Immunoprofiling - Insights of the individual's immune system

The immune system functions as a library, registering and logging an individual's distinct encounters with pathogens and self-antigens. Immunoprofiling involves quantifying these encounters by assessing specific attributes of an individual's immune system, including cell type, activation status, cytokines, and antibody expression. This data can provide insights into diseases, inflammatory conditions, and immune strength.

This information plays a crucial role in advancing precision medicine by enhancing diagnostic precision, facilitating early disease detection, evaluating treatment efficacy, and categorizing patients into suitable treatment protocols.  

Additionally, immunoprofiling offers valuable research prospects, enabling investigators to examine disease subpopulations or endotypes, with the ultimate aim of improving therapies.

While any measurement of immune function aids in immunoprofiling, antibodies offer a cost-effective and informative approach. Antibodies can be easily accessed through a simple blood draw and contain intricate details about an individual's disease status due to their specificity towards unique antigens.

Notably, IgG antibodies serve as robust indicators of immune responses within the body and serve as ideal biomarkers. They circulate in the bloodstream and often manifest before physical symptoms, making them valuable indicators of disease.

Functional protein microarrays can identify a patient's distinct antibody repertoire against thousands of potential antigens. Traditionally, researchers have primarily focused on IgG antibodies. However, IgG antibodies represent only one of many antibody isotypes.

Profiling multiple isotypes offers a more comprehensive understanding of patients and diseases, as each isotype provides distinct information, collectively painting a more detailed picture of the immune system's observations.

Figure 1. The Basic Structure of an Antibody. Left, General overview of antibody structure highlighting location of Fab and Fc regions. Right, 3D reconstruction illustrating antibody shape. Image Credit: Sengenics

Antibodies, which are 10 nm Y-shaped proteins produced by B lymphocytes (see Figure 1), exist in five distinct isotypes: IgG, IgM, IgA, IgD, and IgE. Additionally, IgG has four subclasses, while IgA has two subclasses.  

Certain isotypes can multimerize, such as IgM forming pentamers and IgA forming dimers. Each antibody consists of two heavy chains and two light chains, interconnected by disulfide bonds. The Fc region, located at the stem of the antibody, is comprised of two to three constant domains formed by the heavy chains.

Each isotype has a unique Fc region, which serves various functions, such as anchoring the antibody to the B cell membrane, transducing antigen-induced signaling, and acting as a ligand to Fc receptors that regulate other immune cells.  

The branches on top of the antibody, referred to as the Fab fragment, consist of heavy chains linked to a pair of smaller light chains. Positioned at the amino-terminal end of the Fab fragment are three hypervariable domains generated through somatic mutations and random recombination of gene segments.  

It is within these regions that antigen binding takes place. Unlike the Fc domain, the hypervariable regions exhibit significant diversity, resulting in trillions of possible binding configurations. Each B cell and its descendants are dedicated to producing a single type of antibody, hence termed monoclonal.

Both the Fc and Fab regions play distinct roles in immune system function. During B cell development, immature bone marrow B cells initially express membrane-bound IgM, which forms part of the B cell receptor (BCR) with the Fc region bound to the cell membrane and the Fab region exposed extracellularly to bind with circulating antigens.

CD79, a signal transduction region, associates with IgM to complete the complex. BCRs exhibiting high affinity for self-antigens undergo clonal deletion through apoptosis. Surviving B cells continue to mature in the spleen or lymph nodes, where they can express IgD or IgM transmembrane antibodies.  

Upon stimulation by foreign antigens, class switching, and clonal expansion occur. B cells congregate in germinal centers and produce elevated levels of secreted antibodies specific to the stimulating antigen (LeBien & Tedder, 2008; Mårtensson et al., 2010; Melamed et al., 1998; Tonegawa, 1983).

The expressed isotype depends on the antigen and its location within the body. For instance, B cells activated in the intestinal lumen generate circulating IgA, an antibody with the ability to function effectively in low pH environments and high peptidase activity.  

IgA also hinders pathogen entry into the intestine and is present in mucosal membranes. The most prevalent isotype, IgG, is primarily produced in response to bacteria and viruses, circulating in the lymph and blood.  

IgE triggers mast cells and eosinophils, which act in allergic reactions and safeguard against larger microbes and parasites. The exact function of IgD remains unclear; it is expressed in minimal quantities and contributes to B cell activation and class switching.  

Lastly, IgM is implicated in complement signaling and facilitates cellular immunity (Table 1). Antibodies exhibit high specificity to a particular antigen and possess diverse effector functions mediated through their Fc regions.

The Fc regions can act as ligands for Fc receptors and interact with complement to regulate the activity of other immune cells, such as neutrophils, natural killer cells, macrophages, and microglial cells, thereby recruiting and activating them.

This activation of other immune cells of the innate immune system represents a common function of IgM antibodies. As demonstrated below, the antibody isotype plays a pivotal role in immunotherapies.

Table 1. Antibody Isotypes. Source: Sengenics

Numerous organ systems are involved in complex diseases like cancer, neurodegenerative diseases, and autoimmune diseases, leading to the expression of multiple isotypes and subclasses of antibodies. Some antibodies may be present even before the target organ is affected.  

For instance, autoimmune diseases not only demonstrate autoantibodies with antigen specificity but also exhibit isotype specificity.  

While IgM and IgG are the most prevalent in almost all autoimmune diseases, antiphospholipid syndrome of the vascular system is characterized by prominent IgA autoantibodies, whereas SLE (Suurmond & Diamond, 2015) is associated with high levels of IgE.  

This additional identification of isotypes, along with antibody specificity, enhances disease detection and understanding of pathology.  

In rheumatoid arthritis, combining the quantification of IgG, IgM, and IgA isotypes with specific antigens (RF, ACPA, and RA33) has improved the identification of the disease by up to 30% (Sieghart et al., 2018).  

Understanding subclasses aids in identifying pathogens, determining disease status, and guiding therapeutic development. For instance, IgG2a antibodies are predominantly stimulated by the CNS-infecting yeast C. neoformans, while Mycobacterium tuberculosis induces higher levels of IgG3 than any other antibody (Suurmond & Diamond, 2015).

Consequently, antibody isotypes can provide valuable insights into challenging diagnostic cases.  

In the context of SARS-CoV-2, researchers at the University of Cape Town demonstrated a deficiency in anti-Spike Protein IgA production among HIV patients who were also infected with SARS-CoV-2, using a dual-color IgA and IgG protein microarray (Smith et al., 2023).

This information underscores how previous immunological experiences can modify patients' immunoprofiles, emphasizing the importance of precision medicine in guiding treatment decisions. The choice of antibody isotype is significant when designing immunotherapies.  

In vitro studies have shown that microglial cells stimulated by antibody Fc receptors exhibit increased uptake and degradation of tau and α-synuclein particles, which is an encouraging finding for the development of monoclonal antibody therapies for neurodegenerative diseases involving protein aggregates like Parkinson's, Alzheimer's, and Huntington's.  

However, IgG1 antibodies have a higher likelihood of inducing neuroinflammation and adverse events compared to IgG4 antibodies, which do not react with complement (Katsinelos et al., 2019).

In this scenario, immunoprofiling could aid in determining the most suitable antibody subtype for safety and efficacy purposes. Traditionally, isotype screening has been conducted using immunochemistry and ELISA technology, along with isotype-specific secondary antibodies.

However, ELISA's throughput and sensitivity fall short compared to modern protein microarrays. Functional protein microarrays, which offer high throughput capabilities, have emerged as a superior option for quickly profiling the diverse range of antibodies found in an individual's serum.

Until recently, array assays detected IgG, the predominant antibody in serum. Sengenics provides a validated dual color detection analysis assay of exceptional quality. This assay enables the simultaneous identification of specific IgG and IgA antibodies from a small patient sample.

Sengenics' patented KREX technology ensures that only correctly folded proteins with their intact discontinuous epitopes are present on the array. Thus, highly specific antibody-antigen binding is achieved, a critical factor in dual isotype profiling. This is crucial as non-specific binding could compromise the accuracy of the results.

By leveraging this technology, individuals exposed to SARS-CoV-2 were successfully profiled. The findings indicated that healthy vaccinated individuals produced both IgG and IgA anti-spike antibodies. In contrast, HIV patients did not exhibit anti-spike IgA production (Smith et al., 2023).

This information can play a significant role in directing HIV patient care. Incorporating antibody isotypes alongside specificity introduces unique data to antibody immunoprofiling. This approach has the potential to unveil novel, high-resolution biomarker signatures that might be missed when focusing solely on a single isotype.

A dual-color detection assay greatly enhances diagnostic potential, provides a deeper understanding of endotyping, improves predictions of adverse outcomes, and uncovers new disease-related pathways.

References and further reading

1. Katsinelos, T., Tuck, B. J., Mukadam, A. S., & McEwan, W. A. (2019). The Role of Antibodies and Their Receptors in Protection Against Ordered Protein Assembly in Neurodegeneration. Front Immunol, 10, 1139. https://doi.org/10.3389/fimmu.2019.01139  
2. Sieghart, D., Platzer, A., Studenic, P., Alasti, F., Grundhuber, M., Swiniarski, S., Horn, T., Haslacher, H., Bluml, S., Smolen, J., & Steiner, G. (2018). Determination of Autoantibody Isotypes Increases the Sensitivity of Serodiagnostics in Rheumatoid Arthritis. Front Immunol, 9, 876. https://doi.org/10.3389/fimmu.2018.00876  
3. Smith, M., Kwatra, G., Izu, A., Nel, A., Cutland, C., Ahmed, K., Baillie, V., Barnabas, S., Bhorat, Q., Briner, C., Lazarus, E., Dheda, K., Fairlie, L., Koen, A., Madhi, S., & Blackburn, J. (2023). Longitudinal IgA and IgG Response, and ACE2 Binding Blockade, to Full-Length SARS-CoV-2 Spike Protein Variants in a Population of Black PLWH Vaccinated with ChAdOx1 nCoV-19. Viruses, 15(448), 11. https://doi.org/10.3390/v15020448  
4. Suurmond, J., & Diamond, B. (2015). Autoantibodies in systemic autoimmune diseases: specificity and pathogenicity. J Clin Invest, 125(6), 2194-2202. https://doi.org/10.1172/JCI78084

About Sengenics

Sengenics is an immunoproteomics company working to improve patient outcomes through physiologically relevant, data-guided decision making. Our solutions enable the discovery and validation of autoantibody biomarker signatures for patient stratification, therapeutic response prediction and elucidation of disease mechanisms.

The company has a global footprint with multiple corporate and research sites across the world with customers and collaborators that include top pharma, biotech and ivy league academic institutions in North America, Europe, and Asia.

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