Inflammation and Autoimmune B Cell Assays
Assess Your Autoimmune-Targeted Therapy with B Cell Assays
Autoimmunity and inflammation associated diseases are characterized by an immune response against self, driving tissue destruction. As a key driver of this, B cells are an amenable target for those seeking treatments for autoimmune disease and inflammation.
Current broad-spectrum therapies include drugs such as corticosteroids, methotrexate, and leflunomide, though newer biologic therapies such as rituximab aim to deplete B cells. Unfortunately, this treatment indiscriminately depletes B cells regardless of their specificity. There is currently an unmet need for therapies that specifically target autoreactive B cells and leave the remainder of the B cell population untouched.
B Cells as Targets for Therapeutic Development
Antibody, or ‘humoral’ immunity plays a key role in protecting against infection, inhibiting viral or bacterial entry and mobilizing the immune response against a variety of pathogens.
Produced by B cells, each antibody is uniquely specific to a single linear or structural epitope. Initially, foreign antigens are recognized by the surface immunoglobulin (or Ig) molecule, or B cell receptor (BCR) on the surface of a B cell within secondary lymphoid tissue. Following interaction with an activated CD4+ T cell, which also recognizes pathogen-associated peptides, the B cell secretes antibody and undergoes affinity maturation to secrete better and more effective antibodies.
Although they play a key role in pathogen-associated immune responses and have been studied extensively in a vaccination context, B cells can also play an important role in the immunopathogenesis of a range of autoimmune diseases like rheumatoid Arthritis (RA), SLE (lupus), multiple sclerosis, Sjögren’s syndrome, and myasthenia gravis. In these patients, B cells produce antibodies that recognize self-proteins or DNA, causing inflammation and tissue destruction.
B Cell Assays: Antibody Production
Antibodies form immune complexes with antigens, which in turn bind to Fc or complement receptors on the surface of phagocytes and drive their activation.
In an autoimmune setting, the antigen is self-derived, for example citrullinated proteins, in the context of rheumatoid arthritis (RA) and multiple sclerosis (MS) or dsDNA or nuclear antigens in lupus. You can therefore target these pathways in order to better treat autoimmune disease. Peripheral blood mononuclear cells (PBMCs) can be stimulated in vitro, either polyclonally, or with specific antigen to cause antibody production. The ability of novel therapeutics to suppress this process can then be assessed.
In the example shown in figure 3, PBMCs were stimulated with a polyclonal stimulus (Staphylococcus aureus, Cowan I and IL-2) for 6 days before assessing the frequency of antibody secreting cells by ELISpot.
Representative images are shown for unstimulated and stimulated cultures. Total IgG secreting- or antigen-specific antibody secreting B cell frequency can then be determined using ELISpot or quantitative levels by ELISA.
This assay would be applicable to therapeutics targeting lupus, RA or MS.
Evaluating Autoimmune Disease Targeted Therapies That Reprogram the Immune System
Learn a more efficient approach to your efficacy studies. In this webinar, our experts explain how combining disease-relevant models and in vitro inflammation and autoimmune T cell assays can provide a clear picture for your therapy to reprogram the immune system.
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B Cell Assays: Activation and Proliferation
Following recognition of antigen and BCR cross-linking, and in the presence of an activated T cell secreting cytokines, a signaling cascade is initiated within the B cell, ultimately leading to NF-κB activation. This leads to activation and differentiation of the B cell, which can be visualized early in this process by examining surface marker expression and cytokine production.
In the example shown in Figure 4, B cells were co-cultured with a variety of stimuli in the presence or absence of rituximab. CD25 is robustly upregulated following stimulation and the B cells proliferate. This was inhibited in the presence of rituximab.
This assay can be used to test whether novel autoimmune targeted therapies can alter the ability of B cells to respond to stimuli in comparison with a known standard of care biologic, used to treat several autoimmune conditions.
B Cell Assays: Antibody Class Switching
Antibodies on the surface of naïve B cells are usually an IgM/IgD isotype.
If the B cell is exposed to the antigen in an inflammatory environment and in the presence of activated CD4+ T cells, which provide ‘help’ to the B cell via CD40-CD40L interaction and IL-4 production. Then, it undergoes somatic hypermutation, affinity maturation and class switch recombination (CSR); shown in Figure 5.
Figure 5. Naïve B cells can undergo aspects of CSR in vitro, secreting IgG, IgA or IgE antibodies
CSR results in antibodies with increased effector function as the constant portion of the antibody molecule changes to an IgA, IgG or IgE isotype, which better activates phagocytic cells, despite the antigen-binding domain being preserved.
In order to understand whether autoimmune targeted therapies can affect B cell CSR, naïve B cells can be isolated from the blood and cultured with CD40L and cytokines to drive their activation and CSR.
Figure 6. Naïve B cells can be isolated and stimulated to undergo aspects of CSR in vitro
At the end of the assay, levels of IgM, IgG, IgA and IgE produced in the cultures can be determined to understand how effectively the B cell underwent CSR.
Autoimmune therapeutics can be tested in this assay to determine whether they are able to reduce overall antibody production or reduce the production of IgG isotypes more associated with a strong inflammatory and phagocyte activating response.
B Cell Assays: Antigen Presentation
B cells express high levels of major histocompatibility complex (MHC) class II and can internalize and present antigen to T cells. Consequently, they are implicated in driving or perpetuating the expansion of autoreactive T cells in various autoimmune diseases.
Targeting the role of B cells in antigen-presentation is therefore an interesting approach in treating autoimmune disease.
In the example shown in Figure 7, B cells were cultured with autologous CD4+ T cells at a range of ratios in the presence of a recall antigen. The CD4+ T cells proliferated when assessed by cell trace violet dilution. The effect of an MHC class II blocking antibody in this assay was then determined and it was found to completely inhibit T cell proliferation, suggesting the B cells were acting as efficient antigen presenting cells.
Therapeutics targeting the antigen processing or presentation pathways can be tested in this assay alongside the MHC class II control antibody.
Is it better to quantify antibody responses in your B cell assay via ELISA or ELISpot?
They tell you slightly different things. One tells you about the quantity of antibody being produced and the other tells you the frequency of antibody-secreting cells. The right B cell assay is likely to be determined by the question you are trying to ask. Learn more
Is it better to run my therapeutic in a purified B cell assay or a whole PBMC assay?
This is determined by the nature of your therapeutic. If you believe it acts directly on B cells, then it may be simpler to use a purified B cell assay. However, if you believe there could be a bystander mechanism, it is likely to be better to use whole PBMC.
Can you model vaccine efficacy in vitro?
It is difficult to fully model vaccine efficacy in vitro due to a lack of specific cellular constituents and the complexity of lymphoid tissue architecture. However, you can ask specific questions about the initiation of a B cell response and how this might be altered with therapeutics. Alongside relevant rodent B cell assays or models you can obtain a broad understanding of vaccine efficacy.
Can you use antibody isotypes to predict the T cell bias of an adaptive immune response in humans – as you can for mice?
In the mouse, IgG2a isotype antibodies indicate a Th1-biased T cell response whereas IgG1 indicates a Th2 response. In humans, the isotype-associated link to T cell function is slightly different, but the biology is largely consistent. In humans Th1 production of IFNγ drives a predominantly IgG1-biased response.
Are B cells efficient antigen-presenting cells?
Historically, the dogma would suggest that B cells are less efficient at priming naïve T cells but this is somewhat controversial. B cells express all the requisite molecules to take up, process and present antigen; for example, they express considerable amounts of MHC and co-stimulatory molecules and are classed as a professional antigen-presenting cell (APC). They have been shown to be the dominant naïve T cell priming APC under some circumstances. Learn more