial VDJ recombination event. Even unaffected individuals may harbor early B cells that are predisposed to causing pemphigus, but these B cells may never act on their criminal tendencies by actually secreting the pathogenic antibodies that cause disease. More work is needed in order to understand why patients’ immune systems allow these autoreactive B cells to persist and expand, while unaffected individuals are able to weed them out.

Fact Check

Now that we had identified the lineage relationships among subclass-specific B cells in pemphigus vulgaris, we wanted to know how reliable our findings were. One of the drawbacks of human immunology research is that we can’t harvest blood, spleen, lymph nodes, and bone marrow from patients to know that we have fully sampled every compartment where B cells reside. Humans have around 5 liters of blood, and we were drawing 60 milliliters for analysis, a little over 1% of the total blood volume. If we were to draw a different blood sample from the same patients, would we find the same result?

We used two ecological methods to estimate our sampling depth, known as mark-and-recapture and rarefaction analysis. Mark-and-recapture is commonly used to estimate the size of an animal population in a particular area. For example, if you want to know how many birds are on an island, you capture a certain number, tag and release them, then return at a later time and capture another sample to determine how many are identical to the ones you captured before, which allows an estimation of the total population size and the sampling depth.  In our analysis, we drew two 30 milliliter samples of blood from patients at the same time (ie, biological replicates that could not have the same B cell in them, but could have clonal relatives within the same clan or lineage) to determine how many of the same lineages were represented in each sample.  These analyses indicated that we captured 35-82% of all the different B cell families within a patient with a 30 mL blood draw.

In the rarefaction analysis, we used the entire 60 mL pooled blood sample to extrapolate out the number of lineages we would find if our sample was infinitely large.  If we identify new lineages with every additional new sequence analyzed, then we haven’t reached the saturation point. Our calculations revealed that we identified 71%-97% of the total lineages and 90-100% of the dominant lineages within the sample, indicating that our methods were sufficient to capture the vast majority of the B cell lineages within patients.

In closing

This study was unique in that we were able to analyze the evolution of autoreactive B cell subclasses in the model autoimmune disease pemphigus vulgaris and characterize the different lineage pathways that lead to autoimmunity. Our studies suggest that IgG4 is a rogue subclass in pemphigus, subverting its normal protective function to cause disease. We hope our study will facilitate future approaches to better understand the genealogy and pathophysiology of autoimmune diseases beyond pemphigus.

These findings are described in the article entitled Autoreactive IgG and IgA B Cells Evolve through Distinct Subclass Switch Pathways in the Autoimmune Disease Pemphigus Vulgaris, recently published in the journal Cell Reports. This work was conducted by Eric M. Mukherjee, currently at Yale University School of Medicine, and by Christoph T. Ellebrecht, Qi Zheng, Eun Jung Choi, Shantan G. Reddy, Xuming Mao, and Aimee S. Payne from the University of Pennsylvania, Philadelphia, Philadelphia.